3, summary of the invention
The present invention is by the surprised discovery of a large amount of experiments, after paclitaxel and phenylalanine make up with certain proportion, solved the problem of its water solublity and stability, side effect also reduces accordingly, make things convenient for clinical application, and add phenylalanine and can play synergistic function, more can strengthen active anticancer with paclitaxel than single.
The purpose of this invention is to provide the compositions that contains paclitaxel and phenylalanine, said composition strengthens active anticancer and reduces side effect.
Another object of the present invention provides the injection of the compositions that contains paclitaxel and phenylalanine, and the water solublity of this injection and good stability can be stored the long period.
A further object of the present invention provides the preparation method of the injection of the compositions that contains paclitaxel and phenylalanine, and the injection preparation cost of the paclitaxel that this method makes and the compositions of phenylalanine is low, quality controllable.
An also purpose of the present invention provides the application of compositions in the preparation cancer therapy drug that contains paclitaxel and phenylalanine.
The invention provides the composition and method of making the same that contains paclitaxel and phenylalanine, technical scheme is as follows:
The compositions that contains paclitaxel and phenylalanine provided by the invention is characterized in that, the weight ratio of described paclitaxel and phenylalanine is 1: 0.01~0.8.
The described compositions that contains paclitaxel and phenylalanine is characterized in that, the weight ratio of paclitaxel and phenylalanine is preferably 1: 0.05~and 0.5.
The described compositions that contains paclitaxel and phenylalanine is characterized in that, the weight ratio of paclitaxel and phenylalanine more preferably 1: 0.1~0.3.
The described compositions that contains paclitaxel and phenylalanine is characterized in that, a step is preferably 1: 0.2 to the weight ratio of paclitaxel and phenylalanine again.
The described compositions that contains paclitaxel and phenylalanine is characterized in that, the compositions of paclitaxel and phenylalanine is an injection.
The described injection that contains the compositions of paclitaxel and phenylalanine is characterized in that, the solubilizing agent in the injection is a polyoxyethylene castor oil, and solvent is a dehydrated alcohol.
The described injection that contains the compositions of paclitaxel and phenylalanine is characterized in that, the volume ratio of polyoxyethylene castor oil and dehydrated alcohol is 1: 2.
The described injection that contains the compositions of paclitaxel and phenylalanine is characterized in that, the preparation method that contains the injection of paclitaxel and phenylalanine compositions is:
(1) join auxilliary liquid: polyoxyethylene castor oil adds exsiccant molecular sieve, fills nitrogen, seal three days standby; Be sealed in the exsiccator standby after the active carbon activation; It is transparent that molecular sieve is washed till ethanol with dehydrated alcohol, flings to ethanol, drying, cool be sealed in the exsiccator standby; Polyoxyethylene castor oil, ethanol and active carbon are mixed (rustless steel container), ultrasonic agitation.
(2) the auxilliary aseptic filter pressing of liquid: with the aseptic filter pressing of microporous filter membrane, weigh, calculating need be added the dehydrated alcohol amount;
(3) molten principal agent: principal agent is at the vacuum drying oven inner drying, and precision takes by weighing principal agent, joins in the filtrate of step (2), adds dehydrated alcohol flushing, fills high pure nitrogen, sealing, and jog ultrasonicly dissolves fully to sample;
(4) the aseptic filter pressing of solution: solution is ultrasonic, with the aseptic filter pressing of microporous filter membrane;
(5) packing and sealing: liquor separator is cleaned, oven dry; Just filtrate is regulated loading amount, and the rim pure nitrogen gas of leaping high is divided on the limit; Seal rapidly after the packing, promptly.
The described compositions that contains paclitaxel and phenylalanine is characterized in that the application in the preparation cancer therapy drug.
The paclitaxel that contains of the present invention is compared with immediate existing close technology with the composition and method of making the same of phenylalanine, has the following advantages:
The present invention has improved the water solublity of paclitaxel by adding phenylalanine, makes things convenient for clinical application;
Greatly reduce side effect such as allergy, simultaneously, also increased its active anticancer;
The present invention improves prepared paclitaxel injection stability by the improvement to paclitaxel prescription and injection technology, and simultaneously, products obtained therefrom is rapid-action;
Dose controlled, good appearance;
Processing technology is simple, and use is reliable, convenient, safe and practical, can reduce production costs, and is applicable to the realization suitability for industrialized production.
Below example is further set forth the beneficial effect of the compositions of paclitaxel of the present invention and phenylalanine by experiment, but this should be interpreted as that the compositions of paclitaxel of the present invention and phenylalanine only has following beneficial effect.
The proportioning of experimental example 1 etoh solvent and solubilizing agent polyoxyethylene castor oil and stable screening experiment
Whether stable to investigate solution at low temperatures after the ethanol volume ratio mixing different with polyoxyethylene castor oil.
1, experimental technique
Polyoxyethylene castor oil adds the exsiccant molecular sieve of 17.5% (m/v), fill nitrogen, the sealing three days standby.Active carbon is sealed in the exsiccator standby 121 ℃ of activation 2 hours.It is transparent that molecular sieve is washed till ethanol with dehydrated alcohol, flings to ethanol, 150 ℃ of dryings 5 hours, cool be sealed in the exsiccator standby.
The polyoxyethylene castor oil that drying is good and ethanol than mixing, are tested stability of solution at low temperatures by different volumes.
2, experimental result and conclusion
The proportioning of table 1 ethanol and polyoxyethylene castor oil and stable screening experiment
Conclusion: above test shows, ethanol and polyoxyethylene castor oil volume ratio be 2: 1 o'clock the most stable, liquid viscosity is suitable, is easy to filter, and helps producing.
Conditional filtering among experimental example 2 preparation technologies
Prescription and technological basis the analysis showed that preparation technology mainly contains two key conditions: dry, fill nitrogen.Drying mainly is insoluble in water because of principal agent, and solubilizing agent and water unmixing.Fill nitrogen and mainly prevent the oxidation of principal agent and the corruption of polyoxyethylene castor oil.
1, experimental technique
Prepare three kinds of preparations, relatively polyoxyethylene castor oil is being carried out dried and the be untreated outward appearance of prepared paclitaxel injection and its related substances of filling nitrogen and not filling the preparation for preparing under the nitrogen condition respectively.
Preparation one: according to the preparation preparation of mentioning in " preparation method ";
Preparation two: in " joining auxilliary liquid " process, polyoxyethylene castor oil is without molecular sieve drying, and other parts are according to the preparation preparation of mentioning in " preparation method " two;
Preparation three: in " joining auxilliary liquid " process, polyoxyethylene castor oil does not fill nitrogen after adding molecular sieve drying, and other parts are according to the preparation preparation of mentioning in " preparation method " three;
2, experimental result and conclusion
(1) dried
Polyoxyethylene castor oil is carried out dried and the paclitaxel injection preparation one that is untreated prepared and the outward appearance of preparation two investigate, the result is as follows:
The injection of the table 2 polyoxyethylene castor oil dried and the preparation of being untreated relatively
The selection result shows, polyoxyethylene castor oil is carried out dried after, with the comparison of being untreated, no matter place 5 or 10 days, solution appearance all shows clarification.
(2) fill nitrogen
Carry out related substance investigation (10 days) to filling nitrogen with preparation one that does not fill nitrogen and preparation three, the result is as follows:
Table 3 fills nitrogen and compares with the preparation that does not fill nitrogen
The selection result shows, preparation fills nitrogen to be placed 10 days, with do not fill nitrogen place 10 days preparation relatively, 1% contrast area, assorted peak area and its related substances all are significantly less than does not fill the preparation that nitrogen is handled.
Conclusion: The selection result shows that reply polyoxyethylene castor oil preparation process is carried out the intensive drying processing, simultaneously solution is filled nitrogen, to guarantee stability of formulation.
Experimental example 3 formulation and technology screening experiment
1, experimental technique
According to above-mentioned design principle, press the different prescriptions of table 1 preparation.Research paclitaxel related substance, content, medicinal liquid pH and change in color situation.
Table 4 paclitaxel injection prescription screening
2, experimental result and conclusion
Get the solution of aseptic filter pressing, pH and the clarity and the color of the related substance of mensuration paclitaxel, content, medicinal liquid.The results are shown in Table 5.
Table 5 paclitaxel injection prescription screening testing result
Conclusion: by above A, B, C, the D screening of side everywhere, learn that prescription A stickiness height is difficult for filter pressing and unstable at low temperatures, the solution of prescription B, C and D all has ideal physical stability and chemical stability, can be as the prescription of this preparation, because prescription D design less expensive, therefore, with prescription D be the first-selection prescription of this product.
Experimental example 4 influence factors experiment
The injection for preparing the present composition according to the preparation method of mentioning in the patent.Select the injection (lot number 0306011) of one group of present composition to place under the condition of illumination (illumination 4500Lx), high temperature (60 ℃) and low temperature (4 ℃) arbitrarily, the sampling respectively in 5,10 days, investigate character, clarity, pH value, related substance and content, measurement result and 0 month numeric ratio are, investigate prescription and the adaptability of technology and the influence of packaging material, the results are shown in Table 6.
Table 6 influence factor experiment
Conclusion: from table data as can be seen, compositions of the present invention places measurement result under illumination, high temperature and the cryogenic condition and 0 month numeric ratio, its character, pH value and content all do not have obvious change, clarity and related substance are all up to specification.Result of the test shows that this prescription and Technological adaptability are good, stablizes feasiblely, and the packaging material ampoule does not have influence to the stability of principal agent.
The experiment of experimental example 5 compatibilities
The injection of paclitaxel and phenylalanine preparation of compositions and 5% glucose injection and 0.9% sodium chloride injection compatibility are tested its stability and whether are had incompatibility.
The injection of table 7 present composition and the test of 5% glucose injection compatibility
The injection of table 8 present composition and the test of 0.9% sodium chloride injection compatibility
Conclusion: result of the test shows that behind this product and 5% glucose injection or the 0.9% sodium chloride injection compatibility, outward appearance, clarity, pH value and peak area all do not have significant change, and ie in solution is stable in 12 hours, no incompatibility.
Experimental example 6 solubility experiments
This experimental example is to utilize prepared injection of the present composition and commercially available similar drug taxol to carry out dissolubility comparative study.
1, medicine
The injection of the compositions of paclitaxel of the present invention and phenylalanine adopts the proportioning preparation of prescription 6;
Taxol: commercial (30mg/ props up).
2, method
The present composition and taxol are dissolved in respectively in the 5mL water, and sonic oscillation a period of time filters to no longer including the solid dissolving, and the gained solid drying is weighed, and can approximately learn the solubility values of the present composition and taxol.
3, result
The dissolubility of taxol is 1.2mg/mL, and the dissolubility of the present composition is 3.3mg/mL.Experimental result shows that the dissolubility of the present composition is 2.75 times of taxol, has promptly obviously improved water solublity.
Experimental example 7 inside and outside antitumor comparative experimentss
Material and medicine
Medicine: the present composition: with 6 the proportioning self-control of writing out a prescription;
Taxol: commercial.
Animal: ICR strain white mice, 18~22g, male and female half and half.
Tumor strain: tumor-bearing mice S
180, tumor strain oophoroma cell AO
10/17
Instrument: Olympus microscope BHT (Japan makes); PB303 electronic balance (Switzerland's manufacturing); HL-2920P enzyme mark automatic analyzer (Chinese 262 factories make); SHELLAB-2300 CO2 gas incubator (U.S. SHELDON manufacturing).
1,
The extracorporeal anti-tumor comparative experiments-to ovarian cancer cell strain AO 10/17 Cytotoxicity
1.1, method
The employing mtt assay detects.Get tumor strain AO
10/17, regularly go down to posterity in containing the complete RPMI1640 culture fluid of 10% calf serum in the recovery back, treat that cell grows to increased logarithmic phase, with pancreatin EDTA digestion, Hank ' s liquid is washed twice, be suspended in the RPMI1640 culture fluid of 10% calf serum, adjust cell concentration 0.3 * 10
6/ mL gets the flat culture plate in 96 holes, and it is stand-by that every hole adds above-mentioned cell suspension 100 μ L.Solvent control group, taxol group and present composition group are established in experiment.The present composition and taxol divide 6 concentration, all with the initial concentration two-fold dilution of 46.86 μ L/mg.Its concentration is respectively 46.86,23.43,11.72,5.86,2.93,1.47 μ L/mg, and each dosage is established the solvent control under the concentration of the same race.Add for the every hole of culture plate by above-mentioned dosage and tried thing 100 μ L, select 6 holes else and add 100 μ L fresh mediums as background, each dosage is made 3 parallel samples.Under 37 ℃, 5% carbon dioxide conditions, act on 4h, form the hyacinthine crystallization, abandon supernatant, wash twice with fresh complete culture solution, add the lysigenous navy blue granule of 100 μ L/ hole DMSO,, under the 570nm wavelength, read optical density (OD) with HL-2920P enzyme mark automatic analyzer.According to following formula, calculate and kill and wound percentage rate.
Kill and wound percentage rate=(1-tests optical density/bias light density) * 100
Parallel testing 3 times.
1.2, result and conclusion
The present composition and taxol are to AO
10/17The cytotoxicity of tumor strain sees Table 9.
Table 9 present composition and taxol are to AO
10/17The cytotoxicity of tumor strain
The ratio of outflow extremely of the present composition and taxol itself is the ratio of outflow that kills that ratio of outflow deducts corresponding solvent respectively that kills of the present composition and taxol, the results are shown in Table 10.
The ratio of outflow extremely of table 10 present composition and taxol
Conclusion: from table data as seen, 3 times result of the test all shows, the present composition itself kill ratio of outflow than taxol itself to kill ratio of outflow high about 2 times, promptly the present composition is better active.
2, anti-tumor in vivo comparative test-to the influence of mice S180 solid tumor
2.1, method
Get the S of inoculation 7~10d
1801 of solid tumor mice is got the tumor piece under the sterile working, the tumor body is cut into small pieces, and grinds with glass homogenizer, adds the tumor cell suspension that sterile saline is diluted to 1: 4.Other gets 60 of ICR mices, through right side axillary fossa Sc inoculation liquid 0.2mL/ only.The inoculation back is divided into 6 groups next day at random, and 10 every group, male and female half and half.Divide physiology saline negative group, solvent control group, present composition group and taxol group, quiet notes, once a day, the 0.2mL/10g body weight continuous 4 days, was put to death each treated animal on the 9th day in the inoculation back, weigh, separation tumor body is also weighed, and calculates the tumor body than (the contained tumor of every 100g body weight is heavily asked tumour inhibiting rate.Following formula is pressed in therapeutic evaluation):
Tumor control rate=(the average tumor of the average tumor weight-administration of matched group group is heavy)/average tumor of matched group heavy * 100
Parallel testing 3 times.
2.2, result and conclusion
The present composition and taxol are to S
180The experimental result of the influence of tumor strain sees Table 11.
Table 11 present composition and taxol are to S
180The influence of tumor strain
(mean±s)
Conclusion: the present composition and taxol are to mice S
180The tumor body weight of solid tumor all has inhibitory action, and 3 experiments all show, active obviously active good than taxol of the present composition.
4, the specific embodiment
With the specific embodiment of experimental example form, foregoing of the present invention is described in further detail by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following examples.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The preparation of the composite injection of embodiment paclitaxel of the present invention and phenylalanine
1, prescription
Prescription 1:
Paclitaxel 12g
Phenylalanine 0.6g
Polyoxyethylene castor oil 671g
Dehydrated alcohol 1072g
Full dose 2000mL
Make 1000 altogether
Prescription 2:
Paclitaxel 12g
Phenylalanine 2.4g
Polyoxyethylene castor oil 670g
Dehydrated alcohol 1072g
Full dose 2000mL
Make 1000 altogether
Prescription 3:
Paclitaxel 12g
Phenylalanine 6g
Polyoxyethylene castor oil 666g
Dehydrated alcohol 1072g
Full dose 2000mL
Make 1000 altogether
Prescription 4:
Paclitaxel 30g
Phenylalanine 1.5g
Polyoxyethylene castor oil 1679g
Dehydrated alcohol 2680g
Full dose 5000mL
Make 1000 altogether
Prescription 5:
Paclitaxel 30g
Phenylalanine-3,4-quinone g
Polyoxyethylene castor oil 1677g
Dehydrated alcohol 2680g
Full dose 5000mL
Make 1000 altogether
Prescription 6:
Paclitaxel 30g
Phenylalanine 6g
Polyoxyethylene castor oil 1674g
Dehydrated alcohol 2680g
Full dose 5000mL
Make 1000 altogether
Prescription 7:
Paclitaxel 30g
Phenylalanine 9g
Polyoxyethylene castor oil 1671g
Dehydrated alcohol 2680g
Full dose 5000mL
Make 1000 altogether
Prescription 8:
Paclitaxel 30g
Phenylalanine 15g
Polyoxyethylene castor oil 1665g
Dehydrated alcohol 2680g
Full dose 5000mL
Make 1000 altogether
Prescription 9:
Paclitaxel 60g
Phenylalanine-3,4-quinone g
Polyoxyethylene castor oil 3357g
Dehydrated alcohol 5360g
Full dose 10000mL
Make 1000 altogether
Prescription 10:
Paclitaxel 60g
Phenylalanine 6g
Polyoxyethylene castor oil 3354g
Dehydrated alcohol 5360g
Full dose 10000mL
Make 1000 altogether
Prescription 11:
Paclitaxel 60g
Phenylalanine 12g
Polyoxyethylene castor oil 3348g
Dehydrated alcohol 5360g
Full dose 10000mL
Make 1000 altogether
Prescription 12:
Paclitaxel 60g
Phenylalanine 18g
Polyoxyethylene castor oil 3342g
Dehydrated alcohol 5360g
Full dose 10000mL
Make 1000 altogether
Prescription 13:
Paclitaxel 60g
Phenylalanine-3,4-quinone 0g
Polyoxyethylene castor oil 3330g
Dehydrated alcohol 5360g
Full dose 10000mL
Make 1000 altogether
2, preparation method
(1) join auxilliary liquid: polyoxyethylene castor oil adds the exsiccant molecular sieve of 17.5% (m/v), fill nitrogen, the sealing three days standby.Active carbon is sealed in the exsiccator standby 121 ℃ of activation 2 hours.It is transparent that molecular sieve is washed till ethanol with dehydrated alcohol, flings to ethanol, 150 ℃ of dryings 5 hours, cool be sealed in the exsiccator standby.
Polyoxyethylene castor oil, ethanol and 0.05% active carbon are mixed (rustless steel container), ultrasonic agitation 30 minutes (the ultrasonic water bath temperature is no more than 45 ℃, promptly takes out stirring as heat, falls cold continuation again).
(2) the auxilliary aseptic filter pressing of liquid: with the aseptic filter pressing of 2 0.22 μ m microporous filter membrane (nitrogen pot pressure between 0.15-0.3mpa, the filtrate receiving vessel is should be transparent visual and bore is little, with the minimizing solvent evaporates), weigh, calculating need be added the dehydrated alcohol amount.
(3) molten principal agent: principal agent is at vacuum drying oven inner drying 24 hours (vacuum 0.09, temperature 42-45 ℃), and precision takes by weighing principal agent, add in the filtrate of step (2), add the dehydrated alcohol flushing, fill high pure nitrogen, sealing, jog ultrasonicly dissolves (estimating no granule) fully to sample.
(4) the aseptic filter pressing of solution: with ultrasonic 10 minutes of solution, with the aseptic filter pressing of 2 0.22 μ m microporous filter membrane, that receiving vessel is wanted was clean (can not residual alkali liquor, 180 ℃ dry heat sterilization three hours).
(5) packing: the liquor separator wash clean, use earlier the 70-75% soak with ethanol, the flushing of reuse dehydrated alcohol, 100 ℃ of oven dry.Just filtrate is regulated loading amount, and the rim pure nitrogen gas of leaping high is divided on the limit, and pressure is at 0.01-0.05mpa.
(6) seal: seal rapidly, in case nitrogen overflows, promptly.