CN1973826A - Injection containing lipoid microsphere of etoposide and its prepn process - Google Patents
Injection containing lipoid microsphere of etoposide and its prepn process Download PDFInfo
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Abstract
The present invention belongs to the field of medicine technology, and is especially lipoid microsphere injection containing etoposide and its preparation process. The injection contains etoposide, fat soluble medium, water and surfactant; and consists of oil phase 5-30 wt%, etoposide 0.001-0.5 wt%, surfactant 0.5-5 wt%, osmotic regulator 0.5-5 wt%, and water for injection the rest. Etoposide is high pressure homogenized under the action of high speed airflow to form ultrasonic stirring, so as to be dissolved fast and permeated in molecular form into oil/water interface film. By means of medicine carrying interface film principle, the present invention raises the medicine carrying amount of insoluble etoposide and the stability while lowering the toxicity and blood vessel irritation. The preparation of the present invention has low toxicity, low irritation and high curative effect in clinical application.
Description
Technical field
The invention belongs to medical technical field, relate in particular to a kind of preparation technology and prescription that contains the lipide microsphere injection (oil in water emulsion) of etoposide.
Background technology
Cancer is human to be difficult to most to cure and one of common disease, captures this obstinate fort of cancer, promote the well-being of mankind, and be extremely urgent key subjects on the our times.At present, the cancer patient increases year by year, and the mortality rate of cancer is in continuous rising, the China recent years statistics, and annual nearly 800,000 people die from cancer, and the average per minute in the whole nation has 1.5 people to die from cancer.The progress of a host of facts proof treatment of cancer is very little, the threat mankind's that cancer is more and more serious life.So seek anticancer new drug, change dosage form and improve its anticancer curative effect, reduce its toxic and side effects, become the task of top priority.
Etoposide (etoposide, 4-demethyl epipodophyllotoxin-β-D-second pyranglucoside, be called for short Vp16-213) be the carbohydrate metabolism product of podophyllotoxin, belong to the cell cycle specific antitumor drug, has the active and quite high therapeutic index of broad spectrum anticancer, especially respond well to the abominable minicell cancer of early stage diffusion, prognosis, be the strongest activated monomer of treatment small cell carcinoma and carcinoma of testis.Etoposide is by suppressing the activity performance Graft Versus Tumor of mammal DNA topoisomerase II, it is the embedded type inhibitor of topoisomerase, it acts on the DNA topological enzyme, make the fracture of strand or double-stranded DNA, double-stranded DNA fracture number is 2～3 times of single-strand break number, and this shows that cytotoxic mechanism is the mechanism of action of this medicine, this makes the toxic and side effects of this medicine also very big, its main toxicity is the minimizing of bone marrow depression neutrophil, and this medicine dissolubility in water is minimum, awaits improving dosage form.
The dosage form that etoposide is commonly used clinically is injection and oral soft capsule, and dosage is every 50-100mg and every 50-100mg.The dosage of vascular drug delivery is every 3-5 days 300-600mg/m
2(being equivalent to body weight is the adult 450-900mg of 70kg).At present, the injection type of Shi Yonging belongs to the non-aqueous solution system clinically, and is general earlier with the normal saline dilution during intravenously administrable, and unstable in 5% Glucose Liquid, can form fine precipitation, and injection blood vessel place causes stronger local excitation.The application of oral formulations is limited to non-critical patient, its absolute bioavailability is low, have only 50%, blood level only is intravenous 52 ± 8%, oral administration biaavailability widely different (25%-74%) between the different patients, its main cause are the poorly water-solubles of etoposide and are decomposed into the non-activity product in acid and alkaline mediums.In the aqueous solution of pH2-6, dissolubility only is 148.5-167.25 μ g/ml in the water that etoposide is 37 ℃.And the chemistry of aqueous solution poor stability of etoposide, in the solution of pH1.3 and 10, etoposide is degraded rapidly, and degradation half life was respectively 2.88 and 3.83 hours; In the aqueous solution of pH7.30, degradation half life is 27.72 days; Scope at pH5-6.15 is the most stable relatively, and degradation half life was respectively 63 days and 49.5 days.The untoward reaction of etoposide clinical practice mainly is that bone marrow depression is more obvious, and the fastest 2～3 weeks could recover, and can produce digestive tract reaction and alopecia symptom, postural hypotension may occur when instiling fast.
In view of above-mentioned characteristic, bring into play clinical efficacy better for making etoposide, improve the stability of etoposide, the research and development novel form, advantage efficient, low toxicity with novel form is given full play to the etoposide anti-tumor activity and is reduced toxic and side effects, and this undoubtedly will be significant to further promotion and the application of popularization etoposide in clinical.
Lipomul is applied to clinical nearly half a century as the important supply mode of the outer energy of intestinal.Between 1920～nineteen thirty, Japanese scholar was that raw material, lecithin are emulsifying agent with Oleum Ricini once, and at first the synthetic lipomul is tried out in animal; The U.S. has released Lipomul based on cotton seed oil the fifties and has been applied to patient but all fails to promote because of serious toxicity.1964, the U.S. stopped to produce and using lipomul.But in Europe, with Wretling be a collection of scholar of representative to study untiringly and use based on soybean oil, with the lecithin be emulsifying agent, be the lipomul Introlipid of isotonic agent with glycerol.1962, Introlipid was used for clinical in Sweden by official approval.1967, article " lipomul the is applied to complete intravenous nutrition " comprehensive summing up of delivering by Hallberg etc. experiment and the clinical research of Introlipid, prove that it is applied to clinical is safe and reliable, and proposed the criterion of Introlipid clinical practice." obtaining growth, growth and positive nitrogen balance under the long-term complete parenteral nutrition " literary composition that this literary composition and nineteen sixty-eight Dudrick etc. deliver is acknowledged as parenteral nutrition is developed into two pieces of classical works that routine clinical is used significant impact.After this more than 20 year, Introlipid obtained extensive use all over the world, and developed countries such as moral, method, Japan and the United States have developed national lipomul in succession.1976, U.S. appropriate authorities have ratified lipomul again can be in routine clinical application.At present, kind of a lipomul appeared on the market surplus the whole world had 20 at least.China is also joint the end of the eighties and Sweden Kabi company, and mass production Introlipid is for clinical practice.1973, that Solassol etc. have at first introduced is three-in-one, and (All-in-one, the AIO) notion of solution proved that lipomul and all other nutritional solutions can be mixed in the bottle or in the bag, can keep stable and nutritional support effect at certain condition with in the time.After this promoted AIO transfusion plastic bag, the preparation and the infusion of parenteral nutrition liquid simplified in this simple but relevant improvement greatly, for the wide clinical application of lipomul has been widened road.Up to the present, it is oil-in-water type (oil in water, the o/w) submicronized emulsion of emulsifying agent as decentralized photo with the Ovum Gallus domesticus Flavus lecithin that common lipomul is meant with fatty glyceride.Because the Chylomicron that exists behind the composition of lipomul emulsion droplet and particle diameter and the oral fat group food is very similar, therefore it has been generally acknowledged that the emulsion droplet of lipomul is also similar with Chylomicron in the intravital behavior of people in blood.Along with the further investigation to lipomul, it is also just bringing into play more and more important effect at pharmaceutical field.
The special physicochemical property and the hypotoxicity of lipomul have determined it to can be used as fat-soluble medicine, particularly the good carrier of cancer therapy drug, anaesthetic and anti-inflammatory drug.The normal method that adopts is the lipid core part that pharmaceutical pack is wrapped in lipomul because this structure also is similar to microsphere, so lipid microsphere (Lipid Microsphere, title LM) is also arisen at the historic moment.It is generally acknowledged that lipid microsphere is by medicine is dissolved in the fatty oil, and after phospholipid emulsifying is scattered in water, make, be a kind of be soft substrate and the microparticulate system sealed by immobilized artificial membrane with fatty oil, mean diameter is about 200nm.
LM has many physical chemistry and advantage biologically:
1. be the good carrier of fat-soluble medicine.Clinical many medicine poorly water-solubles must rely on the effect of organic solvent competence exertion, and organic solvent not only itself have certain toxicity, also may the interference medicament effect.
2. can effectively increase stability of drug.In the pastille lipid microsphere, quite a few drug distribution is arranged in oil phase or oil-water interfacial film, avoided directly contacting with water.Change responsive medicine for facile hydrolysis or to pH, this " isolation " played the effect that increases stability.
3.LM Chinese medicine partly is wrapped in oil phase or the interfacial film, has avoided contacting with the direct of body fluid, thereby has reduced issuable part of medicine self and blood vessel irritation.In addition, medicine is by slowly discharge the untoward reaction that can avoid medicine to cause owing to the initial stage excessive concentration in the oil phase when injection in vivo.
4. the small particle of particle diameter about 200nm can be engulfed by the phagocyte of the reticular tissue system of body and be trapped in the reticular tissue system (as liver, lung etc.), have targeting, this characteristics for antitumor drug improve drug effect, to reduce toxic and side effects particularly important.
People's such as Takahashi relevant bibliographical information: manufacture experimently into LM with bleomycin, and compare, test with tumor-bearing rat with water preparation.Carry out administration in vein or the tumor, measure tumor size and tumor Chinese medicine concentration then respectively, the result shows: the tumor of LM group is dwindled more than 50%, still can detect medicine in 10 hours after the administration; And the tumor of water preparation group do not have and obviously dwindles, and can not detect medicine after 30 minutes to 1 hour.The above dosage form difference of explanation medicine, effect is also different, has shown that LM has the advantages that selectivity distributes and slowly discharges, and all is suitable carrier dosage form to the disease and the cancer chemotherapy of liver, lymphoid tissue system.Studies have shown that this characteristics for antitumor drug improve drug effect, to reduce toxic and side effects particularly important.
In recent years, be subject to people's attention gradually as the targeting transferance of pharmaceutical carrier with LM.The medicine lipomul is once exploitation, and good benefit has been received in just extensive use clinically.A series of medicine carrying lipomul Products Development listings such as the Lipo PGE1 of the Yutaka Mizushima development of Japan and Lipo PGI2 have indicated this type of preparation wide prospect.Especially the application of sophisticated Emulsion production technology and synthetic or natural surfactant and the raising of Emulsion production technology have been attained, promoted to have the development (stable phase is approximately 2 years) of the Emulsion product of commercial value, stable topography, for the Development and Production of pastille fat milk has been created better condition.As rely on a meter ester, propofol, dexamethasone palmitate lipomul, flurbiprofen ethyoxyl α-ethyl ester liplid emulsions, PGE1 lipid breast etc.
Summary of the invention
The invention provides a kind of lipide microsphere injection (compositions of oil-in-water emulsion) that contains etoposide.And provide preparation method.Be intended to improve the stability of etoposide, clinical use convenience, improve the patient compliance, reduce its zest to blood vessel, better bring into play clinical efficacy.
Said composition comprises etoposide, fat-soluble medium, water and surfactant.
Use the acceptable material of term " oil " indication one big class physiology herein, be mineral oil, vegetable oil, animal oil, quintessence oil or artificial oil, or its mixture.Therefore, use term " oil " in order to refer to the very material of different chemical character that has of a wide region herein.With type or functional classification oil the time, as mineral oil source from oil and comprise fat or the cerul hydrocarbon, aromatic hydrocarbon or blended fat and aromatic radical hydrocarbon.The oil such as the refined paraffin wax wet goods that in the mineral oil classification, also comprise petroleum derivation.In the vegetable oil classification, oil is mainly derived from seed or nut, and comprises drying oil such as Semen Lini and Oleum Verniciae fordii; Semi-drying oil such as safflower oil and soybean oil; Non-drying oil such as Oleum Ricini, Oleum Gossypii semen and Oleum Cocois and the soap stock that can be used as such as Petiolus Trachycarpi oil and Oleum Cocois.In the animal oil classification, oil is usually from as sebum, Adeps Sus domestica and stearic fat.Aqueous animal oil comprises fish oil, oleic acid, spermaceti wet goods.They contain abundant fatty acid usually.Comprise some vegetable oil, as olive oil, Oleum Gossypii semen, corn oil and Oleum Arachidis hypogaeae semen also comprise some special fish oil, and they are used as medicine widely owing to be rich in vitamin, as the morrhua liver, shark liver oil etc.Aqueous fatty oil such as single, double, triglyceride, or its mixture is preferred oil.According to the present invention, the triglyceride of medium chain also are useful oil.Be preferably long-chain fat acid glyceride, medium chain length fatty acid triglyceride, and composition thereof.
Used surfactant can be any surfactant, is generally phospholipid, tween (Tween), pluronic (poloxamer), enuatrol, oleic acid, cholic acid, deoxycholic acid and composition thereof.Described phospholipid is selected from lecithin, fabaceous lecithin, and composition thereof.Described tween is selected from polysorbas20, polysorbate40, and polysorbate60, Tween 80, polysorbate85, and composition thereof.Be preferably lecithin, fabaceous lecithin, enuatrol, oleic acid, Tween 80, pluronic F68, and composition thereof.Selected osmotic pressure regulator is the salt that glycerol, ethylene glycol, isopropyl alcohol and sodium chloride etc. pharmaceutically allow.
Typically, consist of in quality of the pharmaceutical preparations prescription of the present invention:
Etoposide 0.001%～0.5%
Surfactant 0.5%～5%
Osmotic pressure regulator 0.5%～5%
All the other are water for injection
If desired, also can add multiple additives in the compositions.As containing metal-chelator.The common metal chelating agen is disodium edetate (disodium EDTA), sodium calcium edetate (calcium disodium edathamil salt), and composition thereof.Metal-chelator is 0% to 1% in quality of the pharmaceutical preparations consumption.Also can contain antioxidant, general antioxidant is vitamin E, vitamin C, sodium sulfite, sodium pyrosulfite, cysteine, the antioxidant that one or more Mixtards are commonly used.Antioxidant is 0.001% to 0.2% in quality of the pharmaceutical preparations consumption.The pH regulator agent is a sodium chloride, sodium hydroxide, and commonly used buffer salt etc., one or more mix the pH regulator agent.The pH regulator agent is 0.005% to 2% in quality of the pharmaceutical preparations consumption.
The present invention also relates to contain the preparation method of the lipide microsphere injection (oil in water emulsion) of etoposide.Specifically comprise one or more of following operating procedure:
1. etoposide solution or powder are added to the blank Emulsion that do not contain etoposide through mixed etoposide lipide microsphere injection and oil in water emulsion.
2. or with etoposide solution or powder be added in the Emulsion that contains the part etoposide through mixed etoposide lipide microsphere injection and the oil in water emulsion of getting.
3. or with etoposide be added in the water-soluble medium that contains surfactant, mix with oil phase then, prepare colostrum through high-speed stirred.
4. or with etoposide solution or powder be added in the oil-soluble medium that contains surfactant, mixed with water then, prepare colostrum through high-speed stirred.
5. in view of the chemistry of aqueous solution poor stability of etoposide, preparation process should be controlled aqueous pH values and remain between the 4-8.
6. the preparation method of preparation mainly comprises high-speed stirred process and high pressure homogenize process or ultra-sonic dispersion and emulsion process.
7. steam rotation sterilization is adopted in the sterilization of preparation.
Etoposide is the powder of ultramicro powder suspension or ultra micro.
The principle of preparation application interface film medicine carrying of the present invention is wrapped in etoposide in oil phase and the interfacial film, reduces its blood vessel irritation and toxicity, strengthens its curative effect.Because etoposide is all insoluble medicine of profit, and almost insoluble in water, through high pressure homogenize, the high speed airflow bump forms a kind of supersonic speed and stirs, and makes medicine dissolving and be penetrated in the oil-water interfacial film with molecular forms rapidly.Wherein, etoposide has certain distribution (about 20～80%) in the biphase interfacial film of profit.This preparation is a kind of preparation of antitumor drug, and in vivo feature mainly shows as higher chemical physical stability, hypotoxicity, low irritant and high efficiency behind the administrated by injection.
The etoposide lipide microsphere injection (oil in water emulsion) of the present invention preparation has reduced the blood vessel irritation of etoposide, has improved pharmaceutical chemistry stability and curative effect and has reduced toxic and side effects, thereby had certain innovation and stronger practicality.
Description of drawings
Fig. 1 is the LM structural representation
Fig. 2 is a rat plasma Chinese medicine concentration-time curve among the embodiment 6
Fig. 3 is a rats'liver Chinese medicine concentration-time curve among the embodiment 7
Fig. 4 is a rat spleen Chinese medicine concentration-time curve among the embodiment 7
Fig. 5 is a kidney of rats Chinese medicine concentration-time curve among the embodiment 7
Fig. 6 is an induced lung Chinese medicine concentration-time curve among the embodiment 7
Fig. 7 is a rat pharmaceutical concentration-time curve in the heart among the embodiment 7
Fig. 8 is a rat brain Chinese medicine concentration-time curve among the embodiment 7
■ etoposide lipid microsphere; Zero etoposide normal injection
■ etoposide lipid microsphere; ◆ the etoposide normal injection
The specific embodiment:
The prescription preparation technology of embodiment 1 etoposide preparations
Prescription 1:
Injection safflower oil 20g
Etoposide 0.2g
Lecithin 1.8g
Glycerol 2.5g
Water for injection adds to 100ml
Preparation method 1:
(1) glycerol with recipe quantity mixes with an amount of water for injection that is preheated to 40-100 ℃, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin is added to 40-100 ℃ of heating for dissolving in the recipe quantity safflower oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (3) the etoposide drug powder is added this Ruzhong just, stirred 10 minutes, adding is preheated to 40-100 ℃ water for injection to full dose; (4) be transferred in the high pressure homogenizer, high pressure homogenize 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (5) embedding is filled nitrogen in infusion bottle, places steam rotation steriliser to sterilize.
Prescription 2:
Injection soybean oil 20g
Etoposide 0.2g
Fabaceous lecithin 1.2g
Glycerol 2.5g
Tween-80 0.02g
Enuatrol 0.05g
Water for injection adds to 100ml
Preparation method 2:
(1) with Tween-80, enuatrol, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously fabaceous lecithin is added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (3) the etoposide drug powder is added this Ruzhong just, stirred 10 minutes, adding is preheated to 40-100 ℃ water for injection to full dose; (4) be transferred in the high pressure homogenizer, high pressure homogenize 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (5) embedding is filled nitrogen in infusion bottle, places steam rotation steriliser to sterilize.Prescription 3:
Oil phase (the 20g of soybean oil/MCT)
Etoposide 0.1g
Lecithin 1.8g
Glycerol 2.5g
Oleic acid 0.03g
Tween-80 0.02g
EDTA 0.05g
Water for injection adds to 100ml
Preparation method 3:
(1) with Tween-80, EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place steam rotation steriliser, sterilization.Prescription 4:
Oil phase (the 30g of soybean oil/MCT)
Etoposide 0.1g
Lecithin 1.8g
Glycerol 2.5g
Oleic acid 0.06g
EDTA 0.05g
Water for injection adds to 100mL
Preparation method 4:
(1) with EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place steam rotation steriliser, sterilization.Prescription 5:
Oil phase (the 20g of soybean oil/MCT)
Etoposide 0.1g
Lecithin 1.5g
Glycerol 2.5g
Oleic acid 0.06g
EDTA 0.05g
Water for injection adds to 100mL
Preparation method 5:
(1) with EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, span 20, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser, sterilization.
In above listed prescription the compositions, by above method 4 preparations of preparation in addition, they composed as follows:
| Etoposide | 0.2g |
| Soybean oil, safflower oil, Semen Coicis oil, Oleum Fructus Bruceae, medium chain length fatty acid triglyceride | 5-30g |
| Lecithin, fabaceous lecithin | 0-5g |
| Oleic acid, enuatrol | 0-1g |
| Cholic acid, sodium deoxycholate | 0-1g |
| Glycerol | 0-5g |
| EDTA | 0-1g |
| Poloxamer F68 | 0-5g |
| Tween 80 | 0-1g |
The investigation of embodiment 2 etoposide lipid microsphere stability
According to the bibliographical information experimental technique: with etoposide lipid microsphere sample respectively with redistilled water, buffer salt (Na
2HPO
4, 0.017M; KH
2PO
4, 0.0014M; NaCl, 0.1370M pH7.4) and glycerol (2.21%w/w) dilution, measures dilution back 1h, 2h, 4h, the particle diameter behind 24h and the 48h, Zeta-potential and medicine encapsulation ratio.The etoposide lipid microsphere was preserved 6 months respectively at 4-8 ℃ and 25 ℃, the particle diameter of different time points working sample and encapsulation ratio, result of the test shows that the particle diameter of etoposide lipid microsphere is not subjected to the influence of redistilled water, buffer salt (pH7.4) and glycerol (2.21%w/w) dilution.Behind the 48h, the particle diameter in redistilled water and the glycerol does not almost change, and particle diameter has increased 50nm in buffer salt (pH7.4).4-8 ℃ and 25 ℃ six months experimental result that keep sample shows that the particle diameter of etoposide lipid microsphere and entrapment efficiency significantly do not change.
Embodiment 3 etoposide lipide microsphere injection irritation tests
(1) blood vessel irritation experiment
Two kinds of different dosage forms of etoposide inj are carried out body surface area by clinical application amount (100mg/ time) to convert and draws experimental rabbit with dosage (5.2mg/kg).The dosage of pressing 2ml/kg before the test is with the fresh preparation of aseptic normal saline solution.Select 6 of the healthy new zealand white rabbits of body weight 2.5-3.0kg for use, male and female have concurrently.After iodine tincture and ethanol disinfection were used in the injection site, 3 white rabbits were in auris dextra auricular vein injection etoposide liquid drugs injection injection (Cs), and left ear is injected the aseptic normal saline solution of same dose in contrast; 3 white rabbits are in auris dextra auricular vein injection etoposide lipide microsphere injection (Cz) in addition, and left ear is injected the aseptic normal saline solution of same dose in contrast, and injection speed is 2.8ml/min (being equivalent to people's clinical injection speed).Once a day, continuous three days, the last administration was after 24 hours, inject air by auricular vein and put to death white rabbit, the response situation of perusal injection site, and dissect rabbit ear blood vessel and surrounding tissue is done paraffin section (entad holding 1cm and 5cm place under the injection site), HE dyeing, light microscopy checking.The result of perusal injection site reaction situation is shown in table-1, and pathological section is checked by disease prevention and control center, Liaoning Province, and presented audit report.
The result: shown in table-1, two kinds of dosage form blood vessel irritation tests of etoposide inj perusal is the result show: the blood vessel irritation of Cz is weaker than Cs; Microscopy report shows: the Cs group has to a certain degree blood vessel irritation to New Zealand white rabbit ear blood vessel, and the Cz group is not seen obvious irritation to New Zealand white rabbit ear blood vessel.Microscopy the results are shown in report.
Table-1 blood vessel irritation test perusal
| Dosage form | Sequence number | Sex | Auris dextra | Left side ear |
| Cs Cz | 1 2 3 1 2 3 | ♂ ♂ ♀ ♂ ♂ ♀ | Congested congested normal normal | Slight congested normal slight congested normal slight congested |
(2) muscle irritation experiment
The dosage of etoposide inj converts, the selection of medicine preparation, New Zealand white rabbit the same (totally 4, every kind dosage form 2).Cut off the rabbit hair at quadriceps femoris position, white rabbit both sides, behind iodine tincture and ethanol disinfection, respectively at right lateral thigh musculus quadriceps injection Cs and Cz injection 1ml, the physiological saline solution injection of left side quadriceps femoris injection equivalent is injected after 48 hours in contrast, goes into air by auricular vein and puts to death white rabbit, dissect and take out quadriceps femoris, vertically cut, observe the response situation of injection site muscular tissue, determine the order of reaction.
0 grade: no change.
1 grade: mild hyperaemia, its scope is below 0.5cm * 1.0cm.
2 grades: moderate hyperemia, its scope is more than 0.5cm * 1.0cm.
3 grades: severe hyperemia, with myodegeneration.
4 grades: necrosis occurs, the brown degeneration is arranged.
5 grades: the popularity necrosis occurs.
Calculate 4 quadriceps femoris order of reaction summations then, test again if the difference of the peak of the quadriceps femoris order of reaction and minimum, then should be got 2 healthy rabbits in addition greater than 2.After obtaining the result, if 4 quadriceps femoris order of reaction summations, think then that the local irritation test of test sample is up to specification less than 10.
The result: the muscle irritation result of the test of two kinds of dosage form injection of etoposide is shown in table-2.The result shows: the muscle irritation of Cz is weaker than Cs.
Table-2 muscle irritation result of the tests
| Dosage form | Sequence number | Sex | The right lateral thigh musculus quadriceps | The left side quadriceps femoris |
| Cs Cz | 1 2 1 2 | ♂ ♀ ♂ ♀ | 2 grades 2 | 0 |
The experiment of embodiment 4 hemolytics
Get blood 20ml from the common carotid artery of New Zealand white rabbit, place in the flask, stir gently with Glass rod, after several minutes, remove and defibrinate, take out blood, add the equivalent normal saline solution, centrifugal (1500r/min 10min), removes supernatant; Sedimentary erythrocyte adds normal saline solution again and cleans, and is centrifugal.Transparent until supernatant so repeatedly, be made into 2% suspension with normal saline by erythrocytic capacity.
Get 7 of clean tube, numbering adds each liquid in the following table successively respectively, the 6th pipe does not add test liquid as the blank pipe, and the 7th effective distilled water replaces normal saline, shakes up, whether place 37 ℃ of water-baths, observing respectively at 0.5 hour, 1 hour, 2 hours, 3 hours has haemolysis to take place.
The result shows: Qs, Qz do not see that haemolysis takes place, and the hemolytic experiment of two kinds of dosage forms is all qualified.
The experiment of embodiment 5 anaphylaxis
Get 8 of healthy guinea pigs, body weight 250-280g, male and female half and half, two kinds of dosage forms of Qs, Qz each 4 (male and female half and half), prepare by clinical practice dosage (100mg/ time) the conversion Cavia porcellus consumption (2.0mg/200g) of etoposide inj and with normal saline solution, the corresponding test liquid 0.5ml of every Cavia porcellus ip (lumbar injection), the next day once, totally three times.The the 14th, the 21 day corresponding reagent liquid 1ml (the dosage conversion is the same) that supplies of lateral vein injection outside the hind paw of every Cavia porcellus after injection first attacks.Each intravenously administrable was observed 2 hours, as two or more person who occurs grabbing in nose, perpendicular hair, cough, the dyspnea is judged to the positive; Be judged to the positive if any one of spasm, gatism, collapse, shock, phenomena of mortality person.The negative of above-mentioned symptom do not appear.
The result shows: two kinds of dosage forms of Qs, Qz all meet the anaphylaxis experimental standard.
Embodiment 6 rat plasma pharmacokinetics
Dosage regimen
12 rats are divided into two groups at random, and 6 every group, one night of fasting before the experiment.The first group is a matched group, in right back vena femoralis injection etoposide normal injection (1mgmL
-1), the second group is organized for being subjected to examination, in right back vena femoralis injection etoposide lipid microsphere, respectively at 5min, 10min, 15min, 30min, 45min, 1h, 2h, 4h, the 6h eye socket is got 0.5mL blood, puts in the centrifuge tube of the sharp end of heparinization in advance, centrifugal, the accurate upper plasma 200 μ L that draw add 20 μ L waters for injection, after certain method processing, get 20 μ L sample introductions, calculate the concentration of etoposide in each time point sample with the standard curve on the same day.
Experimental result: compartment model pharmacokinetic parameter
Average blood drug level data with 3P97 pharmacokinetics routine processes etoposide lipid microsphere and injection.Judge that according to AIC and degree of fitting both models belong to, the result shows that the blood drug level data of etoposide lipid microsphere and injection all meet two compartment models.
Non-compartment model pharmacokinetic parameter
ETP etoposide lipid microsphere wherein; EPT etoposide normal injection:
| Group | T 1/2α(h) | T 1/2β(h) | AUC 0-a (μgmL -1h) a | CL (lh -1kg -1) a | V aa(lkg -1) | MBT(h) b | C max (μgmL -1) b |
| ETP EPE | 0.13±0.1 0.23±0.1 | 1.5±0.2 6.7±5.6 | 14.7±1.5 38.1±9.2 | 0.7±0.1 0.3±0.1 | 1.0±0.2 1.8±1.7 | 1.5±0.1 5.7±1.5 | 32.1±1.9 43.4±4.2 |
Values are mean±SD.
ap＜0.01.
bp＜0.05.
The distributed power research of embodiment 7 rat tissues
Dosage regimen
84 rats are divided into two groups at random, 42 every group.The first group is a matched group, and in right back vena femoralis injection etoposide normal injection, the second group is organized for being subjected to examination, in right back vena femoralis injection etoposide lipid microsphere, respectively at 0.5h, 1h, 2h, 4h, 6h, 12h, 24h (every 12 rats, first, each 6 of second groups) sacrificed by decapitation animal, take out the heart, liver, spleen, lung, kidney, brain, clean with normal saline flushing, blot with filter paper, take by weighing 0.2g, add 1mL buffer salt (pH7.4) homogenate, centrifugal, get supernatant, after handling down by " tissue sample is handled and measured " item, get 20 μ L sample introductions, calculate the concentration of etoposide in each time point sample with the standard curve on the same day.
Experimental result
The AUC measurement result sees Table 3 after laboratory animal intravenous injection etoposide lipid microsphere and the etoposide normal injection, and the meansigma methods and the time relation of each remedy,tissue's substrate concentration of each time point of laboratory animal are seen Fig. 1～6.
The AUC of table-3 ETP and EPE tissue distribution curve
0.05-24h
| Tissues | AUC 0.05-24h(μgg -1h)±SD | pvalues a | |
| ETP | EPE | ||
| Liver Spleen Lungs Heart Kidney Brain Liver uptake(mL h -1) Spleen uptake(mL h -1) | 51.8±6.0 35.3±3.5 121.2±3.3 21.3±1.9 23.3±2.0 9.3±0.3 2.6±1.0 1.0±0.3 | 24.1±0.7 25.0±1.0 33.6±1.1 41.2±2.0 19.6±1.1 20.4±1.0 0.65±0.2 0.27±0.1 | ＜0.001 ＜0.05 ＜0.001 ＜0.001 ＜0.05 ＜0.001 ＜0.001 ＜0.001 |
Data are preaented as mean±SD(n＝6).
aAUC
0.05-24h of EPE was compared with that of ETP in correeponding tissue.
Show by blood plasma pharmacokinetics result of the test in the rat body, all be higher than common commercially available injection in the blood Chinese medicine concentration of each time point.Illustrate that lipide microsphere injection has high efficiency.Tissue distribution kinetics is investigated experimental result and is shown, the distribution situation of etoposide in each tissue is descending to be successively: liver, lung, kidney, heart and brain.Wherein, the AUC of heart and brain
0.05-24hAll be higher than general formulation.Studies show that recently pulmonary's small cell carcinoma often shifts and diffusion to brain, so the etoposide lipide microsphere injection is in the gathering of brain, probably the vertigo for tumor cell moves the generation curative effect, thereby has antitumor efficacy widely.
Claims (10)
1. lipide microsphere injection that contains etoposide, it is characterized in that: this injection comprises: etoposide, fat-soluble medium, water and surfactant consist of in quality of the pharmaceutical preparations prescription of the present invention:
Oil phase 5%～30%,
Etoposide 0.001%～0.5%,
Surfactant 0.5%～5%,
Osmotic pressure regulator 0.5%～5%,
All the other are water for injection.
2. a kind of lipide microsphere injection that contains etoposide according to claim 1 is characterized in that: grease separation is from mineral oil, vegetable oil, and animal oil, quintessence oil and artificial oil, and composition thereof; Surfactant is selected from phospholipid, tween, span, pluronic, enuatrol, oleic acid, cholic acid, deoxycholic acid and composition thereof.
3. a kind of lipide microsphere injection that contains etoposide according to claim 2 is characterized in that: described grease separation is from safflower oil, soybean oil, and Semen Maydis oil, MCT Oil (MCT), Semen Coicis oil, Oleum Fructus Bruceae, and composition thereof.
4. a kind of lipide microsphere injection that contains etoposide according to claim 2, it is characterized in that: described oil is soybean oil, MCT Oil (MCT), or the two mixture.
5. a kind of lipide microsphere injection that contains etoposide according to claim 2, it is characterized in that: described tween is selected from polysorbas20, polysorbate40, polysorbate60, Tween 80, polysorbate85, or the mixture of above different size; Described pluronic is pluronic F68; Described phospholipid is selected from lecithin, fabaceous lecithin, or the two mixture.
6. a kind of lipide microsphere injection that contains etoposide according to claim 1 is characterized in that: also can contain glycerol, and sorbitol, mannitol, glucose, and composition thereof; Sodium hydroxide, hydrochloric acid, and composition thereof; Buffer salt; Metal-chelator; Antioxidant.
7. a kind of lipide microsphere injection that contains etoposide according to claim 6 is characterized in that: buffer salt is acetic acid, sodium acetate, citric acid, sodium citrate, sodium hydrogen phosphate, sodium dihydrogen phosphate, and composition thereof; Metal-chelator is a disodium edetate, sodium calcium edetate, and composition thereof; Antioxidant is vitamin E, vitamin C, and sodium sulfite, sodium pyrosulfite, cysteine, and composition thereof.
8. a kind of lipide microsphere injection that contains etoposide according to claim 6 is characterized in that: described glycerol is 0.5% to 5% in the preparation consumption; Buffer salt is 0.005% to 2%; Metal-chelator is 0% to 1%; Antioxidant is 0.001% to 0.2%.
9. preparation method that contains the lipide microsphere injection of etoposide is characterized in that: comprise following steps:
A) powder of etoposide ultramicro powder suspension or ultra micro is added to the blank Emulsion that does not contain etoposide or contains in the Emulsion of part etoposide oil in water emulsion through mixed etoposide lipide microsphere injection or etoposide;
B) etoposide is added in the water-soluble medium that contains surfactant, mixes with oil phase then, prepare colostrum through high-speed stirred;
C) etoposide solution or powder are added in the oil-soluble medium that contains surfactant, mixed with water then, prepare colostrum through high-speed stirred, in view of the chemistry of aqueous solution poor stability of etoposide, preparation process should be controlled aqueous pH values and remain between the 4-8;
The preparation method of preparation mainly comprises high-speed stirred process and high pressure homogenize process, the high-pressure rotary sterilization is adopted in the sterilization of preparation, the compositions that constitutes lipide microsphere injection is identical with the compositions of oil in water emulsion, etoposide has certain distribution in the preparation in the biphase interfacial film of profit, about 20～80%, the remainder etoposide is distributed in water and the oil phase
10. according to the described preparation method that contains the lipide microsphere injection of etoposide of claim 9, it is characterized in that:
(1) with EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, span 20, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser, sterilization;
(1) with Tween-80, enuatrol, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously fabaceous lecithin is added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (3) the etoposide drug powder is added this Ruzhong just, stirred 10 minutes, adding is preheated to 40-100 ℃ water for injection to full dose; (4) be transferred in the high pressure homogenizer, high pressure homogenize 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (5) embedding is filled nitrogen in infusion bottle, places steam rotation steriliser to sterilize;
(1) with Tween-80, EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place steam rotation steriliser, sterilization;
(1) with EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place steam rotation steriliser, sterilization;
(1) with EDTA, the glycerol of recipe quantity and be preheated to 40-100 ℃ an amount of water for injection and mix, change in the tissue mashing machine, stirred for several minute, 1-5 time, until each composition dissolving, water; Simultaneously lecithin, oleic acid, span 20, etoposide are added to 40-100 ℃ of heating for dissolving in the recipe quantity soybean oil, get oil phase; (2) oil phase is added aqueous phase, changes in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, colostrum, adjust pH is to 4-8; (4) colostrum is added in the high pressure homogenizer homogenizing 1-10 time, the sampling microscopy, to oil droplet below 0.5 micron; (6) get the embedding of etoposide submicronized emulsion in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser, sterilization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010139278A1 (en) * | 2009-06-04 | 2010-12-09 | 江苏恒瑞医药股份有限公司 | Preparation method of drug loaded emulsion |
| CN102247320A (en) * | 2010-05-21 | 2011-11-23 | 天津药物研究院 | Etoposide long-circulating emulsion and preparation method thereof |
| CN102106866B (en) * | 2009-12-25 | 2013-04-03 | 四川科伦药物研究有限公司 | Pharmaceutical composition and preparation method thereof |
| CN104324000A (en) * | 2010-07-29 | 2015-02-04 | 湖南康都制药有限公司 | Etoposide liposome combined medicine, and large-scale production process and application thereof |
-
2006
- 2006-12-18 CN CN 200610169149 patent/CN1973826A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010139278A1 (en) * | 2009-06-04 | 2010-12-09 | 江苏恒瑞医药股份有限公司 | Preparation method of drug loaded emulsion |
| CN102106866B (en) * | 2009-12-25 | 2013-04-03 | 四川科伦药物研究有限公司 | Pharmaceutical composition and preparation method thereof |
| CN102247320A (en) * | 2010-05-21 | 2011-11-23 | 天津药物研究院 | Etoposide long-circulating emulsion and preparation method thereof |
| CN102247320B (en) * | 2010-05-21 | 2013-03-13 | 天津药物研究院 | Etoposide long-circulating emulsion and preparation method thereof |
| CN104324000A (en) * | 2010-07-29 | 2015-02-04 | 湖南康都制药有限公司 | Etoposide liposome combined medicine, and large-scale production process and application thereof |
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