CN1985851A - Lipoid microsphere injection containing toad cake extract and its preparing method - Google Patents

Abstract

The present invention relates to lipoid microspere injection composition containing liposoluble toad cake extract and its preparation process. The injection composition contains liposoluble toad cake extract 0.0001-5 wt%, liposoluble medium 1-30 wt%, surfactant 0.5-10 wt%, isoosmotic regulator 0.5-10 wt%, and water for the rest. The injection has anticancer and analgetic effects, less blood vessel irritation caused by toad cake, no toxicity on heart and capacity of raising the medicine concentration inside tumor.

Description

Contain lipide microsphere injection of Venenum Bufonis extract and preparation method thereof

Technical field

The present invention relates to medical technical field, exactly it is a kind of lipide microsphere injection (oil in water emulsion) and preparation method that contains the Venenum Bufonis lipoclastic.

Background technology

For many years, though people have carried out many effort and obtained bigger progress, the harm of tumor up to now still fails effectively to be contained.Have data to show, 2002 domestic because of malignant tumor and the lethal number of cancer account for dead population's then 25.37%, and tumor has become first killer of harm humans health.Over the past two years, the morbidity of tumor was in rising trend, and age of onset also obviously moved down from average 55 years old.Can foretell that in from now on two, 30 years, the sickness rate of cancer and mortality rate will be the trend that continues rising.

Cancerous pain is the most common and symptom the most rambunctious of cancer patient, also is the key factor that influences patients ' life quality.China has cancer patient 2,000,000 now, annual New Development patient 1,600,000, and the cancer pain incidence rate is 51.5%.The World Health Organization (WHO) has set up World Health Organization (WHO) cancer pain treatment committee in nineteen eighty-two, and proposes to reach in 2000 the target that " makes cancer patient not bitterly " in the worldwide.Yet about 30%～50% cancer pain patient does not still obtain satisfied alleviation at present regrettably.At present, the key factor that life quality is effectively carried out, influenced to cancer pain as the anticancer plan of influence has been subjected to extensive attention, and the correlational study of cancerous pain has become global important subject.The scheme of " three ladder medicine analgesias " cancer pain control that application WHO widelys popularize, though curative effect is more definite, life-time service analgesics toxic and side effects is big, and addiction, dependency are strong, and be subjected to the restriction of patient tolerability, cause part patient analgesic effect not good enough.

Because most antitumor drug untoward reaction are serious, body and mind to the patient in therapeutic process causes very major injury, therefore how when improving curative effect of medication, alleviate the patient suffering, reduce the zest and the toxicity of medicine, thereby improve patient's life quality, the life that prolongs the patient as far as possible becomes new direction and the emphasis of present antitumor drug research gradually.Chinese medicine has its unique effect in the oncotherapy field, no matter suppress or killing tumor cell aspect, still postoperative conditioning, alleviate put, chemotherapy adverse effect, improve sings and symptoms, important effect has all been brought into play in aspects such as raising life quality.

Venenum Bufonis has another name called Bufo siccus eyebrow fat (" property of medicine opinion "), Bufo siccus eyebrow shortcake (" Japan hanako materia medica "), toad slurry (" Xinjiang medical material "), Oviductus Ranae shortcake (" Shandong Chinese medicine "), the Oviductus Ranae slurry (" the Chinese crude drug handbook), for the ear rear gland of Bufonidae animal Bufo siccus Bufo Bufo gargarizans Cantor or Bufo melanostictus Bufo melanostictusSchneide etc. and the white serosity of skin gland secretion, form through dry processing.The property sweet suffering, temperature (also have medical book to propose the Venenum Bufonis flavor and be hot cool viewpoint), poisonous.Can detoxify detumescence, heart tonifying, pain relieving.The treatment skin ulcer, carbuncle, diseases such as carbuncle on the back.Bufo siccus and related preparations treatment tumor have very long history in China.Just putting down in writing in first monograph on materia medica Shennong's Herbal of China: " the Rana limnocharis acrid in the mouth is cold, main that gas, and the hard blood of broken lump, carbuncle, the erosion of vulva, that obeys does not fever ".

Modern study shows and comprises the number of chemical composition in the Bufo siccus body, can be divided into two big classes by the difference of dissolution properties, and fat-soluble part accounts for 20%, has the steroidal structure, is referred to as bufanolide class (Bufosterol).Water-soluble portion is a certain amount of indoles alkaloid, and part polysaccharide, organic acid and aminoacid etc.

Studies confirm that the Venenum Bufonis anticancer effective component mostly is fat-soluble compound, wherein bufogenin, Toadpoison Medicine, cinobufotalin content is more, activity is stronger.The pharmacological action and the molecular pharmacology Study on Mechanism thereof anticancer, that alleviate cancer pain of Venenum Bufonis liposoluble constituent have become in recent years the focus and the focus of research both at home and abroad.

Toadpoison Medicine R

Cinobufacin OCOCH ₃

Bufogenin H

Pharmaceutical research shows that Venenum Bufonis main mechanism anticancer, that alleviate cancer pain is: the biosynthesis of (1) anticancer DNA and RNA.(2) mitochondrion and the rough surface endoplasmic reticulum of destruction oncocyte.(3) inducing cancer cell differentiation, apoptosis.(4) value of raising cAMP and cAMP/cGMP.(5) improve mice serum IgG and total white blood cells.(6) strengthen macrophage phagocytic effect, the stimulation body release antitumor cell factor.Renewal studies show that Toadpoison Medicine can suppress tumor vascular endothelium and blood vessel hyperplasia, compares with traditional oncotherapy, suppresses the tumor tissues angiogenesis drug and is primarily aimed at the new vessels that has started, so have specificity.Simultaneously it can also human body immunity improving power, alleviates the toxicity of chemotherapy, reduces the cancer pain incidence rate, and enhancing body is to the toleration of radiotherapy, chemotherapy, and in optimal dose, toxicity is minimum.But the Venenum Bufonis liposoluble extract is widely distributed in vivo, causes allergic reaction easily after the intravenous injection, has zest for blood vessel, can cause heart condition reactions such as ventricular premature contraction simultaneously.

HUACHANSU ZHUSHEYE, Venenum Bufonis Injection development listing are early, with new drug approval standard at that time, only require that it provides stable repeatably composition as quality control standard, so be that to be no less than 0.50% (mg/ml) in 5-hydroxy tryptamine indoles alkaloid content be quality control standard.Indoles alkaloid class composition is proved the report that antitumor action is studied in the Bufo siccus but do not see so far, on the contrary, give birth to the generation that too much 5-hydroxy tryptamine can cause carcinoid syndrome in the tumor patient body, clinical manifestation is the congested flushing of women's head-ornaments portion paroxysmal, tachycardia and hypertension, also can cause dyspnea, asthma, the patient who has can cause intractable diarrhea.Bufotenine is the derivant of 5-hydroxy tryptamine, and pharmacological action is similar to the hallucinogen LSD (lysergicaciddiethylamide).Have lower content to distribute in the human body, but content detection is negative in urine, and excretion obviously raise in autism or obsession patient's the urine.Therefore it is still not improper to do the Quality Control composition of medicine with 5-hydroxy tryptamine, bring potential risk but also use for the Venenum Bufonis clinical safety.

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 (LipidMicrosphere, 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) is the good carrier of fat-soluble medicine, has avoided the use of organic solvent.(2) quite a few drug distribution is in oil phase or oil-water interfacial film; change responsive medicine for facile hydrolysis or to pH; this " isolation " can protect medicine to exempt from destruction, avoided simultaneously contacting with the direct of body fluid, reduced issuable part of medicine self and blood vessel irritation.(3) particulate carrier can be concentrated contained medicine and be transported to specific part release and the performance curative effect.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 reticular tissue system (as liver, lung etc.), and be significant to treatment for cancer.

This research will be developed the Venenum Bufonis novel form with the effect of medicine bomb targeted therapy--Venenum Bufonis lipide microsphere injection, reduce medicine for blood vessel irritation, avoid effective ingredient to be absorbed by heart, improve medicine tumor bulk concentration.This will reduce the cancer pain incidence rate to giving full play to its anti-tumor activity undoubtedly, further promote and to promote Venenum Bufonis significant in Clinical Application.

Summary of the invention

The purpose of this invention is to provide a kind of lipide microsphere injection (oil in water emulsion) and preparation method that contains the Venenum Bufonis lipoclastic.

This injection comprises the Venenum Bufonis lipoclastic, fat-soluble medium, water and surfactant.Wherein the Venenum Bufonis lipoclastic is the bufanolide class (Bufosterol) with steroidal structure, and such material derives from the Venenum Bufonis medical material, perhaps directly extracts conversion via Bufo siccus, is perhaps prepared by synthetic.

The acceptable material of fat-soluble medium indication one big class physiology, no matter be mineral oil, vegetable oil, animal oil, quintessence oil or artificial oil, or its mixture.Therefore, fat-soluble medium is in order to refer to the very material of different chemical character that has of a wide region.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, polyoxyethylene sorbitan fatty acid ester (tween, Tween), the poloxalkol class (pluronic, Poloxamer, Pluronic), enuatrol, oleic acid, cholic acid, sodium cholate, deoxycholic acid, sodium deoxycholate 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.

Typically, forming mass percent prescription of the present invention in preparation consists of:

Fat-soluble medium 1%～30%

Venenum Bufonis lipoclastic 0.0001%～5%

Surfactant 0.5%～10%

Isoosmotic adjusting agent 0.5%～10%

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 a disodiumedetate, calcium disodium chelate, ethylenediamine tetraacetic acid disodium magnesium salt and composition thereof.Metal-chelator is about 0% to about 1% in the preparation consumption.

The present invention also relates to contain the preparation method of the lipide microsphere injection and the oil in water emulsion of Venenum Bufonis lipoclastic.Comprise in the following steps one or more:

1. the powder of Venenum Bufonis lipoclastic ultramicro powder suspension or ultra micro is added to the blank Emulsion that does not contain the Venenum Bufonis lipoclastic or contain in the Emulsion of part Venenum Bufonis lipoclastic through the high pressure homogenize process mixed Venenum Bufonis lipoclastic lipide microsphere injection.

The heating of Venenum Bufonis lipoclastic and fat-soluble medium is mixed 2., treat that effective ingredient is dissolved in fat-soluble medium after, by filtering or the centrifugal impurity of removing, redispersion prepares colostrum at aqueous phase through high-speed stirred.

With Venenum Bufonis lipoclastic and surfactant dissolves in organic solvent, remove organic solvent then, surplus materials is mixed with fat-soluble medium heating, redispersion passes through high-speed stirred and prepares colostrum at aqueous phase.

4. the preparation method of said preparation mainly comprises high-speed stirred process and high pressure homogenize process.

5. the high-pressure rotary sterilization is adopted in the sterilization of preparation.

This research will be prepared the Venenum Bufonis novel form with the effect of medicine bomb targeted therapy--Venenum Bufonis lipide microsphere injection, the clinical anticancer analgesic that is used for, this injection will reduce the blood vessel irritation of Venenum Bufonis, avoid effective ingredient to be absorbed by heart, improve medicine tumor bulk concentration.

Description of drawings:

Fig. 1 is the lipid microsphere structural representation.

Fig. 2 is that different concentration ethanol is to extracting result's influence.

(wherein,

Expression extract dry thing middle finger index composition content and,

The expression index components rate of transform)

Fig. 3 is that the different grain size powder is to extracting result's influence.

(wherein, Expression extract dry thing middle finger index composition content and, The expression index components rate of transform)

Fig. 4 is for extracting return time to extracting result's influence.

(wherein, the expression index components rate of transform,

Expression extract dry thing middle finger index composition content and)

Fig. 5 is the mensuration of Venenum Bufonis extract apparent oil water partition coefficient (Log P).

(wherein,

The expression Toadpoison Medicine, The expression cinobufagin,

The expression bufogenin)

Fig. 6 is the influence of Venenum Bufonis lipide microsphere injection effect HGC cell 24h to survival rate

(wherein, Represent blank Emulsion group, expression The expression negative control group, Expression Venenum Bufonis lipid microsphere group)

Fig. 7 is the influence of Venenum Bufonis lipide microsphere injection effect A549 cell 24h to survival rate

(wherein, Represent blank Emulsion group, expression The expression negative control group, Expression Venenum Bufonis lipid microsphere group)

Fig. 8 is the influence of Venenum Bufonis lipide microsphere injection effect U87 cell 24h to survival rate

(wherein, Represent blank Emulsion group, ■ represents Venenum Bufonis lipid microsphere group)

Fig. 9 is the influence of Venenum Bufonis lipide microsphere injection effect U251 cell 24h to survival rate

(wherein, Represent blank Emulsion group, ■ represents Venenum Bufonis lipid microsphere group)

The specific embodiment

Embodiment 1

The prescription preparation technology of Venenum Bufonis lipide microsphere injection

Prescription 1:

Semen Coicis oil 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Lecithin 0.5-10g

Glycerol 0.5-10g

Water for injection adds to 100ml

Preparation method 1:

(1) lecithin, the glycerol of recipe quantity is added in an amount of water for injection 40-100 ℃ mix water; (2) the Venenum Bufonis lipoclastic is added in the Semen Coicis oil, be heated at 40-100 ℃ and must clarify oil phase through 0.45 μ m after effective ingredient dissolves fully; (3) will water and oil phase change in the tissue mashing machine after mixing, stirred for several minute 1-5 time, is prepared into colostrum; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 2:

Soybean oil 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Fabaceous lecithin 0.5-10g

Glycerol 0.5-10g

Oleic acid 0.5-10g

Water for injection adds to 100ml

Preparation method 2:

(1) recipe quantity glycerol is added in an amount of water for injection 40-100 ℃ mix water; (2) with the Venenum Bufonis lipoclastic, fabaceous lecithin, oleic acid add in the soybean oil, are heated at 40-100 ℃ and must clarify oil phase through 0.45 μ m after effective ingredient dissolves fully; (3) will water and oil phase change in the tissue mashing machine after mixing, stirred for several minute 1-5 time, is prepared into colostrum; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 3:

Medium chain length fatty acid triglyceride 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Fabaceous lecithin 0.5-10g

Glycerol 0.5-10g

Enuatrol 0.5-10g

Water for injection adds to 100ml

Preparation method 3:

(1) with recipe quantity glycerol, enuatrol add in an amount of water for injection 40-100 ℃ mix water, regulating water pH is 4-7; (2) with the Venenum Bufonis lipoclastic, fabaceous lecithin adds in the medium chain length fatty acid triglyceride, is heated at 40-100 ℃ and must clarifies oil phase through centrifugal after effective ingredient dissolves fully; (3) will water and oil phase change in the tissue mashing machine after mixing, stirred for several minute 1-5 time, is prepared into colostrum; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 4:

Oleum Fructus Bruceae 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Fabaceous lecithin 0.5-10g

Glycerol 0.5-10g

Oleic acid 0.5-10g

Water for injection adds to 100ml

Preparation method 4:

(1) recipe quantity glycerol is added in an amount of water for injection 40-100 ℃ mix water; (2) with the Venenum Bufonis lipoclastic, fabaceous lecithin, oleic acid add in the Oleum Fructus Bruceae, are heated at 40-100 ℃ and must clarify oil phase through 0.45 μ m after effective ingredient dissolves fully; (3) will water and oil phase change in the tissue mashing machine after mixing, stirred for several minute 1-5 time, is prepared into colostrum; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 5:

Soybean oil 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Fabaceous lecithin 0.5-10g

Glycerol 0.5-10g

Oleic acid 0.5-10g

Tween 80 0.5-10g

EDTA-2Na 0-1g

Water for injection adds to 100ml

Preparation method 5:

(1) with recipe quantity glycerol, Tween 80, EDTA-2Na add in an amount of water for injection 40-100 ℃ mix water; (2) with the Venenum Bufonis lipoclastic, fabaceous lecithin, oleic acid add in the soybean oil, are heated at 40-100 ℃ and must clarify oil phase through 0.45 μ m after effective ingredient dissolves fully; (3) will water and oil phase change in the tissue mashing machine after mixing, stirred for several minute 1-5 time, is prepared into colostrum; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 6:

Soybean oil/medium chain length fatty acid triglyceride 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Lecithin 0.5-10g

Glycerol 0.5-10g

Tween 80 0.5-10g

F68 0.5-10g

EDTA-2Na 0-1g

Water for injection adds to 100ml

Preparation method 6:

(1) recipe quantity Venenum Bufonis lipoclastic is scattered in the Tween-80 solution, high speed agitator stirred for several minute, 1-5 time, use 1-10 (50-100MP of high pressure dispersing emulsification machine homogenize then, 20-100 ℃) medicine dispersion soln (2) with lecithin, the glycerol of recipe quantity and be preheated to 40-100 ℃ medicine dispersion soln and mix, change in the tissue mashing machine stirred for several minute over to, 1-5 time, disperse until each uniform ingredients; (3) in above-mentioned dispersion liquid, add 40-100 ℃ the soybean oil of being preheated to of recipe quantity, change in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, the water for injection that adds 20-100 ℃ is to full dose; (4) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (5) get homogenizing after the Emulsion embedding in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser to sterilize.

Prescription 7:

Safflower oil 1-30g

Venenum Bufonis lipoclastic 0.0001g-5g

Lecithin 0.5-5g

Glycerol 0-5g

Tween-80 0-1g

Water for injection adds to 100ml

Preparation method 7:

(1) recipe quantity Venenum Bufonis lipoclastic is scattered in the Tween-80 solution, high speed agitator stirred for several minute, 1-5 time, use 1-10 (50-100MP of high pressure dispersing emulsification machine homogenize then, 20-100 ℃) medicine dispersion soln (2) with lecithin, the glycerol of recipe quantity and be preheated to 40-100 ℃ water for injection and mix, change in the tissue mashing machine stirred for several minute over to, 1-5 time, until each uniform ingredients disperse water; (3) water is added 40-100 ℃ the soybean oil of being preheated to of recipe quantity, change in the tissue mashing machine, stirred for several minute, 1-5 time, until the oil phase homodisperse, the water for injection that adds 20-100 ℃ is to full dose; (4) Venenum Bufonis lipoclastic ultramicro powder suspension being added blank Emulsion high speed stirs; (5) get above-mentioned colostrum and add water for injection, regulate pH5-8, be transferred in the high pressure homogenizer to full dose, homogenize 1-10 time, the microscopy of taking a sample, to oil droplet below 0.5 μ m; (6) get the embedding of Venenum Bufonis lipoclastic submicronized emulsion in infusion bottle, fill nitrogen, place the high-pressure rotary steriliser, sterilization.

Prescription 8:

Oil phase (the 5-30g of soybean oil/MCT)

Venenum Bufonis lipoclastic 0.0001g-5g

Lecithin 0.5-5g

Glycerol 0-5g

Enuatrol 0-1g

EDTA 0-1g

Water for injection adds to 100mL

Preparation method 8:

(1) glycerol for injection, enuatrol, EDTA, F68 are scattered in an amount of water for injection, are heated to 40-100 ℃ to the magnetic stirring apparatus to be stirred to whole dissolvings; (2) injection lecithin, Tween-80, be added in the mixing oil phase of forming by injection MCT, injection soybean oil, be heated to and be stirred to lecithin under 40-100 ℃ and dissolve fully, add the Venenum Bufonis lipoclastic, carry out high pressure homogenize.(3) oil phase is added to aqueous phase, places high-speed tissue mashing machine's stirred for several minute, 1-5 time.(4) regulate pH value to 4-9, the water for injection dilution is settled to recipe quantity, is transferred to the high pressure homogenizer homogenizing 3-10 time.(5) bottling, sealed cans nitrogen places the high-pressure rotary steriliser, sterilization.

In above listed prescription, the compositions, prepare 5 preparations in addition by above method, their quality percentage composition following (all the other are water):

The Venenum Bufonis lipoclastic

0.0001-5％

Soybean oil, safflower oil, Semen Coicis oil, Oleum Fructus Bruceae, medium chain length fatty acid triglyceride	1％-30％
		Lecithin, fabaceous lecithin	0.5％-10％
Oleic acid, enuatrol	0.5％-10％
		Cholic acid, sodium deoxycholate	0.5％-10％
Glycerol, sorbitol, mannitol, glucose	0.5％-10％
		EDTA	0-1％
Poloxamer F68	0.5％-10％
		Tween
80	0.5％-10％

Embodiment 2

The Venenum Bufonis Study on extraction process

1 different concentration ethanol is to extracting result's influence

Choose Venenum Bufonis medical material fine powder, (1g: 10mL) the different proportion alcohol heating reflux is 1 hour, puts to room temperature sucking filtration, filtrate evaporate to dryness, the dry thing of filtrate and each vacuum drying of residue one day to adopt 10 times of amounts.Measure index sexual element content in the dry thing respectively.The results are shown in Figure 2.

Along with the increase of concentration of alcohol, the content of index components in dry extract increases, and 90% ethanol extract color and luster is near colourless as can be seen in appearance, and it is yellow that 70% ethanol extract then is.Also because 90% ethanol remove impurity ability is strong, the dry extraction amount is starkly lower than the dry thing of low concentration ethanol extraction simultaneously, so extract yield is minimum, the medicine rate of transform that calculates is minimum.Bibliographical information is arranged in addition, detect various toad spider toxin extract and chromatography purification thing to HL60 cell inhibitory effect intensity difference by cell viability, what its inhibitory action was the strongest is 80% alcohol reflux product.Take all factors into consideration, this test initial option 80% ethanol carries out the investigation of other indexs as extracting solvent.

2 different grain size powder are to extracting result's influence

Get three parts of different grain size Venenum Bufonis medicinal powders, adopt 10 times of amounts (1g: 10mL) 80% alcohol heating reflux is 1 hour, put to room temperature, sucking filtration, the filtrate evaporate to dryness, the dry thing of filtrate and each vacuum drying of residue one day are measured index sexual element content in the dry thing respectively.The results are shown in Figure 3.

The pulverizer that is adopted in this test operation at short notice can be with pulverizing medicinal materials below 60 orders, and wherein majority is distributed in the 80-100 order.Therefore we are only to the 60-80 order, and the 80-100 order reaches less than 100 purpose powder and carried out extracting investigation.The result shows that three kinds of scope powder do not have notable difference in the extraction result of 80% alcohol reflux after 1 hour.

3 extract return time to extracting result's influence

Choose Venenum Bufonis medical material fine powder, adopt 10 times of amounts (1g: 10mL) 80% alcohol heating reflux is 0.5,1,2 hour, put to room temperature, sucking filtration, the filtrate evaporate to dryness, the dry thing of filtrate and each vacuum drying of residue one day are measured index sexual element content in the dry thing respectively.The results are shown in Figure 4.No matter found out that by figure extraction time surpassed after 1 hour, be index sexual element content or its rate of transform does not all have significant change in the extract.Therefore determine that tentatively under the constant prerequisite of other extraction conditions, the alcohol reflux time is 1 hour.

4 single factors are investigated back extraction process demonstration test

Investigate the extraction process result of the test according to single factor, determine that tentatively Venenum Bufonis lipoclastic extraction process is: choose Venenum Bufonis medical material fine powder, (1g: 10mL) 80% alcohol heating reflux is 1 hour to adopt 10 times of amounts, put to room temperature, sucking filtration, filtrate evaporate to dryness, the dry thing vacuum drying of gained one day.Get three parts of Venenum Bufonis medical materials, carry out demonstration test, measure the index sexual element content and the rate of transform in the dry thing according to said extracted technology.The result shows that the extraction process repeatability is good, and the meansigma methods of extract yield index sexual element percentage composition is 27.5 4%, and rate of transform meansigma methods is 84.63%.

The single factor of table 1 is investigated back extraction process demonstration test

Number 123 mean RSD (%)	Extract yield	Extract index composition percentage composition and (%)				The rate of transform
							(％) 36.7 36.3 36.2 36.4 0.7	B 7.05 7.06 7.13 7.08 0.6	C 9.15 9.28 9.12 9.18 0.9	R 11.52 11.38 11.42 11.44 0.6	Σ 27.72 27.22 27.67 27.54 1.0	(％) 85.0 84.6 84.3 84.63 0.4

Embodiment 3

The mensuration of Venenum Bufonis lipide microsphere injection particle diameter and Zeta-potential

Particle diameter and particle size distribution are one of most important characteristics of lipid microsphere, the granularity requirements of used for intravenous injection lipid microsphere still there is not unified regulation at present, the general particle diameter of the product of having reported is even, on average less than 1 μ m, do not have to assemble and merge phenomenon, in 1 Emulsion liquid (0.05mL), no more than 2 of the emulsion droplet of 10-15 μ m does not have the emulsion droplet greater than 15 μ m.About the normal method that adopts of the emulsion droplet granulometry of lipid microsphere microscopic method, dynamic light scattering method, laser scattering method, Coulter counting method etc. are arranged at present.The particle size determination of LM is to adopt optical microscope and photon correlation spectroscopy method (Photon-Correlation Spectroscopy in this test, PCS), also claim dynamic light scattering principle (Dynamic light scatter, DLS, NicompTM PSS 380).

The operational approach of using NicompTM PSS 380 to carry out granulometry is: used the water for injection of 0.22 μ m microporous filter membrane to dilute about 5000 times in sample, put into sample cell immediately, (Intensity) is adjusted to about 300 with light intensity, and light source is HeNe laser (λ ₀=633nm), room temperature when temperature in the operating parameter is made as mensuration begins to measure, and keeps measuring stopping to measure when Time history curve is tending towards straight line, preserves data.Experimental result is seen Fig. 5.

Embodiment 4

The mensuration of Venenum Bufonis lipide microsphere injection median lethal dose(LD 50)

Experiment material

1 laboratory animal: Kunming kind white mice, body weight (20 ± 2) g, male and female half and half.

2 experimental drugs and reagent

Venenum Bufonis lipide microsphere injection (crude drug content: 40mg/50ml); Blank Emulsion.

3 experimental techniques

Venenum Bufonis lipide microsphere injection stock solution by 1: 0.7 dilution proportion, is made into the solution of 4 concentration.Get 50 of 24 hours mices of fasting, per 10 one group, the 1-4 group is by the dilution solution tail vein injection of above difference, and the 5th group is the blank group, and injection capacity is the 0.24ml/10g body weight.Observe immediately after the administration, every subsequently 4h observes 1 time, and observe 1 every day behind the 24h, finishes until 14d.Observe various toxic reactions, central nervous system's symptom and other unusual performance; The death time and the number of elements of record dead animal.And in time dissect dead animal, and take out heart, lung, when finishing, experiment dissects all animals, take out heart, lung, normal saline flushing is placed in 10% formalin fixing, does pathological examination.

4 experimental results

The central nervous system's of Venenum Bufonis lipide microsphere injection high dose group acute toxicity shows as the tic of laboratory animal whole body, the appearance of part laboratory animal is tetanic, opisthotonus deadly; Respiratory system show as accelerated breathing; Cardiovascular system show as tachycardia; Symptoms such as visible in addition upper eyelid is sagging, exophthalmos and incontinence; In, low dose group central nervous system's performance is not obvious, most animals is in the state of reposing, sagging, the rapid breathing in upper eyelid, tachycardia.Dead animal carries out gross anatomy, the visible pulmonary of naked eyes congestion.Its approximate LD ₅₀See Table 2.

Table 2: Venenum Bufonis lipide microsphere injection LD ₅₀Measurement result

Dosage (mg/kg) (d)	Dosage logarithm value (x)	Mortality rate (p)	Probit (y)	Weight (w)
					9.41 13.44 19.20	0.974 1.128 1.283	2/10(20％) 5/10(50％) 9/10(90％)	4.16 5.0 6.28	4.90 6.39 3.43

Σ15.44 Σ14.72

Adopt and simplify the median lethal dose(LD 50) that the probability method is calculated the Venenum Bufonis lipide microsphere injection.

${\mathrm{IgLD}}_{50}-\frac{2i(y_x-\stackrel{-}{y})}{y_3-{\mathrm{<figure-callout\; id="1"\; label="Y"\; filenames="A20061004740300171.png"\; state="\{\{state\}\}">Y</figure-callout>}}_1}+{\mathrm{<figure-callout\; id="2"\; label="X"\; filenames="A20061004740300171.png"\; state="\{\{state\}\}">X</figure-callout>}}_2$

＝2×0.1549(5-5.147)/(6.28-4.16)+1.128

＝1.107

LD ₅₀＝10 ^-11.107＝12.794mg/kg

$S_{x50}\frac{2i}{{(y_3-y_1)}^2}\sqrt{\frac{6{(y_k-\stackrel{-}{y})}^2+{(y_3-y_1)}^2}{\mathrm{\&Sigma;W}}}$

＝0.0689×0.56

＝0.0216

LD ₅₀95% average fiducial limit=LD ₅₀± 4.5 * Sx ₅₀* LD ₅₀

＝12.794±1.244mg/kg

Embodiment 5

The Venenum Bufonis lipide microsphere injection is to the influence of tumor cell survival rate

Medicine: Venenum Bufonis lipide microsphere injection (in crude drug 0.8mg/ml, medicament teaching and research room of Shenyang Pharmaceutical University provides)

Reagent: 1640, DMEM, FBS, CS, MTT

Cell line: HGC stomach carcinoma (human), A549 lung carcinoma (human), U87 astrocytoma (human), U251 astrocytoma (human)

Method (MTT colorimetry):

Take the logarithm the cell of trophophase with 10 * 10 ⁴The density of/ml is added in 96 well culture plates, 37 ℃, 5%CO ₂Cultivate 24h under the condition, after changing serum-free medium, the Venenum Bufonis lipide microsphere injection is diluted to variable concentrations with serum-free medium to add in the experimental port respectively, other establishes not dosing negative control and the blank Emulsion contrast of each respective concentration, and each concentration is established 5 multiple holes, continues to cultivate 24h, add MTT (final concentration 0.25%) after cultivation finishes and continue to hatch 3h, change DMSO100 μ l/ hole after hatching end, effect 10min surveys the absorbance A value at microplate reader 492nm place.Suppression ratio=(1-dosing group absorbance/blank group absorbance) * 100%.

Statistical method: adopt one-way ANOVA in conjunction with the difference (* P＜0.05) between blank Emulsion of each concentration of dunnett test comparison and the culture fluid matched group; Adopt Independent sample t-test relatively administration group and the blank Emulsion matched group (#P＜0.05) of respective concentration.

The result:

1, Venenum Bufonis lipide microsphere injection effect HGC cell 24h is to the influence of survival rate

As seen from Figure 6, the blank Emulsion pair cell of each concentration survival rate has some influences, but does not have significant difference, may cause more greatly owing to standard deviation.Compare with its Emulsion matched group, but 8 and 80 μ g/ml group significance reduces the survival rate of HGC.

2, Venenum Bufonis lipide microsphere injection effect A549 cell 24h is to the influence of survival rate

As seen from Figure 7, the blank Emulsion pair cell of each concentration survival rate has some influences, and 10 times of groups of blank Emulsion dilution can significantly suppress cell survival rate.Compare with its Emulsion matched group, but 8 and 80 μ g/ml group significance reduces the survival rate of A549.

3, Venenum Bufonis lipide microsphere injection effect U87 cell 24h is to the influence of survival rate

As seen from Figure 8, the blank Emulsion matched group of each concentration does not all influence the survival rate of U87, but 0.08,0.8,8,80 μ g/ml significances reduce cell survival rate, and maximum can suppress about 50%.

4, Venenum Bufonis lipide microsphere injection effect U251 cell 24h is to the influence of survival rate

As seen from Figure 9, dilute blank Emulsion 10 ³, 10 ⁴Survival rate that doubly can appreciable impact U251, but 0.08,0.8,8,80 μ g/ml significances reduce cell survival rate, and maximum can suppress about 40%.

Embodiment 6

Venenum Bufonis lipide microsphere injection mice acetic acid twisting test report

Equipment: 1ml syringe, balance, stopwatch.

Medicine: 0.7% acetum; Venenum Bufonis lipide microsphere injection (in crude drug 0.8mg/ml, medicament teaching and research room of Shenyang Pharmaceutical University provides)

Animal: Kunming kind white mice (18～22g).

Method: the investigation of (1) Venenum Bufonis lipide microsphere injection dose-effect relationship: white mice is divided into 8 groups at random, and 12 every group, the abdominal cavity gives the Venenum Bufonis lipide microsphere injection of variable concentrations, and the administration volume is 0.2ml/10g.Each Mus lumbar injection 0.7% acetum 0.2ml of 1h after the administration.Observe and write down the number of times that occurs writhing response in the 15min, calculate and turn round the body suppression ratio, carry out group difference relatively.(2) investigation of Venenum Bufonis lipide microsphere injection time-effect relationship: white mice is divided into 5 groups at random, every group 6, the abdominal cavity gives the Venenum Bufonis lipide microsphere injection, and dosage is the lowest dose level that significant difference is arranged with the solvent control group among the dose-effect result, and the administration volume is 0.2ml/10g.Each treated animal is injected 0.7% acetum 0.2ml at administration 0.5h, 1h, 1.5h, 2h, 2.5h pneumoretroperitoneum respectively, observes and write down the number of times that occurs writhing response in the 15min, calculates and turns round the body suppression ratio.

The result:

(1) mice acetic acid twisting method is investigated Venenum Bufonis lipide microsphere injection dose-effect relationship, as following table.

Group	Dosage is (by giving birth to amount approximately, mg/ml)	Example number (only)	Turn round body number of times (X ± SD) in the 15min	Suppression ratio (%)
					1 2	0.4 0.16	12 12	6.33±4.479** 6.50±3.754**	69.00 68.17

3 4 5 6 7 8

0.08 0.04 0.02 0.01 0.008 solvent control group

12 12 12 12 12 12

7.17±4.549** 7.92±4.441** 9.25±5.446* 11.67±5.015 15.08±4.719 20.42±7.489

64.89 61.21 54.70 42.85 26.85 0.00

Annotate: compare * P＜0.05, * * P＜0.01 with the solvent control group.

(2) mice acetic acid twisting method is investigated Venenum Bufonis lipide microsphere injection time-effect relationship, as following table.

Group	Dosage (by crude drug) mg/ml	Example number (only)	Time (h)	Turn round body number of times (X ± SE) in the 15min	Suppression ratio (%)
						1 2 3 4 5	0.02 solvent control group 0.02 solvent control group 0.02 solvent control group 0.02 solvent control group 0.02 solvent control group	6 6 6 6 6 6 6 6 6 6	0.5 0.5 1.0 1.0 1.5 1.5 2.0 2.0 2.5 2.5	11.50±3.658 20.50±2.930 3.83±1.682 21.07±3.439 3.83±1.352 21.67±3.084 1.33±0.494 19.00±2.793 5.33±1.406 19.83±2.496	43.90 81.91 82.33 93.00 73.12

The result of the test of [conclusion] dose-effect relationship shows, all can significantly suppress the mouse writhing reaction that acetic acid causes when drug level is 0.4mg/ml, 0.16mg/ml, 0.08mg/ml, 0.04mg/ml, 0.02mg/ml.As calculated, the Venenum Bufonis lipide microsphere injection is to the median effective dose ED of mice acetic acid twisting reaction ₅₀=0.0253mg/ml.The result of the test of time-effect relationship shows that behind the administration 2h, it is the most remarkable that the writhing response that Dichlorodiphenyl Acetate causes suppresses.

Claims (9)

1, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic is characterized in that: it comprises Venenum Bufonis lipoclastic, fat-soluble medium, water, surfactant and isoosmotic adjusting agent, and the mass percent of its composition is:

Fat-soluble medium 1% to 30%,

Venenum Bufonis lipoclastic 0.0001% to 5%,

Surfactant 0.5% to 10%,

Isoosmotic adjusting agent 0.5% to 10%,

Surplus is a water.

2, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1, it is characterized in that: described injection comprises the oil in water emulsion that contains the Venenum Bufonis lipoclastic that intravenous transfusion is used, and the compositions that constitutes this type of oil in water emulsion is identical with the compositions that constitutes lipide microsphere injection.

3, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2, it is characterized in that: described Venenum Bufonis lipoclastic is the bufanolide class (Bufosterol) with steroidal structure, such material derives from the Venenum Bufonis medical material, perhaps directly extract conversion, perhaps prepare by synthetic via Bufo siccus.

4, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2, it is characterized in that: also contain metal-chelator in the preparation, it forms mass percent is 0% to 1%, described metal-chelator (EDTA) is a disodiumedetate, calcium disodium chelate, ethylenediamine tetraacetic acid disodium magnesium salt or its mixture; Described isoosmotic adjusting agent is glycerol, sorbitol, mannitol, glucose, and composition thereof; Also contain sodium hydroxide in this external preparation, hydrochloric acid, and composition thereof.

5, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2, it is characterized in that: described fat-soluble medium is selected from mineral oil, vegetable oil, animal oil, quintessence oil and artificial oil, or its mixture; Described surfactant is selected from phospholipid, and polyoxyethylene sorbitan fatty acid ester (tween, Tween), poloxalkol class (pluronic, Poloxamer, Pluronic), enuatrol, oleic acid, cholic acid, sodium cholate, deoxycholic acid, sodium deoxycholate and composition thereof.

6, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2 is characterized in that: described grease separation is from safflower oil, the safranine caul-fat, soybean oil, Semen Maydis oil, Oleum Ricini, Oleum Gossypii semen, Oleum Cocois, Petiolus Trachycarpi oil, MCT Oil (MCT), Semen Coicis oil, Oleum Fructus Bruceae, and composition thereof; Wherein said phospholipid is selected from lecithin, fabaceous lecithin, synthetic phospholipid, or the arbitrary proportion mixture of above different size; Described tween is selected from polysorbas20, polysorbate40, polysorbate60, Tween 80, polysorbate85, or the arbitrary proportion mixture of above different size.

7, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2 is characterized in that: comprise following processing step: Venenum Bufonis lipoclastic ultramicro powder suspension or ultra-micro powder are added to the blank Emulsion that does not contain the Venenum Bufonis lipoclastic or contain in the Emulsion of part Venenum Bufonis lipoclastic through the high pressure homogenize process mixed Venenum Bufonis lipoclastic lipide microsphere injection.

8, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2, it is characterized in that: comprise following processing step: with Venenum Bufonis lipoclastic and surfactant dissolves in organic solvent, remove organic solvent then, surplus materials is mixed with fat-soluble medium heating, redispersion is at aqueous phase, after the process high-speed stirred prepares colostrum, carry out the high pressure homogenize process, the high-pressure rotary sterilization is adopted in the sterilization of preparation.

9, a kind of lipide microsphere injection that contains the Venenum Bufonis lipoclastic according to claim 1 and 2, it is characterized in that comprising following processing step: Venenum Bufonis lipoclastic and the heating of fat-soluble medium is mixed, after treating that effective ingredient is dissolved in fat-soluble medium, by filtering or the centrifugal impurity of removing, redispersion is at aqueous phase, after the process high-speed stirred prepares colostrum, carry out the high pressure homogenize process, the high-pressure rotary sterilization is adopted in the sterilization of preparation.

Images