WO2006076826A1

WO2006076826A1 - An ultrasonic contrast composition having phospholipid as film-former and the preparation method thereof

Info

Publication number: WO2006076826A1
Application number: PCT/CN2005/000075
Authority: WO
Inventors: Xingguo Mei; Sicheng Li; Yingzheng Zhao; Jie Tang; Yan Zhang; Xinhui Luan
Original assignee: Institute Of Pharmacology Toxicology, Academy Of Military Medical Sciences; Cheng Du Yiping Medical Technology Co. Tld.
Priority date: 2005-01-18
Filing date: 2005-01-18
Publication date: 2006-07-27

Abstract

A liposomal ultrasonic contrast composition and its preparation.The composition consists of film-forming materials and fluorocarbon gas. paid film-forming materials consist of 1-10 wt% of phospholipid, 5-15 wt% of foaming agent, 0.5-10 wt% of stabilizer, 70-90 wt% of polymer. The vesicular ultrasonic contrast composition is in a single dosage form, which contains 0.15-0.5 ml of fluorocarbon gas.

Description

Ultrasound using phospholipid component as film forming material

Contrast composition and preparation method thereof

The present invention relates to an ultrasonic contrast agent composition, and more particularly to an ultrasonic contrast agent composition coated with a fluorocarbon-based inert gas using a phospholipid component as a film-forming material and a preparation method thereof. technical background

The new ultrasound contrast agent combined with new ultrasound technology can effectively enhance the two-dimensional ultrasound image and blood flow Doppler signal of the normal organs such as myocardium, liver, kidney and brain, reflecting the different blood of normal tissues and diseased tissues (tumor, ischemic myocardium). Flow perfusion significantly improves the sensitivity and specificity of ultrasound diagnosis. In addition, ultrasound contrast agents carrying genes and drugs have broad application prospects in therapy. The ideal new ultrasound contrast agent has the following characteristics: high scattering, low dispersion, low solubility, no biological activity (no harm to human body, self-extraction through capillaries, uniform size of microbubbles, good tissue development, effective enhancement Tissue development is sufficient for inspection time).

A new generation of ultrasound contrast agents mostly use fluorine-containing gas as the core of microbubbles. Because of the inert gas of fluorine-containing carbon gas, the molecular weight is large, the solubility and dispersion in the blood are poor, and the stability is good. There are albumin, surfactants, bricks, and polymers depending on the material of the fluorocarbon gas. Compared with the enhanced imaging of contrast-enhanced ultrasound, the use of phospholipids as a film-forming material contrast agent has more advantages due to: (1) targeting. After the phospholipid contrast agent is introduced into the body, it is preferentially taken up by tissues rich in reticuloendothelial cells such as liver, spleen and bone marrow. (2) Good stability. Phospholipids Contrast agents are chemically stable, can be stored for several months at room temperature, and are easy to commercialize; (3) Safe. The phospholipid membrane constituting the phospholipid contrast agent is biodegradable and harmless to the human body; and the albumin-based contrast agent has a risk of transmitting blood diseases due to human albumin as a carrier. Summary of the invention

It is an object of the present invention to provide an improved contrast agent composition comprising a phospholipid component as a film forming material; the present invention also provides an optimized method of preparing a contrast agent; It can increase the yield of microbubbles and prolong the effective enhancement time of contrast agent in tissue development.

To achieve one of the above objectives, the present invention adopts the following technical solutions:

The contrast agent composition provided with the lipid component as a film-forming material is a composition which uses a phospholipid substance as a main component to form a film-forming material, and the film-forming material and fluorocarbon type. The inert gas is mixed together for the purpose of imaging. The composition of the film-forming material includes components such as a phospholipid component, a foaming agent, a polymer component, a stabilizer, etc.; the weight percentage of these components in the contrast agent composition is, the ratio of the phospholipid substances is 1 to 10 The weight %, the proportion of the foaming agent is 5 to 15 parts by weight. /. The stabilizer ratio is 0.5 to 10 parts by weight. /. The polymer composition ratio is 70 to 90% by weight, and the rest is a fluorocarbon inert gas.

The above contrast agent composition may be present in unit dosage form, for example, as a vial, and when administered, different doses may be applied according to the needs of the patient, and each dosage unit may contain a fluorocarbon inert gas of 0.15 to 0.5 ml.

The phospholipid component is selected from at least one of the following: lecithin (PC), hydrogenated soy phosphatidylcoline (HSPC), hydrogenated egg phosphatidylcoline (HSPC), two palms Acylphosphatidylethanol (DPPE), dipalmitoylphosphatidylcholine (DPPC), dioleylphosphatidylethanolamine (DOPE), polyethylene glycol-distearoylphosphatidylethanolamine (PEG-DSPE), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic acid glyceryl-sodium salt (DPPG), 1,2-distearoyl-sn-glyceryl-3-phosphatidylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphatidic acid-sodium salt (DPPA), 1,2-dipalsyl-sn-glyceryl-3-phosphatidylcholine (DPPC).

Said foaming agent is selected from at least one nonionic surfactant, nonionic surfactants such as Tween, Span; the purpose of using a foaming agent is to increase the preparation of contrast microbubbles The yield of microbubbles at the same time has a stabilizing effect on the lipid membrane.

The polymer is selected from at least one high molecular polymer, such as a poloxamer, and the high molecular weight polymer is used as a lipid component and a foaming agent to form a contrast film. Provides a support structure while contributing to the stability of the membrane.

The stabilizer used is selected from the group consisting of polyethylene glycol, glyceryl monostearate, palmitic acid, preferably polyethylene glycol 1500 (PEG1500), and the purpose of applying stabilizers is to reduce the mutual interaction of contrast microbubbles. The tendency of fusion improves the hydrophilic lipophilic effect of the lipid bilayer and enhances the stability of the contrast microbubbles. The role of the fluorocarbon-based inert oxidizing gas in the contrast agent is to provide a high-intensity reflection interface for the ultrasonic waves together with the film-forming material, the fluorocarbon-based inert gas being selected from the group consisting of fluoride gas, fluoride gas, specifically It includes perfluorocarbon gas, sulfur fluoride gas, etc. In the application, it is mainly perfluoropropane and sulfur hexafluoride.

The contrast agent composition of the present invention can be prepared by the following method:

Step (a):

The film-forming material phospholipid component, foaming agent, polymer component, stabilizer and other components (such as HEPC; Poloxamer 188, Tween 80 and PEG 1500) and a small amount of anhydrous or water-containing nonaqueous solvent Contact, using ultrasonic and heating to make it a uniform solution system, using a temperature of 45~65 ° C, the time is 20~40 minutes.

The nonaqueous solvent described in the above step is a linear or branched alcohol such as butanol which is anhydrous or contains a trace amount of water. The nonaqueous solvent described in the more preferred embodiment is tert-butanol. The temperature required for the film material to be dispersed and dissolved in a linear or branched alcohol such as t-butanol is 45 to 65 ° C for 20-40 minutes.

Step (b):

Slowly cool the (a) solution to 0-4 °C, allow the liquid to solidify, or quickly refrigerate at 0-4 °C, then use ultrasound (room temperature 15-22 °C) to emulsion or repeated freeze-thaw method. The precipitated particles are uniformly dispersed. The ultrasonic mode may be a water bath or probe type ultrasound, and the ultrasonic mode described in the more preferred embodiment is probe type ultrasound.

Step (c):

Freeze-drying with a freeze dryer, freeze-drying time is 20~25 hours, and the vacuum suction pressure is 50~ in freeze-drying; 120 X l (T ³ mBar, freeze-drying temperature control is -40~- 50 °C.

Step (d):

The freeze-dried lyophilized product is pulverized in a strictly controlled sterile chamber, and dispensed into a vial to prepare a powder injection, and a fluorocarbon-based inert gas is injected into the bottle to cover the cake.

The superiority of the invention is as follows: 1. The microbubble yield is high, and the microbubble concentration reaches lx l0 ⁹ /ml, and And the uniformity of microbubbles is good, the microbubbles with diameter of 2~6 μ ιη account for 50~70% (see Figure 1); 2. The effective enhancement time of contrast agent in tissue development is long; 3. The cost is low, the product cost is much lower than Similar products. DRAWINGS

Figure 1 shows the morphology of the microbubbles at different times after the configuration (the contrast agent is diluted 4 times and observed under a 400-fold light microscope). a configuration after 300min; b configuration after 5min; c configuration after lOmin; d configuration 20min; e configuration 30min; f configuration 50min; g configuration 90min; h configuration 120min.

Figure 2 is a contrast-enhanced image of contrast agent after renal artery injection into the rabbit ear vein; the figure shows the dose of O.lmL/kg before the bolus (a), contrast agent bolus Kidney development after 30 seconds (b), 1 minute (c).

Figure 3 is a contrast-enhanced image of the contrast agent after contrast-enhanced administration of the rabbit ear vein vein. The figure shows the dose of 0.1 mL/kg at 10 seconds (a), 30 seconds after the bolus injection. (b), 1 minute (c) liver parenchymal development. detailed description

Example 1: Preparation of a contrast agent using a phospholipid component as a film-forming material

1.5 mg of hexadecanoic acid, 2 mg of monostearic acid glycerate, 120 mg of polyethylene glycol 1500 (PEG1500), and 120 m of poloxamer 188 (Pooxamer 188) were dispersed in 3 ml of tert-butanol, and dissolved in a water bath at 60 °C. Also, take egg yolk lecithin (PC) 20mg ^ t in lml t-butanol, sonicate in a water bath at 60 ° C, mix with the upper liquid, quickly set at 0 ~ 4 ° C for more than 30 minutes, the liquid is solidified, Freeze for 20 hours. The 200m lyophilized sample was weighed into a 5ml vial, filled with perfluoropropane gas, and sealed to obtain a solid ultrasonic contrast agent.

When using the plug, inject 2ml of normal saline, gently shake to form a microbubble suspension, sample and observe the microbubble concentration is 2 X 10 ⁷ /ml, the particle size distribution is 2~10 μ m, and the bubble wall thickness is 300-1000 nm. .

Rabbits were used as simulated animals, and the effect of ultrasonic imaging in vivo was measured by intravenous bolus injection. The results showed that the microbubbles of this product have similar development effects to the commercially available ultrasound contrast agent Levovist, indicating that the product has excellent in vivo ultrasound development. effect. Example 2: Effect of Tween

Weigh 1.5 mg of hexadecanoic acid, 2 mg of glyceryl monostearate, 120 mg of polyethylene glycol 1500 (PEG1500), and poloxamer 188 (POLoxamer 188 ) 1201 ^ tert-butanol dispersed in 3 1111, dissolved in a water bath at 60 ° C . Also known as polyegrose lecithin (PC) 20mg and different amounts of Tween 80 (Tween 80) dispersed in lml t-butanol, sonicated in a water bath at 60 ° C, mixed with the supernatant, quickly chilled at 0 ~ 4 ° C For more than 30 minutes, the liquid was solidified and lyophilized for 20 hours. 200 mg of the lyophilized sample was weighed and placed in a 5 ml vial, and the fluoropropane gas was sufficiently condensed to prepare a solid ultrasonic contrast agent.

When using the plug, 2 ml of physiological saline was injected, and the microbubble suspension was formed by shaking gently. The concentration of the microbubble microbubbles, the average particle diameter, and the percentage of microbubbles of 2 to 8 μm were observed. The results are shown in Table 1.

Table 1 Effect of different content of Tween80 on microbubble concentration, particle size and particle size distribution

Tween80 rounds (% by weight)

3 6 8 10 14 18 Microbubble concentration ( 10 ⁷ /ml ) 4 3 5 5 7 5

Average particle size ( μ ηι ) 7 7 6 6 6 6

Percentage of microvesicles of 2-8 μ m 30 25 30 35 26 20 It can be seen from Table 1 that different contents of Tween80 have significant effects on the concentration, particle size and particle size distribution of microbubbles, and the content of Tween80 is 8- At 14% by weight, the combined results of microbubble concentration, average particle size and percentage of microbubbles of 2~8 μιη were the best. It has been found that the higher the Tween 80 content, the corresponding increase in the viscosity of the lyophilized sample, so that the content of Tween 80 described in the more preferred embodiment does not exceed 15 wt ^% . . The development effect of the microbubbles in which the content of Tween 80 was 5 to 15% by weight in Example 2 was better than that in Example 1. '

Example 3: Effect of hydrogenation of lecithin

Studies have shown that long-circulating liposomes prepared by hydrogenated egg phosphatidylcholine (HEPC) are slower in vivo than long-circulating liposomes prepared by non-hydrogenated egg phosphatidylcholine (EPC). We hydrogenated egg yolk lecithin: powdered egg yolk lecithin (PC) was dissolved in ethanol, 5% palladium on carbon, and hydrogenated on a hydrogenator. After the hydrogenation treatment is completed, the filtrate is filtered, and the filtrate is rotated under reduced pressure to remove ethanol and frozen. It is dried with hydrogenated egg phosphatidylcholine (HEPC). Substituting 5 mg of hydrogenated lecithin for egg yolk egg yolk (PC) in Example 1 20 mg, Tween 80

The remaining steps of 25 m _g were the same as in Example 1, and an ultrasonic contrast agent was prepared. Sampling to observe the microbubble concentration is 1

X 10 ⁸ /ml, the particle size distribution is 2~8μιη, and the cell wall thickness is 200-800 nm.

The microbubbles of Example 3 had stronger development strength and longer development time than Examples 1 and 2, indicating that the product exhibited better development effects than those of Examples 1 and 2.

Example 4: Effects of different freezing methods

Weigh 1.5mg of hexadecanoic acid, 2mg of glyceryl monostearate, 75mg of PEG1500 (PEG1500), 150mg of Poloxamer 188, 5m of hydrogenated lecithin and 25mg of Tween80 in 2ml of tert-butyl In the alcohol, the water bath was sonicated at 60 ° C. Different freezing methods (1) -10 °C rapid freezing; (2) rapid freezing; (3) repeated freezing and thawing: "0-4 °C coagulation - water bath 15-22 ° C ultrasonic to emulsion - Solidification at 0~4 °C"; (4) Place at 15-22 °C until the liquid solidifies, and refrigerate at 0-4 °C for 10 min. Freeze for 20 hours. 200 mg of the lyophilized sample was weighed into a 5 ml vial, filled with perfluoropropane gas, and sealed to obtain a solid ultrasonic contrast agent lyophilized powder.

When using the plug, inject 2 ml of physiological saline and gently shake to form a microbubble suspension. The concentration of the microbubbles, the average particle size, and the percentage of microbubbles of 2 to 8 μm were observed by sampling. The results are shown in Table 2.

Table 2 Effect of different freezing methods on microbubble concentration, particle size and particle size distribution

Freezing method

(4) 15~22. C item (1) -10 °c (2) 0~4. C (3) repeated

Place at room temperature to liquid, freeze quickly, refrigerate quickly, freeze 3⁄4 way

Solidification, 0~4 °C refrigeration lOmin microbubble concentration

1 3 6 7

(l0 ⁷ /ml)

The average particle size

7 7 6 6

( μιη)

2 to 8 μ m

30 40 55 55

Microbubble percentage

It can be seen from 2 that the freezing method is different for the concentration, particle size and particle size distribution of the microbubbles. Significant effect, mode (3) (repeated freeze-thaw mode: 0-4 °C coagulation - water bath 15_ ² 2 °C ultrasound to emulsion-like -4 Ό coagulation) and mode (4) (15-22 'C room temperature placed The concentration of microbubbles, the average particle size and the percentage of microbubbles of 2~8 μηη obtained from 0-4 C refrigerated lOmin after liquid solidification were the best. The development effects of the modes (3) and (4) are better than those of the embodiment 3.

Example 5

Weighing poloxamer 188 (POLoxamer 188) 150 mg, hydrogenated egg fat 5 mg and Tween 80 25 mg ^ t in 2 ml of tert-butanol, and sonicating in a water bath at 60 °C. 18-22 Place at room temperature until the liquid solidifies (40 min), 0-4. C refrigerated for 10 min. Freeze for 20 hours. The lOOmg lyophilized sample was weighed into a 5 ml vial and filled with perfluoropropane gas and condensed to prepare a solid ultrasonic contrast agent.

When using the plug, inject 1ml of normal saline, gently shake to form a microbubble suspension, sample and observe the microbubble concentration is 1 X 10 ⁹ /ml, the particle size distribution is 2~6 μ m, and the bubble wall thickness is 200-500 nm. .

Application Verification: Ultrasound imaging of liver and kidney in rabbits

The liver and kidney of rabbits were subjected to contrast-enhanced ultrasound using the contrast agent prepared in Example 5, and administered to the rabbit ear vein at a dose of 0.03 ml/k _g . The liver and kidneys showed significant enhanced imaging in about 10 seconds. , 30 seconds to reach the peak, effective enhancement time of more than 1 minute.

Example 6:

Poloxamer 188 (POLoxamer 188) 150 mg, hydrogenated lecithin 8 mg and Tween 80 20 mg ^t were weighed into 2 ml of anhydrous tert-butanol, and sonicated in a water bath at 60 °C. Refrigerate at 0-4 ° C until the liquid is solidified, ultrasonically to an emulsion at 37-39 ° C, and set to solidify at room temperature. Freeze for 20 hours. A lOOmg lyophilized sample was weighed into a 5 ml vial, filled with perfluoropropane gas, and sealed to obtain a solid ultrasonic contrast agent.

When using the plug, inject 1ml of normal saline, gently shake to form a microbubble suspension, sample and observe the microbubble concentration is 1 X 10 ⁹ /ml, the particle size distribution is 2~5 μ ιη, and the bubble wall thickness is 200-500 nm. .

Application Verification: Ultrasound imaging of liver and kidney in rabbits

The contrast agent prepared in Example 6 was better than that in Example 5. The effective enhancement of renal imaging was more than 2 minutes, and the gray scale of liver parenchyma was still higher than that before non-contrast at 10 minutes.

Claims

Rights request

1. An ultrasonic contrast agent composition comprising a phospholipid component as a film forming material, comprising a film forming material and a fluorocarbon inert gas, wherein the film forming material is composed of a phospholipid component, a foaming agent, a polymer, and a stabilizer. It is characterized in that: the weight percentage of each component in the film-forming material, the phospholipid component is 1 to 10%, the foaming agent is 5 to 15%, the stabilizer is 0.5 to 10%, and the polymer is 70 to 90%.

The composition of claim 1 in the form of a unit dosage form, wherein the amount of the fluorocarbon-containing inert gas per unit dose is 0.15 to 0.5 ml.

3. The composition of claim 1 wherein the phospholipid component is selected from the group consisting of at least one lecithin, hydrogenated soybean phospholipid, hydrogenated egg yolk phospholipid, dipalmitoyl phosphatidyl alcohol, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidyl amide Ethanolamine, polyethylene glycol-distearoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid glyceryl-sodium salt, 1,2-distearoyl-sn-glycerol 3-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid-sodium salt, 1,2-dipalmitoyl-SH-glycero-3-phosphatidylcholine; Wherein the blowing agent is selected from the group consisting of at least one nonionic surfactant; wherein the polymer is selected from the group consisting of at least one high molecular polymer; wherein the stabilizer is selected from the group consisting of polyethylene glycols, glyceryl monostearate, palmitic acid.

4. The composition of claim 3 wherein the nonionic surfactant is a Tween-like substance and a Span substance; the high molecular polymer is a poloxamer; and the polyethylene glycol is a polyethylene glycol 1500-6000. »

The composition of claim 4 wherein the Tween is Tween 80; the poloxamer is poloxamer 188; and the polyethylene glycol 1500 6000 is polyethylene glycol 1500.

6. A method of preparing a composition according to claim 1, comprising the steps of: (a)

The film-forming material is contacted with a small amount of an anhydrous or aqueous organic solvent, and is made into a uniform solution system by ultrasonication and heating;

Step (b)

Slowly cool the solution by (a) the solution, or quickly refrigerate, then use ultrasound to the milk. The turbid liquid or repeated freeze-thaw method makes the precipitated particles evenly dispersed;

Step (C)

Freezing the product of step (b);

Step (d)

The lyophilized product of the step (c) is pulverized, dispensed into a vial to prepare a powder injection, and a fluorine-carbon-based inert gas is injected into the bottle to cover.

The preparation method according to claim 6, wherein the nonaqueous solvent is anhydrous or a linear or branched alcohol having a trace amount of water, and the ultrasonic and heating conditions are at a temperature of 45 to 65°. C, time is 20~40 minutes; the ultrasonic mode of step (b) is selected from water bath or probe type ultrasonic; the freeze-drying condition of step (c) is freeze-drying time is 20~25 hours, said in freeze-drying The vacuum suction pressure is 50 120 X 10 ³ mBar, and the freeze-drying temperature is controlled from -40 to -50. C.

The preparation method according to claim 7, wherein the non-aqueous solvent in the step (a) is anhydrous or tert-butanol containing a trace amount of water; and the ultrasonic mode of the step (b) is probe-type ultrasonic.

The preparation method according to claim 8, characterized in that the tert-butanol is dehydrated before use, and the water content per liter of t-butanol is less than 0.02 ml.

The preparation method according to claim 6, characterized in that: poloxamer 188 150 mg> hydrogenated lecithin 8 m and Tween 80 20 m are dispersed in 2 ml of anhydrous t-butanol, and dissolved in a water bath at 60 ° C; 0- Refrigerate at 4 °C until liquid solidifies, sonicate to emulsion at 37-39 °C, solidify at room temperature, freeze-dry for 20 hours; weigh 100 mg of lyophilized sample in 5 ml vial, filled with perfluoropropane gas, densely packed, That is, a solid ultrasound contrast agent is prepared.