JP4808842B2 - Vesicle preparation method - Google Patents

Description

[0001]
The present invention relates to a method for preparing a gas-containing vesicle (for example, an ultrasound contrast agent or a precursor thereof).
[0002]
In ultrasound diagnostic imaging, the use of contrast agents that include vesicles (eg, microballoons, liposomes, or micelles) containing a gas or mixture of gases trapped within a membrane has been widely proposed. For such purposes, the membrane can be a single layer or multiple layers of amphiphilic materials such as, for example, lipids.
[0003]
Vesicles containing such gases can be readily produced by shaking or sonicating a liquid containing film-forming material in the presence of a suitable gas or mixture of gases. (Gas or a mixture of gases includes materials that become gaseous at body temperature (eg, 37 ° C.)).
[0004]
However, the size of vesicles produced by such techniques varies from batch to batch and is distributed over a wide range. In addition, the yield (ie, the proportion of film-forming material that results in appropriately sized gas-containing vesicles) can also vary from batch to batch.
[0005]
It is desirable that the vesicles produced have a narrow size distribution (eg 3 ± 1 μm) around the desired vesicle size, generally from 1 to 7 μm.
[0006]
The inventor believes that the relative rotation of the rotor-stator mixer (ie the starting mixture is on two surfaces, one on a member called the rotor and the other on the member called the stator). It has been found that the production of vesicles using a mixer that passes through a zone where shear forces occur increases the yield and does not unnecessarily produce oversized vesicles.
[0007]
A rotor-stator mixer is typically used to form an emulsion from a mixture of immiscible liquids. The accompanying FIG. 1 is a schematic of a standard laboratory scale rotor-stator. Such a rotor-stator with a rotor outer diameter of 15 mm is used at a rotational speed of 23000 rpm and contains a gas as described in Example 2 (b) of WO 97/29783 (Nycomed). Formed vesicles.
[0008]
However, the inventor has found that the yield of appropriately sized gas-containing vesicles is improved when the relative speed of the rotor and stator surfaces is at least 20 m / s.
[0009]
Thus, according to one aspect of the present invention, in a method for preparing a gas-containing vesicle, a mixture of gas or gas precursor, liquid and vesicle membrane-forming material is at least 20 m / s, preferably at least 25 m. / S, particularly preferably at least 30 m / s, and more particularly preferably at a speed of at least 35 m / s (for example up to 100 m / s, more particularly up to 60 m / s and especially up to 50 m / s) A method is provided for preparing vesicles that pass through a zone subjected to shear forces generated by a relatively moving surface.
[0010]
The inventor also provides that the vesicle-forming mixture has a plurality of shear force zones (eg, rotor: stator stage), such as at least two shear force zones, preferably at least three shear force zones, and more preferably At least 4 shear zones, particularly preferably at least 12 shear zones (eg up to 90, more particularly up to 44 shear zones (eg up to 20 or up to 12 shear zones)) ) Was found to improve the size distribution of the vesicles. In this way, the range of residence time in the shear zone is a narrower distribution and the occurrence of excessive vesicles is reduced. Certainly, by providing multiple successive shear zones through which the mixture passes, the mixture does not need to be recirculated through the open shear zone, so the mixing conditions (e.g., residual time and temperature) of the entire mixture. Moderate mixing is ensured so that the profile is substantially the same. With such uniform and configurable mixing parameters, the product properties do not vary much from batch to batch. This is very important in mixing processes that are upgraded from a laboratory bench to a pilot plant or mass production.
[0011]
According to another aspect of the invention, a method for preparing a gas-containing vesicle, wherein a mixture of gas or gas precursor, liquid and vesicle membrane-forming material is produced by surfaces that move relative to each other. A method is provided for preparing vesicles that are successively passed through a plurality of different zones subject to shear forces. In this second method, the mixture successfully passes through at least one shear force zone, where the relative speed of the surface is at least 10 m / s, preferably at least 15 m / s, especially at least 30 m. / S, more particularly up to 50 m / s, and particularly preferably according to the first method of the invention. It has been found advantageous to operate at a relative surface velocity of 46 m / s when using an outer zone with a diameter of 110 mm.
[0012]
In the method of the invention, it is desirable that the surfaces that move relative to each other to form a shear force are less than 2 mm, preferably less than 1 mm, particularly preferably less than 500 μm (eg 100 to 300 μm) apart from each other. . The optimal separation interval depends on the viscosity of the mixture passing through the shear zone and the minimum separation interval can be determined by manufacturing constraints. In general, however, for aqueous mixtures, the separation interval is desirably in the range of 200 to 300 μm. Such a separation interval refers to a value that can be measured when the surface is not moving, since the surface may be deformed while it is moving.
[0013]
For convenience, the shear zone is generally formed between the moving surface and the stationary surface, preferably (in the rotor-stator mixer) between the rotating surface and the stationary surface.
[0014]
Where the mixture passes through multiple shear zones, for example, this can be accomplished by providing multiple rotor-stator devices, but a device arranged in this way is formed by the previous device. Receive the mixture. Such devices may have separate rotational drives or may share a common drive (ie, be arranged coaxially about a common drive shaft). However, it is more efficient to use one or more rotor-stator combinations, each providing a plurality of radially separated shear force zones.
[0015]
According to another aspect of the present invention, in a method for preparing a gas-containing vesicle, at least a mixture of a gas or a gas precursor, a liquid, and a vesicle membrane-forming material is moved relative to at least one stator. Providing two or more radially separated zones of action of one rotor, preferably more than one (eg 2 to 20 (eg 2 to 10), but more convenient (eg 13)) There is provided a method for preparing vesicles that are successively passed through a plurality of different zones subjected to shear forces due to the action of two or more (eg 2 to 20 (eg 2 to 10)) coaxial rotors.
[0016]
The relative speed of the rotor surface and the stator surface in the third method of the invention is such that the rotor surface and the stator surface are at least 10 m / s, preferably at least 15 m / s, more preferably in at least one zone. It is preferably set to move at least 30 m / s (eg up to 50 m / s, particularly preferably according to the first method of the invention).
[0017]
The rotor-stator mixer apparatus suitable for use in the third method of the present invention is novel and constitutes another aspect of the present invention.
[0018]
According to this aspect of the invention, there is provided a rotor-stator mixer apparatus comprising a mixing chamber having gas and liquid inlet ports and a mixture outlet port, wherein the chamber is provided with a rotor and its driving means, The stator has a stator so as to face the rotor, and the stator and the rotor have an axially extending raised ridge and a groove provided with a fluid passage means extending in the radial direction. Thereby providing a rotor-stator mixer apparatus that defines a plurality of shear force zones such that fluid passes radially from the inlet port between the rotor and the stator.
[0019]
In the mixer device of the present invention, the inlet port is preferably located radially inward of the shear force zone on or near the rotational axis of the rotor. The inlet port is adjacent to mixing means (eg, an axially extending flange or “propeller”) provided on the drive shaft for the rotor and gas and liquid are mixed before entering the shear zone. Is desirable.
[0020]
To ensure proper mixing, a second rotor (further (eg up to 5) rotors as desired) is provided and is driven by the same drive means, preferably a rotary drive shaft. It is preferable. Where a second rotor is provided, the mixer preferably has a second mixing chamber having an inlet port in communication with the outlet port of the first chamber and having its own outlet port. The inlet port of the second chamber is also preferably radially inward of the shear zone between the second rotor and its stator.
[0021]
Multiple stators may be in the mixing chamber, or one stator may be provided on the walls of the mixing chamber.
[0022]
The rotor: stator combination provides multiple (eg, 2 to 25, preferably 7 to 20, especially 9 to 15) radially separated shear force zones per combination. . Thus, it is desirable that a total of 18-30 shear zones can be provided by a device having two rotor: stator combinations.
[0023]
In the rotor: stator combination, the interlocked ridges and grooves are provided by engaging a cylindrical extension on the base of the rotor and stator, within the cylindrical extension. It is preferred to have fluid passing means provided by slots (eg, cuts) extending radially and axially spaced apart.
[0024]
The rotor and stator may be formed of any suitable material or combination of materials, but is preferably a metal or a metal, particularly a metal such as steel. Further, the rotor and stator surfaces can be coated or treated as desired to obtain a final product with optimal yield or properties. The dimensions of the rotor and stator components depend on the rotor material, the upper limit of the desired vesicle size, the speed of rotation, the diameter of the rotor, and the viscosity of the mixture. However, in general, for stainless steel components, the rotational speed is 5000 to 10,000 rpm, an aqueous mixture is used, and the rotor diameter is up to 25 cm (eg 7.5 to 15 cm), Any rotor with a cylindrical extension of 2 to 3 mm in radial direction, a slot axial depth of 5 to 6 mm, and a circumferential width of 0.3 to 2 mm, separated by at least 1.5 mm. : Even in the stator combination, there are a total of 10 to 50 slots for each cylindrical extension, preferably used when there are the same number of slots on each extension obtain. However, the mixer device according to the present invention is not limited to these parameters and may be manufactured with other dimensions, materials and operating speeds.
[0025]
The apparatus of the present invention provides a significant heating effect on the mixture of gas, gas precursor, liquid, and film-forming material, and the size and stability of the vesicles can be affected by temperature, so that temperature control means (e.g., Thermostat-controlled heating or cooling means such as a cooling jacket surrounding the mixing chamber), or a cooling element in or in addition to or in the stator or rotor, or rotor drive It is particularly desirable to provide an apparatus with a cooling element that conducts heat in or between a mechanical seal surrounding a shaft or rotor drive shaft. Such a cooling element is, for example, a cooling coil that surrounds or is embedded in the component to be cooled, or is disposed within such a component (eg in the drive shaft, away from the corresponding rotor) It may take the form of a cooling fluid conduit through which heat is conducted between such components (eg, on the stator side, around the stator, etc.). The temperature of the mixture can be monitored at the outlet of each mixing chamber or at the edge of the rotor and used to control such temperature control means. In general, the temperature can thus be controlled with an accuracy of ± 2 ° C., preferably ± 0.5 ° C. or more. The determined temperature can preferably be maintained below 45 ° C., in particular below 40 ° C. after the first or single rotor, in particular below 30 ° C. after the final rotor in a multi-rotor device. . If the stator is provided with cooling means and the rotor drive shaft and / or seal is cooled, the temperature of the mixture can be maintained at 35 ° C. after each rotor.
[0026]
If the device is to be used for drug production, it must be aseptic and can be sterilized without removing the mixing chamber. Thus, a double mechanical seal on the drive shaft that enters the mixing chamber (or the first mixing chamber, or more preferably the final mixing chamber for a multi-rotor device), for example, a fixed ceramic surface and a rotating ceramic surface. It is particularly desirable to be provided with a mechanical seal provided between the two. Further, each mixing chamber is preferably provided with a discharge port so that fluid can be discharged from the chamber. Such an exhaust port may be provided by a chamber exit port, but is generally provided separately from the exit port.
[0027]
The gas inlet port to the first mixing chamber is preferably large enough for the sterilization medium to pass through, but is generally smaller than the liquid inlet port. The inlet port is preferably provided with a multi-way valve which acts as a gas inlet port and in other cases as an inlet port for the sterilization medium. Thus, the “gas” inlet port serves the dual function as a gas inlet and a liquid inlet for gases and gas mixtures (gas or liquid state, ie in the form of a liquid precursor), by operating the valve. It can have different diameters for the two functions. Thus, for example, the liquid inlet port has a diameter of 3 to 8 mm, whereas the gas inlet port has a diameter of 0.2 to 2 mm (eg, 0.5 mm) when acting as a gas inlet. And when acting as a liquid inlet, the diameter may be 3 to 8 mm.
[0028]
In a preferred embodiment, the gas and liquid inlets open to a premixing chamber in a first mixing chamber having a wall defined by an end of the drive shaft and a recess in the wall of the mixing chamber, and at the end of the drive shaft. The surrounding annular opening opens to the main part of the mixing chamber. Mixing means (eg, flanges, eccentric pins, propellers, etc.) are attached to the end of the drive shaft so that gas and liquid premixing takes place in this premixing chamber.
[0029]
The gas introduced into the gas inlet may be any material that becomes a gas at a mixing temperature (for example, 10 to 45 ° C.), but preferably air, oxygen, nitrogen, helium, carbon dioxide, sulfur hexafluoride. A physiologically acceptable gas or gas mixture, such as a low molecular weight hydrocarbon or a low molecular weight fluorinated hydrocarbon, or a mixture of two or more thereof. If desired, the gas or gas mixture may be a material that is liquid upon introduction into a temperature history within the device or after administration to a living body. Such materials that are not initially gaseous but that act to produce a gas when mixed and / or administered (eg, at temperatures up to 45 ° C.) are referred to herein as gaseous precursors. ) When the gas or gas mixture is in liquid (gas precursor) form when introduced into the rotor-stator, this “liquid gas” is the liquid into which the film-forming material is introduced through the liquid inlet port. It is preferable not to mix. In general, however, the gases and gas mixtures described in WO 97/29783, in particular perfluorocarbons such as perfluorobutane or perfluoropentane, are particularly preferred. The material introduced into the rotor-stator mixer may optionally be more than one gas component or gas forming component (eg, liquid at ambient temperature during mixing but forming gas at physiological temperature). And components that are in gaseous form at ambient temperature during mixing). Thus, when administered to a living body, vesicle compositions that increase in vesicle size as described, for example, in WO 98/17324, the contents of which are incorporated herein by reference, Can be formed. The liquid gas-forming component is conveniently an emulsifying liquid having a low boiling point, such as, for example, perfluoropentane, as described in WO 94/16379 and listed in WO 98/17324.
[0030]
The film-forming material introduced into the liquid inlet is preferably an amphiphilic material, such as an ionic or non-ionic surfactant, or more preferably a lipid (eg, phospholipid). Since one particular use of gas-containing vesicles includes injectable contrast agents, it is preferred that the film-forming material be physiologically acceptable. Particularly preferred are phospholipids (particularly charged phospholipids and mixtures thereof) as disclosed in WO 97/29783.
[0031]
The liquid into which the film forming material is introduced may be any liquid as long as it can form gas-containing vesicles. Preferably, the liquid is a sterilized aqueous liquid as described, for example, in WO 97/29783.
[0032]
The absolute and relative flow rates of gases, liquids and film-forming materials to the mixing device used according to the present invention depend on the specific materials and devices used as the operating parameters of the mixing device. In general, however, the device is operated to form vesicles having an average particle size with an average particle size ranging from 1 to 10 μm, in particular from 2 to 7 μm, in particular from 2.5 to 5 μm by volume.
[0033]
The device according to the invention can also be used to produce emulsions. In this case, a separate gas inlet port is not necessary. Such an apparatus constitutes another aspect of the present invention. However, the emulsion forming apparatus of the present invention preferably has at least one of a double acting seal on the drive shaft, temperature control means, and a plurality of stators.
[0034]
The disclosures of the documents mentioned herein are hereby incorporated by reference.
[0035]
Hereinafter, the present invention will be further described with reference to the accompanying drawings.
[0036]
Please refer to FIG. FIG. 1 shows a stator 1 in the form of an open cylinder with an opening 2 on the side. The rotor 3 is disposed in the stator 1 and has a cylindrical opening tip with a groove 4 extending in the axial direction. In use, the fluid is discharged from the opening 2 through the groove 4 from within the rotor 3.
[0037]
Please refer to FIG. In the apparatus of the present invention, gas and liquid are introduced into the premixing chamber 7 through inlets 5 and 6, respectively. The wall of the premixing chamber 7 is defined by the recess of the housing 8, the first stator member 9, and the tip 10 of the rotor drive shaft 11.
[0038]
The tip 10 of the rotor drive shaft 11 carries a flange 12 (shown attached to the side) that acts to mix gas and liquid in the premix chamber 7.
[0039]
The housing 8 provides a cylindrical chamber, has a cup-shaped portion 8 and an end cap 13, and the rotor drive shaft 11 enters through the base of the cup-shaped portion 8 and is sealed with a double-acting seal 14. Yes. The drive shaft 11 is rotated by a motor 15 disposed outside, and the first rotor 16 and the second rotor 17 engaged with the first stator 18 and the second stator 19 are engaged with each other. Rotate. The rotor and stator are gold-plated and have an outer diameter of about 110 mm.
[0040]
The premixing chamber 7 is in communication with the first mixing chamber 20, which is defined by the surface of the housing 8, the first stator 18, and the second stator 19, and the second mixing chamber It has an outlet 21 corresponding to the inlet to the chamber 22. The second mixing chamber 22 is defined by the second stator 19 and the surface of the housing 8.
[0041]
In the first mixing chamber 20, material from the premixing chamber 7 passes through the fluid passage defined by the axial slots 26, 27 in the first rotor and the first stator to the first stator 18. And passes radially outward through the shear force zones 23a, b, c, etc. between the cylindrical extensions 24, 25 of the first rotor 16. Each rotor-stator assembly defines about 12 to 14 such shear zones. Around the mixing chamber, the material being mixed passes radially inward and from the first mixing chamber through the outlet 21 to the second mixing chamber.
[0042]
The cylindrical extensions of the rotor and stator are not shown in the lower part of FIG. The slots in the cylindrical extensions of the second rotor and second stator are likewise not shown.
[0043]
In the second mixing chamber 22, the mixture passes radially outward between the second rotor and the second stator before being discharged through the outlet 28.
[0044]
The first and second mixing chambers 20 and 22 are provided with discharge ports 29 and 30 along the lower boundary line. These discharge ports can be connected to a vapor trap and a discharge, for example, as in a conventional drug manufacturing device. Further, around the periphery, the first and second mixing chambers are provided with annular temperature control means (for example, water cooling jackets 31 and 32 whose temperature is controlled by monitors 33 and 34 and control means 35 and 36).
[0045]
In a typical use case of a mixer device, the drive shaft 11 is at a speed such that the 8000 rpm or external shear zone 23 has a relative rotor: stator speed of at least 32 m / s (eg 46 m / s). It is rotated. Gas (eg, perfluorobutane) is introduced through inlet 5 at a flow rate of 50 to 200 mL / min. An aqueous liquid containing hydrogenated egg phosphatidylserine (5 mg / mL) and 5.4% w / w propylene glycol and glycerol (3:10 w / w) has a rate of 50 to 200 mL / min. Preferably, it is introduced through the inlet 6 in a volume ratio to gas of 0.5: 1 to 2: 1. Gases and liquids are introduced at 20 ± 5 ° C., the first water cooling jacket 31 is controlled to maintain the temperature of the mixture at 38 ° C. in the monitor 33, and the second water cooling jacket 32 is mixed in the monitor 34. The volume size of the intermediate vesicles in the mixture discharged from the outlet 28 is about 3 ± 1.5 μm.
[0046]
FIG. 3 shows a rotor-stator array of a rotor-stator mixing device. Cylindrical extensions (flanges) 24, 25 and axial slots 26, 27 of the stator and rotor extend in the axial direction and are spaced axially and radially in the circumferential direction. It can be seen that it consists of a plurality of axially extending “teeth” 37 separated by a plurality of corresponding openings 38 extending in the direction. In use, the material being mixed passes through these openings to a continuous shearing zone defined by the adjacent cylindrical extensions and the peripheral sides of the axial slot.
[0047]
FIG. 4 shows the individual flanges 24, 25.
FIG. 1 is a schematic partial cutaway view of a conventional rotor-stator mixer.
FIG. 2 is a schematic axial sectional view of a rotor-stator mixer device according to the present invention.
FIG. 3 is a diagram of a rotor-stator assembly of a mixer device according to the present invention.

Claims (9)

A process for the preparation of an ultrasound contrast agent consisting of vesicles containing gas, and said gas or gaseous precursor, and a liquid, a mixture of vesicle membrane forming material comprising a lipid, at a rate of at least 20 m / s A method of preparing vesicles that pass through a zone subjected to shear forces generated by surfaces that move relative to one another.   The method of claim 1, wherein the surfaces move relative to each other at a speed of at least 30 m / s.   The method according to claim 1 or 2, wherein the surfaces move relative to each other at a speed of up to 60 m / s.   4. A method according to any one of claims 1 to 3, wherein the mixture passes through a plurality of different zones in succession and is subjected to shear forces from a surface in which the mixture moves relative to one another.   The method of claim 4, wherein the mixture is successively passed through a plurality of different zones subjected to shear forces caused by at least one rotor moving relative to the at least one stator.   6. A method according to claim 5, wherein the mixture is successively passed through a plurality of different zones subjected to shear forces caused by at least two rotors each moving relative to the corresponding stator.   7. A method according to any one of claims 4 to 6, wherein the mixture is passed through at least 18 of the zones in succession.   8. A method according to any one of claims 4 to 7, wherein the mixture is continuously passed through up to 90 zones.   9. A method according to any one of the preceding claims, wherein the moving surfaces are spaced from 100 to 500 [mu] m.

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