Ultrasonography contrast agents and methods of using them

Abstract

This document provides an ultrasound contrast agent and its use. [Solution] The present invention provides an ultrasound contrast agent comprising (a) perfluorocarbon microbubbles stabilized by a phospholipid membrane, wherein the perfluorocarbon is perfluorobutane and the phospholipid is hydrogenated egg yolk phosphatidylserine; and (b) a buffer, which is tris(hydroxymethyl)aminomethane (Tris); and having a bulk pH of about 7.5 to about 8.5 when measured at a temperature of 5°C. The ultrasound contrast agent is for long-term storage and is immediately usable in vivo. Furthermore, the present invention provides a method for preparing such an ultrasound contrast agent, pharmaceutical compositions, and the use of the ultrasound contrast agent for manufacturing pharmaceuticals.

Description

Technical Field

[0001] The present disclosure relates to the field of in vivo imaging and diagnosis of a subject, particularly an ultrasonic contrast agent that is immediately available for in vivo use and can withstand long-term storage before in vivo use. The present disclosure further relates to a method for preparing an ultrasonic contrast agent and a method for using the ultrasonic contrast agent at the clinical site.

Background Art

[0002] Ultrasonic contrast agents based on lipid-stabilized microbubbles of perfluorocarbon are well known in the art (see, for example, Wheatley et al, J. Drug Del. Sci. Technol., 23(1), 57-72, 2013). A single microbubble can consist of a gaseous core, about 2-10 μm in size, encapsulated by a shell or membrane of a layer of lipid molecules for stabilization. The compressible gaseous core can expand and contract when subjected to ultrasonic waves. The expansion and contraction of the microbubbles when exposed to ultrasonic waves cause backscattering of the sound waves, which is used for diagnostic imaging purposes. The surface of the microbubbles can be further functionalized with a target drug portion released when the microbubbles rupture and / or cavitation occurs during ultrasonic application, and such ultrasonic contrast agents can thereby also be used for therapeutic applications (Upadhyay et al, RSC Adv., 6, 15016-15026, 2016).

[0003] Sonazoid (trademark) is a lipid-stabilized microbubble of perfluorocarbon This is an example of an ultrasound contrast agent based on [the specified compound]. More specifically, Sonazoid (trademark) is [the specified compound]. Perfluorobutane stabilized by a hydrogenated egg yolk phosphatidylserine membrane It is formulated as a powder consisting of freeze-dried sucrose with clobubbles encapsulated, and perfluoro It is stored under butane headspace. Sonazoid (trademark) is perfluoro Tan (PFB) is continuously dispersed in an aqueous dispersion of hydrogenated egg yolk phosphatidylserine (HEPS). It is manufactured aseptically by homogenizing it. After the initial generation of microbubbles, The concentration and particle size distribution of the chlorobubbles are controlled by a series of separation steps. The goal is to create 8 μl of microbubbles per ml in the formulated product. The final dispersion is made isotonic by adding sucrose. 2 ml of dispersion is mixed with 10 ml of gas. The vials are filled and freeze-dried. After freeze-drying, the heads of the vials are removed before capping. Perfluorobutane is backfilled into the space. In other words, Sonazoid (Trademark) is a freeze-dried product and must be reconstituted with water before use. For more details, before administering the product to the subject, the supplied vented filter (5μm) Spike (Codan Chemoprotect®, Codan) Reconstructed by GmbH&Co., Germany by adding 2 ml of sterile water for injection. This is done, and then mixed by hand for 1 minute. After recomposition, the product appears as a milky white homogeneous dispersion. Because the dispersion is opaque, visual inspection of dissimilar particles is difficult. To ensure there are no issues, draw the product into the syringe through the filter spike before administration. If left undisturbed after recomposition, the microspheres will begin to separate by suspension and will rise to the top of the liquid phase. It forms a creamy layer. Unless used immediately after recombination, the product should be handled by hand before use. It should be homogenized again by mixing for 10 seconds (Sontum, Ultrasound Med. & Biol., 34(5), 824-833, 2008).

[0004] The active ingredient in ultrasound contrast agents is not a chemical substance but a physical state (microbubbles). Therefore, two types of stability must be considered: physical stability and chemical stability. In other words, not only the chemical composition of the constituent components, but also the concentration of microbubbles The focus must be on how to control and maintain the particle size distribution. Cloroxalobubbles are generally thermodynamically unstable systems and undergo physical changes during preparation and storage. This can occur (for example, in the international publication pamphlet No. 2015150354(A1); Segers et al, Langmuir, 33, 10329-10339, 2017; Borden et al, Advances in Colloid See also Interface Science 262, 39-49, 2018). In addition, microbubbles The phospholipid membrane that stabilizes the product undergoes hydrolysis in solution, producing impurities in the final product. This can happen. Phospholipids readily undergo hydrolytic cleavage in acidic and alkaline media. Lipids are only sufficiently stable at pH 7. This is because esterification occurs under these conditions. This is because the hydrolysis of the compound does not proceed very much (Phospholipids Handbook, 1993, edited by Gregor Cevc; see Chapter 9 “Chemical stability”, Evstigneeva, pp. 323-324. (Please be illuminated). Temperature and pH greatly affect the reaction rate of hydrolysis (Phospholipid). s Handbook, 1993, edited by Gregor Cevc; Chapter 9 “Chemical stability”, Cromm See Elin et al, pp. 338-339. Furthermore, once hydrolysis is initiated at a low pH, It is known that this lowers the pH and accelerates decomposition. Therefore, So A major challenge during the initial development of nazoid (trademark) was how to achieve an acceptable shelf life. The goal was to obtain a product having the following: Sonazoid (trademark) in the form of freeze-dried powder. Lyophilization, which results in the formulation, has until now been considered the only way to obtain such a product.

[0005] Freeze-drying can result in products with excellent shelf-life stability and quality. Because it is also time and resource-intensive, it significantly increases manufacturing costs and the end user It reduces the "ease of use" for (typically healthcare professionals). Therefore, in this field, A perfluorocarbon phospholipid-stabilized microphone that combines excellent storage stability with ease of use. There is a need for improved ultrasound contrast agents based on Robalubelle. [Overview of the Initiative]

[0006] The objective above is to provide an ultrasound contrast agent that combines high storage stability and ease of use. This disclosure achieves, surprisingly, that it can withstand long-term storage before use. It is possible and immediately available for use, i.e., immediately injectable into the target, in the form of a stable and immediate dispersion. Regarding usable ultrasound contrast agents.

[0007] For more details, this disclosure is: (a) Perfluorocarbon microbubbles stabilized by a phospholipid membrane; and and (b) a buffer; comprising, having a bulk pH of about 7.5 or greater, preferably about 8.5 or greater, a contrast agent for ultrasound imaging.

[0008] The present disclosure also relates to (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of phospholipid to produce phospholipid-stabilized microbubbles of perfluorocarbon dispersed in the aqueous dispersion; <00ooo100>step; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 - 6 μm, preferably 2 - 5 μm step; (iii) optionally, adding an isotonic agent to the aqueous dispersion; (iv) adding a buffer to the aqueous dispersion to adjust the bulk pH of the aqueous dispersion to a pH of about 7.5 or greater, preferably about 8.5 or greater; (v) adjusting the concentration of the microbubbles in the aqueous dispersion to achieve a target concentration of about 6 - 10 μl / ml of microbubbles; step; (vi) dispensing the aqueous dispersion into vials and flushing the headspace of the vials with perfluorocarbon; step; A method for preparing a contrast agent for ultrasound imaging.

[0009] Furthermore, the present disclosure relates to a method for improving the contrast of ultrasonic images of tissues in a subject, a method for in vivo imaging of tissues in a subject, and a method for diagnosing a subject, and these methods include injecting the contrast agent for ultrasound imaging as described above into the subject. step.

[0010] This disclosure also relates to ultrasound contrast agents for use in the manner described herein. .

[0011] Furthermore, this disclosure relates to manufacturing pharmaceuticals for use in the manner disclosed herein. This relates to the use of ultrasound contrast agents as disclosed herein.

[0012] Preferred embodiments of this disclosure are described below in the detailed description and dependent claims. [Brief explanation of the drawing]

[0013] [Figure 1] This figure shows the chemical stability of Sonazoid bulk products after being collected before freeze-drying, prepared as a non-buffered aqueous dispersion, and stored at 5°C for 8 months. [Figure 2] This figure shows the chemical stability of freeze-dried sonazoid powder stored at 5°C for 6 months, prepared as a non-buffered aqueous dispersion, prepared as a buffered aqueous dispersion containing a buffer and having a bulk pH of 7 at room temperature, and prepared as a buffered aqueous dispersion containing a buffer and having a bulk pH of 8 at room temperature. [Figure 3] This figure shows the physical stability of freeze-dried sonazoid powder stored at 5°C for 6 months, prepared as a non-buffered aqueous dispersion, prepared as a buffered aqueous dispersion containing a buffer and having a bulk pH of 7 at room temperature, and prepared as a buffered aqueous dispersion containing a buffer and having a bulk pH of 8 at room temperature. [Modes for carrying out the invention]

[0014] This disclosure provides a liquid ultrasound contrast agent that is immediately usable with good storage stability. By including a buffering agent in the product, the powder can be reconstituted before use of the claimed product. Because it is no longer necessary, the freeze-dried powder of the conventionally known Sonazoid (trademark) is used. Compared to the previous method, a liquid formulation that is easier for the end user to handle was achieved. Here, perfluoro A buffer is added to an ultrasound contrast agent based on carbon phospholipid-stabilized microbubbles. This could mean that electrolytes (for example, those present in the buffer) could change the composition of the dispersion. Therefore, it is not clear to those skilled in the art whether or not it will function as a functional ultrasound contrast agent. I would like to keep this. Nevertheless, the inventors of this invention have surprisingly found that microbubbles We managed to maintain the volume concentration and distribution of at the desired level. In other words, compared to conventional methods, Physical stability of dispersions of known reconstituted Sonazoid™ freeze-dried powders. Compared to physical and chemical stability, the chemical stability of the product is improved while maintaining physical stability. We managed to maintain qualitative accuracy. Therefore, the storage stability of the product claimed here is stable. The performance has been improved compared to conventionally known products.

[0015] By including a buffer in the formulation, the ultrasound contrast agent according to this disclosure is aldehyde at a temperature of 5°C. It has a bulk pH in the potassium range. As will be further shown in the following examples, it is alkaline. The pH significantly reduces the rate of hydrolysis of phospholipids present in the ultrasound contrast agent, and Therefore, it maintains a chemically stable state for a much longer period. The phospholipid membrane stabilizes the microbubbles. To clarify, the chemical stability of phospholipids further influences the physical stability of microbubbles. To give a sound.

[0016] For more details, this disclosure is: (a) Microbubbles stabilized by a phospholipid membrane, perfluorocarbon Microbubble; and (b) buffering agent; It contains and has a bulk pH of about 7.5 or higher, preferably about 8.5 or higher. By providing an ultrasound contrast agent, phospholipid stabilization micro-perfluorocarbons To solve or at least mitigate the problems associated with existing bubble-based ultrasound contrast agents. ru.

[0017] The term "contrast agent" has traditionally been used in the field of in vivo medical imaging. An agent that has meaning and is in a form suitable for administration to mammals, and is intended for use in mammals. It helps to provide clearer images of regions or organs of interest than those obtained with the ping alone. It refers to an agent that exerts an effect. The term "target" refers to mammals in vivo, preferably in This refers to the intact mammalian body in vivo, more preferably a living human subject. The phrase "a form suitable for administration to mammals" means that it is sterilized and free of pyrogenic substances. It is free of toxic or harmful compounds and has a biocompatible pH (approximately pH 4.0-10). 5) This refers to compositions formulated in vivo. Such compositions induce embolisms. It does not contain any particulate matter that could potentially cause precipitation, and precipitates when it comes into contact with bodily fluids (e.g., blood). They are formulated to avoid complication. Such compositions are also biocompatible excipients. It contains only and is preferably isotonic.

[0018] Like other in vivo imaging agents, the contrast agent is used to image the limbs that are being imaged. It is designed to exert minimal pharmacological effects on dairy animals. Preferably, the contrast agent is minimal. When performed in an extremely invasive manner, that is, under the medical expertise of a specialist, infant feeding It can be administered to mammals without posing any substantial health risk to the target substance. Such minimally invasive administration preferably does not require local or general anesthesia. This involves intravenous administration of the subject into a peripheral vein.

[0019] The term "microbubble" is used in the field of in vivo ultrasound imaging. A gas with a conventional meaning, having an inner diameter between 0.1 and 10 μm, typically between 0.5 and 5 μm. This refers to microbubbles in the body. Such microbubbles are similar in size to red blood cells. As a result, microbubbles exhibit similar properties in the microvessels and capillaries throughout the mammalian body. This can be demonstrated (Sirsi et al, Bubble Sci. Eng. Technol, 1(1-2), 3-17, 2009). In this specification, the terms "microbubble" and "microsphere" are interchangeable. It may be used.

[0020] The term "perfluorocarbon" has its conventional chemical meaning, and formula C x F y of This is a general term for the group of organofluorine compounds that contain (i.e., only carbon and fluorine). (IUPAC, Compendium of Chemical Terminology, 2nd ed., 1997 (2006~ is online) See the revised version. All compounds with the prefix perfluoro hydrocarbons including those with heteroatoms, in which the CH bond is replaced by a CF bond. Perfluorocarbons include perfluoroalkanes, fluoroalkenes, and fluorine It contains roalkynes and perfluoroaromatic compounds. "Perfluorocarbons" The terms "fluorocarbon" and "container" are sometimes used interchangeably. Suitable perfluorocarbons according to the diagram include perfluoroalkanes, for example, perfluoro This includes lobutane, perfluoropropane, and perfluoropentane. In the context of medical applications, preferred perfluorocarbons have their standard chemical meaning. Then there is perfluorobutane ("PFB"), also known as perflubutane. The chemical formula for oro-n-butane is CF3CF2CF2CF3 or C4F 10 And -2. It has a boiling point of 2°C. Commercially available perfluoro-n-butane contains small amounts (typically 2-4%). It contains the perfluoroisobutane isomer of ) namely C4HF9.

[0021] Appropriate microbubbles as described herein include, for example, Sontum (above) and Sirsi et al. Perfluorocarbons stabilized by a phospholipid membrane, as described above Contains microbubbles. Appropriate phospholipid membrane (or shell or ) according to this disclosure The coating has an effective negative charge. Currently preferred phospholipids are hydrogenated egg yolk phosphatase. The phospholipids present in tidylserine (HEPS), that is, mainly phosphatidylserine And phosphatidic acid (Hvattum et al, J. Pharm. Biomed. Anal., 42(4), 506-5 (12, 2006). Phospholipid membranes typically have a thickness of 10–100 nm.

[0022] In this specification, the term "buffer" refers to a buffer solution, which is a weak acid and its salts. Alternatively, it is a solution containing either a weak base or a salt thereof, and whose pH does not change easily. For example, a buffer solution is either a weak acid and its conjugate base or a weak base and its conjugate acid. It is an aqueous solution. A buffer can neutralize small amounts of additional acid or base, thus maintaining the solution. Used to maintain a stable pH in (or in suspensions or dispersions). Buffering agent This is any buffer that is physiologically compatible and suitable for in vivo injection into the target. Selected from the following. An example of a suitable buffer according to this disclosure is tris(hydroxymethyl)amino Methane (abbreviated as Tris), sodium phosphate, ammonium chloride, diethanolamine, glycerin These are lysine, triethanolamine, and sodium carbonate.

[0023] Currently, the preferred buffering agent is Tris. The pH of Tris is temperature-dependent. At low temperatures, Tris The pH of Tris buffer is higher at high temperatures. For example, Tris buffer has a pH of 8.26 at 5°C. Therefore, the pH of Tris buffer solution will be 7.7 at 25°C and 7.4 at 37°C. The agent should preferably be stored in a refrigerator, which helps maintain the physical and chemical stability of the ultrasound contrast agent. This is carried out in space, i.e., at a temperature of approximately 3-6°C. Further details and explanations are provided elsewhere in this specification. Similarly, an alkaline pH is also important for maintaining the chemical and physical stability of ultrasound contrast agents. It is useful. Furthermore, when ultrasound contrast agents are injected into the subject in vivo, they are preferred. Therefore, it should have a pH close to the physiological pH of 7.4. The fact that it is temperature-dependent means that in refrigerated storage, the pH becomes higher than the pH of body temperature, so This can be used as an advantage of ultrasound contrast agents due to disclosure.

[0024] The term "bulk pH" refers to the pH of the center of the solution's volume or its vicinity, rather than the pH of the solution's surface. The solution (or suspension or dispersion) when measured within the bulk or volume of the solution. This refers to the pH of the body. Bulk pH may differ from the pH of the solution surface. The ultrasound contrast agent has a bulk pH within the alkaline range at a temperature of 5°C. That is, At a temperature of 5°C, the value should be approximately 7.5 or higher, and ideally 10.0 or lower at 5°C. For example, at a temperature of 5°C... Approximately 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5 It has a bulk pH of 9.75 or 10.0. Currently, the preferred bulk pH is at 5°C. The temperature range is approximately 8.25 to 9.25; for example, at 5°C, the values ​​are 8.25, 8.5, 8.75, and 9. It is 0 or 9.25. In this context, throughout the text, the term "about" is used. All pH values ​​mentioned in the specification are generally around 0.1 to 0.5, i.e., ±0.1 to 0. 0.5, for example, can vary by ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5. This is intended to mean that.

[0025] The ultrasound contrast agent of the present invention is preferably stored at low temperatures, and especially over a longer storage period. It is stored. For storage periods of up to about one month, a temperature up to room temperature may be appropriate. The storage temperature is Preferably, the freezing point is not lower than that of the ultrasound contrast agent, and the solution is above the freezing point. It is more preferable that the typical temperature range for storing the ultrasound contrast agent of the present invention is It is likely to be above the freezing point and around 5°C. When using, the ultrasound contrast agent of the present invention It is brought to ambient temperature before being administered to the target.

[0026] The ultrasound contrast agent disclosed herein is for long-term storage, that is, it can withstand long-term storage. In other words, ultrasound contrast agents can preserve their physical and chemical stability over the long term. It maintains within, that is, has an acceptable, or even excellent, shelf life. In this context, throughout the text, "long term" refers to a period of several months or several years, for example, 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 It is intended to mean a period of 8, 19, 20, 21, 22, 23, or 24 months. For long-term storage, it is preferable to store at a temperature of approximately 3-6°C, for example, 3, 4, 5, or 6°C. It will be done there.

[0027] The ultrasound contrast agent described herein is ready for immediate use directly from the vial at the time of purchase. Therefore, it differs from freeze-dried formulations currently on the market. Here, "ready to use immediately" means It can be used immediately in clinical settings, for example, for in vivo imaging and diagnosis of the target. And / or it means it is ready for immediate use to inject into a patient for treatment.

[0028] The ultrasound contrast agents described herein are in liquid form, i.e., liquid formulations, and in particular, as described herein. It is in the form of a dispersion as defined elsewhere, for example, an aqueous dispersion. Liquid formulations are for long-term storage. It is designed for practical use and can be immediately used in clinical settings.

[0029] The buffering agent contained in the ultrasound contrast agent according to this disclosure is tris(hydroxymethyl)aminomethyl Tan (Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, The following may be selected: triethanolamine and sodium carbonate.

[0030] Furthermore, the buffering material is approximately 1 mM to 10 mM, for example, 1, 2, 3, 4, 5, 6, 7, 8 It may have a concentration of 9 or 10 mM. In this context, throughout the text, "about" The term refers to a concentration value of approximately 0.1 to 0.5 mM for all concentration values ​​mentioned herein. That is, ±0.1 to 0.5, for example, ±0.1, ±0.2, ±0.3, ±0.4, or This is intended to mean that it can fluctuate by ±0.5mM.

[0031] The phospholipid membrane contained in the ultrasound contrast agent according to this disclosure preferably has an effective negative charge. .

[0032] The currently preferred ultrasound contrast agent according to this disclosure is as described by Sontum (above). Sonazoid (trademark) (GE Healthcare AS) (formerly NCI 00) Stabilized by hydrogenated egg yolk phosphatidylserine (known as 100%). It contains microbubbles of perfluorobutane and tris(hydroxymethyl)amino It further contains methane (i.e., Tris) as a buffer.

[0033] The ultrasound contrast agent disclosed herein is an isotonic agent, that is, to make the ultrasound contrast agent isotonic. It may further contain excipients to be added. An example of an isotonic agent is the biocompatibility of plasma cations. Salts with sex-opposites, sucrose, physiological saline, dextrose, glycerol, and man. It is nitrate.

[0034] The ultrasound contrast agents described herein may, alternatively or additionally, be viscous agents, i.e., ultrasound contrast agents. Excipients added to alter the viscosity of the contrast agent, and / or floatation-reducing agents. educating agent), for example, propylene glycol, glycerol, glycerin, and / or it may contain polyethylene glycol.

[0035] This disclosure also, (i) Perfluorocarbons are continuously homogenized in a sterile aqueous dispersion of phospholipids. A phospholipid-stabilized microbubble of perfluorocarbon dispersed in a dispersion is generated. Step; (ii) The particle size distribution of microbubbles in the aqueous dispersion is 1 to 6 μm, preferably 2 to 5 μm. A step to adjust the median diameter to within the range of m; (iii) Optionally, an isotonic agent is added to the aqueous dispersion; (iv) Add a buffer to the aqueous dispersion to bring the bulk pH of the aqueous dispersion to approximately 7.5 or higher, preferably. Alternatively, a step to adjust the pH to approximately 8.5 or higher; (v) Adjust the concentration of microbubbles in the aqueous dispersion to approximately 6-10 μl / ml, for example , approximately 6, 7, 8, 9, or 10 μl / ml, currently preferably about 8 μl / ml micro Steps to achieve the target bubble concentration; (vi) Dispense the aqueous dispersion into the vial, and fill the headspace of the vial with perfluorocarbon Steps to flash with a bonnet This report covers methods for preparing ultrasound contrast agents, including those mentioned above.

[0036] In other words, this disclosure means that (i) Perfluorocarbons are continuously homogenized in a sterile aqueous dispersion of phospholipids. A phospholipid-stabilized microbubble of perfluorocarbon dispersed in a dispersion is generated. Step; (ii) The particle size distribution of microbubbles in the aqueous dispersion is 1 to 6 μm, preferably 2 to 5 μm. A step to adjust the median diameter to within the range of m; (iii) Optionally, an isotonic agent is added to the aqueous dispersion; (iv) Add a buffer to the aqueous dispersion to bring the bulk pH of the aqueous dispersion to approximately 7.5 or higher, preferably. Alternatively, a step to adjust the pH to approximately 8.5 or higher; (v) Adjust the concentration of microbubbles in the aqueous dispersion to approximately 6-10 μl / ml, for example , approximately 6, 7, 8, 9, or 10 μl / ml, currently preferably about 8 μl / ml micro Steps to achieve the target bubble concentration; (vi) Dispense the aqueous dispersion into the vial, and fill the headspace of the vial with perfluorocarbon Steps to flash with a bonnet; Includes, Long-term storage of ultrasound contrast agents and / or lyophilization of dispersions before in vivo injection Subject to the condition that it is not done or is not required, This study focuses on methods for preparing ultrasound contrast agents.

[0037] In the context of "aqueous dispersions," the term "dispersion" refers to a mixture of one substance and another. It is intended to mean a dispersed composition. There are various ways in which dispersions are classified. It is possible, but the two main approaches for classification are (1) the internal and external phases of the dispersion (1) the properties (e.g., solid, liquid, or gas), and (2) the range of the size of the dispersed particles. (Colloidal particles vs. coarse particles)

[0038] The term "suspension" is used to describe Sonazoid preparations that are already known. It has been used for this purpose (for example, in the pamphlet International Publication No. 2015150354(A1) (See [reference]) However, the term "suspension" is used when used as a medicine. Therefore, since it is generally used for solid particles dispersed in an external phase, in this specification we will refer to it as a "dispersion". The term is preferably used for newly disclosed liquid formulations that include a gas dispersed in the external phase. However, the terms “dispersion” and “suspension” are used interchangeably herein. This may happen.

[0039] The term "aqueous dispersion" refers to a miscible solvent containing water and / or a water-miscible solvent. This refers to a dispersion of chlorobubbles. The aqueous solvent is preferably a biocompatible carrier. The term "sexual carrier" means that the composition is physiologically tolerable, i.e., toxic or non-toxic. This refers to fluids, especially liquids, that can be administered to the body of a mammal without causing excessive discomfort. The biocompatible carrier is appropriately an injectable carrier solution, such as sterile water for injection that does not contain pyrogenic substances. Body; aqueous solutions such as physiological saline (preferably equilibrated so that the final product for injection is isotonic) (This may be done.) A biocompatible carrier may be one of the types known in the art or Multiple excipients, for example: a buffered aqueous solution containing a biocompatible buffer (e.g., phosphate buffer); One or more isotonic substances (e.g., salts of plasma cations with biocompatible counterions), Sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or (This refers to mannitol), glycols (e.g., glycerol), or other nonionic polio An aqueous solution of a glycol material (e.g., polyethylene glycol, propylene glycol, etc.) is added. It may be added. Preferably, the biocompatible carrier is water for injection free of pyrogenic substances or isotonic water. This is physiological saline. Therefore, the aqueous dispersion should appropriately exclude water-immiscible organic solvents.

[0040] The phrase "adjust the bulk pH of the aqueous dispersion to a pH of approximately 7.5 or higher" is preferable. When measured at a specific temperature, for example 5°C, the bulk pH of the aqueous dispersion is approximately 7.5 or higher. This is intended to mean adjusting the pH.

[0041] In this specification, “target concentration” refers to the concentration after long-term storage and / or in the subject. Defined as the concentration at the time of injection in vivo. During the preparation of ultrasound contrast agents, microbubbles The concentration of the substance initially decreases and may stabilize at slightly lower temperatures. The target concentration is based on the known particle size. The degree distribution stabilization is obtained based on the appropriate dilution of microbubbles.

[0042] The target concentration of microbubbles is approximately 6-10 μl / ml, preferably approximately 8 μl / ml. In this context, throughout this text, the term "about" refers to all references herein. The concentration values ​​are generally about 0.1 to 0.5 μl / ml, i.e., ±0.1 to 0.5 μl / ml l, for example, ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5 μl / ml variation. It is intended to mean that it is possible.

[0043] When dispensing the aqueous dispersion into the vial by step (vi) above, Typically, the top is not filled completely but only partially, thereby creating a head on the dispersion. Leave space and flush it with headspace gas (i.e., fill it). This is possible. The term "headspace" has its original meaning, and in the vial Refers to the gas on top of an aqueous dispersion. A suitable type of via that can store the aqueous dispersion. The container or bottle may have a surface coating (for example, to prevent ionic leaching). Injection vials such as plastic or glass, opaque or It includes (which is transparent). The intention is also to use pre-filled ultrasound contrast agents. It is a syringe, which is used to draw the ultrasound contrast agent from the vial before injecting it into the target. This eliminates the need to do so.

[0044] In the above method for preparing an ultrasound contrast agent, steps (iii) and (iv) are performed at the discretion of the user. It may be done in any order of intent.

[0045] In the above method for preparing an ultrasound contrast agent, step (v) is performed after step (i) Step (v) is performed on the condition that it is performed before step (vi). ii), (iii), and (iv) may be performed before or after any one of them.

[0046] Ultrasonographic contrast agents prepared by the above method are for long-term storage and / or clinical use. It is immediately usable on-site, meaning it can be used for in vivo imaging, diagnosis, and other purposes. It can be used immediately in vivo for ingestion / treatment, etc.

[0047] This disclosure further provides a method for improving contrast enhancement of ultrasound images of tissue in a subject, the above Injecting an ultrasound contrast agent into the subject according to any one of the embodiments and models thereof, Furthermore, the method includes performing an ultrasound scan of the aforementioned tissue.

[0048] This disclosure also relates to a method for in vivo imaging of tissue in a subject, Injecting an ultrasound contrast agent into the subject according to any one of the embodiments and models described above, This includes performing an ultrasound scan of the tissue and generating an image of the tissue. This method will be the focus.

[0049] Furthermore, this disclosure relates to methods for diagnosing a subject, such as in vivo diagnosis of the subject, Injecting an ultrasound contrast agent into the subject according to any one of the above embodiments and models. , performing an ultrasound scan of the region of interest in the subject, and generating an image of the region of interest This includes methods for performing and evaluating the aforementioned images in order to make a diagnosis. .

[0050] This disclosure also relates to a method for improving contrast enhancement of ultrasound images of tissue in a subject, as described above. Injecting an ultrasound contrast agent into the subject according to either one of the embodiments or models, Ultrasonography for use in a method which includes performing an ultrasound scan of the said tissue The target is pharmaceuticals.

[0051] Furthermore, this disclosure relates to a method for in vivo imaging of tissue in a subject. Injecting an ultrasound contrast agent according to any one of the above embodiments and models into the subject and to perform an ultrasound scan of the tissue and generate an image of the tissue. This refers to ultrasound contrast agents for use in methods that include [specific methods].

[0052] This disclosure also relates to a method for in vivo diagnosis of a subject, in the manner and implementation described above. Injecting an ultrasound contrast agent into the subject using one of the following methods, and the interest of the subject. Performing an ultrasound scan of the region, generating an image of the region of interest, and diagnosing A method for use in ultrasound contrast agents, which includes evaluating the aforementioned images in order to make a diagnosis. do.

[0053] This disclosure further improves contrast enhancement of ultrasound images of tissue in the subject (as described above and Injecting an ultrasound contrast agent into the subject according to any one of the embodiments, and the set The above embodiments for manufacturing pharmaceuticals (including performing an ultrasound scan of the tissue) This includes the use of ultrasound contrast agents according to any one of the embodiments.

[0054] Furthermore, this disclosure provides for in vivo imaging of tissue in the subject (as described above). Injecting an ultrasound contrast agent into the subject according to any one of the embodiments, and the tissue (including performing an ultrasound scan and generating an image of the tissue) Ultrasonography contrast agent according to any one of the above embodiments and models for manufacturing pharmaceuticals Regarding use.

[0055] Furthermore, this disclosure provides for in vivo diagnostics of subjects (any of the above aspects and embodiments). Injecting one of the ultrasound contrast agents into the subject, or ultrasound of the region of interest in the subject. Performing an acoustic scan, generating an image of the region of interest, and making a diagnosis. The above embodiments and This applies to the use of ultrasound contrast agents according to any one of the embodiments.

[0056] A composition "containing" one or more enumerated elements is a composition containing other elements not specifically enumerated. It can also include elements. The term "includes" includes, as a subset, "essentially becoming from". This refers to a composition whose constituent components are listed without the presence of other features or components. It means to include.

[0057] The singular forms "a" and "an" are to be interpreted as including the plural form. ru.

[0058] The main impurities found in conventionally known injectable Sonazoid® powder are: The two components of the HEPS-Na excipient are phosphatidylserine sodium salt (P The phospholipid components resulting from the hydrolysis of S) and sodium phosphatidic acid salt (PA) This is the hydrolysis product. The hydrolysis of the excipient HEPS-Na is mainly due to the autologous hydrated phospholipid suspension. This occurs during craving. The main breakdown products are free fatty acids (FFAs) and lysophosphatidyl Lucerin sodium salt (LysoPS) and lysophosphatidic acid sodium salt (LysoPA) PA exists as a component of HEPS-Na, but it is also a degradation product of PS. It's possible. Diacylglycerol (diacyl G) is another phospholipid-related degradation product. In the following examples, the main parameters used as measures of the chemical stability of the product are: The percentage of phosphatidylserine (PS) and phosphatidic acid (PA) present at each measurement. This is the free fatty acid (FFA) as a percentage. The presence of lyso-PS and lyso-PA was also measured. The corresponding data is shown only in relation to one of the examples below. The main parameters used as measures of physicost stability are the volume concentration of microbubbles and This is the median diameter. [Examples]

[0059] [Example 1] Samples were produced using residue from the pre-freeze-drying filling line of commercially available Sonazoid manufacturing. Success. Bulk products in a 20L Sartorius Stedim Flexboy bag. The vials are temporarily stored in a container and then filled into four different sterile vial types within the LAF bench. Then the headspace was flushed with perfluorobutane (PFB). Two different We tested the following Sonazoid batches (batch 1, batch 2).

[0060] The physical stability of microbubbles is the parameter most susceptible to influence by liquid formulations. Therefore, the microbubble content and microbubble size in relation to the storage time The primary response was evaluated from the parameters of assay analysis by Coulter counting; number and The parameters were volume concentration, as well as number and volume-weighted average diameter / distribution. In addition, microbubbles Morphology (shape, structure, aggregation, foreign matter, etc.) is evaluated by microscopic analysis / image analysis, and lipid content is assessed. Chemical analysis of quantity and purity of samples stored for 6 months (Batch 1) or 8 months (Batch 2) Each sample was tested using a ring point. All samples were stored at 5°C.

[0061] Stability results The physical stability of microbubbles is surprisingly good, as is the manufacturing process of Sonazoid aqueous dispersions. It remained stable even 6 months after production (Batch 1, Batch 2). However, as shown in Figure 1... Hydrolysis of phospholipids in sea urchins was significant at 6 months (Batch 1) and 8 months (Batch 2). Figure 1 shows the results after sampling before freeze-drying, preparing as a non-buffered aqueous dispersion, and drying at 5°C for 6-8 hours. Phosphate lipids due to hydrolysis in a sample of Sonazoid bulk product stored for several months. This shows the degree of degradation of the substance (the x-axis represents the number of months). The degree of hydrolysis is determined by the three degradation products, namely , free fatty acids (FFA), lysophosphatidylserine (lysoPS), and lysophospha The presence of tidic acid (lyso-PA) indicates the presence of phosphatidylserine at each analysis. (PS) and phosphatidic acid (PA) are shown as the total percentages, respectively (on the y-axis) (Percentage of FFA which is PS + PA).

[0062] Furthermore, after 6 months of storage (Batch 1, Batch 2), the pH changed from approximately 6-7 to approximately The levels decreased from 4.9 to 6.4, meaning they decreased in all samples, which is due to phosphatidylsin. This was predicted in light of the significant hydrolysis of phosphorus.

[0063] The conclusion drawn from this study is that in order to obtain an acceptable shelf life for an immediately usable formulation, The hydrolysis must be significantly slowed, and the critical level of hydrolysis is slow It is likely based on the recorded effects of hydrolyzed impurities on the properties of microbubbles. The fact that they are not there.

[0064] From Example 1 above, the immediately usable Sonazoid is already undergoing significant chemical degradation. It was suggested that this cannot be obtained with existing formulations (i.e., when stored in water). Existing sonazoid formulations do not contain buffers, and their pH is typically around 6-7. After significant hydrolysis according to Example 1 above, the pH decreases to 4.9-6.4.

[0065] Literature data related to liposome dispersions suggests that phospholipid hydrolysis is affected by pH. This is suggested (Grit et al, Biochim. Biophys. Acta, 1167, 49-55, 1993). However, because phospholipid-stabilized microbubbles differ from liposomes in several respects, The buffer used with positories may not be compatible with microbubbles, and liposomes The stability data obtained from this method may not necessarily be applicable to phospholipid-stabilized microbubbles. Microbubbles contain a single, stabilized single layer, with water molecules in addition to gas molecules between the outer and inner phases. There is no transportation. Physically, microbubbles differ greatly between their internal and external phases. While they tend to float, small monolayer liposomes retain without any visible sedimentation. It can be physically homogeneous during storage. Therefore, microbubbles prevent coagulation during storage. This may require additional surface stabilization or charge. Adding ions shields the surface charge. This is expected to reduce the physical stability of the dispersion.

[0066] Nevertheless, the inventors of this invention intend to conduct a second study on shelf-life stability. Determined, the research involved raising the pH to make it neutral or basic, and initially By adding a buffer solution that prevents the decrease in pH due to hydrolysis, the degradation is significantly delayed. We tested whether this would work. The research plan and the results of the stability of the research are shown in the following examples. Disclosed in section 2.

[0067] [Example 2] To control and stabilize the pH, add 5 mM tris(hydroxymethyl)aminomethane. Using (Tris) buffer, two different test dispersions were obtained; each at 7.5°C. As described elsewhere in this specification, the pH of Tris is temperature-dependent, so at room temperature it is approximately pH A buffered aqueous dispersion of Sonazoid having a bulk pH equivalent to 7, Sonazoid has a bulk pH of 8.5 at 5°C (equivalent to approximately pH 8 at room temperature). It is a buffered aqueous dispersion. Furthermore, Sonazoid in a non-buffered aqueous dispersion Used as a reference.

[0068] Freeze-dried Sonazoid was used for sample preparation. Stock buffer was used in In Vitrogen buffer kit (Thermo Fisher Scientific) It was prepared from a 1 M Tris solution having pH 7.0 and 8.0 (at room temperature). RIS buffer in 100 ml of B. Braun Water For Injection (WFI) Diluted in an Ecoflac bottle.

[0069] Preparation of Sonazoid vials with 5 mM Tris buffer, pH 7 (room temperature): • Using a syringe with a sterile filter, use a 1M Tris pH 7.0 buffer kit (In vitrogen, manufactured by Thermo Fisher Scientific, AM985 Aspirate 0.5 ml from 0G Ambion, B. Braun Medical SA GE Healthcare Ecoflac WFI 100m manufactured by . Inject into 1. Shake the bottle to obtain a homogeneous buffer. 20 Sonazoid vials Reconfigure the vial and remove air from it using a sterile filter.

[0070] Preparation of Sonazoid vials with 5 mM Tris buffer, pH 8 (room temperature): • Using a syringe with a sterile filter, use a 1M Tris pH 8.0 buffer kit (In vitrogen, manufactured by Thermo Fisher Scientific, AM985 Aspirate 0.5 ml from 0G Ambion, B. Braun Medical SA GE Healthcare Ecoflac WFI 100m manufactured by . Inject into 1. Shake the bottle to obtain a homogeneous buffer. 20 Sonazoid vials Reconfigure the vial and remove air from it using a sterile filter.

[0071] For comparison, 15 vials of Sonazoid from B. Braun Medical GE Healthcare Ecoflac WFI 10 manufactured by SA. The solution was reconstituted with sterile water for injection starting from 0 ml. All vials were stored at 5°C after reconstitution.

[0072] Stability results The selected response is based on the size and concentration of microbubbles (measured by the Coolter count), and The parameters were purity (by thin-layer chromatography, TLC) and pH.

[0073] Figure 2 shows unbuffered water for injection, buffer at pH 7 (room temperature), or buffer at pH 8 (room temperature). Each component was reconstituted with a different agent and freeze-dried, then stored at 5°C for 6 months. This shows the degree of phospholipid degradation due to hydrolysis in powder samples (x-axis: number of months). The degree of water decomposition is indicated by the decomposition product, free fatty acids (FFA), which are present at the time of each analysis. It is expressed as the percentage of the total of phosphatidylserine (PS) and phosphatidic acid (PA). These are shown (the y-axis represents the percentage of FFA, which is (PS + PA)).

[0074] Figure 3 shows the volume concentration of microbubbles stored at 5°C up to 6 months (x-axis represents months). The y-axis represents volume concentration (in μl / ml). This figure shows the average values ​​of 10 samples for each data point. The average results are shown. The variation between data points is normal analytical variation, and the volume concentration after 6 months of storage is also shown. No trend was observed regarding the changes. Therefore, the volume concentration remained stable during storage for 6 months. Ta.

[0075] The median diameter of the microbubbles was also found to be stable during storage for 6 months (data (Not shown).

[0076] The pH values ​​of the two test dispersions at time zero, 3 months, and 6 months are as follows: This is shown in Table 1.

[0077] [Table 1]

[0078] [Example 4] The ultrasound contrast agent according to this disclosure is prepared as follows: Perfluorobutane is prepared using HEPS Microbubbles are formed by homogenizing a sterile aqueous dispersion of sodium, HEPS-stabilized microbubbles of dispersed PFB are generated. Repeated flotation is performed to reduce their size. By removing the microbubbles of the opposite type, the particle size distribution of the microbubbles is adjusted, and 1~ Obtain a median diameter between 6 μm. Dilute the dispersion with water. Optionally, add sucrose. Which isotonic agent is used to adjust the tension?

[0079] The pH of the dispersion can be increased to approximately 7.5 or higher by adding Tris to a concentration of 5 mM. Adjust the pH to the desired alkaline level.

[0080] The target concentration is obtained based on the appropriate dilution of stabilized microbubbles with a known particle size distribution. This can be done, for example, as follows: The concentration of microbubbles is, for example, micro By adjusting the concentration of Robubble between 8 and 20 μl / ml, approximately 6 to 10 days of storage can be achieved. It is adjusted to achieve the target concentration of microbubbles in μl / ml.

[0081] Fill 2-10 ml vials with the dispersion, and before capping, remove the heads. Flash the pace with PFB.

[0082] Store the vials in the refrigerator.

[0083] Consideration The percentage of FFA was used as a marker for differences in hydrolysis between samples. (Example 2) The percentage of FFA in the sample containing Riss buffer is the unbuffered aqueous solution of Examples 1 and 2. Compared to the percentage of FFA in the initial study, it was significantly lower after 6 months of storage. (Example 2 study) All other purity parameters measured were stable after 6 months (data not shown). ). From the data, raising the pH using a buffer during storage and stabilizing it reduces hydrolysis. A significant effect is demonstrated. At 5°C and pH 8.5, depending on the reaction rate, 1 to 2 A shelf life of several years is possible (based on the results shown in Figure 2, if the rate of hydrolysis is linear). (For example, 30 months or older).

[0084] From Example 1, phospholipid-stabilized perfluorocarbons stored in water at 5°C for 6 months. Microbubbles undergo significant hydrolysis of phospholipids, and as a result, the microbubbles Physical stability was also affected, and it was shown that the volume concentration decreased after 6 months. In comparison, The microbubbles were stored at 5°C for 6 months, but in an aqueous dispersion containing a buffer solution, the result was 7. From Example 2, which was stored at a pH above 5 or 8.5, hydrolysis was significantly less, and physical It was shown that stability was not affected in any way. The addition of ions between individual microbubbles It is known to reduce the repulsive effect, but even when a small amount of buffer is added, visible clumping occurs. It did not result in a cluster. Also, physical microbubbles such as volume concentration and median diameter. The parameters were unaffected after 6 months.

[0085] From these results, it can be seen that adding a buffer to raise the pH of ultrasound contrast agents in the form of a dispersion is effective. It has been shown to be a feasible method that significantly reduces the degradation rate of negatively charged phospholipids. At the same time, this result indicates that the appearance or volume concentration of microbubbles is affected by the addition of a buffering agent. This shows that it is not affected. Therefore, both the physical and chemical stability of the dispersion This was shown to be maintained at a physiologically acceptable level during storage. Therefore, This disclosure is immediately available (meaning it can be immediately injected into the subject in vivo). Furthermore, the present invention provides an ultrasound contrast agent that can withstand long-term storage before such use.

[0086] This disclosure is not limited to the exemplary embodiments described above, and the appended claims Please understand that some possible changes to this disclosure are possible within the scope of this disclosure.

Claims

1. (a) Perfluorocarbon microbubbles stabilized by a phospholipid membrane; and (b) Buffering agent; It contains and has a bulk pH of about 7.5 or higher, preferably about 8.5 or higher. Ultrasonography contrast agent.

2. The ultrasound contrast agent according to claim 1, which is for long-term storage.

3. In clinical settings, for example, in vivo imaging, diagnosis, and / or treatment of subjects. An ultrasound contrast agent according to claim 1 or 2, which is ready for immediate use.

4. The buffering agent is tris(hydroxymethyl)aminomethane (Tris), sodium phosphate. ammonium chloride, diethanolamine, glycine, triethanolamine, and carbon Ultrasonic fabrication according to any one of claims 1 to 3, selected from the group consisting of sodium acid Contrast agent.

5. The buffering agent has a concentration of about 1 mM to about 10 mM, according to any one of claims 1 to 4. The ultrasound contrast agent described.

6. The phospholipid membrane has an effective negative charge, as described in any one of claims 1 to 5. Wave contrast agent.

7. An ultrasound contrast agent according to any one of claims 1 to 6, further comprising an isotonic agent.

8. The present invention further comprises a viscous agent and / or a flotation-reducing agent, as described in any one of claims 1 to 7. Ultrasound contrast agent.

9. The perfluorocarbon is perfluorobutane, perfluoropropane, and perfluorocarbon. Ultrasonographic contrast agent according to any one of claims 1 to 8, selected from fluoropentane 。

10. The perfluorocarbon is perfluorobutane, and the phospholipid is hydrogenated egg yolk. An ultrasound contrast agent according to any one of claims 1 to 9, wherein the contrast agent is phosphatidylserine.

11. (i) Perfluorocarbons are continuously homogenized in a sterile aqueous dispersion of phospholipids. A phospholipid-stabilized microbubble of perfluorocarbon dispersed in a dispersion is generated. Step; (ii) The particle size distribution of microbubbles in the aqueous dispersion is 1 to 6 μm, preferably 2 to Steps to adjust the median diameter to within the range of 5 μm; (iii) an optional step of adding an isotonic agent to the aqueous dispersion; (iv) Add a buffer to the aqueous dispersion to bring the bulk pH of the aqueous dispersion to approximately 7.

5. The above step preferably involves adjusting the pH to approximately 8.5 or higher; (v) Adjust the concentration of microbubbles in the aqueous dispersion to approximately 6 to 10 μl / ml Steps to achieve the target concentration of Iclobubbles; (vi) Dispense the aqueous dispersion into a vial, and fill the headspace of the vial with Perful Steps to flash Orocarbon A method for preparing an ultrasound contrast agent, including [the specified ingredient].

12. The method according to claim 11, wherein steps (iii) and (iv) are performed in any order. 。

13. Step (v) is performed after step (i) and before step (vi). For example, step (v) is any of steps (ii), (iii), and (iv) The method according to claim 11 or 12, wherein the method is performed before or after one of the steps.

14. The aforementioned ultrasound contrast agent is for long-term storage and / or, In clinical settings, for example, in vivo imaging, diagnosis, and / or treatment of subjects. It is immediately available for use. The method according to any one of claims 11 to 13.

15. The method according to any one of claims 11 to 14, wherein the freeze-drying step is not included.

16. An ultrasound contrast agent prepared by the method described in any one of claims 11 to 15.

17. A method for improving contrast enhancement in ultrasound images of tissue in a subject, according to claims 1 to 10 or Injecting the ultrasound contrast agent described in any one of item 16 into the subject, and the tissue A method that includes performing an ultrasound scan.

18. A method for in vivo imaging of tissue in a target, according to claims 1 to 10. Or, injecting the ultrasound contrast agent described in any one of item 16 into the subject, the ultrasound contrast agent of the tissue A method comprising performing an acoustic scan and generating an image of the tissue.

19. A method for diagnosing a subject, the ultrasonic method according to any one of claims 1 to 10 or 16. Injecting a contrast agent into the subject, and performing an ultrasound scan of the region of interest in the subject. To do so, generate an image of the region of interest, and evaluate the image to make a diagnosis. A method that includes doing something.

20. An ultrasound contrast agent for use in the method according to any one of claims 17 to 19.

21. For manufacturing a pharmaceutical product for use in the method according to any one of claims 17 to 19 The use of an ultrasound contrast agent according to any one of claims 1 to 10 or 16.

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