Ultrasound contrast agents and methods of using same

Abstract

The present disclosure relates to an ultrasound contrast agent comprising perfluorocarbon microbubbles stabilized by a phospholipid membrane and a buffering agent, and having a bulk pH of about 7.5 or greater, preferably about 8.5 or greater. The ultrasound contrast agent is suitable for long-term storage and immediate in vivo use. Also disclosed are methods for preparing such ultrasound contrast agents and methods for using them in clinical settings.

Description

[Technical Field]

[0001] The present disclosure relates to the field of in vivo imaging and diagnostics, and in particular to ultrasound contrast agents that are immediately usable in vivo and can withstand long-term storage prior to use in vivo. The present disclosure further relates to methods of preparing the ultrasound contrast agents and methods of using the ultrasound contrast agents in clinical settings. [Background technology]

[0002] Ultrasound contrast agents based on perfluorocarbon phospholipid-stabilized microbubbles are well known in the art (see, e.g., Wheatley et al., J. Drug Del. Sci. Technol., 23(1), 57-72, 2013). A single microbubble consists of a gas core, which can be approximately 2-10 μm in size, encapsulated in a shell or membrane of a layer of stabilizing phospholipid molecules. The compressible gas core can expand and contract when subjected to ultrasound. The expansion and contraction of the microbubbles upon exposure to ultrasound produces backscattering of acoustic waves, which can be used for diagnostic imaging purposes. The surface of the microbubbles can be further functionalized with targeting drug moieties that are released upon microbubble rupture and / or cavity formation upon ultrasound application, thereby enabling such ultrasound contrast agents to be used for therapeutic purposes (Upadhyay et al., RSC Adv., 6, 15016-15026, 2016).

[0003] Sonazoid™ is an example of an ultrasound contrast agent based on perfluorocarbon phospholipid-stabilized microbubbles. More specifically, Sonazoid™ is formulated as a powder consisting of lyophilized sucrose encapsulating perfluorobutane microbubbles stabilized by a membrane of hydrogenated egg yolk phosphatidylserine and stored under a perfluorobutane headspace. Sonazoid™ is aseptically manufactured by sequentially homogenizing perfluorobutane (PFB) in an aqueous dispersion of hydrogenated egg yolk phosphatidylserine (HEPS). After initial microbubble generation, the microbubble concentration and size distribution are adjusted through a series of controlled separation steps. The final dispersion, targeted to yield 8 μl of microbubbles per ml in the reconstituted product, is made isotonic by the addition of sucrose. 2 ml of the dispersion is filled into 10 ml glass vials and lyophilized. After lyophilization, the headspace of the vials is backfilled with perfluorobutane before stoppering. In other words, Sonazoid™ is a freeze-dried product that must be reconstituted with water before use. More specifically, before administration to a subject, the product is reconstituted by adding 2 ml of sterile water for injection through the provided vented filter (5 μm) spike (Codan Chemoprotect® Spike, Codan GmbH & Co., Germany), followed by hand mixing for 1 minute. After reconstitution, the product appears as a milky, homogeneous dispersion. Because the dispersion is opaque, visual inspection for extraneous particles is difficult. To ensure the absence of such particles, the product is drawn into a syringe through a filter spike before administration. If left unstirred after reconstitution, the microspheres begin to separate by floating, forming a creamy layer on top of the liquid phase. Unless used immediately after reconstitution, the product should be homogenized again by hand mixing for 10 seconds before use (Sontum, Ultrasound Med. & Biol., 34(5), 824-833, 2008).

[0004] Because the active ingredient of ultrasound contrast agents is their physical state (microbubbles) rather than a chemical substance, two types of stability must be considered: physical stability and chemical stability. In other words, the focus must be on how to control and maintain the concentration and size distribution of the microbubbles, as well as the chemical composition of the components. Microbubbles are generally thermodynamically unstable systems and may undergo physical changes during preparation and storage (see, e.g., WO 2015150354(A1); Segers et al., Langmuir, 33, 10329-10339, 2017; Borden et al., Advances in Colloid and Interface Science, 262, 39-49, 2018). In addition, the phospholipid membrane that stabilizes microbubbles can undergo hydrolysis in solution, resulting in impurities in the final product. Phospholipids readily undergo hydrolytic cleavage in acidic and alkaline media. Phospholipids are only sufficiently stable at pH 7. This is because hydrolysis of ester bonds does not proceed significantly under these conditions (see Phospholipids Handbook, 1993, edited by Gregor Cevc; Chapter 9 "Chemical stability", Evstigneeva, pp. 323-324). Temperature and pH greatly affect the hydrolysis kinetics (see Phospholipids Handbook, 1993, edited by Gregor Cevc; Chapter 9 "Chemical stability", Crommelin et al., pp. 338-339). Furthermore, it is known that once hydrolysis is initiated at low pH, a decrease in pH will accelerate degradation. Therefore, a major challenge during the early development of Sonazoid™ was how to obtain a product with an acceptable shelf life. Lyophilization, which results in Sonazoid™ formulations in the form of a freeze-dried powder, has been considered the only way to obtain such a product.

[0005] While lyophilization can result in products with excellent shelf-life stability and quality, it is also time- and resource-intensive, thereby significantly increasing manufacturing costs and reducing "ease of use" for the end user (typically a healthcare professional). Thus, there is a need in the art for improved ultrasound contrast agents based on perfluorocarbon phospholipid-stabilized microbubbles that combine high storage stability with ease of use. Summary of the Invention

[0006] The above object of providing an ultrasound contrast agent that combines high storage stability with ease of use is achieved by the present disclosure, which relates to a stable, ready-to-use ultrasound contrast agent formulation in the form of a dispersion that can surprisingly withstand long-term storage prior to use and is ready-to-use, i.e., immediately injectable into a subject.

[0007] More particularly, the present disclosure provides: (a) perfluorocarbon microbubbles stabilized by a phospholipid membrane; and (b) buffering agents; and having a bulk pH of about 7.5 or greater, preferably about 8.5 or greater; This applies to ultrasound contrast agents.

[0008] The present disclosure also provides (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of phospholipids to produce phospholipid-stabilized microbubbles of the perfluorocarbon dispersed in the aqueous dispersion; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 to 6 μm, preferably 2 to 5 μm; (iii) optionally adding a tonicity agent to the aqueous dispersion; (iv) adding a buffering agent 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 microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μl / ml; (vi) dispensing the aqueous dispersion into vials and flushing the headspace of the vials with perfluorocarbon; The present invention relates to a method for preparing an ultrasound contrast agent, comprising:

[0009] Furthermore, the present disclosure is directed to methods for improving contrast in ultrasound images of tissue in a subject, methods for in vivo imaging of tissue in a subject, and methods for diagnosing a subject, which methods comprise injecting into a subject an ultrasound contrast agent as described above.

[0010] The present disclosure also relates to ultrasound contrast agents for use in the methods as described herein.

[0011] Additionally, the present disclosure relates to the use of an ultrasound contrast agent as disclosed herein for the manufacture of a medicament for use in a method as disclosed herein.

[0012] Preferred aspects of the present disclosure are set forth hereinafter in the detailed description and dependent claims. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the chemical stability of Sonazoid bulk product taken prior to lyophilization, prepared as an unbuffered aqueous dispersion, and stored at 5° C. for 8 months. [Figure 2] FIG. 1 shows the chemical stability of freeze-dried powder of Sonazoid stored at 5° C. for 6 months, prepared as an unbuffered aqueous dispersion, a buffered aqueous dispersion containing a buffer and having a bulk pH of 7 at room temperature, and a buffered aqueous dispersion containing a buffer and having a bulk pH of 8 at room temperature. [Figure 3]FIG. 1 shows the physical stability of freeze-dried powder of Sonazoid stored at 5° C. for 6 months, prepared as an unbuffered aqueous dispersion, a buffered aqueous dispersion containing a buffer and having a bulk pH of 7 at room temperature, and a buffered aqueous dispersion containing a buffer and having a bulk pH of 8 at room temperature. DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure provides a shelf-stable, ready-to-use liquid ultrasound contrast agent. By increasing the pH and including a buffer in the product, reconstitution of the powder prior to use of the claimed product is unnecessary, resulting in a liquid formulation that is easier for end users to handle than the previously known Sonazoid™ freeze-dried powder. It should be noted that adding a buffer to a perfluorocarbon phospholipid-stabilized microbubble-based ultrasound contrast agent would not be known to those skilled in the art as electrolytes (e.g., those present in the buffer) could alter the structure of the dispersion, resulting in a functional ultrasound contrast agent. Nevertheless, the present inventors surprisingly managed to maintain the desired microbubble volume concentration and distribution. That is, they managed to maintain the physical stability while improving the chemical stability of the product compared to the physical and chemical stabilities, respectively, of the previously known reconstituted Sonazoid™ freeze-dried powder dispersion. Therefore, the shelf-stability of the claimed product is improved compared to the previously known product.

[0015] By including a buffer in the formulation, ultrasound contrast agents according to the present disclosure have a bulk pH in the alkaline range at a temperature of 5° C. As further shown in the Examples below, the alkaline pH significantly reduces the rate of hydrolysis of the phospholipids present in the ultrasound contrast agent, thereby rendering it chemically stable for a much longer period of time. Because the phospholipid membrane stabilizes the microbubbles, the chemical stability of the phospholipids also impacts the physical stability of the microbubbles.

[0016] More particularly, the present disclosure provides: (a) perfluorocarbon microbubbles, where the microbubbles are stabilized by a membrane of phospholipids; and (b) buffering agents; and having a bulk pH of about 7.5 or greater, preferably about 8.5 or greater; An ultrasound contrast agent is provided that solves or at least alleviates problems associated with existing ultrasound contrast agents based on perfluorocarbon phospholipid-stabilized microbubbles.

[0017] The term "imaging agent" has its conventional meaning in the field of in vivo medical imaging and refers to an agent in a form suitable for administration to a mammal that assists in providing a clearer image of a region or organ of interest than can be obtained by imaging the mammalian subject alone. The term "subject" refers to an in vivo mammal, preferably an intact mammalian body in vivo, and more preferably a living human subject. The phrase "form suitable for administration to a mammal" refers to a composition that is sterile, pyrogen-free, free of toxic or harmful compounds, and formulated at a biocompatible pH (approximately pH 4.0-10.5). Such compositions are free of particulates that may pose an embolic risk in vivo and are formulated to prevent precipitation upon contact with bodily fluids (e.g., blood). Such compositions also contain only biologically compatible excipients and are preferably isotonic.

[0018] As with other in vivo imaging agents, contrast agents are designed to have minimal pharmacological effects on the mammalian subject to be imaged. Preferably, the contrast agent can be administered to the mammalian body in a minimally invasive manner, i.e., without substantial health risks to the mammalian subject when performed under the medical expertise of a specialist. Such minimally invasive administration is preferably intravenous administration into a peripheral vein of the subject, without the need for local or general anesthesia.

[0019] The term "microbubble" has its conventional meaning in the field of in vivo ultrasound imaging and refers to gas microbubbles with internal diameters between 0.1 and 10 μm, typically between 0.5 and 5 μm. Such microbubbles are similar in size to red blood cells, which allows them to exhibit similar properties in microvasculature and capillaries throughout the mammalian body (Sirsi et al., Bubble Sci. Eng. Technol, 1(1-2), 3-17, 2009). As used herein, the terms "microbubble" and "microsphere" may be used interchangeably.

[0020] The term "perfluorocarbon" has its conventional chemical meaning and is represented by the formula C x F y (i.e., containing only carbon and fluorine) (see IUPAC, Compendium of Chemical Terminology, 2nd ed., 1997 (2006-present online revised version)). Compounds with the prefix perfluoro are hydrocarbons, including those with heteroatoms, in which all C-H bonds have been replaced with C-F bonds. Perfluorocarbons include perfluoroalkanes, fluoroalkenes, fluoroalkynes, and perfluoroaromatic compounds. The terms "perfluorocarbon" and "fluorocarbon" may be used interchangeably. Suitable perfluorocarbons according to the present disclosure include perfluoroalkanes, such as perfluorobutane, perfluoropropane, and perfluoropentane. A currently preferred perfluorocarbon of the present disclosure is perfluorobutane ("PFB"), which has its standard chemical meaning and is also called perfluorobutane in the context of medical applications. The chemical formula for perfluoro-n-butane is CF3CF2CF2CF3 or CF4F 10 and has a boiling point of -2.2 °C. Commercially available perfluoro-n-butane contains small amounts (typically 2-4%) of the perfluoro-iso-butane isomer, C4HF9.

[0021] Suitable microbubbles according to the present disclosure include perfluorocarbon microbubbles stabilized by a phospholipid membrane, as described, for example, in Sontum (supra) and Sirsi et al. (supra). Suitable phospholipid membranes (or shells or coatings) according to the present disclosure have a net negative charge. Currently preferred phospholipids are those present in hydrogenated egg yolk phosphatidylserine (HEPS), i.e., primarily phosphatidylserine and phosphatidic acid (Hvattum et al., J. Pharm. Biomed. Anal., 42(4), 506-512, 2006). The phospholipid membrane typically has a thickness of 10-100 nm.

[0022] As used herein, the term "buffer" refers to a buffer solution, which contains either a weak acid and its salt or a weak base and its salt, and is resistant to pH changes. In other words, a buffer solution is an aqueous solution of either a weak acid and its conjugate base or a weak base and its conjugate acid. Buffers are used to maintain a stable pH in a solution (or suspension or dispersion) because they can neutralize small amounts of added acid or base. The buffer can be selected from any buffer that is physiologically compatible and suitable for in vivo injection into a subject. Examples of suitable buffers according to the present disclosure are tris(hydroxymethyl)aminomethane (abbreviated as Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, triethanolamine, and sodium carbonate.

[0023] The currently preferred buffer is Tris. The pH of Tris is temperature-dependent. At lower temperatures, the pH of Tris is higher than at higher temperatures. For example, if a Tris buffer has a pH of 8.26 at 5°C, the pH of the Tris buffer will be 7.7 at 25°C and 7.4 at 37°C. Storage of the ultrasound contrast agent is preferably performed in a refrigerated space, i.e., at a temperature of approximately 3-6°C, which helps maintain the physical and chemical stability of the ultrasound contrast agent. As explained and demonstrated elsewhere herein, an alkaline pH also helps maintain the chemical and physical stability of the ultrasound contrast agent. Additionally, the ultrasound contrast agent should have a pH close to physiological pH, preferably 7.4, when injected in vivo into a subject. Thus, the fact that the pH of Tris is temperature-dependent can be used to the advantage of the ultrasound contrast agent of the present disclosure, since refrigerated storage results in a pH higher than that at body temperature.

[0024] The term "bulk pH" refers to the pH of a solution (or suspension or dispersion) as measured within the bulk or volume of the solution, such as at or near the center of the volume of the solution, rather than at the surface of the solution. The bulk pH may differ from the pH at the surface of the solution. Ultrasound contrast agents according to the present disclosure have a bulk pH in the alkaline range at a temperature of 5°C, i.e., a bulk pH of about 7.5 or greater at a temperature of 5°C, suitably 10.0 or less at a temperature of 5°C, e.g., about 7.5, 7.75, 8.0, 8.25, 8.5, 8.75, 9.0, 9.25, 9.5, 9.75, or 10.0 at a temperature of 5°C. Currently preferred bulk pHs are about 8.25-9.25 at a temperature of 5°C, e.g., 8.25, 8.5, 8.75, 9.0, or 9.25 at a temperature of 5°C. In this context, and throughout the text, the term "about" is intended to mean that all pH values ​​referred to herein may generally vary by about 0.1 to 0.5, i.e., ±0.1 to 0.5, e.g., ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5.

[0025] The ultrasound contrast agents of the present invention are preferably stored at low temperatures, especially for longer storage periods. For storage periods of up to about one month, temperatures up to room temperature may be appropriate. The storage temperature is preferably not lower than the freezing point of the ultrasound contrast agent, and more preferably above the freezing point as a solution. A typical temperature range for storage of the ultrasound contrast agents of the present invention would be above the freezing point and up to around 5°C. When used, the ultrasound contrast agents of the present invention are allowed to reach ambient temperature before administration to a subject.

[0026] The ultrasound contrast agents disclosed herein are intended for long-term storage, i.e., can withstand long-term storage. In other words, the ultrasound contrast agents maintain their physical and chemical stability during long-term storage, i.e., have acceptable or even excellent shelf life. In this context, throughout the text, "long-term" is intended to mean a period of several months or years, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months. Long-term storage is preferably carried out at a temperature of approximately 3-6°C, e.g., 3, 4, 5, or 6°C.

[0027] The ultrasound contrast agents of the present disclosure differ from currently commercially available freeze-dried formulations in that they are ready to use directly from the vial in which they are purchased, where "ready to use" means ready to use in a clinical setting, e.g., ready to be injected into a patient for in vivo imaging, diagnosis, and / or treatment of a subject.

[0028] The ultrasound contrast agents according to the present disclosure are in liquid form, i.e., liquid formulations, particularly in the form of dispersions, e.g., aqueous dispersions, as defined elsewhere herein, which are intended for long-term storage and ready for use in the clinical setting.

[0029] Buffers included in ultrasound contrast agents according to the present disclosure may be selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, triethanolamine, and sodium carbonate.

[0030] Further, the buffering agent may have a concentration of about 1 mM to about 10 mM, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. In this context, and throughout the text, the term "about" is intended to mean that all concentration values ​​referred to herein may vary generally by about 0.1 to 0.5 mM, i.e., ±0.1 to 0.5, e.g., ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5 mM.

[0031] The phospholipid membrane contained in the ultrasound contrast agent according to the present disclosure preferably has a net negative charge.

[0032] A presently preferred ultrasound contrast agent according to the present disclosure comprises perfluorobutane microbubbles stabilized by hydrogenated egg yolk phosphatidylserine, such as Sonazoid™ (GE Healthcare AS) (formerly known as NCI 00100) as described by Sontum (supra), and further comprises tris(hydroxymethyl)aminomethane (i.e., Tris) as a buffering agent.

[0033] Ultrasound contrast agents according to the present disclosure may further comprise an isotonicity agent, i.e., an excipient added to make the ultrasound contrast agent isotonic. Examples of isotonicity agents are salts of plasma cations with biocompatible counterions, sucrose, saline, dextrose, glycerin, and mannitol.

[0034] Ultrasound contrast agents according to the present disclosure may alternatively or additionally include viscosity agents, i.e., excipients added to alter the viscosity of the ultrasound contrast agent, and / or floatation-reducing agents, such as propylene glycol, glycerol, glycerin, and / or polyethylene glycol.

[0035] The present disclosure also provides (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of phospholipids to produce phospholipid-stabilized microbubbles of the perfluorocarbon dispersed in the aqueous dispersion; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 to 6 μm, preferably 2 to 5 μm; (iii) optionally adding a tonicity agent to the aqueous dispersion; (iv) adding a buffering agent 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 microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μl / ml, e.g., about 6, 7, 8, 9, or 10 μl / ml, currently preferably about 8 μl / ml; (vi) dispensing the aqueous dispersion into a vial and flushing the headspace of the vial with a perfluorocarbon; The present invention relates to a method for preparing an ultrasound contrast agent, comprising:

[0036] In other words, the present disclosure: (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of a phospholipid to produce phospholipid-stabilized microbubbles of the perfluorocarbon dispersed in the aqueous dispersion; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 to 6 μm, preferably 2 to 5 μm; (iii) optionally adding a tonicity agent to the aqueous dispersion; (iv) adding a buffering agent 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 microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μl / ml, e.g., about 6, 7, 8, 9, or 10 μl / ml, currently preferably about 8 μl / ml; (vi) dispensing the aqueous dispersion into a vial and flushing the headspace of the vial with a perfluorocarbon; Including, provided that lyophilization of the dispersion prior to long-term storage of the ultrasound contrast agent and / or in vivo injection is not performed or required; A method for preparing an ultrasound contrast agent is provided.

[0037] The term "dispersion," as in "aqueous dispersion," is intended to mean a composition in which one substance is dispersed within another. How dispersions are classified can vary, but two main approaches to classification are (1) the nature of the dispersion's internal and external phases (e.g., solid, liquid, or gas), and (2) the size range of its dispersed particles (colloidal versus coarse particles).

[0038] The term "suspension" has been used to describe previously known formulations of Sonazoid (see, e.g., WO2015150354A1). However, since the term "suspension," when used pharmaceutical, is now generally used primarily for solid particles dispersed in an external phase, the term "dispersion" is preferably used herein for the newly disclosed liquid formulations comprising a gas dispersed in an external phase. However, the terms "dispersion" and "suspension" may be used interchangeably herein.

[0039] The term "aqueous dispersion" refers to a dispersion of microbubbles in an aqueous solvent, including water and / or water-miscible solvents. The aqueous solvent is preferably a biocompatible carrier. The term "biocompatible carrier" refers to a fluid, especially a liquid, in which the composition is physiologically tolerable, i.e., can be administered to a mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid, such as pyrogen-free sterile water for injection; an aqueous solution, such as saline (which may be advantageously balanced so that the final product for injection is isotonic). The biocompatible carrier may contain one or more excipients known in the art, such as a buffered aqueous solution containing a biocompatible buffer (e.g., phosphate buffer); one or more isotonicity agents (e.g., salts of plasma cations with biocompatible counterions), sugars (e.g., glucose or sucrose), sugar alcohols (e.g., sorbitol or mannitol), glycols (e.g., glycerol), or other non-ionic polyol materials (e.g., polyethylene glycol, propylene glycol, etc.). Preferably, the biocompatible carrier is pyrogen-free water for injection or isotonic saline. Thus, the aqueous dispersion suitably excludes water-immiscible organic solvents.

[0040] The phrase "adjusting the bulk pH of the aqueous dispersion to a pH of about 7.5 or greater" is intended to mean adjusting the bulk pH of the aqueous dispersion to a pH of about 7.5 or greater, preferably when measured at a particular temperature, e.g., 5°C.

[0041] As used herein, "target concentration" is defined as the concentration after long-term storage and / or the concentration upon in vivo injection in a subject. During preparation of the ultrasound contrast agent, the concentration of microbubbles may initially decrease and stabilize at a slightly lower temperature. The target concentration is achieved based on the appropriate dilution of stabilized microbubbles of a known size distribution.

[0042] The target concentration of microbubbles is about 6-10 μl / ml, preferably about 8 μl / ml. In this context, and throughout the text, the term "about" is intended to mean that all concentration values ​​referred to herein may generally vary by about 0.1-0.5 μl / ml, i.e., ±0.1-0.5 μl / ml, e.g., ±0.1, ±0.2, ±0.3, ±0.4, or ±0.5 μl / ml.

[0043] When dispensing the aqueous dispersion into a vial by step (vi) above, the vial is typically only partially filled, rather than to the top, thereby leaving a headspace above the dispersion, which can be flushed (i.e., filled) with headspace gas. The term "headspace" has its conventional meaning and refers to the gas above the aqueous dispersion in the vial. Suitable types of vials or containers in which the aqueous dispersion can be stored include injection vials (e.g., plastic or glass, opaque or transparent), such as vials with surface coatings (e.g., to prevent ionic leachables). Also contemplated are ready-made syringes pre-filled with ultrasound contrast agents, thereby eliminating the need to aspirate the ultrasound contrast agent from the vial before injecting it into a subject.

[0044] In the above method of preparing an ultrasound contrast agent, steps (iii) and (iv) may be performed in any order.

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

[0046] The ultrasound contrast agents prepared by the above methods may be for long-term storage and / or ready for clinical use, i.e., ready for in vivo use in the in vivo imaging, diagnosis, and / or treatment of a subject.

[0047] The present disclosure is further directed to a method of improving contrast in an ultrasound image of tissue in a subject, the method comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject and performing an ultrasound scan of the tissue.

[0048] The present disclosure is also directed to a method of in vivo imaging of tissue in a subject, the method comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject, performing an ultrasound scan of the tissue, and generating an image of the tissue.

[0049] Furthermore, the present disclosure is directed to a method of diagnosing a subject, such as in vivo diagnosis of a subject, comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject, performing an ultrasound scan of a region of interest in the subject, generating an image of the region of interest, and evaluating the image to make a diagnosis.

[0050] The present disclosure is also directed to an ultrasound contrast agent for use in a method of improving contrast in an ultrasound image of tissue in a subject, the method comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject and performing an ultrasound scan of the tissue.

[0051] The present disclosure is further directed to an ultrasound contrast agent for use in a method of in vivo imaging of tissue in a subject, the method comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject, performing an ultrasound scan of the tissue, and generating an image of the tissue.

[0052] The present disclosure also relates to an ultrasound contrast agent for use in a method for in vivo diagnosis of a subject, the method comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into the subject, performing an ultrasound scan of a region of interest in the subject, generating an image of the region of interest, and evaluating the image to make a diagnosis.

[0053] The present disclosure is further directed to a use of an ultrasound contrast agent according to any one of the above aspects and embodiments for the manufacture of a medicament for improving contrast in an ultrasound image of a tissue in a subject, comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into said subject and performing an ultrasound scan of said tissue.

[0054] The present disclosure also relates to the use of an ultrasound contrast agent according to any one of the above aspects and embodiments for the manufacture of a medicament for in vivo imaging of tissue in a subject, comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into said subject, performing an ultrasound scan of said tissue, and generating an image of said tissue.

[0055] Furthermore, the present disclosure is directed to the use of an ultrasound contrast agent according to any one of the above aspects and embodiments for the manufacture of a medicament for in vivo diagnosis of a subject (comprising injecting an ultrasound contrast agent according to any one of the above aspects and embodiments into said subject, performing an ultrasound scan of a region of interest in said subject, generating an image of said region of interest, and evaluating said image to make a diagnosis).

[0056] A composition "comprising" one or more recited elements may also include other elements not specifically recited. The term "comprising" includes "consisting essentially of" as a subset, which means that the composition contains the recited components without the presence of other features or components.

[0057] The singular forms "a" and "an" are intended to be construed as including the plural forms as well.

[0058] The primary impurities found in the previously known Sonazoid™ powder for injection are phospholipid degradation products resulting from the hydrolysis of two components of the HEPS-Na excipient, phosphatidylserine sodium salt (PS) and phosphatidic acid sodium salt (PA). Hydrolysis of the excipient HEPS-Na occurs primarily during autoclaving of hydrated phospholipid suspensions. The primary degradation products are free fatty acids (FFA), lysophosphatidylserine sodium salt (lyso-PS), and lysophosphatidic acid sodium salt (lyso-PA). PA is present as a component of HEPS-Na but can also be a degradation product of PS. Diacylglycerol (diacyl-G) is another phospholipid-related degradation product. In the following examples, the primary parameter used as a measure of the chemical stability of the product is free fatty acids (FFA) as a percentage of the phosphatidylserine (PS) and phosphatidic acid (PA) present at each measurement. The presence of lyso-PS and lyso-PA was also measured, but the corresponding data is only presented in connection with one of the examples. The main parameters used as a measure of the physical stability of the product in the following examples are the volume concentration and median diameter of the microbubbles. [Example]

[0059] [Example 1] Samples were generated using leftovers from the filling line prior to lyophilization of the commercial Sonazoid production. The bulk product was temporarily stored in 20 L Sartorius Stedim Flexboy bags and then filled into four different sterile vial types in the LAF bench, after which the headspace was flushed with perfluorobutane (PFB). Two different Sonazoid batches were tested (Batch 1, Batch 2).

[0060] Because microbubble physical stability is the parameter most sensitive to liquid formulations, microbubble content and microbubble size were evaluated over storage time. Primary responses were parameters from Coulter count assay analysis: number and volume concentration, and number- and volume-weighted mean diameter / distribution. In addition, microbubble morphology (shape, structure, aggregation, foreign matter, etc.) was assessed by microscopy / image analysis, and chemical analysis of lipid content and purity was performed at sampling points of 6 months (batch 1) or 8 months (batch 2), respectively. All samples were stored at 5°C.

[0061] Stability Results Surprisingly, the physical stability of the microbubbles remained stable even 6 months after the preparation of Sonazoid aqueous dispersions (Batch 1 and Batch 2). However, as shown in Figure 1, phospholipid hydrolysis was significant after 6 months (Batch 1) and 8 months (Batch 2). Figure 1 shows the degree of phospholipid degradation due to hydrolysis in samples of Sonazoid bulk product taken before lyophilization, prepared as unbuffered aqueous dispersions, and stored at 5°C for 6–8 months (months on the x-axis). The degree of hydrolysis is indicated by the presence of three degradation products: free fatty acids (FFA), lysophosphatidylserine (lyso-PS), and lysophosphatidic acid (lyso-PA), each expressed as a percentage of the sum of phosphatidylserine (PS) and phosphatidic acid (PA) present at each analysis (percentage of FFA, which is (PS + PA) on the y-axis).

[0062] Furthermore, after 6 months of storage (Batch 1, Batch 2), the pH decreased from approximately 6-7 to approximately 4.9-6.4, i.e., in all samples, which was expected given the significant hydrolysis of phosphatidylserine.

[0063] The conclusion drawn from this study is that hydrolysis must be significantly slowed to obtain an acceptable shelf life for ready-to-use formulations, and that a critical level of hydrolysis must likely be based on the documented effect of hydrolytic impurities on microbubble properties.

[0064] Example 1 above suggests that ready-to-use Sonazoid cannot be obtained with existing formulations (i.e., when stored in water) due to significant chemical degradation. Existing Sonazoid formulations do not contain buffers and typically have a pH of about 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 (Grit et al., Biochim. Biophys. Acta, 1167, 49-55, 1993). However, because phospholipid-stabilized microbubbles differ from liposomes in some respects, buffers used with liposomes may not be compatible with microbubbles, and stability data obtained with liposomes may not be transferable to phospholipid-stabilized microbubbles. Microbubbles contain a single stabilized monolayer, and there is no water transport between the external and internal phases other than gas molecules. Physically, microbubbles tend to float due to the large difference between the internal and external phases, while small unilamellar liposomes can be physically homogeneous during storage without visible sedimentation. Therefore, microbubbles may require additional surface stabilization or charge to prevent coalescence during storage. The addition of ions would be expected to mask the surface charge and reduce the physical stability of the dispersion.

[0066] Nevertheless, the inventors of the present invention decided to conduct a second shelf-life stability study, in which they tested whether degradation could be significantly delayed by raising the pH to neutral or basic and by adding a buffer that would prevent the pH decrease due to initial hydrolysis. The study design and stability results of said study are disclosed below in Example 2.

[0067] [Example 2] To control and stabilize the pH, 5 mM tris(hydroxymethyl)aminomethane (Tris) buffer was used to prepare two different test dispersions: one buffered aqueous dispersion of Sonazoid with a bulk pH of 7.5 at 5° C. (corresponding to approximately pH 7 at room temperature, since the pH of Tris is temperature dependent, as described elsewhere herein), and one buffered aqueous dispersion of Sonazoid with a bulk pH of 8.5 at 5° C. (corresponding to approximately pH 8 at room temperature). Additionally, Sonazoid in an unbuffered aqueous dispersion was used as a reference.

[0068] Freeze-dried Sonazoid was used for sample preparation. Stock buffer was made from 1 M Tris solution with pH 7.0 and 8.0 (at room temperature) from an Invitrogen buffer kit (Thermo Fisher Scientific). This Tris buffer was diluted in a 100 ml Water For Injection (WFI) Ecoflac bottle from B. Braun.

[0069] Preparation of Sonazoid vials in 5 mM Tris buffer, pH 7 (at room temperature): Using a syringe with a sterile filter, aspirate 0.5 ml from a 1 M Tris pH 7.0 buffer kit (Invitrogen, Thermo Fisher Scientific, AM9850G Ambion) and inject it into 100 ml of GE Healthcare Ecoflac WFI (manufactured by B. Braun Medical SA). Shake the bottle to obtain a homogenous buffer solution. Reconstitute 20 Sonazoid vials and evacuate the vials with a sterile filter.

[0070] Preparation of Sonazoid vials in 5 mM Tris buffer, pH 8 (at room temperature): Using a syringe with a sterile filter, aspirate 0.5 ml from a 1 M Tris pH 8.0 buffer kit (Invitrogen, Thermo Fisher Scientific, AM9850G Ambion) and inject it into 100 ml of GE Healthcare Ecoflac WFI (manufactured by B. Braun Medical SA). Shake the bottle to obtain a homogenous buffer solution. Reconstitute 20 Sonazoid vials and evacuate the vials with a sterile filter.

[0071] For comparison, 15 vials of Sonazoid were reconstituted with water for injection from GE Healthcare Ecoflac WFI 100 ml manufactured by B. Braun Medical SA. All vials were stored at 5°C after reconstitution.

[0072] Stability Results The responses selected were microbubble size and concentration (by Coulter counting), as well as purity (by thin layer chromatography, TLC) and pH.

[0073] Figure 2 shows the degree of phospholipid degradation due to hydrolysis in samples of freeze-dried Sonazoid powder reconstituted with unbuffered water for injection, pH 7 buffer (at room temperature), or pH 8 buffer (at room temperature) and stored at 5°C for 6 months (months on the x-axis). The degree of hydrolysis is indicated by the degradation products, free fatty acids (FFA), and is expressed as a percentage of the sum of phosphatidylserine (PS) and phosphatidic acid (PA) present at each time of analysis (percentage of FFA, which is (PS + PA) on the y-axis).

[0074] Figure 3 shows the volume concentration of microbubbles during storage at 5°C for up to 6 months (months on the x-axis; volume concentration in μl / ml on the y-axis). The figure shows the average results of 10 samples for each data point. The variation between data points is normal analytical variation, and no trend was observed regarding the change in volume concentration after 6 months of storage. Therefore, the volume concentration was stable during 6 months of storage.

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

[0076] The pH values ​​of the two test dispersions at time zero, after three months, and after six months are shown in Table 1 below, respectively.

[0077] [Table 1]

[0078] [Example 4] Ultrasound contrast agents according to the present disclosure are prepared as follows: Microbubbles are formed by homogenizing perfluorobutane in a sterile aqueous dispersion of sodium HEPS to produce HEPS-stabilized microbubbles of PFB dispersed in water. The microbubble size distribution is adjusted by repeated flotation to remove smaller microbubbles, resulting in a median diameter between 1 and 6 μm. The dispersion is diluted with water. Optionally, tonicity is adjusted with a tonicity agent such as sucrose.

[0079] The pH of the dispersion is adjusted to a desired alkaline pH, such as about 7.5 or higher, by adding Tris to a concentration of 5 mM.

[0080] The target concentration is achieved by appropriate dilution of stabilized microbubbles of known size distribution. This may be done, for example, as follows: The concentration of microbubbles is adjusted to achieve a target concentration of approximately 6-10 μl / ml of microbubbles after storage, for example, by adjusting the concentration of microbubbles to between 8-20 μl / ml.

[0081] The dispersion is filled into 2-10 ml vials and the headspace is flushed with PFB before stoppering and capping.

[0082] Store the vial refrigerated.

[0083] Consideration The percentage of FFA was used as a marker of differences in hydrolysis between samples. The percentage of FFA in the Tris-buffered sample in Example 2 was significantly lower after 6 months of storage compared to the percentage of FFA in the unbuffered aqueous solution in Examples 1 and 2. All other purity parameters measured in the Example 2 study were stable after 6 months (data not shown). The data indicate that increasing and stabilizing the pH with a buffer during storage has a significant effect on reducing hydrolysis. At 5°C and pH 8.5, a shelf life of 1-2 years may be possible, depending on the reaction rate (based on the results shown in Figure 2, 30 months or more if the rate of hydrolysis is linear).

[0084] Example 1 showed that phospholipid-stabilized perfluorocarbon microbubbles stored in water for 6 months at 5°C underwent significant hydrolysis of the phospholipids, which affected the physical stability of the microbubbles and resulted in a decrease in volumetric concentration after 6 months. In comparison, Example 2, in which microbubbles were stored for 6 months at 5°C but in a buffered aqueous dispersion at a pH above 7.5 or 8.5, showed significantly less hydrolysis and no impact on physical stability. Although the addition of ions is known to reduce repulsion between individual microbubbles, the addition of small amounts of buffer did not result in visible aggregation. Furthermore, physical microbubble parameters such as volumetric concentration and median diameter were unaffected after 6 months.

[0085] These results indicate that adding a buffer to increase the pH of an ultrasound contrast agent in the form of a dispersion is a feasible method for significantly reducing the decomposition rate of negatively charged phospholipids. At the same time, these results indicate that the appearance or volume concentration of microbubbles is not affected by the addition of a buffer. Thus, both the physical and chemical stability of the dispersion are maintained at physiologically acceptable levels during storage. Thus, the present disclosure provides an ultrasound contrast agent that is both ready for immediate use (meaning it can be immediately injected into a subject in vivo) and can withstand long-term storage prior to such use.

[0086] It should be understood that the present disclosure is not limited to its exemplary embodiments described above, and that several modifications of the present disclosure are possible that are within the scope of the appended claims. The present invention includes the following aspects. <1> (a) perfluorocarbon microbubbles stabilized by a phospholipid membrane; and (b) buffering agents; and having a bulk pH of about 7.5 or greater, preferably about 8.5 or greater; Ultrasound contrast agents. <2> It is for long-term storage, <1> The ultrasound contrast agent according to claim 1. <3> ready for use in a clinical setting, e.g., for in vivo imaging, diagnosis, and / or treatment of a subject; <1> or <2> The ultrasound contrast agent according to claim 1. <4> the buffering agent is selected from the group consisting of tris(hydroxymethyl)aminomethane (Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, triethanolamine, and sodium carbonate; <1> ~ <3> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <5> The buffer has a concentration of about 1 mM to about 10 mM. <1> ~ <4> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <6> The phospholipid membrane has a net negative charge. <1> ~ <5> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <7> further comprising a tonicity agent, <1> ~ <6> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <8> further comprising a viscosity agent, and / or a flotation reducing agent; <1> ~ <7> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <9> the perfluorocarbon is selected from perfluorobutane, perfluoropropane, and perfluoropentane; <1> ~ <8> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <10> The perfluorocarbon is perfluorobutane, and the phospholipid is hydrogenated egg yolk phosphatidylserine. <1> ~ <9> 10. The ultrasound contrast agent according to any one of claims 1 to 9. <11> (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of a phospholipid to produce phospholipid-stabilized microbubbles of the perfluorocarbon dispersed in the aqueous dispersion; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 to 6 μm, preferably 2 to 5 μm; (iii) optionally adding a tonicity agent to the aqueous dispersion; (iv) adding a buffering agent 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 microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μl / ml; (vi) dispensing the aqueous dispersion into vials and flushing the headspace of the vials with a perfluorocarbon A method for preparing an ultrasound contrast agent, comprising: <12> Steps (iii) and (iv) are performed in any order; <11> The method described below. <13> step (v) occurs before or after any one of steps (ii), (iii), and (iv), provided that step (v) occurs after step (i) and before step (vi); <11> or <12> The method described below. <14> the ultrasound contrast agent is for long-term storage; and / or ready for use in a clinical setting, e.g., for in vivo imaging, diagnosis, and / or treatment of a subject; <11> ~ <13> A method according to any one of the preceding claims. <15> No freeze-drying step is involved <11> ~ <14> A method according to any one of the preceding claims. <16> <11> ~ <15> An ultrasound contrast agent prepared by any one of the methods described above. <17> 1. A method for improving contrast in an ultrasound image of tissue in a subject, comprising: <1> ~ <10> or <16> and performing an ultrasound scan of the tissue. <18> 1. A method of in vivo imaging of tissue in a subject, comprising: <1> ~ <10> or <16> 10. A method comprising injecting the ultrasound contrast agent of any one of claims 1 to 9 into the subject, performing an ultrasound scan of the tissue, and generating an image of the tissue. <19> 1. A method of diagnosing a subject, comprising: <1> ~ <10> or <16> 1. A method comprising injecting an ultrasound contrast agent according to any one of claims 1 to 10 into the subject, performing an ultrasound scan of a region of interest in the subject, generating an image of the region of interest, and evaluating the image to make a diagnosis. <20> <17> ~ <19> 10. An ultrasound contrast agent for use in any one of the methods described above. <21> <17> ~ <19> For producing a pharmaceutical product for use in any of the methods described above, <1> ~ <10> or <16> Use of the ultrasound contrast agent according to any one of 。

Claims

1. (a) perfluorocarbon microbubbles stabilized by a membrane of phospholipids, wherein the perfluorocarbon is perfluorobutane and the phospholipid is hydrogenated egg yolk phosphatidylserine; and (b) a buffering agent 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.

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

3. The ultrasound contrast agent according to claim 1 or 2, which is ready for clinical use.

4. 4. The ultrasound contrast agent of claim 3, wherein the clinical setting is for in vivo imaging, diagnosis, and / or treatment of a subject.

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

6. The ultrasound contrast agent of any one of claims 1 to 5, wherein the phospholipid membrane has a net negative charge.

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

8. The ultrasound contrast agent of any one of claims 1 to 7, further comprising a viscosity agent and / or a flotation-reducing agent.

9. (i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of phospholipids to produce phospholipid-stabilized microbubbles of the perfluorocarbon dispersed in the aqueous dispersion; (ii) adjusting the particle size distribution of the microbubbles in the aqueous dispersion to a median diameter within the range of 1 to 6 μm, preferably 2 to 5 μm; (iii) optionally adding a tonicity agent to the aqueous dispersion; (iv) adding a buffering agent to the aqueous dispersion to adjust the bulk pH of the aqueous dispersion to a pH of about 7.5 to about 8.5, measured at a temperature of 5° C.; (v) adjusting the concentration of microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μl / ml; (vi) dispensing the aqueous dispersion into vials and flushing the headspace of the vials with perfluorocarbon. A method for preparing the ultrasound contrast agent of any one of claims 1 to 8, comprising:

10. 10. The method of claim 9, wherein steps (iii) and (iv) are performed in any order.

11. 11. The method of claim 9 or 10, wherein step (v) occurs before or after any one of steps (ii), (iii), and (iv), provided that step (v) occurs after step (i) and before step (vi).

12. the ultrasound contrast agent is for long-term storage; and / or Immediately usable in clinical settings, The method according to any one of claims 9 to 11.

13. 13. The method of claim 12, wherein the clinical setting is for in vivo imaging, diagnosis, and / or treatment of a subject.

14. The method according to any one of claims 9 to 13, which does not include a freeze-drying step.

15. 10. A pharmaceutical composition for use in a method for improving contrast in ultrasound images of tissue in a subject, said method comprising injecting into said subject an ultrasound contrast agent according to any one of claims 1 to 8 and performing an ultrasound scan of said tissue, A pharmaceutical composition comprising the ultrasound contrast agent according to any one of claims 1 to 8.

16. 10. A pharmaceutical composition for use in a method for in vivo imaging of tissue in a subject, said method comprising injecting into said subject an ultrasound contrast agent according to any one of claims 1 to 8, performing an ultrasound scan of said tissue, and generating an image of said tissue; A pharmaceutical composition comprising the ultrasound contrast agent according to any one of claims 1 to 8.

17. 10. A pharmaceutical composition for use in a method of diagnosing a subject, the method comprising injecting the ultrasound contrast agent of any one of claims 1 to 8 into the subject, performing an ultrasound scan of a region of interest in the subject, generating an image of the region of interest, and evaluating the image to make a diagnosis; A pharmaceutical composition comprising the ultrasound contrast agent according to any one of claims 1 to 8.

18. 10. Use of an ultrasound contrast agent according to any one of claims 1 to 8 for the manufacture of a medicament for use in a method for improving contrast in ultrasound images of tissue in a subject, comprising: The method comprises injecting the subject with an ultrasound contrast agent according to any one of claims 1 to 8 and performing an ultrasound scan of the tissue; 9. Use wherein the pharmaceutical product comprises an ultrasound contrast agent according to any one of claims 1 to 8.

19. 10. Use of an ultrasound contrast agent according to any one of claims 1 to 8 for the manufacture of a medicament for use in a method for in vivo imaging of tissue in a subject, comprising: The method comprises injecting the subject with an ultrasound contrast agent according to any one of claims 1 to 8, performing an ultrasound scan of the tissue, and generating an image of the tissue; 9. Use wherein the pharmaceutical product comprises an ultrasound contrast agent according to any one of claims 1 to 8.

20. 10. Use of an ultrasound contrast agent according to any one of claims 1 to 8 for the manufacture of a medicament for use in a method of diagnosing a subject, comprising: The method comprises injecting the ultrasound contrast agent of any one of claims 1 to 8 into the subject, performing an ultrasound scan of a region of interest in the subject, generating an image of the region of interest, and evaluating the image to make a diagnosis; 9. Use wherein the pharmaceutical product comprises an ultrasound contrast agent according to any one of claims 1 to 8.

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