HK40062095A

HK40062095A - Ultrasound contrast agent and methods for use thereof

Info

Publication number: HK40062095A
Application number: HK62022050171.7A
Authority: HK
Inventors: Henriksen Ingrid; Krog Tamnes Eva; Sontum Per; Kvale Svein
Original assignee: Ge Healthcare As
Priority date: 2018-12-21
Filing date: 2019-12-20
Publication date: 2022-06-10

Description

Ultrasound contrast agents and methods of use thereof

Technical Field

The present disclosure relates to the field of in vivo imaging and diagnosis of subjects, and in particular to an ultrasound contrast agent that is immediately available in vivo and can survive long-term storage prior to in vivo use. The present disclosure also relates to a method of preparing an ultrasound contrast agent and a method of using an ultrasound contrast agent in a clinical setting.

Background

Ultrasound contrast agents based on phospholipid-stabilized perfluorocarbon 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 may be about 2-10 μm in size, encapsulated in a layer of shell or membrane of stabilized phospholipid molecules. The gaseous core is compressible and can expand and contract when subjected to ultrasound. The expansion and contraction of the microbubbles upon exposure to ultrasound produces acoustic backscatter, which is used for diagnostic imaging purposes. The microbubble surface can be further functionalized with a targeted drug moiety that will be released when the microbubble is disrupted and/or cavitated upon application of ultrasound, and thus such ultrasound contrast agents can also be used in therapeutic applications (Udahyay et al, RSC adv., 6, 15016-15026, 2016).

Sonazoid @ is an example of an ultrasound contrast agent based on phospholipid-stabilized perfluorocarbon microbubbles. More particularly, Sonazoid @ is formulated as a powder consisting of lyophilized sucrose-entrapped perfluorobutane microbubbles stabilized by a membrane of hydrogenated egg phosphatidylserine stored under the perfluorobutane headspace. Sonazoid @ is produced aseptically by continuous homogenization of Perfluorobutane (PFB) in an aqueous dispersion of Hydrogenated Egg Phosphatidylserine (HEPS). After initial microbubble generation, the concentration and size distribution of the microbubbles is adjusted through a series of controlled isolation steps. The final dispersion (aimed at generating 8 μ l of microbubbles per ml of reconstituted product) was made isotonic by the addition of sucrose. 2ml of this dispersion were filled into 10ml glass vials and lyophilized. After lyophilization, the vial headspace was backfilled with perfluorobutane and then stoppered. In other words, the Sonazoid cell is a lyophilized product that must be reconstituted with water prior to use. More specifically, prior to administration to the subjects, the product was reconstituted by the addition of 2ml of sterile water for injection by a supplied vent filter (5 μm) piercer (Codan Chemoprotect Spike, Codan GmbH & Co., Germany) followed by manual mixing for 1 minute. Upon reconstitution, the product appeared as a milky white uniform dispersion. Since the dispersion is opaque, it is difficult to visually inspect the foreign particles. To ensure that no such particles are present, the product is drawn into the syringe through the filter piercer prior to administration. Upon reconstitution, the microspheres will begin to separate by floating and form a cream layer on top of the liquid phase if not stirred. If not used immediately after reconstitution, the product should be re-homogenized by manual mixing for 10 seconds prior to use (Sontum, ultrasonic Med. & biol., 34(5), 824-833, 2008).

Since the effective element of ultrasound contrast agents is the physical state (microbubbles) rather than the chemical substance, two types of stability must be considered: physical and chemical stability. In other words, the emphasis must be on how the microbubble concentration and size distribution, as well as the chemical composition of the components, are controlled and maintained. Microvesicles are generally thermodynamically unstable systems that may undergo physical changes during preparation and storage (see, e.g., WO2015150354A 1; Segers et al, Langmuir, 33, 10329-. In addition, phospholipid membranes that stabilize the microbubbles may undergo hydrolytic degradation in solution, thereby creating impurities in the final product. Phospholipids are susceptible to hydrolytic cleavage in acidic and basic media. Phospholipids are sufficiently stable only at pH 7, since hydrolysis of the ester bond does not proceed to any significant extent under these conditions (Phospholipids Handbook, 1993, edited by Gregor Cevc; cf. Chapter 9 "Chemical stability", Evsigneeva, p. 323-324). The hydrolysis kinetics are strongly influenced by temperature and pH (Phospholipids Handbook, 1993, edited by Gregor Cevc; cf. Chapter 9 "Chemical stability", Crommelin et al, p. 338-339). Furthermore, it is known that once hydrolytic degradation starts at low pH, the pH drops and leads to accelerated degradation. Thus, one of the major challenges in the early development of Sonazoid cells is how to obtain a product with an acceptable shelf life. The freeze-drying process to produce a Sonazoid preparation in the form of a lyophilized powder has heretofore been considered the only way to obtain such a product.

Although freeze-drying provides products with excellent shelf-life stability and quality, it also significantly increases manufacturing costs and reduces the "ease of use" of the end user (typically a healthcare professional) because it is time and resource intensive. There is therefore a need in the art for improved ultrasound contrast agents based on phospholipid-stabilized perfluorocarbon microbubbles, which are both highly storage-stable and easy to use.

Disclosure of Invention

The above object of providing an ultrasound contrast agent having both high storage stability and ease of use is achieved by the present disclosure, which relates to a stable and ready to use ultrasound contrast agent formulation in the form of a dispersion, which surprisingly can withstand long term storage prior to use and is ready for use, i.e. ready for injection into a subject.

More particularly, the present disclosure relates to an ultrasound contrast agent comprising:

(a) perfluorocarbon microbubbles, said microbubbles stabilized by a phospholipid membrane; and

(b) a buffering agent;

wherein the bulk pH of the ultrasound contrast agent is about 7.5 or higher, preferably about 8.5 or higher.

The present disclosure also relates to a method of preparing an ultrasound contrast agent comprising the steps of:

(i) continuously homogenizing a perfluorocarbon in a sterile aqueous dispersion of a phospholipid to produce phospholipid-stabilized perfluorocarbon microbubbles dispersed in the aqueous dispersion;

(ii) adjusting the size distribution of the microbubbles in the aqueous dispersion to a median size in 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 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 microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μ l/ml;

(vi) the aqueous dispersion was dispensed into the vial and the headspace of the vial was flushed with perfluorocarbon.

Furthermore, the present disclosure relates to a method for improving the contrast of an ultrasound image of tissue in a subject, a method for in vivo imaging of tissue in a subject and a method for diagnosis of a subject, the method comprising injecting into the subject an ultrasound contrast agent as described above.

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

Furthermore, 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.

Preferred aspects of the present disclosure are described in the following detailed description and dependent claims.

Drawings

Figure 1 illustrates the chemical stability of the Sonazoid bulk product taken prior to lyophilization, prepared as a non-buffered aqueous dispersion and stored at 5 ℃ for 8 months.

Figure 2 illustrates the chemical stability of a lyophilized powder of Sonazoid prepared as a non-buffered aqueous dispersion, as a buffered aqueous dispersion comprising a buffer and having a bulk pH of 7 at room temperature, and as a buffered aqueous dispersion comprising a buffer and having a bulk pH of 8 at room temperature, respectively, and stored for 6 months at 5 ℃.

Figure 3 illustrates the physical stability of a lyophilized powder of Sonazoid prepared as a non-buffered aqueous dispersion, as a buffered aqueous dispersion comprising a buffer and having a bulk pH of 7 at room temperature, and as a buffered aqueous dispersion comprising a buffer and having a bulk pH of 8 at room temperature, respectively, and stored for 6 months at 5 ℃.

Detailed Description

The present disclosure provides a liquid ultrasound contrast agent that is storage stable and ready-to-use. By increasing the pH and including a buffer in the product, a liquid formulation is achieved which will be easier for the end user to handle than previously known Sonazoid freeze-dried powders, since reconstitution of the powder is not required prior to use of the presently claimed product. Here, we wish to point out that it is not obvious to the skilled person that the addition of a buffer to ultrasound contrast agents based on phospholipid-stabilized perfluorocarbon microbubbles results in a functional ultrasound contrast agent, since electrolytes (e.g. present in the buffer) may alter the composition of the dispersion. However, the inventors of the present application have surprisingly sought to maintain the volume concentration and distribution of microvesicles at a desired level, i.e., have sought to improve the chemical stability of the product while maintaining the physical stability, as compared to the physical stability and chemical stability, respectively, of a previously known dispersion of a Sonazoid reconstituted lyophilized powder. Thus, the storage stability of the presently claimed product is improved compared to previously known products.

By including a buffer in the formulation, the ultrasound contrast agent according to the present disclosure has a bulk pH in the alkaline range at a temperature of 5 ℃. As further shown below in the examples, an alkaline pH will significantly reduce the rate of hydrolysis of phospholipids present in ultrasound contrast agents, which will thus remain chemically stable for a much longer time. The chemical stability of phospholipids also affects the physical stability of the microvesicles, since phospholipid membranes stabilize the microvesicles.

More particularly, the present disclosure solves or at least alleviates the problems associated with existing ultrasound contrast agents based on phospholipid-stabilized perfluorocarbon microbubbles by providing an ultrasound contrast agent comprising:

(b) a buffering agent;

The term "contrast agent" has its conventional meaning in the field of in vivo medical imaging and refers to an agent in a form suitable for mammalian administration that helps provide a sharper image in a target area or organ than can be obtained by imaging a mammalian subject alone. The term "subject" refers to a mammalian body, preferably an intact mammalian body, more preferably a living human subject. The phrase "in a form suitable for mammalian administration" refers to a composition that is sterile, pyrogen-free, devoid of compounds that produce toxicity or side effects, and formulated at a biocompatible pH (about pH 4.0 to 10.5). Such compositions lack microparticles that may risk causing embolisms in vivo and are formulated so as not to precipitate upon contact with biological fluids (e.g., blood). Such compositions also contain only biocompatible excipients and are preferably isotonic.

Like 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 may be administered to the mammalian body in a minimally invasive manner, i.e. without substantial health risk to the mammalian subject when performed by a professional medical professional. Such minimally invasive administration is preferably intravenous into a peripheral vein of the subject, without the need for local or general anesthesia.

The term "microbubble" has its conventional meaning in the field of in vivo ultrasound imaging and refers to a gas microbubble having an inner diameter of 0.1-10 μm, typically between 0.5 and 5 μm. Such microbubbles are similar in size to erythrocytes, which makes them display similar characteristics throughout the capillaries and capillaries of the mammalian body (Sirsi et al, Bubble sci. eng. Technol, 1(1-2), 3-17, 2009). The terms "microbubble" and "microsphere" are used interchangeably herein.

The term "perfluorocarbons" has its conventional chemical meaning and is a group of compounds of formula C_xF_yI.e. they contain only carbon and fluorine (see IUPAC, complex of Chemical terminologies (compilation of Chemical terms), 2 nd edition, 1997; online revised edition, 2006-). Compounds with a prefix perfluoro-being hydrocarbonsIncluding those having heteroatoms wherein all C-H bonds are replaced with C-F bonds. Perfluorocarbons include perfluoroalkanes, fluoroolefins, fluoroalkynes, and perfluoroaromatics. The terms "perfluorocarbon" and "fluorocarbon" are used interchangeably. Suitable perfluorocarbons in accordance with the present disclosure include perfluoroalkanes such as perfluorobutane, perfluoropropane, and perfluoropentane. The presently preferred perfluorocarbon of the present disclosure is perfluorobutane ("PFB"), which has its standard chemical meaning and is also known in the context of medical use as decafluorobutane (perfluutane). The chemical formula of perfluoron-butane is CF₃CF₂CF₂CF₃Or C₄F₁₀The boiling point is-2.2 ℃. Commercial perfluoron-butane contains a small amount (typically 2-4%) of perfluoroisobutane isomer, C₄HF₉。

Suitable microvesicles according to the present disclosure include perfluorocarbon microvesicles stabilized by phospholipid membranes, as described, for example, in Sontum (above) and Sirsi et al (above). Suitable phospholipid membranes (or shells or coatings) according to the present disclosure have a net negative charge. Presently preferred phospholipids are phospholipids present in Hydrogenated Egg Phosphatidylserine (HEPS), i.e. mainly phosphatidylserine and phosphatidic acid (Hvattum et al, j. pharm. biomed. anal., 42(4), 506-512, 2006). The thickness of the phospholipid membrane is usually 10 to 100 nm.

Herein, the term "buffer" refers to a buffer, which is a solution containing a weak acid and its salt or a weak base and its salt, and which is resistant to a change in pH. In other words, the buffer is an aqueous solution of a weak acid and its conjugate base or a weak base and its conjugate acid. Buffering agents are used to maintain a stable pH in solution (or suspension or dispersion) because they can neutralize small amounts of additional acids or bases. The buffer is 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.

The currently preferred buffer is Tris. The pH of Tris is temperature dependent. At low temperatures, the pH of Tris is higher than at higher temperatures. For example, if the Tris buffer has a pH of 8.26 at 5 ℃, the Tris buffer has a pH of 7.7 at 25 ℃ and a pH of 7.4 at 37 ℃. The storage of the ultrasound contrast agent is preferably performed in a refrigerated space, i.e. at a temperature of about 3-6 ℃, which helps to maintain the physical and chemical stability of the ultrasound contrast agent. As explained and shown elsewhere herein, the alkaline pH also helps to maintain the chemical and physical stability of the ultrasound contrast agent. Meanwhile, when injected into a subject in vivo, the ultrasound contrast agent should preferably have a pH close to physiological pH 7.4. The fact that the pH of Tris is temperature dependent may therefore be used as an advantage of ultrasound contrast agents according to the present disclosure, since the pH of refrigerated storage will be higher than the pH at body temperature.

The term "bulk pH" refers to the pH of a solution measured within the bulk or volume of the solution (or suspension or dispersion), 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 be different from the surface pH of the solution. Ultrasound contrast agents according to the present disclosure have a bulk pH in the alkaline range at a temperature of 5 ℃, i.e. a bulk pH of about 7.5 or higher at a temperature of 5 ℃, suitably at most 10.0 at a temperature of 5 ℃, such as 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 ℃. Currently preferred bulk pH is about 8.25-9.25 at a temperature of 5 ℃, such as 8.25, 8.5, 8.75, 9.0, or 9.25 at a temperature of 5 ℃. In this context, throughout the text, the term "about" is intended to mean that all pH values mentioned herein can generally vary by about 0.1 to 0.5, i.e. ± 0.1 to 0.5, such as ± 0.1, ± 0.2, ± 0.3, ± 0.4 or ± 0.5.

The ultrasound contrast agents of the present invention are preferably stored at low temperatures, especially for a longer storage period. For storage periods of up to about 1 month, temperatures up to room temperature may be suitable. The storage temperature is preferably not lower than the freezing point of the ultrasound contrast agent, more preferably higher than the freezing point, as a solution. An exemplary temperature range for storing the ultrasound contrast agent of the present invention will be above its freezing point and up to about 5 ℃. In use, the ultrasound contrast agents of the present invention will be brought to ambient temperature prior to administration to a subject.

Ultrasound contrast agents according to the present disclosure are useful for, i.e., can withstand, long-term storage. In other words, the ultrasound contrast agent retains its physical and chemical stability during long-term storage, i.e. has an acceptable or even excellent shelf-life. In this context, "long-term" is intended to refer throughout to a period of months or years, such as a period of 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 about 3-6 deg.C, such as at 3, 4, 5 or 6 deg.C.

The ultrasound contrast agent according to the present disclosure differs from currently commercially available lyophilized formulations in that it is immediately available from a vial in the purchased state, wherein "immediately available" means immediately available in a clinical setting, such as immediately available for injection into a patient, for in vivo imaging, diagnosis and/or treatment of a subject.

Ultrasound contrast agents according to the present disclosure are in liquid form, i.e. are liquid formulations, in particular in the form of dispersions, such as aqueous dispersions, as defined elsewhere herein. The liquid formulation is for long term storage and ready for use in a clinical setting.

The buffer included in the ultrasound contrast agent according to the present disclosure may be selected from Tris (hydroxymethyl) aminomethane (Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, triethanolamine, and sodium carbonate.

Further, the concentration of the buffer can be about 1mM to about 10mM, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mM. In this context, throughout the text, the term "about" is intended to mean that all concentration values mentioned herein may generally vary by about 0.1-0.5mM, i.e. + -. 0.1-0.5, such as. + -. 0.1,. + -. 0.2,. + -. 0.3,. + -. 0.4 or. + -. 0.5 mM.

The phospholipid membrane comprised in the ultrasound contrast agent according to the present disclosure preferably has a net negative charge.

Currently preferred ultrasound contrast agents according to the present disclosure comprise perfluorobutane microbubbles stabilized by hydrogenated egg phosphatidylserine, such AS Sonazoid @ (GE Healthcare AS), previously referred to AS NCI 00100, AS described by Sontum (supra), and further comprise Tris (hydroxymethyl) aminomethane (i.e., Tris) AS a buffer.

Ultrasound contrast agents according to the present disclosure may also comprise tonicity agents, i.e. excipients added to make the ultrasound contrast agent isotonic. Examples of tonicity agents are salts of plasma cations with biocompatible counterions, sucrose, physiological saline, dextrose, glycerol and mannitol.

Alternatively or additionally, ultrasound contrast agents according to the present disclosure may comprise: viscosity agents, i.e. excipients added to modify the viscosity of the ultrasound contrast agent, and/or flotation-reducing agents, such as propylene glycol, glycerol and/or polyethylene glycol.

(iii) optionally adding a tonicity agent to the aqueous dispersion;

(v) adjusting the concentration of microbubbles in the aqueous dispersion to achieve a target concentration of microbubbles of about 6-10 μ l/ml, such as about 6, 7, 8, 9, or 10 μ l/ml, with about 8 μ l/ml currently preferred;

In other words, the present disclosure relates to a method of preparing an ultrasound contrast agent, comprising the steps of:

(iii) optionally adding a tonicity agent to the aqueous dispersion;

(vi) dispensing the aqueous dispersion into a vial and flushing the headspace of the vial with a perfluorocarbon;

provided that freeze-drying of the dispersion is not performed or required prior to long-term storage and/or in vivo injection of the ultrasound contrast agent.

The term "dispersion", as in "aqueous dispersion", is intended to refer to a composition in which one material is dispersed within another material. The manner in which dispersions are classified can vary, with the two main classification approaches being (1) the nature of the internal and external phases of the dispersion (e.g., solid, liquid, or gas) and (2) the size range of their dispersed particles (colloidal particles versus coarse particles).

The term "suspension" has been used to describe previously known formulations of Sonazoid (see, e.g., WO2015150354a 1). However, since the term "suspension" is now commonly used in pharmaceutical applications primarily for solid particles dispersed in an external phase, the term "dispersion" herein is preferably used for the newly disclosed liquid formulations comprising a gas dispersed in an external phase. However, the terms "dispersion" and "suspension" are used interchangeably herein.

The term "aqueous dispersion" refers to a dispersion of microbubbles in an aqueous solvent, the aqueous solvent comprising water and/or a water-miscible solvent. The aqueous solvent is preferably a biocompatible carrier. The term "biocompatible carrier" refers to a fluid, especially a liquid, such that the composition is physiologically tolerable, i.e. can be administered to the mammalian body without toxicity or undue discomfort. The biocompatible carrier is suitably an injectable carrier liquid, such as sterile, pyrogen-free water for injection; an aqueous solution such as physiological saline (which may be advantageously balanced so that the final product for injection is isotonic). One or more excipients may be added to the biocompatible carrier as is well known to those skilled in the art, such as: an aqueous buffer solution comprising a biocompatible buffer (e.g., phosphate buffer); an aqueous solution of one or more tonicity-adjusting substances (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.

The phrase "adjusting the bulk pH of the aqueous dispersion to a pH of about 7.5 or higher" is intended to mean adjusting the bulk pH of the aqueous dispersion to a pH of about 7.5 or higher, as preferably measured at a particular temperature, e.g., 5 ℃.

Herein, "target concentration" is defined as the concentration after long term storage and/or the concentration when injected into a subject in vivo. After the ultrasound contrast agent is prepared, the concentration of microbubbles may initially decrease and stabilize at a slightly lower concentration. The target concentration is obtained based on an appropriate dilution of stabilized microbubbles of known size distribution.

The target concentration of microbubbles is about 6-10. mu.l/ml, preferably about 8. mu.l/ml. In this context, throughout the text, the term "about" is intended to mean that the concentration values mentioned herein may typically vary by about 0.1-0.5 μ l/ml, i.e. + -. 0.1-0.5 μ l/ml, such as + -0.1, + -. 0.2, + -. 0.3, + -. 0.4 or + -0.5 μ l/ml.

When the aqueous dispersion is dispensed into a vial according to step (vi) above, the vial is typically not filled to the top but is only partially filled, leaving a headspace above the dispersion that can be flushed (i.e., filled) with a 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 may be stored include injection vials (e.g., opaque or transparent plastic or glass), such as vials with surface coatings (e.g., to prevent ionic leachates). Pre-prepared syringes pre-filled with ultrasound contrast agent are also contemplated, which would avoid the need to draw ultrasound contrast agent from the vial prior to injecting the ultrasound contrast agent into the subject.

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

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

Ultrasound contrast agents prepared according to the above-described methods are for long-term storage and/or ready-to-use in a clinical setting, i.e., ready-to-use in vivo, such as in vivo imaging, diagnosis and/or treatment of a subject.

The present disclosure also relates to a method of improving the contrast of an ultrasound image of tissue in a subject, comprising injecting into the subject an ultrasound contrast agent according to any of the above aspects and embodiments and performing an ultrasound scan of the tissue.

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

Furthermore, the present disclosure relates to a method for diagnosis of a subject, such as in vivo diagnosis of a subject, comprising injecting into the subject an ultrasound contrast agent according to any of the above aspects and embodiments, performing an ultrasound scan of a target region in the subject, generating an image of the target region and evaluating the image to make a diagnosis.

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

Furthermore, the present disclosure relates to an ultrasound contrast agent for use in a method of in vivo imaging of tissue in a subject, the method comprising injecting into the subject an ultrasound contrast agent according to any of the above aspects and embodiments, performing an ultrasound scan of the tissue and generating an image of the tissue.

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

The present disclosure also relates to the use of an ultrasound contrast agent according to any of the above aspects and embodiments for the manufacture of a medicament for improving the contrast of an ultrasound image of tissue in a subject (comprising injecting into the subject an ultrasound contrast agent according to any of the above aspects and embodiments and performing an ultrasound scan of the tissue).

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

Furthermore, the present disclosure relates to the use of an ultrasound contrast agent according to any 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 of the above aspects and embodiments into the subject, performing an ultrasound scan of a target region in the subject, generating an image of the target region and evaluating the image to make a diagnosis).

A composition that "comprises" one or more of the recited elements may also comprise additional, unrecited elements. The term "comprising" includes "consisting essentially of … … as a subset, the latter meaning that the composition has the listed components and no other features or components are present.

The singular forms "a", "an" and "the" are to be understood to include the plural forms as well.

The major impurity present in the previously known Sonazoid powder for injection is the degradation product of phospholipids, which results from the hydrolysis of the two components phosphatidylserine sodium salt (PS) and phosphatidic acid sodium salt (PA) of the HEPS-Na excipient. Hydrolytic degradation of the excipient HEPS-Na occurs primarily during autoclaving of the hydrated phospholipid suspension. The major degradation products are Free Fatty Acids (FFA), lyso-phosphatidylserine sodium salts (lyso-PS) and lysophosphatidic acid sodium salts (lyso-PA). PA is present as a component in HEPS-Na, but may also be a degradation product of PS. Diacylglycerol (diacyl-G) is another degradation product associated with phospholipids. In the following examples, the main parameter used as a measure of the chemical stability of the product is the Free Fatty Acid (FFA) present at each measurement, as a percentage of Phosphatidylserine (PS) and Phosphatidic Acid (PA). The presence of lyso-PS and lyso-PA was also measured, but the corresponding data is shown for only 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 size of the microbubbles.

Example 1

The remainder from the pre-lyophilization fill line from commercial Sonazoid production was used to generate samples. The bulk product was temporarily stored in 20L Sartorius Stedim Flexboy bags and then filled into four different sterile vial types in the LAF bench followed by flushing of the headspace with Perfluorobutane (PFB). Two different Sonazoid batches (batch 1, batch 2) were tested.

Since the physical stability of the microbubbles is the parameter most likely to be affected by the liquid formulation, the relationship of microbubble content and microbubble size to storage time was evaluated. The primary response is a parameter from the assay analysis by coulter count; number and volume concentrations and number and volume weighted mean diameters/distributions. In addition, microbubble morphology (shape, structure, agglomeration, foreign matter, etc.) was evaluated by microscopy/image analysis and chemical analysis of lipid content and purity was performed at sampling times of 6 months (batch 1) or 8 months (batch 2), respectively, of storage. All samples were stored at 5 ℃.

Stability results

The physical stability of the microbubbles was surprisingly stable even 6 months after manufacture of the aqueous dispersion of Sonazoid (batch 1, batch 2). However, as shown in figure 1, hydrolysis of phospholipids was significant after 6 months (batch 1) and 8 months (batch 2). Figure 1 shows the extent of phospholipid degradation caused by hydrolysis in samples of the Sonazoid bulk product taken prior to lyophilization prepared as non-buffered aqueous dispersions and stored at 5 ℃ for 6-8 months (months on the x-axis). The extent of hydrolysis is illustrated by the presence of three degradation products: free Fatty Acid (FFA), lysophosphatidylserine (lyso-PS) and lysophosphatidic acid (lyso-PA), respectively, were present at each assay as a percentage of the sum of Phosphatidylserine (PS) and Phosphatidic Acid (PA) (% FFA of (PS + PA) on the y-axis).

Furthermore, after 6 months of storage (batch 1, batch 2), the pH had dropped from about 6-7 to about 4.9-6.4, i.e. decreased in all samples, which was expected in view of significant hydrolysis of phosphatidylserine.

The conclusion drawn from this study is that hydrolysis must be slowed significantly to obtain acceptable shelf life for the instant formulation, and that critical levels of hydrolysis must most likely be based on literature documented effects of hydrolysis impurities on microbubble properties.

Example 1 above shows that due to significant chemical degradation, no ready-to-use Sonazoid is available with the existing formulation (i.e., when stored in water). The existing Sonazoid formulations do not contain buffer and the pH is typically about 6 to 7. After significant hydrolysis according to example 1 above, the pH dropped to 4.9 to 6.4.

Literature data relating to liposomal dispersions indicate that phospholipid hydrolysis is affected by pH (Grit et al, Biochim. Biophys. Acta, 1167, 49-55, 1993). However, the buffer used with liposomes is not necessarily compatible with the microbubbles, and the stability data obtained with liposomes is not necessarily transferable to phospholipid-stabilized microbubbles, as phospholipid-stabilized microbubbles differ from liposomes in several respects. They contain a single stabilizing monolayer and there is no water transport between the outer and inner phases other than the gas molecules. Physically, microbubbles tend to float due to the large difference between the internal and external phases, whereas small unilamellar liposomes may be physically homogeneous during storage without significant sedimentation. Thus, the microbubbles may require additional surface stabilization or charge to avoid coalescence during storage. The added ions shield the surface charge and are expected to reduce the physical stability of the dispersion.

However, the inventors of the present application decided to conduct a second study of shelf-life stability, in which it was tested whether degradation would be slowed significantly by increasing the pH to neutral or alkaline, and by adding buffers that prevent the pH from decreasing due to initial hydrolysis. The study design and stability results of the study are disclosed in example 2 below.

Example 2

For pH control and stabilization, two different test dispersions were obtained separately using 5mM Tris (hydroxymethyl) aminomethane (Tris) buffer; a Sonazoid buffered aqueous dispersion having a bulk pH of 7.5 at 5 ℃ (corresponding to a pH of about 7 at room temperature since the pH of Tris is temperature dependent, as described elsewhere herein), and a Sonazoid buffered aqueous dispersion having a bulk pH of 8.5 at 5 ℃ (corresponding to a pH of about 8 at room temperature). Furthermore, Sonazoid in a non-buffered aqueous dispersion was used as reference.

Samples were prepared using lyophilized Sonazoid. Stock buffer solutions were prepared from 1M Tris solutions from Invitrogen buffer kit (term Fisher Scientific) at pH 7.0 and 8.0 (at room temperature). This Tris buffer was diluted in a 100ml water for injection (WFI) ecoflex bottle from b.

Construction of 5mM Tris buffer 7 pH (at room temperature) Sonazoid vials:

0.5ml was extracted from the 1M Tris pH 7.0 buffer kit Invitrogen of Thermo Fisher Scientific AM9850G Ambion using a syringe with a sterile filter and injected into 100ml of GE Healthcare Ecoflac WFI manufactured by B. Braun Medical SA. The bottle was shaken to obtain a homogeneous buffer solution. 20 Sonazoid vials were reconstituted and the vials were degassed with a sterile filter.

Construction of 5mM Tris buffer 8 pH (at room temperature) Sonazoid vials:

0.5ml was extracted from the 1M Tris pH 8.0 buffer kit Invitrogen of Thermo Fisher Scientific AM9850G Ambion using a syringe with a sterile filter and injected into 100ml of GE Healthcare Ecoflac WFI manufactured by B. Braun Medical SA. The bottle was shaken to obtain a homogeneous buffer solution. 20 Sonazoid vials were reconstituted and the vials were degassed with a sterile filter.

For comparison, 15 Sonazoid vials were reconstituted with 100ml of water for injection from GE Healthcare ecoflex WFI produced by b. Braun Medical SA. After reconstitution all vials were stored at 5 ℃.

Stability results

The responses selected were microbubble size and concentration (by coulter count) and purity (by thin layer chromatography, TLC) and pH.

Figure 2 shows the extent of phospholipid degradation resulting from hydrolysis in samples of lyophilized Sonazoid powder reconstituted with non-buffered water for injection, buffer pH 7 (at room temperature), or buffer pH 8 (at room temperature), respectively, and stored for 6 months (months on the x-axis) at 5 ℃. The extent of hydrolysis is illustrated by the degradation product Free Fatty Acids (FFA) present at each analysis as a percentage of the sum of Phosphatidylserine (PS) and Phosphatidic Acid (PA) (% FFA on the y-axis (PS + PA)).

Figure 3 shows the volume concentration of microvesicles during storage at 5 ℃ to 6 months (number of months on the x-axis; volume concentration in μ l/ml on the y-axis). Each data point of the figure shows the average results for 10 samples. The change between data points is a normal analytical change and there is no visible trend of volume concentration change after 6 months of storage. Thus, the volume concentration was stable during 6 months of storage.

The median size of the microbubbles was also found to be stable over a 6 month storage period (data not shown).

The pH values of the two test dispersions at time zero, after 3 months and after 6 months, respectively, are shown in table 1 below.

Table 1: pH value

Vial numbering	Zero	3 months old	6 months old
				pH 8 Start	8.04	7.97	8.08
pH 7 Start	7.28	7.26	7.38

Example 4

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

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

The target concentration is obtained based on an appropriate dilution of stabilized microbubbles of known size distribution. This can be done, for example, as follows. The concentration of microvesicles is adjusted, for example by adjusting the concentration of microvesicles to between 8-20 μ l/ml, to reach a target microvesicle concentration of about 6-10 μ l/ml after storage.

The dispersion was filled into 2-10ml vials and the headspace was flushed with PFB before stoppering and capping.

The vials were stored refrigerated.

Discussion of the related Art

% FFA was used as an indicator of hydrolysis differences between samples. The% FFA in the Tris buffer containing samples in example 2 after 6 months of storage was significantly lower compared to the% FFA in the non-buffered aqueous solutions of examples 1 and 2. All other purity parameters measured in the study of example 2 were stable after 6 months (data not shown). The data indicate that increasing pH and stabilizing pH with a buffer during storage has a significant effect on reducing hydrolysis. At pH 8.5 at 5 ℃, depending on the kinetics, a shelf life of 1-2 years is possible (based on the results shown in fig. 2, if the hydrolysis rate is linear, the shelf life is more than 30 months).

Example 1 shows that phospholipid-stabilized perfluorocarbon microbubbles stored in water at 5 ℃ for 6 months undergo significant hydrolysis of the phospholipids, which also affects the physical stability of the microbubbles, with the volume concentration decreasing after 6 months. In contrast, example 2, where microvesicles were stored in aqueous dispersions containing buffer at 5 ℃ but at pH higher than 7.5 or 8.5 for 6 months, showed significantly less hydrolysis and had no effect on physical stability. Although it is known that the addition of ions reduces the repulsive forces between individual microbubbles, the addition of a small amount of buffer does not produce visible aggregation. In addition, physical microbubble parameters such as volume concentration and median size were unaffected after 6 months.

The results show that adding a buffer to increase the pH of an ultrasound contrast agent in dispersion form is a viable way to significantly reduce the degradation rate of negatively charged phospholipids. At the same time, the results indicate that the visual appearance or volumetric concentration of the microbubbles is not affected by the addition of the buffer. Thus, both the physical and chemical stability of the dispersion has been demonstrated to remain at physiologically acceptable levels during storage. Thus, the present disclosure provides an ultrasound contrast agent that is both instantly available (meaning ready for in vivo injection in a subject) and that can withstand long term storage prior to such use.

It is to be understood that the disclosure is not limited to the exemplary embodiments thereof described above, and that several conceivable modifications of the disclosure are possible within the scope of the appended claims.

Claims

1. An ultrasound contrast agent, comprising:

(b) a buffering agent;

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

3. The ultrasound contrast agent according to any of the preceding claims, which is immediately usable in a clinical setting, such as for in vivo imaging, diagnosis and/or treatment of a subject.

4. The ultrasound contrast agent according to any one of the preceding claims, wherein the buffer is selected from Tris (hydroxymethyl) aminomethane (Tris), sodium phosphate, ammonium chloride, diethanolamine, glycine, triethanolamine and sodium carbonate.

5. The ultrasound contrast agent according to any one of the preceding claims, wherein the buffer is at a concentration of about 1mM to about 10 mM.

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

7. The ultrasound contrast agent according to any of the preceding claims, wherein the ultrasound contrast agent further comprises a tonicity agent.

8. The ultrasound contrast agent according to any one of the preceding claims, wherein the ultrasound contrast agent further comprises a viscosity agent and/or a flotation reducing agent.

9. The ultrasound contrast agent according to any preceding claim, wherein the perfluorocarbon is selected from perfluorobutane, perfluoropropane and perfluoropentane.

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

11. A method of preparing an ultrasound contrast agent, the method comprising the steps of:

(ii) adjusting the size distribution of microbubbles in the aqueous dispersion to a median size in the range of 1 to 6 μm, preferably 2 to 5 μm;

(iii) optionally adding a tonicity agent to the aqueous dispersion;

(vi) the aqueous dispersion is dispensed into a vial, and the headspace of the vial is flushed with perfluorocarbon.

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

13. The method of any one of claims 11-12, wherein step (v) is performed before or after any of steps (ii), (iii), and (iv), provided that step (v) is performed after step (i) and before step (vi).

14. The method according to any one of claims 11-13, wherein the ultrasound contrast agent is for long term storage and/or ready for use in a clinical setting, such as for in vivo imaging, diagnosis and/or treatment of a subject.

15. The method of any one of claims 11-14, which does not comprise a lyophilization step.

16. An ultrasound contrast agent prepared according to the method as defined in any one of claims 11-15.

17. A method of improving the contrast of an ultrasound image of tissue in a subject, the method comprising injecting into the subject an ultrasound contrast agent according to any of claims 1-10 or 16 and performing an ultrasound scan of the tissue.

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

19. A method of diagnosing a subject, the method comprising injecting into the subject an ultrasound contrast agent according to any one of claims 1-10 or 16, performing an ultrasound scan of a target region in the subject, generating an image of the target region, and evaluating the image to make a diagnosis.

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

21. Use of an ultrasound contrast agent according to any of claims 1-10 or 16 for the manufacture of a medicament for use in a method according to any of claims 17-19.