EA042049B1 - METHODS AND DEVICES FOR OBTAINING CONTRAST MEDIA FOR ULTRASONIC EXAMINATION - Google Patents

Description

Related application

This application claims priority under section 35 U.S.C. §119(e). US Provisional Applications Serial 62/331968, filed May 4, 2016, and Serial 62/332462, filed May 5, 2016, both titled Methods and Apparatus for Producing Ultrasound Contrast Agents, Complete the contents of each of which are incorporated herein by reference.

Prior Art

Contrast-enhanced ultrasound imaging is an established medical imaging technique. Most, if not all, ultrasound contrast agents (UCAs) are gas-filled microspheres that are suitable for amplifying the ultrasound signal. One such UCA is activated DEFINITY® containing perflutren lipid microspheres (ie gaseous perflutren encapsulated in lipid microspheres). The DEFINITY® formulation is packaged in a vial containing lipids in an aqueous suspension with perflutren gas in the headspace. Before use, DEFINITY® is activated by vigorous shaking of the vial, which forms lipid microspheres containing gaseous perflutren suspended in an aqueous liquid. Proper activation ensures that the formed microspheres are of the appropriate size and concentration to be both diagnostically effective and safe for the subject. Given the importance of proper size and concentration, activation should ideally be performed in such a way as to minimize the possibility of human error.

The essence of the invention

The present invention contemplates methods and devices for ensuring that activation-dependent UCA formulations, such as, without limitation, DEFINITY® formulation, are properly distinguished from one another and thus are properly activated. As additional activation-dependent UCA formulations enter the market, it will be imperative to distinguish between them to ensure that each is handled and activated in the correct intended manner. By way of example, each activation-dependent UCA will be characterized by its own unique activation parameters, including, for example, activation time and/or rate of activation (e.g., intensity of shaking), and thus it will be imperative that with each UCA formulation manipulated in a particular way. The use of incorrect activation parameters for the UCA formulation may result in UCA being unsuitable for diagnosis (eg, due to very low concentration or inappropriate microsphere size), requiring the subject to undergo an ultrasound procedure again. In the worst case, this can lead to the formation of microspheres that are too large, and this increases the risk of ischemia by blocking the capillaries.

One such new and improved UCA formulation is a non-aqueous UCA formulation, referred to herein as DEFINITY-II. This UCA formulation is surprisingly more stable than prior liquid UCA formulations. Specifically, unlike prior liquid UCA formulations that must be refrigerated prior to use, this new non-aqueous UCA formulation is stable at room temperature for extended periods of time. Even more surprising is that a given UCA formulation can be prepared and used without complicated manipulations. Given these added benefits, this new non-aqueous UCA formulation is expected to be easily approved. However, the formulation activates over a different time period than the DEFINITY® formulation, and therefore it is important to ensure that each UCA formulation activates within its own specific optimal time period. Activation for a different period of time may result in the formation of microspheres that are too large or too small, and/or at such a low concentration as to be clinically unusable. Given the significant implications if UCA is improperly prepared (e.g., activated inappropriately), it is important to have methods and devices that identify specific UCA formulations, such as an aqueous DEFINITY® formulation and a non-aqueous DEFINITY-II formulation, and optionally distinguish between them. , and apply the correct activation parameters of such UCA formulations, preferably with minimal reliance on human intervention.

Thus, in one aspect, the present invention provides a method for forming gas-filled microspheres, comprising identifying a UCA composition and causing the UCA composition to be activated for a predetermined (e.g., predetermined) period of time using a device that is configured to select between two or more predetermined periods of time, with the formation of gas-filled microspheres. The device is configured to automatically select between two or more predetermined time periods. In some embodiments, the tool may also identify the UCA formulation and/or its container (e.g., container,

- 1 042049 such as a vial) and it is not necessary to distinguish between such a UCA formulation and/or container and one or more other UCA formulations or containers.

In another aspect, the present invention provides a method for forming gas-filled microspheres, comprising identifying a UCA composition and causing the UCA composition to be activated using a predetermined shaking intensity value using a device that is configured to select between two or more predetermined shaking intensity values, to form gas-filled microspheres.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising providing activation of a UCA composition to form gas-filled microspheres using a tool (e.g., device) that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition (and/or vice versa). An aqueous UCA formulation can be distinguished from a non-aqueous UCA formulation (or vice versa) based on the type of container, including its shape or size, in which such UCA formulations are stored.

In some embodiments of any of the above aspects, the fixture (eg, device) comprises a detector. In some embodiments of any of the above aspects and embodiments, the device (eg, device) is configured to provide activation for a predetermined time period selected from two or more predetermined time periods.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a UCA composition that requires activation for a predetermined period of time, using a device (for example, a device) that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition, and allowing the UCA formulation to activate for a predetermined period of time to form gas-filled microspheres.

In some embodiments of any of the above aspects and embodiments, the UCA formulation is an aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the UCA formulation is a non-aqueous UCA formulation.

In some embodiments of any of the above aspects and embodiments, the predetermined time period is a shorter time period in case the UCA formulation is an aqueous UCA formulation and a longer time period in the case the UCA formulation is a non-aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the predetermined time period is about 45 seconds if the UCA formulation is an aqueous UCA formulation and 60-120 seconds or about 75 seconds if the UCA formulation is a non-aqueous UCA formulation. UCA.

In some embodiments of any of the above aspects and embodiments, the device comprises a first holder configured to hold a vial containing an aqueous UCA formulation and not able to hold a vial containing a non-aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the device comprises a first holder configured to hold a vial containing a non-aqueous UCA formulation and not able to hold a vial containing an aqueous UCA formulation.

In some embodiments of any of the above aspects and embodiments, the device is configured to distinguish between an aqueous UCA formulation and a non-aqueous UCA formulation based on a unique identifier.

In some embodiments of any of the above aspects and embodiments, the device comprises a detector. In some embodiments, the detector is an RFID reader and the UCA is in a container that contains (includes) or is associated with or labeled with an RFID tag/label. In some embodiments, the detector is a barcode scanner and the UCA is in a container that contains (includes) or is associated with or labeled with a barcode. In some embodiments, the detector is a color scanner and the UCA formulation is in a container that contains a colored label.

In some embodiments of any of the above aspects and embodiments, the device reciprocates the vial containing the UCA formulation.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a labeled vial containing a UCA formulation that requires activation for a predetermined period of time, and providing activation of the UCA formulation using a shaking device containing a detector and pre-installed a certain period of time or the possibility of automatic

- 2 042049 the first implementation of the selection of a predetermined period of time based on the identification information about the bottle with the formation of gas-filled microspheres.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a labeled vial containing a UCA formulation that requires activation for a predetermined period of time, using a shaker device containing a detector, and which is set to a predetermined period of time or capability. automatically making a selection of a predetermined time period based on vial identification information; and allowing the UCA formulation to be activated to form gas-filled microspheres.

In some embodiments of any of the above aspects and embodiments, the labeled vial is labeled with a unique identifier.

In some embodiments of any of the above aspects and embodiments, the predetermined time period is approximately 45 seconds.

In some embodiments of any of the above aspects and embodiments, the detector is an RFID reader and the tagged vial contains an RFID tag/label. In some embodiments of any of the above aspects and embodiments, the detector is a barcode scanner and the labeled vial contains a barcode. In some embodiments of any of the above aspects and embodiments, the detector is a color scanner and the labeled vial contains a colored label.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising providing activation of the UCA composition for a predetermined period of time to form gas-filled microspheres, where the UCA composition is activated using a shaking device, which is parameterized to provide activation for at least at least two different predetermined time periods, or with the possibility of automatic selection from at least two different predetermined time periods based on the identification information about the composition of the UCA.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a UCA composition that requires activation for a predetermined period of time, and providing activation of the UCA composition for a predetermined period of time to form gas-filled microspheres, where the UCA composition is activated with using a shaking device, which is parameterized to provide activation for at least two different predefined time periods or with the ability to automatically select from at least two different predefined time periods based on identification information about the composition of the UCA.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a non-aqueous UCA composition that requires activation for a predetermined period of time, and causing the UCA composition to be activated for a predetermined period of time to form gas-filled microspheres.

In some embodiments of any of the above aspects and embodiments, the UCA composition is identified and activated using a shaking device that is parameterized to provide activation for a predetermined period of time or with the ability to automatically select a predetermined period of time based on identification information about part of the UCA.

In some embodiments of any of the above aspects and embodiments, the predetermined time period is approximately 45 seconds. In some embodiments of any of the above aspects and embodiments, the predetermined time period is in the range of 60-120 seconds, or about 75 seconds.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a non-aqueous UCA composition that requires activation for a predetermined first period of time, and causing said UCA composition to be activated to form gas-filled microspheres by agitation using a shaking device configured with the ability to automatically select a first predetermined time period from at least two different predetermined time periods based on the identification information about the composition of the UCA.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a non-aqueous UCA formulation that requires activation for a predetermined first period of time, based on its container, and causing said UCA formulation to be activated to form gas-filled microspheres by shaking using a shaking device configured to automatically select a first predetermined period of time from at least

- 3 042049 two different predetermined time periods based on container identification information. In some embodiments, the container is a vial.

In some embodiments of any of the above aspects and embodiments, at least two different predefined time periods are about 45 seconds and about 75 seconds.

In some embodiments of any of the above aspects and embodiments, a device, such as a shaker, reciprocates the container (eg, vial) when it is in the holder.

In some embodiments of any of the above aspects and embodiments, the shaking device includes a detector. The detector may be an RFID reader and the vial may contain, include, be associated with, or be labeled with an RFID tag/label. The detector may be a barcode scanner and the vial may contain, include, be associated with, or be labeled with a barcode. The detector may be a color scanner and the vial may contain, include, be associated with, or be labeled with a color indicator. The color indicator may include a color label. The color indicator may include a color cap.

In another aspect, the present invention provides a method for forming gas-filled microspheres, comprising providing activation of a UCA composition using a shaking device that is configured to identify the UCA composition and automatically select an activation time based on information about it, where the UCA composition is identified based on a unique identifier other than the shape or size of the vial in which the UCA formulation is stored.

In another aspect, the present invention provides a method for activating a first UCA formulation using a shaking device that is configured to distinguish between a container, such as a vial containing a first UCA formulation, and a container, such as a vial containing a second UCA formulation.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a labeled vial containing a UCA formulation that requires activation for a predetermined first period of time, and providing activation of the UCA formulation using a shaking device that has been set to a predetermined period of time. time or the ability to automatically select a first predetermined time period from at least two different predetermined time periods based on vial identification information to form gas-filled microspheres.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a labeled vial containing a UCA formulation that requires activation for a predetermined first period of time, and providing activation of the UCA formulation using a shaking device containing a detector and in which the a predetermined period of time or the ability to automatically select a first predetermined period of time from at least two different predetermined periods of time based on identification information about the vial, with the formation of gas-filled microspheres.

In another aspect, the present invention provides a method for forming gas-filled microspheres, comprising identifying a vial containing a UCA formulation that requires activation for a predetermined first period of time, and providing activation of the UCA formulation using a shaking device containing a detector and pre-installed a predetermined period of time or the possibility of automatically selecting a first predetermined period of time from at least two different predetermined periods of time based on the vial identification information to form gas-filled microspheres.

In some embodiments of any of the above aspects and embodiments, at least two different predefined time periods are about 45 seconds and about 75 seconds.

In some embodiments of any of the above aspects and embodiments, substantially similar gas-filled microspheres are produced by the method in the first vial and the second vial, provided that the first vial is shaken for a first time period and the second vial is shaken for a second different time period.

In another aspect, the present invention provides a method for forming gas-filled microspheres, comprising identifying an aqueous UCA composition that requires activation for a predetermined period of time, using a device that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition, and providing activation of the aqueous composition of UCA for a predetermined period of time with the formation of gas-filled microspheres.

- 4 042049

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a UCA composition that requires activation for a predetermined period of time, and providing activation of the UCA composition for a predetermined period of time to form gas-filled microspheres, where the UCA composition is identified and activated using a shaking device configured to automatically select a predetermined time period from at least 2 predetermined time periods based on identification information about the composition of the UCA.

In some embodiments of any of the above aspects and embodiments, the UCA formulation is an aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the UCA formulation is a non-aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the aqueous UCA formulation is activated over a shorter period of time than that of the non-aqueous UCA formulation. In some embodiments of any of the above aspects and embodiments, the predetermined time period is approximately 45 seconds. In some embodiments of any of the above aspects and embodiments, the predetermined time period is approximately 75 seconds.

In another aspect, the present invention provides a method of forming gas-filled microspheres, comprising identifying a vial containing an ultrasound contrast agent formulation that requires activation for a predetermined first period of time, using a shaking device configured to select a first period of time from two predefined time periods based on vial identification information.

In some embodiments of any of the above aspects and embodiments, the method is automated.

In some embodiments of any of the above aspects and embodiments, the two predetermined time periods are about 45 seconds and about 75 seconds.

In some embodiments of any of the above aspects and embodiments, substantially similar gas-filled microspheres are produced by the method in the first vial and the second vial, provided that the first vial is shaken for a first time period and the second vial is shaken for a second different time period.

In another aspect, the present invention provides a method for imaging a subject, comprising administering to a subject in need thereof gas-filled microspheres obtained according to any one of the preceding claims and obtaining one or more images of the subject using ultrasound.

In another aspect, the present invention provides an apparatus capable of being used to form gas-filled microspheres according to any of the above methods. In some embodiments, the device further comprises a counter that counts the number of times the device has been used, the number of times the device has been shaken during the first time period, and/or the number of times the device has been shaken during the second time period.

In another aspect, the present invention provides a device that provides activation of a UCA formulation and that is capable of distinguishing between an aqueous UCA formulation and a non-aqueous UCA formulation. In some embodiments, the device provides activation of the aqueous UCA formulation for a shorter period of time than that of the non-aqueous UCA formulation.

In another aspect, the present invention provides a shaking device comprising a holder, a holder shaking device, wherein the holder shakes the vial containing the UCA formulation for various predetermined periods of time.

In some embodiments, the implementation of the device further comprises a device for automatically identifying a predetermined period of time during which the vial must be shaken to form gas-filled microspheres.

In some embodiments, the shaking device reciprocates the vial while it is in the holder.

In some embodiments, the first predetermined time period is approximately 45 seconds. In some embodiments, the second predetermined time period is approximately 75 seconds.

In some embodiments, the vial identification tool provides an RFID reader that responds to a first RFID tag by shaking the vial for a first time period, and that responds to a second RFID tag by shaking the vial for a second time period, where the first and second periods times are different. In some embodiments, the vial identification tool provides a

- 5 042049 microchip dispenser which responds to the first microchip by shaking the vial for a first period of time and which responds to the second microchip by shaking the vial for a second period of time. In some embodiments, the vial identification tool includes a barcode scanner that responds to a first barcode by shaking the vial for a first period of time and that responds to a second barcode by shaking the vial for a second period of time. In some embodiments, the implementation of the RFID label, microchip or barcode is on the vial.

In some embodiments, the UCA formulation is a non-aqueous UCA formulation.

In another aspect, the present invention provides a shaking device for forming gas-filled microspheres, comprising an identification device configured to identify and distinguish between a first vial and a second vial, each vial containing a UCA formulation, and an automated shaking device configured to perform shaking for only one of at least two different predetermined time periods based on vial identification information.

In some embodiments, at least two different predefined time periods are about 45 seconds and about 75 seconds.

In some embodiments, the identification tool includes an RFID reader, a microchip reader, or a barcode scanner.

In another aspect, the present invention provides an agitator for forming gas-filled microspheres, comprising a holder configured to identify and distinguish between a first vial and a second vial, each vial containing a UCA formulation, and an automated device for shaking the vial in the holder for one of two predetermined time periods based on whether the first vial or the second vial was identified.

In some embodiments of any of the above aspects and embodiments, the shaking device reciprocates the vial when it is in the holder.

In some embodiments, the first predetermined time period is approximately 45 seconds. In some embodiments, the second predetermined time period is approximately 75 seconds.

In some embodiments, the implementation of the holder contains an RFID reader.

In some embodiments, the carrier accepts a first configuration if a first vial is present and a second configuration if a second vial is present, wherein the first configuration indicates the presence of the first vial and the second configuration indicates the presence of the second vial.

In some embodiments, the device further comprises a counter that counts the number of times the shaker is applied, the number of times the shaker shakes during the first time period, and/or the number of times the shaker shakes during the second time period.

In another aspect, the present invention provides a shaking device comprising a holder capable of identifying a vial containing a UCA formulation, and a means for shaking the holder, wherein the holder is configured to shake only for a predetermined period of time to form gas-filled microspheres based on the identification. vial information.

In another aspect, the present invention provides a shaking device comprising a holder, a device for shaking the holder, wherein the holder is configured to perform shaking for only a predetermined period of time, and a device for identifying a vial containing a UCA formulation when it is in the holder, and then shaking the identified vial for a predetermined period of time to form gas-filled microspheres, the vial identification device comprising an RFID reader, a microchip reader, or a barcode scanner.

In another aspect, the present invention provides a kit containing any of the above shaking devices with instructions regarding the activation of the UCA composition. In some embodiments, the kit further comprises a container, such as a vial, containing the UCA formulation. In some embodiments, the UCA formulation is a non-aqueous UCA formulation. In some embodiments, the kit further comprises a first vial or a second vial, each vial containing a UCA formulation.

In another aspect, the present invention provides a persistent computer-readable medium programmed with a plurality of instructions that, when executed by at least one shaker processor, performs a method, the method comprising determining based at least in part on type identification information about

- 6 042049 samples in a vial containing the composition of UCA, inserted into the holder of the shaking device, at least one performed action and issuing a command to the shaking device to perform a certain at least one action based, at least in part, on identification information.

In another aspect, the present invention provides a shaking device comprising a holder configured to identify the type of sample in a vial containing a UCA formulation inserted into the holder;

at least one storage device configured to store at least one data structure that provides identification of one or more actions performed in relation to each of the plurality of sample types;

at least one processor programmed to access at least one data structure to determine one or more actions to be performed on the vial based on the identified sample type; and at least one component configured to perform one or more actions defined by at least one processor.

These and other aspects and embodiments of the present invention will be described in more detail in this document.

Brief description of the drawings

Various embodiments will now be described by way of example with reference to the accompanying drawings, in which FIG. 1 is a schematic representation of a sample handling device and a sample vial in one aspect;

fig. 2 is a schematic representation of one embodiment of a sample handling device and a sample vial;

fig. 3 is a schematic diagram of a second embodiment of a sample handling device and a sample vial;

fig. 4 is a schematic diagram of a third embodiment of a sample handling device and a sample vial;

fig. 5 is a flowchart of a method for determining the action(s) to be performed on a sample vial based on its identification information, in one aspect;

fig. 6 is an example of the method described in FIG. 5, in which the vial is identified based on the RFID tag associated with the vial; and fig. 7 is a schematic diagram of a computing system that may be included as part of a sample vial handling apparatus, in accordance with one aspect.

Detailed description of the invention

This document provides new and improved UCA formulations that have one or more advantages over previously developed UCA formulations. One such improved UCA formulation is a non-aqueous UCA formulation containing lipids and perfluorocarbon gas in a non-aqueous solution. Another such improved UCA formulation is an aqueous UCA formulation containing lipids and gaseous perfluorocarbon in an aqueous solution. Each of these UCA formulations provides specific advantages over existing UCA formulations, including, for example, stability at elevated temperatures (eg, room temperature) or improved safety profiles.

However, some of these new formulations have specific activation requirements, some of which differ substantially from the activation requirements for existing formulations. For example, it has been found that the non-aqueous UCA formulations described herein must be activated for longer periods of time than the current UCA formulation, despite the fact that both can be activated using the same device. Ensuring that each UCA compound is activated within its specific required time can be a challenge.

Provided herein are methods and devices (eg, devices) for facilitating the proper and accurate production of activation-dependent UCAs. These methods and devices reduce the risk of improper production of such UCA. UCAs that are obtained inappropriately and inaccurately contain a very small number of gas-filled microspheres, thereby reducing the signal received from such UCAs at best. In the worst case, UCAs that are obtained inappropriately and inaccurately can cause tissue ischemia by clogging the capillaries and even death of the patient. Thus, it is imperative that activation-dependent UCAs be manipulated and appropriately obtained. The present invention provides methods and devices, including devices, that facilitate accurate production of activation-dependent UCAs. Unless otherwise indicated, the UCAs of the present invention are an activation-dependent UCA and thus the terms UCA and

- 7 042049 dependent on UCA activation are used interchangeably.

In view of the development of new and improved non-aqueous UCA formulations that require different activation times or in some cases different activation rates (or shaking rates), a reliable, reliable and error-free system for distinguishing between products is required. The methods and devices (eg, devices) provided herein are unique in distinguishing between different activation-dependent formulations of UCA. As will be described in more detail below, each activation-dependent UCA compound will be characterized by its own specific activation criteria (or parameters) and, therefore, each such UCA compound should only be activated in a certain way. The methods and devices (eg, devices) provided herein generally identify and thus distinguish between an activation-dependent UCA formulation and other activation-dependent UCA formulations, and cause the identified UCA formulation to be activated appropriately. This ensures that activation dependent UCA formulations are activated for their specific predetermined and provided time periods, in some cases using specific shaking parameters. In some embodiments, the methods are performed and the devices (eg, devices) are operated in a relatively self-contained manner such that there is little risk of end user error during the activation process.

The FDA-approved activation-dependent formulation of UCA is DEFINITY®. As described in more detail below, DEFINITY® is provided in a vial as an aqueous lipid suspension with gaseous perflutren in the headspace. When activated for its prescribed time period of 45 seconds using VIALMIX® (or VIALMIX® device, as the terms are used interchangeably), activated DEFINITY® contains no more than 1.2x101° perflutren lipid microspheres per ml of suspension. Activation at inappropriate shaking time or speed will affect the microsphere profile and render the UCA sub-optimal or unsuitable in some cases. With the creation of at least one additional activation-dependent UCA formulation in the form of a non-aqueous activation-dependent UCA formulation described below, it is important to ensure that there is no confusion between the various activation-dependent UCA formulations and that each is handled and activated appropriately, considering adverse consequences that might otherwise arise.

Also provided herein are improved devices (eg, devices) to provide activation of the UCA formulation. As an example, certain advanced devices may contain counters that can monitor device usage, including device life, which can be useful in avoiding equipment failure at critical times. They may also contain temperature sensors that can measure the temperature of the container prior to activation. As described in more detail herein, some of these devices may also provide for the activation of more than one UCA formulation and therefore may identify and optionally distinguish between two or more UCA formulations. With regard to the latter, the device may automatically recognize a container containing a UCA formulation and, based on such identification information, which may be provided, for example, by the label, the shape, color or size of the container, or the optical properties of its contents, may cause the UCA formulation to be activated within a predetermined time period, which in turn can be selected from two or more different predefined time periods.

The device can perform such recognition without user intervention or with minimal user involvement.

Dependent on UCA activation.

As used herein, UCA refers to gas-filled microspheres that are suitable for amplifying an ultrasonic signal. In most cases, UCA is provided in a solution, such as a pharmaceutically acceptable solution. Depending on the concentration of microspheres in UCA, it may be diluted with a pharmaceutically acceptable carrier prior to administration to a subject, although this may not be required in some cases.

Activation-dependent UCA composition, as used herein, refers to a composition that must be activated to form gas-filled microspheres. The UCA formulation typically does not contain such gas-filled microspheres (or such a low concentration as to be clinically unsuitable) and must be activated to form microspheres of sufficient diameter and concentration to be clinically useful.

Activation-dependent UCA formulations typically require vigorous shaking prior to use to form gas-filled microspheres. Such activation is performed by the end user or reseller, not by the supplier or manufacturer of the UCA formulation. Activation-dependent UCA formulations are typically packaged in vials that contain, at a minimum, a lipid solution and a gas.

- 8 042049

Shaking the lipid and gas solution results in the formation of gas-filled microspheres that act as a contrast agent in the ultrasound imaging procedure.

Unless otherwise indicated, the UCA formulations of the present invention are activation dependent UCA formulations and thus the terms UCA formulation and activation dependent UCA formulation are used interchangeably.

By gas-filled, as used herein, is meant that the microspheres contain a gas, such as gaseous perfluorocarbon, including, but not limited to, gaseous perflutrene, in their internal cavity. The lipid envelope that encapsulates the gas can be arranged as a monolayer or bilayer, including single-lamellar or multi-lamellar bilayer structures. The microspheres may have an average diameter of less than 10 microns, or less than 6 microns, or less than 3 microns, or more preferably less than 2 microns. By these mean diameters, it is meant that when analyzing a population of microspheres, the average diameter of the population is less than 10 microns, or less than 6 microns, or less than 3 microns, or more preferably less than 2 microns. The microspheres may have an average diameter ranging from 0.5 to 3 microns, or from 1 to 2 microns, or from 1.4 to 1.8 microns, or from 1.4 to 1.6 microns. The average diameter may be approximately 1.6 microns.

Prior to use, the activation-dependent UCA formulation must be shaken vigorously to form gas-filled microspheres. In some cases, the microspheres may be combined with, for example, an aqueous solution before being removed from their container. This applies in particular to microspheres made from non-aqueous UCA formulations. In the context of the present invention, this step is called resuspension. In some instances, the microspheres, whether resuspended or not, can be removed from their container and combined in another solution, such as an aqueous solution, prior to administration to a subject. In the context of the present invention, this step is called dilution. The resuspended microsphere pool may be used neat or after dilution in a pharmaceutically acceptable solution. Such a dilution may be from about 10-fold to about 50-fold, although it is not limited to these values.

As used herein, gas-filled microspheres and gas-encapsulated lipid microspheres are used interchangeably.

UCA formulations contain at least one or more types of lipids and a gas, such as gaseous perfluorocarbon, such as gaseous perflutren. As described in more detail herein, UCA formulations include aqueous UCA formulations such as DEFINITY® and non-aqueous UCA formulations such as DEFINITY-II. DEFINITY® contains DPPA, DPPC and MPEG5000-DPPE lipids, propylene glycol and glycerin in an aqueous solution along with perflutrene gas. In contrast, DEFINITY-II contains DPPA, DPPC, and MPEG5000-DPPE lipids, as well as propylene glycol and glycerin, along with gaseous perfluorocarbon (eg, gaseous perflutrene).

DEFINITY.

DEFINITY® is an example of an aqueous formulation of UCA. Activated DEFINITY® is FDA approved for use in subjects with suboptimal echocardiograms, which is designed to opaque the left ventricular chamber and improve the delineation of the left ventricular endocardial border. DEFINITY® is provided in a vial containing a single phase solution containing DPPA, DPPC and MPEG5000-DPPE at a ratio of 10:82:8 mole % in an aqueous solution with perfluoropropane gas headspace. Prior to its administration to a subject, DEFINITY® is activated by vigorous shaking, such as vigorous mechanical shaking, and is thereafter referred to as activated DEFINITY®. Activation results in the formation of a sufficient number of gas-encapsulated lipid microspheres having an average diameter of 1.1 to 3.3 microns. DEFINITY®, however, must be refrigerated prior to direct use. This limits its practicality, in particular in situations in which adequate cooling is not available, in particular during a storage period.

In other aqueous UCA formulations, DPPA, DPPC and DPPE can be used at mole percentages of about 77-90 mole% DPPC, about 5-15 mole% DPPA and about 5-15 mole% DPPE, including DPPE-MPEG5000 . Preferred ratios of each lipid include a wt% ratio of 6.0 to 53.5 to 40.5 (DPPA:DPPC:MPEG5000-DPPE) or a mole% ratio of 10 to 82 to 8 (10 :82:8) (DPPA:DPPC:MPEG5000-DPPE).

DEFINITY-II and other non-aqueous UCA formulations.

This document contemplates a range of non-aqueous UCA formulations. Some such formulations contain a non-aqueous mixture of one or more lipids and propylene glycol (PG) or glycerol (G) or propylene glycol and glycerol (PG/G). These formulations can be stored at higher temperatures (eg, room temperature) for longer periods of time than previously thought possible without significant degradation. Non-aqueous UCA formulations, such as DEFINITY-II, may contain less than 10%, less than 5%, or less than 2% impurities when stored at room temperature for a period of time, including, for example, approximately

- 9 042049 months, about 2 months, about 3 months, about 6 months or longer, including about 1 year or about 2 years. It is important to note that non-aqueous UCA formulations may contain less impurities than DEFINITY® when both formulations are stored at room temperature (ie, when non-aqueous UCA and DEFINITY® are stored at room temperature). This impurity reduction may be a difference of about 1%, about 2%, about 3%, about 4%, or about 5% or more.

The non-aqueous mixture of lipids in propylene glycol or glycerin or propylene glycol and glycerin may be a mixture containing water in an amount less than or equal to 5% by weight (i.e. the ratio of the weight of water to the weight of the combination of lipids and propylene glycol and/or glycerin). In some cases, the non-aqueous mixture contains less than 5% water (w/w), 1-4% water (w/w), 1-3% water (w/w), 2-3% water (w/w), or 1 -2% water (w/w). In some cases, the non-aqueous mixture contains less than 1% water (w/w). The water content can be measured at the end of manufacture (and before long-term storage), or it can be measured after storage, including long-term storage, and just before use.

The non-aqueous mixture may also be free of salts, which means that it does not contain any salts other than lipid counterions. More specifically and by way of example, lipids such as DPPA and DPPE are typically provided as sodium salts. As used herein, a salt-free non-aqueous mixture may contain such counterions (eg, sodium if DPPA and/or DPPE are used), but they do not contain other ions. In some cases, the non-aqueous mixture does not contain sodium chloride or chloride.

The non-aqueous mixture may contain a buffer. The buffer may be an acetate buffer, a benzoate buffer, or a salicylate buffer, although it is not limited thereto. Buffers other than phosphate are preferred in some cases due to their dissolution profiles in non-aqueous mixtures provided herein. In some cases, a phosphate buffer may be used (eg, after or simultaneously with the addition of an aqueous diluent, such as during the resuspension or dilution step, as discussed previously).

In some embodiments, the non-aqueous mixture contains (a) one or more lipids;

(b) propylene glycol or glycerin or propylene glycol/glycerin; and (c) the buffer, other than phosphate, consists of them or consists mainly of them.

Such non-aqueous mixtures may be provided with a gas, such as gaseous perfluorocarbon, or they may be provided separately (ie, in the absence of gas). Such non-aqueous mixtures may be provided in single use amounts and/or in single use containers with or without gas. Such containers will typically be sterile.

The buffer other than phosphate may be, but is not limited to, acetate buffer, benzoate buffer, salicylate buffer, diethanolamine buffer, triethanolamine buffer, borate buffer, carbonate buffer, glutamate buffer, succinate buffer, malate buffer, tartrate buffer, glutarate buffer, aconite buffer, citrate buffer, lactate buffer, glycerate buffer, gluconate buffer and tris buffer. Determining and optimizing the buffer concentration for each type of buffer is within the skill of the average person skilled in the art.

DPPA, DPPC and DPPE can be used at mole percentages of about 77-90 mole% DPPC, about 5-15 mole% DPPA and about 5-15 mole% DPPE, including DPPE-PEG5000. Preferred ratios of each lipid include a wt% ratio of 6.0 to 53.5 to 40.5 (DPPA:DPPC:MPEG5000-DPPE) or a mole% ratio of 10 to 82 to 8 (10 :82:8) (DPPA:DPPC:MPEG5000-DPPE).

In some cases, the lipid concentration may range from about 0.1 mg to about 20 mg per ml of non-aqueous mixture, including from about 0.9 mg to about 10 mg per ml of non-aqueous mixture and from about 0.9 mg to about 7.5 mg per ml non-aqueous mixture. In some embodiments, the lipid concentration may range from about 0.94 mg to about 7.5 mg lipid per ml of non-aqueous mixture, including from about 1.875 mg to about 7.5 mg lipid per ml of non-aqueous mixture, or from about 3 .75 mg to about 7.5 mg lipid per ml non-aqueous mixture. In some cases, the lipid concentration is from about 0.94 mg to about 1.875 mg per ml of a non-aqueous mixture, from about 1.875 mg to about 3.75 mg per ml of a non-aqueous mixture, or from about 3.75 mg to about 7.5 mg of total lipid. per ml of non-aqueous mixture.

By way of example, the lipid concentration may range from about 0.1 mg to about 10 mg lipid per ml propylene glycol/glycerol (combined), including from about 1 mg to about 5 mg lipid per ml propylene glycol/glycerol (combined). In some cases, the lipid concentration is from about 0.94 mg to about 3.75 mg of lipid per ml of propylene glycol/glycerol (combined).

- 10 042049

As another example, the lipid concentration may range from about 0.1 mg to about 20 mg of lipid per ml of propylene glycol, including from about 1 mg to about 10 mg of lipid per ml of propylene glycol, or from about 2 mg to about 7, 5 mg lipid per ml propylene glycol, or about 3.75 mg to about 7.5 mg lipid per ml propylene glycol. In some embodiments, the lipid concentration is from about 1.875 mg to about 7.5 mg of lipid per ml of propylene glycol, including from about 3.75 mg to about 7.5 mg of lipid per ml of propylene glycol.

As another example, the lipid concentration may range from about 0.1 mg to about 20 mg lipid per ml of glycerol, including from about 1 mg to about 10 mg of lipid per ml of glycerol, or from about 2 mg to about 7 .5 mg of lipid per ml of glycerol, or from about 3.75 mg to about 7.5 mg of lipid per ml of glycerol. In some cases, the lipid concentration is from about 1.875 mg to about 7.5 mg of lipid per ml of glycerol, including from about 3.75 mg to about 7.5 mg of lipid per ml of glycerol.

DEFINITY-II contains lipids DPPA, DPPC and MPEG5000-DPPE in a 10:82:8 (10:82:8) mole % ratio and a total lipid content of 3.75 mg/ml, and propylene glycol ( 517.5 mg/ml), glycerin (631 mg/ml), sodium acetate (0.370 mg/ml), acetic acid (0.030 mg/ml) together with gaseous perfluoropropane (perflutren) (6.52 mg/ml) in the free above product space.

The microspheres can be resuspended or diluted in an aqueous diluent, and such an aqueous diluent may contain salts, such as but not limited to sodium chloride, and thus may be considered a saline solution. The aqueous diluent may contain a buffer, such as a phosphate buffer, and thus may be considered a buffered aqueous diluent. The aqueous diluent may be buffered saline. The non-aqueous mixture may contain a buffer, such as a buffer other than phosphate, examples of which are provided herein. Both the non-aqueous mixture and the aqueous diluent may contain a buffer. In typical embodiments, the buffer contains either a non-aqueous mixture or an aqueous diluent, but not both. The buffer concentration will vary depending on the type of buffer used, as will be appreciated and determined by one of ordinary skill in the art. The buffer concentration in the non-aqueous lipid formulation may range from about 1 mM to about 100 mM. In some instances, the buffer concentration may be from about 1 mM to about 50 mM, or from about 1 mM to about 20 mM, or from about 1 mM to about 10 mM, or from about 1 mM to about 5 mM, including about 5 mm.

The final composition to be administered, usually intravenously, to a subject, including a human subject, may have a pH value in the range of 4-8 or in the range of 4.5-7.5. In some cases, the pH value may be in the range from about 6 to about 7.5, or in the range from 6.2 to about 6.8. In yet other cases, the pH value may be approximately 6.5 (for example, 6.5±0.5 or ±0.3). In some cases, the pH value may be in the range of 5 to 6.5, or in the range of 5.2 to 6.3, or in the range of 5.5 to 6.1, or in the range of 5.6 to 6, or in the range of 5.65 to 5.95. In another case, the pH value may be in the range from about 5.7 to about 5.9 (for example, ±0.1, or ±0.2, or ±0.3 from either or both ends of the range). Alternatively, the pH may be approximately 5.8 (eg 5.8±0.15 or 5.8±0.1).

In some embodiments, the aqueous diluent comprises glycerol, a buffer such as phosphate buffer, salt(s), and water. Such an aqueous diluent can be used with a non-aqueous mixture that does not contain glycerin. In some embodiments, the lipid solution further comprises saline (salt(s) and water combined) and glycerol in a weight ratio of 8:1.

In some embodiments, the aqueous diluent comprises propylene glycol, a buffer such as phosphate buffer, salt(s), and water. Such an aqueous diluent can be used with a non-aqueous mixture that does not contain propylene glycol.

In some embodiments, the implementation of the aqueous diluent contains a buffer, such as phosphate buffer, salt(s) and water. Such an aqueous diluent can be used with a non-aqueous mixture that contains both propylene glycol and glycerin.

The microspheres can be resuspended and applied directly (as is), or they can be resuspended and diluted. Resuspension and dilution involve combining the microspheres with an aqueous solution, such as a pharmaceutically acceptable solution. Either or both of the two steps can provide microsphere concentrations of at least 1 x 10 ⁷ microspheres per ml of solution, or at least 5 x 10 ⁷ microspheres per ml of solution, or at least 7.5 x 10 ⁷ microspheres per ml of solution, or at least 1x10 ⁸ microspheres per ml of solution, or at least 1x10 ⁹ microspheres per ml of solution, or about 5x10 ⁹ microspheres per ml of solution. The range of values for the concentration of microspheres may in some cases be from 1x10 ⁷ to

- 11 042049

1x101° microspheres per ml solution and more typically 5x10 ⁷ to 5x1° ⁹ microspheres per ml solution. The resuspended set of microspheres can be further diluted by a factor of from about 1° times to about 5° times, but not limited to these values.

In some cases, activation of the non-aqueous UCA formulation followed by resuspension provides approximately 4-5x1° ⁹ microspheres per ml of solution, which can be diluted approximately 1° to give approximately 4-5x1° ⁸ microspheres per ml of solution.

DEFINITY-II is described in more detail in the PCT application, PCT/US2°15/°67615, the entire content of which is incorporated herein by reference.

DEFINITY-II is designed to be used in the same way as DEFINITY®. Thus, for example, DEFINITY-II can be used in subjects with suboptimal echocardiograms to opaque the left ventricular chamber and improve left ventricular endocardial margin delineation, among other imaging applications.

Other aqueous formulations of UCA.

Other aqueous UCA formulations are currently being developed. Compared to DEFINITY®, some of the new aqueous UCA formulations contain a lower volume of an aqueous lipid solution (ie, an aqueous solution containing lipids) and a higher headspace occupied by gas. Compared to DEFINITY®, other new aqueous formulations of UCA contain a lower concentration of lipids in an aqueous solution. Yet another aqueous formulation of UCA is provided in containers of different shapes and sizes (and thus volumes) compared to DEFINITY®. All of these new aqueous UCA formulations can be activated to produce gas-filled microspheres corresponding to those of activated DEFINITY®, including the mid-diameter profile, without compromising the acoustic properties of the microspheres. The ability to form gas-encapsulated lipid microspheres suitable for clinical use using significantly less lipid by reducing either the volume of the lipid solution or the concentration of the lipid is advantageous for a number of reasons, including reducing wastage of the material and the likelihood of overdose in the subject. The choice of container will allow the end user to select the most convenient shape and size (volume) for their desired application.

An example of one such novel aqueous UCA formulation, referred to herein as DEFINITY-III, contains the lipids DPPA, DPPC, and PEG5°°°-DPPE (where PEG5°°° includes, without limitation, hydroxyPEG5°°° or MPEG5°°°) in aqueous solution. together with gaseous perfluorocarbon (eg, gaseous perflutren) in a container, where the gaseous perfluorocarbon occupies approximately 6°-85% of the volume of the container. In contrast, DEFINITY® is provided in a container (ie vial) where gaseous perfluorocarbon (ie gaseous perflutrene) occupies approximately 54% of the volume of the container.

Another example of a novel aqueous formulation of UCA, herein referred to as DEFINITY-IV, contains an aqueous lipid solution containing from about °.1 mg to about °.6 mg of DPPA, DPPC and PEG5°°°-DPPE (combined) per ml of solution, and gaseous perfluorocarbon in a container.

These new aqueous formulations of UCA, including DEFINITY-III and DEFINITY-IV, are described in more detail in the PCT application, PCT/US2°14/°63267, the entire content of which is incorporated herein by reference.

Activation.

UCA formulations are shaken vigorously to form gas-filled microspheres which will typically be used as UCA. Such gas-filled microspheres may be formed directly, or they may be obtained through a process that includes the formation of microspheres and the incorporation of gas into such microspheres. Activation is usually performed by vigorous shaking of the container (eg vial) containing the UCA formulation. The UCA formulation contains at least lipids and gas, and thus activation at least results in the formation of gas-filled lipid microspheres. Lipids may be present in an aqueous solution, such as in the case of DEFINITY®, DEFINITY-III and DEFINITY-IV, or they may be present in a non-aqueous solution, such as in the case of new UCA formulations, including, for example, DEFINITY-II, described more details in this document. Thus, in some cases, activation involves shaking an aqueous solution containing lipid in the presence of a gas, such as gaseous perfluorocarbon (eg, perflutren). In other cases, activation involves shaking a non-aqueous lipid-containing solution in the presence of a gas, a gaseous perfluorocarbon (eg, perflutren). It should be understood that perflutren, gaseous perflutren and octafluoropropane are used interchangeably herein.

Agitation, as used herein, refers to the movement by which a solution, whether aqueous or non-aqueous, is agitated so that gas is incorporated from the local environment of the container (eg, vial) into the solution. Any type of motion that agitates the solution and ensures that the gas is turned on can be used for shaking. Shaking must be done with sufficient force or intensity to ensure the formation of foam

- 12 042049 after a certain period of time. Preferably, the agitation is carried out with sufficient force or intensity so that the foam is formed within the short period of time provided for a particular UCA formulation. Thus, in some cases, such shaking is carried out for about 30 seconds, or for about 45 seconds, or for about 60 seconds, or for about 75 seconds, or for about 90 seconds, or for about 120 seconds, including, for example, within 30 seconds, or within 45 seconds, or within 60 seconds, or within 75 seconds, or within 90 seconds, or within 120 seconds. In some cases, the activation may take place over a period of time in the range of 60-120 seconds or in the range of 90-120 seconds.

The present invention contemplates that in some cases the time (or duration) of shaking will vary depending on the type of UCA formulation to be activated. For example, in some instances, an aqueous UCA formulation may be shaken for shorter periods of time than a non-aqueous UCA formulation. The present invention contemplates that in such cases the shaking intensity (or shaking speed, as these terms are used interchangeably herein) can be kept constant. Thus, an activating or shaking device, such as an activating or shaking device, can be set to perform shaking at the same intensity (defined as, for example, shakes per minute) for two or more different predetermined time periods. .

The present invention further contemplates that in some cases the intensity of the shaking will vary depending on the type of UCA composition to be activated. For example, in some cases, an aqueous UCA formulation may be shaken at a lower shaking intensity than a non-aqueous UCA formulation. The present invention contemplates that in such cases the shaking time (or duration, as these terms are used interchangeably herein) can be constant. Thus, an activation or shaking device, such as an activating device or a shaking device, can be set to perform shaking at two or more different predetermined shaking intensities (defined as, for example, shakes per minute) for one set period of time.

The present invention further contemplates that in some cases the shaking time and shaking intensity will vary depending on the type of UCA formulation to be activated. For example, in some cases, an aqueous UCA formulation may be shaken for a first time period at a first shaking intensity, and a non-aqueous UCA formulation may be shaken for a second time period at a second shaking intensity, the first and second time periods being different and the first and second intensity values shaking may vary. Thus, an activating or shaking device, such as an activating or shaking device, can be set to perform shaking at two or more different predetermined shaking intensities (defined as, for example, shakes per minute) for two or more different predefined time periods. For example, in an activating or shaking device such as an activating or shaking device, parameters can be set to perform shaking at (1) a first predetermined shaking intensity for a first predetermined period of time; and (2) a second predetermined shaking intensity for a second predetermined time period, wherein the first and second time periods are different and the first and second shaking intensity values are different.

Vigorous shaking for approximately 45 seconds with VIALMIX® is required to activate DEFINITY®. Unless otherwise indicated, the term approximately in relation to the activation time means a time that is ±20% of the specified time (ie, 45±9 s).

DEFINITY-II can be activated with VIALMIX® for periods ranging from 60s to 120s. In some instances, DEFINITY-II is activated within approximately 75 seconds (ie, 75±15 seconds). DEFINITY-II can be activated for longer periods of time, including 90-120 seconds.

The agitation can be carried out by a swirling motion (such as swirling), a side-to-side motion, or an up-and-down motion. In addition, different types of movement can be combined. Shaking can be done by shaking the container (eg, vial) containing the aqueous or non-aqueous lipid solution, or by shaking the aqueous or non-aqueous solution in the container (eg, vial) without shaking the container (eg, vial). Shaking is performed with the instrument in order to standardize the process. Mechanical agitators are known in the art and their mechanisms or devices

- 13 042049 shaking can be used in the devices of the present invention. Examples include amalgamators such as those used for dental applications. Intensive shaking includes at least 1000, at least 2000, at least 3000, at least 4000, at least 4500, at least 5000 or more shaking movements per minute. In some cases, intensive shaking involves shaking in the range of 4000-4800 shaking movements per minute. In VIALMIX®, for example, target shaking is performed at 4530 RPM in figure 8, while shaking intensity values in the range of 4077-4756 rpm are allowed. Vortex mixing provides at least 250, at least 500, at least 750, at least 1000 or more rpm. Vortex mixing at at least 1000 rpm is an example of vigorous shaking and is more preferred in some cases. Vortex mixing at 1800 rpm is most preferred.

The intensity of shaking can influence the required shaking time. Higher shaking intensity will generally decrease the length of shaking time required to achieve optimal microbubble formation. For example, shaking at 4530 rpm with a duration of 45 seconds will be achieved in 3398 full revolutions on VIALMIX®. Shaking at 3000 rpm will take 68 seconds to reach the same rpm. Therefore, it should also be understood that a lower shaking intensity will generally increase the length of shaking time required to achieve optimal microbubble formation. The required duration and speed of shaking will also be affected by the shape of the trajectory and the amplitude of the shaking. The speed that the liquid reaches in the container and the forces applied when changing direction will affect the inclusion of the gas. These aspects will be affected by the length and path of the shaker arm, the shape and size of the container, the fill volume, and the viscosity of the formulation. Water has a viscosity of approximately 1.14 cP at 15°C (Khattab, I.S. et al., Density, viscosity, surface tension, and molar volume of propylene glycol+water mixtures from 293 to 323 K and correlations by the Jouyban-Acree model, Arabian Journal of Chemistry (2012)). In contrast, propylene glycol has a viscosity of 42 cP at 25°C (Khattab, I.S. et al., Density, viscosity, surface tension, and molar volume of propylene glycol+water mixtures from 293 to 323 K and correlations by the Jouyban- Acree model, Arabian Journal of Chemistry (2012)), and glycerin has a viscosity of 2200 cP at 15°C (Secut, J.B., Oberstak H.E. Viscosity of Glycerol and Its Aqueous Solutions. Industrial and Engineering Chemistry 43, 9, 2117-2120 , 1951). DEFINITY-II has a high viscosity of 1150 cps at 15°C. Because DEFINITY® is predominantly water, it has a much lower viscosity than DEFINITY-II.

The formation of gas-filled microspheres upon activation can be detected by the presence of foam on top of an aqueous or non-aqueous solution and by the solution turning white.

Activation is performed at a temperature below the phase transition temperature of the gel state in the liquid crystal state of the lipid used. The gel-to-liquid crystal phase transition temperature is understood to mean the temperature at which a lipid layer (such as a lipid monolayer or bilayer) will change from a gel state to a liquid crystal state. This transition is described, for example, in Chapman et al., J. Biol. Chem., 1974, 249, 2512-2521. The gel-to-liquid crystal phase transition temperatures of various lipids will be readily apparent to those skilled in the art and are described, for example, in Gregoriadis, ed., Liposome Technology, vol. I, 1-18 (CRC Press, 1984) and Derek Marsh, CRC Handbook of Lipid Bilayers (CRC Press, Boca Raton, Fla. 1990), on p. 139. Vigorous shaking can cause heating of the composition, taking into account the shaking speed, duration, the length of the shaker arm and its trajectory, the shape and size of the container, the filling volume and the viscosity of the composition.

In light of the present invention, one of skill in the art will appreciate that the lipid(s) or lipid microspheres may be manipulated before or after performing the methods provided herein. For example, after shaking is complete, the gas-filled microspheres can be removed from their container (eg, vial). Retrieval can be done by inserting a syringe needle or needleless element (e.g. PINSYNC®) into a container, including foam if necessary, and drawing a predetermined amount of liquid into the syringe barrel by retracting the plunger, or by adding an aqueous liquid, mixing and drawing a predetermined a certain amount of liquid into the syringe barrel by retracting the plunger. As another example, gas-filled microspheres can be filtered to obtain microspheres of substantially uniform size. The filter unit may contain more than one filter, which may or may not be directly adjacent to each other.

Ways.

- 14 042049

Therefore, the present invention provides various methods for forming gas-filled microspheres. In some instances, these methods at least include providing for activation of an activation-dependent UCA formulation to form gas-filled microspheres. Activation can be performed using an activation device (eg, a shaking device). Such devices may provide activation only, or they may identify the UCA formulation (or its container) and enable the formulation to be activated. Thus, some methods involve identifying a UCA compound and then allowing that UCA compound to be activated based on its identification information. A single fixture (eg, device) can perform both the identification step and the activation step. Alternatively, different devices can be used to perform each step. In another embodiment, the device can be used to provide activation of the composition.

In some instances, these methods include providing an activation-dependent UCA formulation to form gas-filled microspheres using a device (eg, device) that identifies the non-aqueous UCA formulation. Identification of the non-aqueous UCA formulation may involve reading a label specific to the non-aqueous UCA formulation. Parameters can be set in the fixture to retain and cause the non-aqueous UCA formulation to be activated for a predetermined period of time. In some embodiments, such a predetermined time period is approximately 75 seconds.

In other instances, these methods include providing an activation-dependent UCA formulation to form gas-filled microspheres using a device that is configured to distinguish between a non-aqueous UCA composition and an aqueous UCA composition (or alternatively a device that is configured to discriminate between an aqueous UCA composition and a non-aqueous UCA composition). UCA).

The aqueous formulation of UCA is an aqueous solution containing one or more lipids and a gas. When activated, lipids and gas together form gas-filled microspheres. Examples of an aqueous formulation of UCA are DEFINITY®, DEFINITY-III and DEFINITY-IV.

The non-aqueous formulation of UCA is a non-aqueous solution containing one or more lipids and a gas. Upon activation, lipids and gas together form gas-filled microspheres, although in this case the microspheres are surrounded by a non-aqueous solution. An example of a non-aqueous formulation of UCA is a room temperature stable formulation referred to herein as DEFINITY-II. As described in more detail herein, DEFINITY-II contains, at a minimum, the lipids DPPA, DPPC, and PEG5000-DPPE in propylene glycol and glycerin, along with a buffer and gaseous octafluoropropane (perflutrene). PEG5000 refers to PEG having a molecular weight of 5000 daltons. It may be hydroxy-PEG or methoxy-PEG. In some embodiments, DEFINITY-II contains MPEG5000-DPPE. Thus, examples of non-aqueous UCA formulations include, for example, DPPA, DPPC and MPEG5000-DPPE lipids, propylene glycol, glycerin, buffer, and gaseous octafluoropropane (perflutren); or lipids DPPA, DPPC and MPEG5000-DPPE, propylene glycol, buffer and gaseous octafluoropropane (perflutren); or lipids DPPA, DPPC and MPEG5000-DPPE, glycerin, buffer and gaseous octafluoropropane (perflutren); or lipids DPPA, DPPC and MPEG5000-DPPE, propylene glycol, glycerin and gaseous octafluoropropane (perflutren). Upon activation, the gas-filled microspheres similarly contain a DPPA/DPPC/MPEG5000-DPPE lipid shell encapsulating gaseous perflutren. However, these microspheres are diluted in an aqueous solution, such as an aqueous saline solution, and then administered to the subject either as a bolus injection or as a continuous infusion.

It is important to note that these aqueous and non-aqueous UCA formulations have been found to have different optimal activation times for obtaining gas-filled microspheres suitable for diagnostics. For example, in some cases where the intensity of shaking is approximately 4530 shaking movements (eg, figure-of-eight movements) per minute, and shaking is performed using VIALMIX®, some aqueous UCA formulations, including DEFINITY®, activate for approximately 45 s, while the non-aqueous formulation of UCA, DEFINITY-II, is activated for 60-120 seconds, and in some cases for about 75 seconds, to achieve a substantially similar microsphere size distribution profile. Therefore, the methods provided herein facilitate the distinction between non-aqueous UCA formulations and aqueous UCA formulations such as DEFINITY®.

Other methods contemplated herein include identifying a labeled vial containing a UCA formulation that requires activation for a predetermined period of time, using a shaking device containing a detector, and which is set to a predetermined period of time or has the ability to automatically perform pre-selection. a certain period of time based on vial identification information, and causing the UCA formulation to activate to form gas-filled microspheres. The predetermined time period may be 45 seconds, or

- 15 042049 may be 75 seconds, although it is not limited to these values.

Other methods contemplated herein include distinguishing between two or more aqueous UCA formulations (such as, for example, DEFINITY®, DEFINITY-III, and DEFINITY-IV) that require different activation times and optionally different shaking strengths. Two or more aqueous UCA formulations may differ based on their fill volume (ie, the amount of liquid in their respective containers) or based on the shape and size of the container. Fill volumes can be estimated, for example, using optical effects based approaches (eg measuring the light absorption of a composition at a particular position along the length of the container). The shape and size of the container can be judged, for example, by a holder that holds the container. Once an aqueous UCA formulation has been identified (by distinguishing from another UCA), it can then be activated for its intended period of time and at its intended shaking intensity. If the methods involve distinguishing between two or more UCA formulations, the activation device (e.g., shaker) may be set to perform shake for a predetermined period of time, or it may be set to perform shake for two or more different predetermined time periods, and they will therefore be able to automatically select one such time period. Such a device may include a detector. Similar methods are contemplated for distinguishing and optionally providing activation of non-aqueous UCA formulations. Similar methods are provided for distinguishing between aqueous and non-aqueous UCA formulations and optionally ensuring activation of one or both UCA formulations.

Other methods contemplated herein include identifying an aqueous UCA formulation that requires activation for a predetermined period of time using a device that is configured to distinguish between an aqueous UCA formulation and a non-aqueous UCA formulation, and allowing the aqueous UCA formulation to be activated within a predetermined period of time with the formation of gas-filled microspheres.

Other methods contemplated herein include identifying a UCA formulation that requires activation for a predetermined period of time using a device that is capable of distinguishing between a non-aqueous UCA formulation and an aqueous UCA formulation (or vice versa) and providing activation of the UCA formulation. for a predetermined period of time to form gas-filled microspheres. The device may be configured to provide activation for only one predetermined period of time (eg, approximately 45 seconds for aqueous UCA or approximately 75 seconds for non-aqueous UCA), or it may be configured to provide activation for two or more various predetermined time periods (eg, about 45 seconds and about 75 seconds). It should be understood that if two or more predetermined time periods are envisaged, such time periods are different from each other.

Still other methods are contemplated which include identifying a UCA formulation that requires activation for a predetermined period of time and allowing the UCA formulation to be activated for a predetermined period of time to form gas-filled microspheres. For example, the methods include identifying a non-aqueous UCA formulation that requires activation for a predetermined period of time and causing the non-aqueous UCA formulation to be activated for a predetermined period of time to form gas-filled microspheres. The composition of the UCA can be identified and activated using a shaking device, which is set to a predetermined period of time or the ability to automatically select a predetermined period of time based on the identification information about the composition of the UCA.

Thus, in some instances, the methods include identifying a UCA formulation that requires activation for a predetermined period of time and causing the UCA formulation to be activated for a predetermined period of time to form gas-filled microspheres using a shaking device configured with two or more than predetermined time periods or the ability to automatically select between two predetermined time periods based on identification information about the composition of the UCA. In some instances, identification information about a UCA formulation is provided by a label or label on the container (eg, vial) in which the formulation is stored. In some cases, identification information about the composition of the UCA is provided by the composition itself or its volume, as described in more detail in this document. The UCA formulation may be an aqueous UCA formulation, or it may be a non-aqueous UCA formulation. The predetermined time period may be approximately 45 seconds. The predetermined time period may be in the range of 60-120 seconds, or approximately 75 seconds.

Alternatively, other methods provided herein include identifying

- 16 042049 a labeled vial containing a UCA formulation that requires activation for a fixed period of time and a predetermined shaking speed, using a shaking device containing a scanner that is set to a fixed time period and a predetermined shaking speed or the possibility of automatic selection a predetermined shaking speed based on vial identification information, and allowing the UCA formulation to activate to form gas-filled microspheres. The predetermined shaking speed may be approximately 4530 rpm.

Still other methods include providing activation of the UCA formulation using a shaking device that is configured to identify the UCA composition and automatically select activation time or shaking speed (or shaking intensity, and these terms are used interchangeably herein) or both based on information about where the UCA formulation is identified based on a unique identifier other than the shape or size of the vial in which the UCA formulation is stored.

Other methods include providing activation of the first UCA formulation using a shaking device that can distinguish between the first vial containing the first UCA formulation and the second vial containing the second UCA formulation.

Still other methods include identifying a labeled vial containing an aqueous UCA formulation that requires activation for a predetermined first period of time, using a shaking device containing a scanner and in which a predetermined time period is set or the ability to automatically select a predetermined first period of two predetermined time periods based on vial identification information, and allowing the UCA formulation to activate to form gas-filled microspheres.

All of these methods can be fully or partially automated. In some cases, the devices first identify the vial containing the UCA formulation and prompt the user to confirm identification. In other cases, devices perform identification and activation without any user intervention.

Devices.

Identification of a UCA composition and/or discrimination between different UCA compositions can be achieved in a number of ways. For example, devices may be used with scanners capable of reading labels on a UCA formulation container (eg, vial). In other instances, identification and/or discrimination between different UCA formulations may be achieved using devices that recognize the shape and size of the container that stores the aqueous UCA formulation as opposed to the container that stores the non-aqueous UCA formulation. These last devices may contain one holder, or they may contain two or more holders. In the case of a single holder, the holder may be configured to hold a container (eg, vial) holding a non-aqueous UCA formulation and not holding a container (eg, vial) holding an aqueous UCA formulation. Alternatively, the holder may be configured to hold a container (eg, vial) holding an aqueous UCA formulation and not holding a container (eg, vial) holding a non-aqueous UCA formulation.

According to one aspect, the device receives a container in which a UCA is stored, detects a UCA composition type, and performs various actions depending on the detected UCA composition type. The device associates certain actions with each type of UCA composition. Upon detection of a particular type of UCA, the device automatically performs the actions associated with that type of UCA.

Based on the type of UCA composition detected, a number of different actions can be performed. In some embodiments, the device shakes the sample. In some embodiments, the device performs shaking at a specific duration, pattern, and/or intensity depending on the sample type detected. Examples of various shaking patterns include, but are not limited to, side-to-side reciprocation, top-down reciprocation, vibration, rotational motion, figure-eight motion, circular motion, and back-and-forth tipping (e.g., rotation of a container at a certain angle and reverse). action). For example, in one illustrative embodiment, the device associates a shake duration of approximately 45 seconds with a type A sample and approximately 75 seconds with a type B sample. . When the device detects a type B sample, the device automatically shakes the sample for approximately 75 seconds.

Thus, the present invention further contemplates devices that are configured to change one or more parameters upon identification (and thus

- 17 042049 way, establishing differences) types of samples. By way of example, one device may shake all types of samples in the same pattern and at the same shaking intensity, but may shake different types of samples for different durations (i.e., different shaking time periods). As another example, one device may shake all types of samples in the same pattern and for the same time, but may shake different types of samples at different intensities (i.e., different shake intensities). As another example, one device may shake all types of samples at the same shaking intensity and for the same time, but may shake different types of shakes in different shaking patterns. Alternatively, the device may respond to each identified sample type by setting, including possibly changing, two parameters such as shaking intensity and shaking time, or shaking intensity and shaking pattern, or shaking time and shaking pattern. In yet another embodiment, the device may respond to each identified sample by setting, including possibly changing, all three of these parameters (ie, shake intensity, shake time, and shake pattern).

It should be understood that many other actions may be associated with a pattern type. Examples of various actions that a device can perform in response to a detected sample type include, but are not limited to, adjusting temperature settings, adjusting humidity settings, adjusting lighting settings (e.g., exposing the sample to different light intensities and/or frequencies), and/or introducing various substances into container (for example, reagents, dyes, or other suitable additives).

Indicators.

In some embodiments, the container in which the sample is stored contains an indicator that indicates the type of sample, and the device includes a detector that reads the indicator to detect the type of sample. The indicator may be an indicator that is readable by the instrument or device. Examples of indicators readable by the instrument or device include magnetic tapes, chips/microchips, barcodes, including linear, matrix, and 2D barcodes, radio frequency identification (RFID) tags, color-coded labels identified by color detection, and the like. Barcodes, such as linear barcodes, may be barcodes that meet or meet the standards of the Uniform Code Council or the standards of the Medical Industry Liaison Committee. Such indicators may in turn be read from, for example, a device such as a magnetic tape reader, a chip reader, a barcode scanner or reader, an RFID tag reader, or the like. In fact, any marking technology that has been used for the purposes of authentication and/or tracking and control of the passage can be used in relation to the containers provided in this document.

The indicator may be located on any suitable part of the sample container, such as the container body or lid. In some embodiments, the indicator is integral with or otherwise part of the sample container. For example, the indicator may be a colored lid or a physical feature such as a protrusion or indentation on a sample container. In other embodiments, the indicator is attached to the container by, for example, a binder, magnets, hook and loop fasteners, a mechanical arrangement such as inserting the indicator into the retention tabs, or any other suitable attachment arrangement.

The indicator may provide the end user or intermediate handling the container with a variety of information including, but not limited to, the source and/or manufacturer of the composition contained therein, including, for example, the name of the company or subsidiary that manufactured the composition. and/or who manufactured the components of the formulation, the date the formulation was manufactured, the actual location where the formulation was manufactured, the date the container was shipped, the handling of the container, including, for example, whether it was stored at a remote location and the conditions and duration of such storage, date the container was delivered, methods of delivery, National Drug Code (NDC) prescribed by the FDA, contents of the container, dosage and method of administration, including route of administration, etc.

The indicator may serve one or more purposes, including, for example, authenticating the container and the composition contained therein. Authentication means the ability to identify or mark a container as received and manufactured by an authorized party, and it allows an end user or other party to identify a container and formulations received by another unauthorized party. The indicator can also be used to track and control the passage of a container. This feature can be used to track the container and the composition contained therein, after production and before administration to the subject. At the same time, information about the movement of the container during a given period of time may be stored in a database, and optionally such a database may be available to the end user in order to

- 18 042049 make sure the composition is intact.

The indicator may also be a combined indicator, which means that it may contain information that is read in two different ways. For example, the indicator may contain information that is obvious and understandable to the naked eye (for example, it may indicate the name of the manufacturer in words), and other information that is machine readable, such as information contained in an RFID tag or barcode.

An indicator can also be a dual-use indicator, meaning that it can serve two or more purposes. For example, the indicator may contain information that identifies the composition, and additional information that identifies the manufacturer and/or date of manufacture. This information may be provided in the same format or with a different format (for example, one may be provided on an RFID indicator and the other may be provided on a barcode label).

The label may also provide the information recorded on it (eg, using RFID technology) to a device used to shake the vial. For example, such information can be used to prevent reactivation of a vial by any suitably equipped device if it has been previously shaken and the time limit for reactivation of previously activated vials has now expired.

The indicator may provide data that is visible and understandable to a human, such as, for example, the name of the manufacturer. Alternatively or additionally, the indicator may contain information which, although easily visible to the human eye, nevertheless does not provide meaningful information in the absence of a lookup table or other form of database to be consulted. Such information may, for example, be provided in the form of an alphanumeric code.

In some embodiments, the UCA formulation is in a container, such as a vial, and such container is labelled. The container may have an indicator in the form of a label that is affixed to one or more of its outer surfaces. In some embodiments, the indicator is a paper label or other such label that is visible to the eye and can be read and understood by the end user without additional aid or device. Alternatively, as discussed above, the indicator is an indicator that is readable by the instrument or device.

Detectors.

The device may include any suitable detector for reading the indicator. In some embodiments, the detector may be operated by visual, photographic, imaging, electromagnetic, visible light, infrared, and/or ultraviolet techniques.

For example, in some embodiments, the indicator is a barcode and the detector is a barcode scanner. In some embodiments, the indicator is an RFID tag and the detector is an RFID reader. In some embodiments, the indicator is a color label and the detector is a color detection scanner. In some embodiments, the indicator is a chip/microchip and the detector is a chip/microchip reader.

In some embodiments, the sample containers include an indexing element that ensures proper alignment of the indicator on the container and the detector on the device. Examples of pointing features include physical notches or protrusions on the container's lid or body that align with corresponding features on the holder such that the container can only be inserted into the holder in one orientation.

In some embodiments, the indicator is a physical component, such as a protrusion or recess on a container. A detector is a button on a device that is pressed or a sensor that is otherwise activated due to physical interaction with a physical component. In one illustrative embodiment, the indicator is a specifically shaped protruding tab on the lid of the sample container and the device includes corresponding slots into which the tabs can be inserted. Each pattern type is associated with a particular tab shape, and each tab shape only corresponds to one of the slots on the device. The L-shaped tab is associated with the Type A sample and the oval tab is associated with the Type B sample. The portion of the device that interacts with the container lid has associated slots; one that accepts an L-shaped eyelet and the other that accepts an oval eyelet. When the L-tab is inserted into the holder, the lug presses the button in the L-slot and this lets the device know that a Type A sample has been received. that type B was accepted.

In some embodiments, the device may detect the type of sample based on one or more properties of the sample container. Example properties include weight, optical properties, and container size. Regarding the weight, the weight of the sample may reflect the type of sample. For example, containers containing Type A samples may have a single weight range, but a container

- 19 042049 Fish containing Type B specimens may have a second different weight range. The device may have a scale or other weight detection device that determines the combined weight of the container and the sample. If the weight value falls within the first range, the device determines that the sample is type A, and if the weight value falls within the second range, the device determines that the sample is type B. The weight detection device may be built into the holder or may be separate scales in the device. In the case of a separate balance, the user places the container on/on the weight detection device, the device measures the weight with sample type detection, and then the user or the device itself moves the sample container into the holder.

Regarding optical properties, each image type can be associated with a known optical property. Examples of optical properties include, without limitation, refractive index, absorption, and fluorescence. The device may contain a suitable instrument for measuring the optical property and, based on the measurement, determine the associated sample type.

Regarding the size of the sample container, each type of sample can be associated with a container of a different size. For example, sample type A may have a container that is larger than the container used for sample type B. The device may detect the size of the container in a number of ways. In some embodiments, the device has more than one holder, each holder being sized to accommodate one of the sample container sizes. Each sample container size can fit only one of the holders. The device detects when and which holder has accepted the container. Knowing which holder contains the container, the device determines the sample container size and the sample type associated with that container size. In another embodiment, the device has a single holder that can accommodate containers of various sizes. For example, the holder may have a spring-loaded end that can be moved to various positions to accommodate larger containers. The device may have buttons or other sensors that detect the receipt of the container and the amount by which the holder has been increased in order to determine the size of the container. As another example, the user must manually adjust the size of the holder by removing spacers, lifting open doors, or otherwise moving components to size the holder so that it fits the sample container properly and tightly. The device will then measure the size of the holder and size the container accordingly. In other embodiments, the implementation of the device may contain visual detectors, such as a camera and/or laser, to detect the size of the container. For example, a camera can take an image of a container and process the image to determine the size of the container. As another example, the laser can be directed to a position that will hit the container if a large container is used, but will pass through a void if a small container is used, and the device will consequently detect the size of the container by determining whether the laser has been obstructed. , or otherwise get in the way.

It should be understood that the device may have a number of different elements that contribute to the operation. In some embodiments, the device may include a counter that can keep track of how many times the device has been used to perform certain actions. Alternatively, the counter may keep track of the number of revolutions/oscillations that the shaking device has performed. Such a sign can be used to prevent repairs and control the operation of the device. The counter can be digital or manual. In some embodiments, a counter may be used to keep track of how many times a particular sample has been acted upon, for example, the counter may record how many times a particular container/vial has been activated. In some embodiments, the implementation of the counter can be used to keep track in general, how many times each type of sample was received and acted upon.

In some embodiments, the implementation of the device may include a display that can convey a number of different messages to the user. The display can show device status, errors, sample type, and can alert the user to any potential problems.

Warnings may be audible and/or visual. Examples of warnings include warning the user that a particular action has been performed on the sample a certain number of times, that the action has not been performed at the proper level or has been performed too long (for example, the shaking time was too long or too short), that the lid has opened, that the container is not properly secured in the holder, and/or that the device requires or will soon require repair. In some embodiments, the device will alert the user that an action that has been performed on a sample or container (eg, vial) has exceeded or is near the boundaries of an acceptable range. For example, the device may alert the user if the operation of the device has exceeded or is near the boundaries of an acceptable range in terms of shaking speed or duration. As an illustrative example, the device could shake the sample at an intensity that was too high, too low, or close to the upper or lower end of the range of shaking intensity values. The device will alert the user to this potential problem.

- 20 042049 me.

In some embodiments, the device will detect if the sample has expired (for example, by reading information from an indicator on the sample container). The device may warn the user of this and/or may prevent operation of the device when an expired sample is received by the device.

In some embodiments, the implementation of the device contains an indicator part separate from the display, which shows the user the type of sample that was detected. The indicator may have lights that indicate the type of sample (eg, aqueous or non-aqueous UCA), or may have a display that shows the name of the sample type.

In some embodiments, although the user is not required to enter the type of sample and/or the specific action to be taken, the device may ask the user to confirm the type of sample that has been detected before the device can act on the sample.

The device may be powered by plugging into a wall outlet and/or may be battery powered. In some embodiments, the battery is rechargeable.

In some embodiments, the holder includes a button or other sensor for detecting whether the container has been properly received. In some cases, the device will not function until it detects the container in the holder.

In some embodiments, the device may be connected to a computer or network, such as via Wi-Fi, USB, or another connection. This connection can be used to remotely maintain the device, such as restoring/updating software and/or monitoring device status and usage. A connection can also be used to deliver data; for example, the data received by the device may be sent to a database and/or to a printer.

In some embodiments, the device may record and transmit information such as frequency of use of the vial, shaking times, temperature and other conditions, frequency of use of the device, analysis results to a database or other data storage location. In some embodiments, the information from the device may be compared to databases of information to detect deviations in the device or sample and/or the comparisons may be used to determine the category of the sample.

In some embodiments, the device may count and monitor the number of samples processed and/or the status of the device and accordingly advise the user on the need to rebuild items such as samples, parts of the device that need to be replaced, etc.

In FIG. 1 is an illustrative schematic diagram showing a device with a detector for reading an indicator from a sample container. The device 1 includes a base 60 and a lid 70. In some embodiments, the lid 70 is opened by rotating the lid relative to the base. In some embodiments, the implementation of the cover can be completely removed and removed from the base. The sample container 100 that can be used with the device includes an indicator 110 that indicates the type of sample in the container. The device includes a holder 10 for receiving and holding the sample container and a detector 30 for reading indicator 110. The device also includes a shaking device 20. The device further comprises a control panel 40 that includes control buttons 41 and a display 44. In some embodiments, the device may further comprise indicator 50 separate from display 44. Indicator 50 may include signals such as lights that indicate the type of sample that has been detected.

In one illustrative embodiment, shown in FIG. 2, the device 1 is used with a sample container 100 which contains an indicator in the form of an RFID tag 112. The device 1 includes an RFID reader 32 which is connected to the device via a wire 33. The sample container 100 is received by a holder 10 which is attached to a lever 20 shaking. The device also includes a control panel 40 with a start button 42 and a cancel button 43, as well as a display 44. The device also includes an indicator 50 with three lights 51, 52, 53 corresponding to the three types of samples. When the device detects a specific sample type, the lamp corresponding to that sample type lights up.

In another illustrative embodiment shown in FIG. 3, device 1 is used with a sample container 100 which contains a barcode indicator 114. Device 1 includes a barcode reader 34 that is connected to the device via wire 35.

In another illustrative embodiment shown in FIG. 4, the device 1 includes a sample holder 10 which is designed to accommodate only one container size and does not allow the insertion of a larger container. The sample holder 10 may have a cap cap 21 to hold the vial in place. The cap cap 21 can receive a vial cap and hold the vial through an interference fit, threaded configuration (e.g., external threads

- 21 042049 on the cap of the vial, which correspond to the internal threads on the cap 21 for cap), mechanical interlock or any other suitable configuration. The sample holder 10 may have a spring 23 at the base of the holder to prevent the vial from moving in the holder and partially falling out of the vial when the cap is removed for ease of vial removal. In some embodiments, holder 10 may expand to accommodate a larger container as well as detecting the size of the holder for sample type detection, as previously discussed.

Temperature sensors.

In some embodiments, the shaking device includes a temperature sensor that measures the temperature of the UCA formulation and/or the vial containing the UCA formulation. In some cases, the device is set to operate only when the temperature of the UCA formulation or vial containing the UCA formulation is at or near room temperature. Room temperature, as used herein, means temperatures between 15-30°C, including 18-25 and 20-25°C, and all temperatures in between.

Containers (eg vials).

UCA formulations may be provided in a container (or container). In some embodiments, the container is a vial. The vial can be made from any material, including but not limited to glass or plastic. The glass may be pharmaceutical grade glass. The container may be sealed with a stopper, such as a rubber stopper. In some embodiments, the container is a 0.5-10 ml container. The container may be a 1-5 ml container or a 1 or 2 ml container. Such volumes refer to the volume of liquid normally placed in a container (called the liquid fill volume). It is contrasted with the total internal volume of the container, which will be greater than the filling volume of the liquid. Examples of liquid fill volume and internal volume are as follows: 2 ml vial (liquid fill volume) with an internal volume of 2.9 ml from Schott; 3 ml vial (liquid fill volume) with an internal volume of 4.5 ml from Schott; and a 1ml v-shaped vial (liquid fill volume) with an internal volume of 1.2ml from Wheaton.

As will be understood from the context of this disclosure, the interior volume of the container may be occupied by lipid composition and gas. An example of a suitable container is a 2 ml glass vial from Wheaton (commercially available, for example, from Nipro, cat. no. 2702, B33BA, 2 cm ³ , 13 mm, type I, silicon cylindrical vial) having an actual internal volume of approximately 3 .75 ml. An example of a suitable stopper is a gray siliconised butyl freeze-dried stopper from West (Cat. No. V50, 4416/50, 13 mm, WS-842). An example of a suitable crimp cap is West's aluminum tear-off crimp cap (p/n 3766, white, 13mm, 13-FA-591). The containers are preferably sterile and/or sterilized after the addition of the lipid solution and/or gas, as described in PCT Published Application WO 99/36104.

In some embodiments, the container is a flat-bottomed container, such as a flat-bottomed vial. Suitable vials include borosilicate flat bottom vials, including vials from Wheaton. In some embodiments, the container is a container or vial with a non-flat bottom. In some embodiments, the container is a V-bottomed container, such as a V-bottomed vial. In some embodiments, the container is a round bottom container, such as a round bottom vial. In some embodiments, the container has converging walls such that its bottom surface area (or bottom surface diameter) is less than the surface area (or diameter) of its top (opening), or less than any diameter of the portion in between (e.g. , body diameter). For clarity, a V-bottomed container or vial has converging walls and its bottom surface area is significantly less than any of the surface areas of its top or body.

It should be understood that while some of the embodiments described herein refer to vials, they should be construed more broadly to include any suitable container unless expressly stated otherwise.

Lipids.

These UCA formulations contain one lipid and usually more than one lipid. As used herein, lipids or total lipid or combined lipids means a mixture of lipids.

The lipids may be provided in their individual solid state (eg, powdered) forms. Alternatively, the lipids may be provided as a mixture of lipids. Methods for preparing a mixture of lipids include those described in US Pat. No. 8,084,056 and PCT Published Application WO 99/36104. A lipid mixture, as used herein, is meant to be two or more lipids that have been mixed to form a more uniform lipid mixture than would be obtained by simply mixing the lipids in their individual powdered form. The lipid mixture is typically in powder form. Lipid Blend

- 22 042049 can be obtained by an aqueous suspension lyophilization method or a method of dissolving the precipitate in an organic solvent using organic solvents. In an aqueous suspension lyophilization method, the desired lipids are suspended in water at an elevated temperature and then concentrated by lyophilization.

The method of dissolution in an organic solvent includes the following steps.

(a) Contacting the desired lipids (eg DPPA, DPPC and MPEG5000-DPPE) with the first non-aqueous solvent system. This system is usually a combination of solvents, such as CHCl ₃ /MeOH, CH ₂ Cl ₂ /MeOH and toluene/MeOH. Preferably the first non-aqueous solvent is a mixture of toluene and methanol. It may be advantageous to heat the lipid solution to a temperature sufficient to achieve complete dissolution. Such a temperature is preferably about 25-75°C, more preferably about 35-65°C. After dissolution, undissolved foreign matter can be removed by hot filtration or cooling to room temperature and then filtration. Known filtration methods (eg gravity filtration, vacuum filtration or pressure filtration) can be used.

(b) The solution is then concentrated to a thick gel/semi-solid. The concentration is preferably carried out by vacuum distillation. You can also use other methods of concentration of the solution, such as rotary evaporation. The temperature of this stage is preferably from about 20 to 60°C, more preferably from 30 to 50°C.

(c) The thick gel/semi-solid is then dispersed in a second non-aqueous solvent. The mixture is suspended preferably at a temperature corresponding to approximately ambient temperature (for example, 15-30°C). Suitable second non-aqueous solvents are solvents which cause precipitation of lipids from the filtered solution. The second non-aqueous solvent is preferably methyl tert-butyl ether (MTBE). Other esters and alcohols may also be used.

(d) Then collect the solids obtained by adding the second non-aqueous solvent. Preferably the collected solids are washed with another portion of the second non-aqueous solvent (eg MTBE). Collection can be done by vacuum filtration or centrifugation, preferably at ambient temperature. After collection, it is preferable to dry the solids in vacuo at a temperature of approximately 20-60°C.

The contents of US Pat. No. 8,084,056 and PCT Published Application WO 99/36104 relating to a process for preparing a mixture of lipids are incorporated herein by reference.

The dissolution-precipitation method in an organic solvent is preferred over the aqueous suspension lyophilization method for a number of reasons outlined in US Pat. organic solvent.

Alternatively, the lipids may be provided as separate powders that are dissolved together or separately directly in propylene glycol, glycerin, or propylene glycol/glycerin to form a non-aqueous mixture.

As used herein, a lipid solution is a solution containing a mixture of lipids. Similarly, a lipid formulation is a formulation containing one or more lipids. The lipids may be cationic, anionic or neutral lipids. Lipids can be of natural, synthetic, or semi-synthetic origin, including, for example, fatty acids, fluorinated lipids, neutral fats, phosphatides, oils, fluorinated oils, glycolipids, surfactants (surfactants and fluorosurfactants) , aliphatic alcohols, waxes, terpenes and steroids.

At least one of the lipids may be a phospholipid, and thus the mixture of lipids may be referred to as a phospholipid mixture. Phospholipid, as used herein, is a fatty substance containing oily(s) (hydrophobic(s)) hydrocarbon(s) chain(s) with a polar (hydrophilic) phosphorus group representing the head. Phospholipids are amphiphilic. They spontaneously form boundaries and closed microspheres in aqueous media.

Preferably all lipids are phospholipids, preferably 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA); and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE). DPPA and DPPE may be provided in the form of monosodium salts.

In some cases, the lipid components can be modified to reduce the reactivity of the microsphere in the environment, including the environment in vivo, thereby increasing its half-life. Lipids containing polymers such as chitin, hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG) can also be used for this purpose. PEG-conjugated lipids are referred to herein as PEGylated lipids. Preferably the PEGylated lipid is DPPE-PEG or DSPE-PEG.

- 23 042049

The conjugation of a lipid to a polymer, such as PEG, may be through a number of compounds or bonds, such as, but not limited to, amide, carbamate, amine, ester, ether, thioether, thioamide, and disulfide (thioester) bonds.

End groups on PEG may be, without limitation, hydroxy-PEG (HO-PEG) (or its reactive derivatives), carboxy-PEG (COOH-PEG), methoxy-PEG (MPEG) or other lower alkyl group, such as in isopropoxy -PEG or tert-butoxy-PEG, amino-PEG (NH2PEG) or thiol (SH-PEG).

The molecular weight of PEG may vary from about 500 to about 10,000, including from about 1,000 to about 7,500 and from about 1,000 to about 5,000. In some important embodiments, the PEG has a molecular weight of about 5,000. Therefore, DPPE-PEG5000 or DSPE-PEG5000 refers to DPPE or DSPE with a PEG polymer attached thereto having a molecular weight of approximately 5000.

The percentage of PEGylated lipids relative to the total lipids in the lipid solution, on a per mole basis, is or ranges from about 2% to about 20%. In various embodiments, the percentage of PEGylated lipids relative to total lipids is or ranges from 5 mol% to about 15 mol%.

Preferably the lipids are 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) monosodium salt, and N-(polyethylene glycol-5000- carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (PEG5000-DPPE). The polyethylene glycol-5000-carbamoyl may be methoxy polyethylene glycol-5000-carbamoyl. In some important embodiments, the lipids may be one, two, or all three of DPPA, DPPC, and PEG5000-DPPE. PEG5000-DPPE may be MPEG5000-DPPE or HO-PEG5000-DPPE.

A wide variety of lipids, such as those described in US Pat. No. 5,469,854 to Unger et al, can be used in the method of the present invention. Suitable lipids include, for example, fatty acids, lysolipids, fluorinated lipids, phosphocholines such as phosphocholines associated with platelet activating factors (PAFs) (Avanti Polar Lipids, Alabaster, Alabama), including 1-alkyl-2-acetoyl-sn -glycero-3-phosphocholines and 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholines; phosphatidylcholine with both saturated and unsaturated lipids, including dioleoylphosphatidylcholine; dimyristoylphosphatidylcholine; dipentadecanoylphosphatidylcholine; dilauroylphosphatidylcholine; 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC); distearoylphosphatidylcholine (DSPC) and diarachidonylphosphatidylcholine (DAPC); phosphatidylethanolamines such as dioleoylphosphatidylethanolamine, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) and distearoylphosphatidylethanolamine (DSPE); phosphatidylserine; phosphatidylglycerols, including distearoylphosphatidylglycerol (DSPG); phosphatidylinositol; sphingolipids such as sphingomyelin; oleic fatty acids; isolauric fatty acids; isomyristic fatty acids; isopalmitic fatty acids; isostearic fatty acids; cholesterol and cholesterol derivatives such as cholesterol hemisuccinate, cholesterol sulfate and cholesteryl (4'-trimethylammonium) butanoate; glycolipids such as ganglioside GM1 and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids such as 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid; stearic acid; arachidonic acid and oleic acid.

Other suitable lipids include phosphatidylcholines such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC) and distearoylphosphatidylcholine; phosphatidylethanolamines such as dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine and N-succinyldioleoylphosphatidylethanolamine; phosphatidylserines; phosphatidylglycerols; sphingolipids; glycolipids such as ganglioside GM1; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids such as dipalmitoylphosphatidic acid (DPPA); palmitic fatty acids; stearic fatty acids; arachidonic fatty acids; lauric fatty acids; myristic fatty acids; lauroleic fatty acids; fiseteric fatty acids; myristinoleic fatty acids; palmitoleic fatty acids; petroselinic fatty acids; esters of polyoxyethylene and fatty acids; alcohols of polyoxyethylene and fatty acids; ethers of polyoxyethylene and fatty acids; esters of polyoxyethylated sorbitan and fatty acids; glycerol polyethylene glycol oxystearate; glycerol polyethylene glycol ricinoleate; ethoxylated soy sterols; ethoxylated castor oil; polyoxyethylene-polyoxypropylene fatty acid polymers; polyoxyethylene fatty acid stearates; 12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid; N-[12-(((7'diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2succinylglycerol and 1-hexadecyl-2-palmitoylglycerophosphoethanolamine and palmitoylhomocysteine; lauryltrimethylammonium bromide (lauryl-=dodecyl-); cetyltrimethylammonium bromide (cetyl-=hexadecyl-);

- 24 042049 myristyltrimethylammonium bromide (myristyl-=tetradecyl-); alkyldimethylbenzylammonium chloride species such as where alkyl is C.sub.12, C.sub.14 or C.sub.16alkyl; benzyldimethyldodecylammonium bromide; benzyldimethyldodecylammonium chloride, benzyldimethylhexadecylammonium bromide; benzyldimethylhexadecylammonium chloride; benzyldimethyltetradecylammonium bromide; benzyldimethyltetradecylammonium chloride; cetyldimethylethylammonium bromide; cetyldimethylethylammonium chloride; cetylpyridinium bromide; cetylpyridinium chloride; N-[1-2,3-dioleoyloxy)propyl]-N,N,Ntrimethylammonium chloride (DOTMA); 1,2-dioleoyloxy-3-(trimethylammonium)propane (DOTAP) and 1,2-dioleoyl-e-(4'-trimethylammonium)butanoyl-sn-glycerol (DOTB).

In some embodiments where DPPA, DPPC, and DPPE are used, their mole percent amounts may be about 77-90 mole% DPPC, about 5-15 mole% DPPA, and about 5-15 mole% DPPE, including DPPE-PEG5000. Preferred ratios of each lipid include the ratios described in the Examples section, such as the ratio in wt.%, representing 6.0 to 53.5 to 40.5 (DPPA:DPPC:MPEG5000-DPPE), or the ratio in mol. % representing 10 to 82 to 8 (10:82:8) (DPPA:DPPC:MPEG5000-DPPE).

Gas.

The gas is preferably substantially insoluble in the lipid solutions provided herein. The gas may be an insoluble fluorinated gas such as sulfur hexafluoride or gaseous perfluorocarbon. Examples of gaseous perfluorocarbons include perfluoropropane, perfluoromethane, perfluoroethane, perfluorobutane, perfluoropentane, perfluorohexane. Examples of gases that can be used in the microspheres of the present invention are described in US patent No. 5656211 and incorporated herein by reference. In an important embodiment, the gas is perfluoropropane.

Example gases include, without limitation, hexafluoroacetone, isopropylacetylene, allene, tetrafluoroallene, boron trifluoride, 1,2-butadiene, 1,3-butadiene, 1,2,3-trichlorobutadiene, 2-fluoro-1,3-butadiene, 2-methyl- 1,3-butadiene, hexafluoro-1,3-butadiene, butadiene, 1-fluorobutane, 2-methylbutane, decafluorobutane (perfluorobutane), decafluoroisobutane (perfluoroisobutane), 1-butene, 2-butene, 2-methyl-1-butene, 3-methyl-1butene, perfluoro-1-butene, perfluoro-1-butene, perfluoro-2-butene, 4-phenyl-3-buten-2-one, 2-methyl-1-buten-3-in, butyl nitrate, 1-butyne, 2-butyne, 2-chloro-1,1,1,4,4,4-hexafluorobutyne, 3-methyl-1-butyne, perfluoro-2-butyne, 2-bromobutyraldehyde, carbonyl sulfide, crotononitrile, cyclobutane, methylcyclobutane, octafluorocyclobutane (perfluorocyclobutane), perfluorisobutane, 3-chlorocyclopentene, cyclopropane, 1,2-dimethylcyclopropane, 1,1-dimethylcyclopropane, ethylcyclopropane, methylcyclopropane, diacetylene, 3-ethyl-3-methyldiaziridine, 1,1,1-trifluorodiazoethan, dimethylamine , hexafluorodimethylamine, dimethylethylamine, bis-(dimethylphosphine)amine, 2,3-dimethyl-2-norbornane, p erfluorodimethylamine, dimethyloxonium chloride, 1,3-dioxolan-2-one, 1,1,1,1,2tetrafluoroethane, 1,1,1-trifluoroethane, 1,1,2,2-tetrafluoroethane, 1,1,2-trichlor -1,2,2-trifluoroethane, 1,1dichloroethane, 1,1-dichloro-1,2,2,2-tetrafluoroethane, 1,2-difluoroethane, 1-chloro-1,1,2,2,2-pentafluoroethane , 2-chloro1,1-difluoroethane, 1-chloro-1,1,2,2-tetrafluoroethane, 2-chloro-1,1-difluoroethane, chloroethane, chloropentafluoroethane, dichlorotrifluoroethane, fluoroethane, nitropentafluoroethane, nitrosopentafluoroethane, perfluoroethane, perfluoroethylamine, simple ethyl vinyl ether, 1,1-dichloroethylene, 1,1-dichloro-1,2-difluoroethylene, 1,2-difluoroethylene, methane, trifluoromethanesulfonyl chloride, trifluoromethanesulfonyl fluoride, (pentafluorothio)trifluoromethane, bromodifluoronitrosomethane, bromofluoromethane, bromochlorofluoromethane, bromotrifluoromethane, chlordifluoronitromethane, chlordinitromethane, chlorofluoromethane, chlorotrifluoromethane, chlorodifluoromethane, dibromodifluoromethane, dichlorodifluoromethane, dichlorofluoromethane, difluoromethane, difluoroiodomethane, disilanomethane, fluoromethane, iodomethane-iodotrifluoromethane, nitrotrifluoromethane, nitrosotrifluoromethane, tetrafluoro methane, trichlorofluoromethane, trifluoromethane, trifluoromethanesulfonyl chloride, 2-methylbutane, methyl ether, methyl isopropyl ether, methyl lactate, methyl nitrite, methyl sulfide, methyl vinyl ether, neopentane, nitrogen (N.sub.2), nitric oxide(I), complex 2- 1,2,3-nonadecanetricarboxylic acid hydroxycrimethyl ester, 1-nonen-3-yne, oxygen (O.sub.2), oxygen 17 (.sup. 17 O.sub.2), 1,4-pentadiene, n-pentane, dodecafluoropentane (perfluoropentane), tetradecafluorohexane (perfluorohexane), perfluoroisopentane, perfluoroneopentane, 2-pentanone-4-amino-4-methyl, 1-pentene, {cis }-2-pentene, {trans}-2-pentene, 3-bromo-1-pentene, perfluoro-1pentene, tetrachlorophthalic acid, 2,3,6-trimethylpiperidine, propane, 1,1,1,2,2,3 -hexafluoropropane, 1,2-epoxypropane, 2,2-difluoropropane, 2-aminopropane, 2-chloropropane, heptafluoro-1-nitropropane, heptafluoro-1-nitrosopropane, perfluoropropane, propene, 1,1,1,2,3,3-hexafluoro -2,3-dichloropropyl, 1-chloropropylene, {trans} chloro-propylene, 2-chloropropylene, 3-fluoropropylene, perfluoropropylene, propine, 3,3,3-trifluoropropine, 3-fluorostyrene, sulfur hexafluoride, sulfur (di) - decafluoro (S.sub.2 F.sub.10), 2,4-diaminotoluene, trifluoroacetonitrile, trifluoromethyl peroxide, trifluoromethyl sulfide, tungsten hexafluoride, vinyl acetylene, vinyl ether, neon, helium, krypton, xenon (especially rubidium-enriched hyperpolarized xenon gas ), carbon dioxide, helium and air.

Preferred are fluorinated gases (i.e., a gas containing one or more fluorine molecules, such as sulfur hexafluoride), gaseous fluorocarbons (i.e., a fluorinated gas, which is a fluorinated carbon or gas), and gaseous perfluorocarbons (i.e., a fluorocarbon gas that is fully fluorinated, such as perfluoropropane and perfluorobutane).

A gas, such as gaseous perfluorocarbon, is usually present at a concentration below its

- 25 042049 of the same concentration at room temperature due to the inclusion of air upon receipt. It is assumed that the concentration of perfluoropropane, when it is in a vial containing a non-aqueous mixture and a gas in the headspace, is approximately 6.52 mg/ml at a pressure of approximately one atmosphere. Concentrations of other gases, as is known in the art, will similarly dilute due to the inclusion of air upon receipt.

A gas, such as gaseous perflutren, may be introduced or otherwise added to a container (eg, vial) containing the solution, or to the solution itself, to provide a gas other than air. Gases that are not heavier than air can be added to a sealed container, while gases that are heavier than air can be added to a sealed or non-pressurized container.

One skilled in the art will appreciate that it is also possible to use a gaseous precursor and then convert the precursor to a gas by changing either temperature or pressure.

Ways of application and practical application.

The present invention provides methods for using the UCA formulations provided herein. Once activated, UCA formulations can be used in vivo for human or non-human subjects, or in vitro. The compositions provided herein can be used for diagnostic or therapeutic purposes, or for diagnostic and therapeutic purposes in combination.

When used as a UCA in human subjects, the formulations are activated as described herein to form a sufficient number of gas-filled microspheres. Such microspheres may be applied directly (as is) or may be further diluted into a solution, including a pharmaceutically acceptable solution, and administered as one or more bolus injections or by continuous infusion. Administration is usually intravenous administration. Shortly thereafter, visualization is performed. The imaging application may be directed to the heart, or it may cover another area of the body that is receptive to ultrasound imaging. The imaging may be an imaging of one or more organs or regions of the body, including but not limited to the heart, blood vessels, cardiovascular system, liver, kidneys, and head.

Subjects of the present invention include, without limitation, humans and animals. In some cases, humans are preferred. Animals include companion animals such as dogs and cats, and farm or valuable animals such as, but not limited to, bulls and horses.

UCA is administered in effective amounts. An effective amount will be that amount which facilitates or mediates the intended response and/or in vivo use. In the context of an imaging application, such as an ultrasound application, an effective amount can be that amount of lipid microspheres that allows imaging of a subject or region of a subject's body.

Software and hardware.

As discussed above, some embodiments relate to a device configured to perform various actions based on identification information about a container and/or the contents of the container. In this regard, the device according to some embodiments may include a computing system containing at least one processor programmed to perform identification of the container and/or its contents and, after determining the identification information, determine the appropriate actions to be performed based on the identification information.

In FIG. 5 illustrates a flow diagram of a method for selectively performing one or more actions on a vial placed in a device, in accordance with some embodiments. In step 510, identifying information about the type of sample placed in the device is determined. Sample type identification information may be determined in any suitable manner, examples of which are described herein. For example, the device may present a user interface that allows the user to manually enter a sample type identifier. Alternatively, the device may be configured to automatically determine sample type identification information by analyzing one or more indicators located on or associated with the vial, or by analyzing one or more properties of the contents of the container. In some embodiments, the device may be configured to initially automatically identify the sample type, and if such automatic identification is not performed, then the device may issue an error message and/or prompt the user of the device to manually enter sample type identification information. Embodiments where sample type identification is performed manually rather than automatically may provide a less expensive and/or simpler device that does not require a detector (eg, RFID reader, optical scanner, etc.).

Once the sample type is identified, the method proceeds to step 520 where one or more

- 26 042049 how many actions to perform on the sample. In some embodiments, the device may include at least one storage device configured to store a look-up table (LUT) or other data structure that stores information about the action(s) performed given the identification information about a particular type of sample. For example, a first set of actions may be performed if the vial is determined to contain a first type of UCA formulation, and a second set of actions may be performed if the vial is determined to contain a second type of UCA formulation. The device may be configured to distinguish between containers with any number of different types of formulations or substances contained therein, and the embodiments are not limited in this regard.

After determining the action(s) to be performed(s), the method proceeds to step 530, where at least one processor embedded in the device instructs the components of the device to perform the action(s) determined in step 520. In some embodiments, the determination regarding execution the action(s) may be based, at least in part, on factors other than sample type identification information. For example, factors such as whether the lid of the device is closed or whether the device is in a particular operating state may be considered in determining whether the action(s) should be performed. The at least one processor may communicate with the various components of the device to implement the specific action(s) in any suitable manner.

In FIG. 6 illustrates a detailed flow diagram of a method for determining the action(s) to be performed on a sample in a vial using RFID, according to the embodiment described in more detail below. As shown in FIG. 6, if the RFID tag is on a container inserted into the device, the device reads the RFID tag identifying the container as containing a first UCA compound type (DEFINITY®) or a second UCA compound type (DEFINITY-II). Based on matching identity information and other conditions, the device provides activation within a specific period of time.

An exemplary implementation of a computing system 700 that may be used in connection with any of the embodiments of the present invention described herein is shown in FIG. 7. Computing system 700 may include one or more processors 710 and one or more computer-readable tangible persistent media (eg, a memory unit 720, one or more non-volatile media 730, or any other suitable storage device). The processor 710 may control writing data to and reading from memory 720 and non-volatile storage 730 in any suitable manner, as aspects of the present invention described herein are not limited in this regard. To perform any of the functionality described herein, the processor 710 may execute one or more instructions stored in one or more computer-readable media (eg, memory 720) that may serve as tangible, persistent computer-readable media that stores the instructions. for execution by the 710 processor.

The above described embodiments of the present invention can be implemented in a number of ways. For example, embodiments may be implemented in hardware, software, or a combination thereof. When implemented by software, the program code may execute on any suitable processor or collection of processors provided either on a single computer or located on a plurality of computers. It should be understood that any component or collection of components that perform the functions described above can generally be considered as one or more controllers that control the functions discussed above. One or more controllers may be implemented in a variety of ways, such as specialized hardware or generic hardware (eg, one or more processors) that is programmed in microcode or software to perform the functions listed above.

In this regard, it should be understood that one implementation of embodiments of the present invention includes at least one permanent computer-readable medium (e.g., computer memory, USB drive, flash memory, CD, tape, etc.) encoded by means of a computer program (i.e., a plurality of instructions) that, when executed by a processor, perform the functions described above in accordance with embodiments of the present invention. The computer-readable medium may be mobile, for example, the program stored thereon may be downloaded to any computing resource to implement the aspects of the present invention discussed herein. In addition, it should be understood that reference to a computer program that, when executed, performs the above functions, is not limited to an application program running on a host computer. Rather, the term computer program is used herein generically to refer to any type of computer code (eg, software or microcode) that can be used to program a processor to implement the above aspects of the present invention.

- 27 042049

Various numbered embodiments of the subject matter provided herein are listed below.

1. The method of formation of gas-filled microspheres, including the identification of the composition of the UCA; and providing activation of the UCA composition for a predetermined period of time using a device that is configured to select between two or more predetermined periods of time, with the formation of gas-filled microspheres.

2. The method according to embodiment 1, wherein the device performs shaking at a predetermined shaking intensity.

3. The method of formation of gas-filled microspheres, including the identification of the composition of the UCA; and providing activation of the UCA composition using a predetermined shaking intensity using a device that is configured to select between two or more predetermined shaking intensity values to form gas-filled microspheres.

4. The method according to embodiment 3, wherein the device performs shaking for a predetermined period of time.

5. A method of forming gas-filled microspheres, comprising providing activation of an ultrasound contrast agent (UCA) composition to form gas-filled microspheres using a device that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition.

6. A method for forming gas-filled microspheres, comprising identifying an ultrasound contrast agent (UCA) composition that requires activation for a predetermined period of time, using a device that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition; and allowing the UCA formulation to activate for a predetermined period of time to form gas-filled microspheres.

7. The method according to embodiments 5 or 6, wherein the device is configured to provide activation for a predetermined time period selected from two or more predetermined time periods.

8. The method according to embodiment 7, wherein the device performs shaking at a predetermined shaking intensity.

9. The method according to embodiments 5 or 6, wherein the devices are configured to provide activation at a predetermined shaking intensity selected between two or more predetermined shaking intensity values.

10. The method according to embodiment 9, wherein the device shakes for a predetermined period of time.

11. The method according to any one of embodiments 5-10, wherein the UCA formulation is an aqueous UCA formulation.

12. The method according to any one of embodiments 5-10, wherein the UCA formulation is a non-aqueous UCA formulation.

13. The method according to any one of embodiments 5-12, wherein the fixture comprises a first holder configured to hold a container containing an aqueous UCA formulation and not to hold a container containing a non-aqueous UCA formulation.

14. The method according to any one of embodiments 5-12, wherein the fixture comprises a first holder configured to hold a container containing a non-aqueous UCA formulation and not to hold a container containing an aqueous UCA formulation.

15. The method according to embodiments 13 or 14 wherein the containers are vials.

16. The method according to any one of embodiments 5-15, wherein the device is configured to distinguish between an aqueous UCA formulation and a non-aqueous UCA formulation based on a unique identifier.

17. The method according to any one of embodiments 5-16, wherein the fixture comprises a detector.

18. The method of embodiment 17 wherein the detector is an RFID reader and the UCA is in a container that contains (includes) or is associated with or marked with an RFID tag/label.

19. The method of embodiment 17, wherein the detector is a barcode scanner and the UCA is in a container that contains (includes) or is associated with or labeled with a barcode.

- 28 042049

20. The method of Embodiment 17 wherein the detector is a color scanner and the UCA formulation is in a container that contains a colored label.

21. The method according to any one of embodiments 5-17, wherein the fixture imparts a reciprocating motion, rotation, or figure-eight motion to a container containing the UCA formulation.

22. The method according to any one of embodiments 5-8 and 11-21, wherein the predetermined time period is about 45 seconds if the UCA formulation is an aqueous UCA formulation and 60-120 seconds or about 75 seconds if the UCA formulation is a non-aqueous UCA formulation.

23. The method according to any one of embodiments 5-22, wherein the attachment is a shaking device.

24. The method of formation of gas-filled microspheres, including the identification of a labeled container containing the composition of the contrast agent for ultrasound (UCA), which requires activation within a predetermined period of time; and providing activation of the UCA composition to form gas-filled microspheres, where the shaking device is used to identify the labeled container and/or activate the UCA composition, and where the shaking device contains a detector, and it is set to a predetermined time period or the ability to automatically select a predetermined period time based on container identification information.

25. A method for forming gas-filled microspheres, comprising identifying a labeled container containing an ultrasound contrast agent (UCA) formulation that requires activation at a predetermined shaking intensity; and providing activation of the UCA composition at a predetermined intensity of shaking to form gas-filled microspheres, where the shaking device is used to identify the labeled container and/or activate the UCA composition and where the shaking device contains a detector, and it is set to a predetermined intensity of shaking or the ability to automatically select a predetermined shaking intensity based on container identification information.

26. The method according to embodiments 24 or 25, wherein the tagged container is tagged with a unique identifier.

27. The method according to any one of embodiments 24-26, wherein the detector is an RFID reader and the tagged container contains an RFID tag/label.

28. The method according to any one of embodiments 24-26, wherein the detector is a barcode scanner and the labeled container contains a barcode.

29. The method according to any one of embodiments 24-26, wherein the detector is a color scanner and the labeled container contains a color label.

30. The method according to any one of embodiments 24-29, wherein the labeled container is a labeled vial.

31. The method according to any one of embodiments 24 and 26-30, wherein the shaking device performs shaking at a predetermined shaking intensity.

32. The method according to any one of embodiments 25-31, wherein the predetermined shaking intensity value is in the range of 4000-4800 strokes per minute or 4100-4700 strokes per minute.

33. The method according to any one of embodiments 25-32, wherein the shaking device performs shaking for a predetermined period of time.

34. The method according to any one of embodiments 24-33, wherein the UCA formulation is an aqueous UCA formulation.

35. The method according to any one of embodiments 25-34, wherein the predetermined time period is approximately 45 seconds.

36. The method according to any one of embodiments 24-33, wherein the UCA formulation is a non-aqueous UCA formulation.

37. The method according to any one of embodiments 25-33 and 36, wherein the predetermined time period is in the range of 60-120 seconds, or about 75 seconds.

38. The method according to any one of embodiments 24-37, wherein the shaking device comprises a first holder configured to hold a container containing an aqueous UCA formulation and not to hold a container containing a non-aqueous UCA formulation.

39. The method according to any one of embodiments 24-37, wherein the shaking device comprises a first holder configured to hold a container containing seine

- 29 042049 UCA composition, and the inability to hold a container containing an aqueous UCA composition.

40. A method of forming gas-filled microspheres, comprising providing activation of the UCA composition for a predetermined period of time with the formation of gas-filled microspheres using a shaking device, in which the parameters are set to provide activation for at least two different predetermined periods of time or with the ability to automatically select from at least two different predetermined time periods based on identification information about the composition of the UCA.

41. The method according to embodiment 40, wherein the shaking device performs shaking at a predetermined shaking intensity.

42. A method of forming gas-filled microspheres, including providing activation of the UCA composition at a predetermined intensity of shaking with the formation of gas-filled microspheres using a shaking device, in which the parameters are set to provide activation at two or more different predetermined values of shaking intensity or with the possibility of automatic selecting from at least two different predetermined shaking intensity values based on the identification information about the composition of the UCA.

43. The method according to embodiment 42, wherein the shaking device performs shaking for a predetermined period of time.

44. The method of any one of embodiments 40-43 further comprising identifying an ultrasound contrast agent (UCA) formulation that requires activation for a predetermined period of time or at a predetermined shaking intensity, where optionally the UCA formulation is identified and activated with using a shaking device.

45. The method according to any one of embodiments 40-44, wherein the UCA formulation is an aqueous UCA formulation.

46. The method according to any one of embodiments 40-44, wherein the UCA formulation is a non-aqueous UCA formulation.

47. The method according to any one of embodiments 40-46, wherein the shaking device comprises a first holder configured to hold a container containing an aqueous UCA formulation and not to hold a container containing a non-aqueous UCA formulation.

48. The method according to any one of embodiments 40-46, wherein the shaking device comprises a first holder configured to hold a container containing a non-aqueous UCA formulation and not able to hold a container containing an aqueous UCA formulation.

49. The method according to embodiments 47 or 48, wherein the containers are vials.

50. The method according to any one of embodiments 40-46, wherein the shaker is configured to distinguish between an aqueous UCA formulation and a non-aqueous UCA formulation based on a unique identifier.

51. The method of any one of embodiments 41-50, wherein the shaking device comprises a detector.

52. The method of embodiment 51 wherein the detector is an RFID reader and the UCA is in a container that contains (includes) or is associated with or marked with an RFID tag/label.

53. The method of embodiment 51, wherein the detector is a barcode scanner and the UCA is in a container that contains (includes) or is associated with or labeled with a barcode.

54. The method of embodiment 51 wherein the detector is a color scanner and the UCA formulation is in a container that contains a color label.

55. The method according to any one of embodiments 40-54, wherein the shaking device reciprocates, rotates, or figures eight to the container containing the UCA formulation.

56. The method according to any one of embodiments 40, 41, and 44-55, wherein the predetermined time period is about 45 seconds if the UCA formulation is an aqueous UCA formulation and 60-120 seconds or about 75 seconds if the UCA formulation is a non-aqueous UCA formulation.

57. The method according to any one of embodiments 42-55, wherein the predetermined shaking intensity value is in the range of 4000-4800 strokes per minute or 4100-4700 strokes per minute.

58. A method of forming gas-filled microspheres, including the identification of a non-aqueous composition of an ultrasound contrast agent

- 30 042049 (UCA), which requires activation within a predetermined period of time; and allowing the UCA formulation to activate for a predetermined period of time to form gas-filled microspheres.

59. The method of Embodiment 58 wherein the UCA formulation is identified and activated using a shaking device configured to enable activation within a predetermined period of time or to automatically select a predetermined period of time based on identification information of the UCA formulation .

60. The method according to embodiments 58 or 59, wherein the predetermined time period is approximately 75 seconds.

61. A method for forming gas-filled microspheres, comprising identifying a non-aqueous ultrasound contrast agent (UCA) formulation that requires activation for a predetermined first period of time; and allowing said UCA composition to be activated to form gas-filled microspheres by shaking using a shaking device configured to automatically select a first predetermined time period from at least two different predetermined time periods based on the UCA composition identification information.

62. The method of Embodiment 61, wherein at least two different predetermined time periods are about 45 seconds and about 75 seconds.

63. A method for forming gas-filled microspheres comprising identifying a non-aqueous ultrasound contrast agent (UCA) formulation that requires activation at a predetermined first shaking intensity; and providing activation of said UCA formulation to form gas-filled microspheres by shaking using a shaking device configured to automatically select a first predetermined shaking intensity from at least two different predetermined shaking intensity values based on the identification information of the UCA composition.

64. The method of embodiment 63 wherein the non-aqueous UCA formulation is identified based on its container.

65. The method of Embodiment 64 wherein the container is a vial.

66. The method according to any one of embodiments 61-65, wherein the method is automated.

67. The method according to any one of embodiments 61-66, wherein the shaking device shakes the non-aqueous UCA formulation with a reciprocating motion.

68. The method according to any one of embodiments 61-67, wherein the shaking device comprises a detector.

69. The method of Embodiment 68 wherein the detector is an RFID reader and the non-aqueous UCA formulation is tagged with an RFID tag/label.

70. The method of embodiment 68, wherein the detector is a barcode scanner and the non-aqueous UCA formulation is labeled with a barcode.

71. The method of embodiment 68 wherein the detector is a color scanner and the non-aqueous UCA formulation is labeled with a color indicator.

72. The method of Embodiment 71 wherein the color indicator provides a color label.

73. The method of Embodiment 71 wherein the color indicator provides a color label.

74. A method for forming gas-filled microspheres, comprising providing activation of an ultrasound contrast agent (UCA) composition using a shaking device that is configured to identify the UCA composition and automatically select the activation time based on information about it, where the UCA composition is identified based on a unique an identifier other than the shape or size of the vial in which the UCA formulation is stored.

75. A method for providing activation of a first ultrasound contrast agent (UCA) composition using a shaking device that is configured to distinguish between a container containing a first UCA composition and a container containing a second UCA composition.

76. A method for forming gas-filled microspheres, comprising identifying a labeled vial containing an ultrasound contrast agent (UCA) formulation that requires activation within a predetermined first period of time; And

- 31 042049 providing activation of the UCA composition using a shaking device in which a predetermined time period is set or the possibility of automatically selecting the first predetermined time period from at least two different predetermined time periods based on vial identification information, with the formation of filled microsphere gas.

77. The method of formation of gas-filled microspheres, including the identification of a container containing the composition of the contrast agent for ultrasound (UCA), which requires activation within a predetermined first period of time; and providing activation of the UCA composition using a shaking device containing a detector and in which a predetermined time period is set or the ability to automatically select a first predetermined time period from at least two different predefined time periods based on container identification information, with the formation gas-filled microspheres.

78. The method of Embodiment 77 wherein the UCA formulation is an aqueous UCA formulation.

79. The method of Embodiment 77 wherein the UCA formulation is a non-aqueous UCA formulation.

80. The method according to any one of embodiments 77-79, wherein at least two different predetermined time periods are about 45 seconds and about 75 seconds.

81. The method according to any one of embodiments 77-80, wherein the method produces substantially similar gas-filled microspheres in a first vial and a second vial, provided that the first container is shaken for a first period of time and the second container is shaken for a second distinct period. time, and optionally the first time period is shorter than the second time period.

82. The method according to any one of embodiments 77-81, wherein the container is a labeled container.

83. The method according to any one of embodiments 77-82, wherein the container is a vial.

84. The method according to any one of embodiments 77-83, wherein the method is automated.

85. A method of forming gas-filled microspheres, comprising identifying an aqueous composition of an ultrasound contrast agent (UCA) that requires activation for a predetermined period of time, using a device that is configured to distinguish between an aqueous UCA composition and a non-aqueous UCA composition; and allowing the aqueous UCA composition to be activated for a predetermined period of time to form gas-filled microspheres.

86. The method of formation of gas-filled microspheres, including the identification of the composition of the contrast agent for ultrasound (UCA), which requires activation within a predetermined period of time; and providing activation of the UCA composition for a predetermined period of time to form gas-filled microspheres, where the UCA composition is identified and activated using a shaking device configured to automatically select a predetermined time period from at least 2 predetermined time periods based on the identification information about the composition of the UCA.

87. The method of Embodiment 86 wherein the UCA formulation is an aqueous UCA formulation.

88. The method of Embodiment 86 wherein the UCA formulation is a non-aqueous UCA formulation.

89. The method according to any one of embodiments 86-88, wherein the aqueous UCA formulation is activated for a shorter period of time than that of the non-aqueous UCA formulation.

90. The method according to embodiments 86 or 87, wherein the predetermined time period is approximately 45 seconds.

91. The method according to embodiments 86 or 88, wherein the predetermined time period is approximately 75 seconds.

92. A method for forming gas-filled microspheres, comprising identifying a vial containing an ultrasound contrast agent composition that requires activation for a predetermined first time period, using a shaking device configured to select a first time period from two predetermined periods time based on identification information about

- 32 042049 vial.

93. The method of embodiment 92, wherein the method is automated.

94. The method according to embodiments 92 or 93, wherein the method produces substantially similar gas-filled microspheres in a first vial and a second vial, provided that the first vial is shaken for a first time period and the second vial is shaken for a second different time period.

95. The method according to any one of embodiments 92-94, wherein the two predetermined time periods are about 45 seconds and about 75 seconds.

96. A method for visualizing a subject, comprising administering to a subject in need thereof, gas-filled microspheres obtained according to any of the above embodiments; and obtaining one or more images of the subject using ultrasound.

97. An apparatus capable of being used to form gas-filled microspheres according to any of the above-described embodiments.

98. The apparatus of Embodiment 97, further comprising a counter that counts the number of times the device has been used, the number of times the device has been shaken during the first time period, and/or the number of times the device has been shaken during the second time period.

99. A device that provides activation of an ultrasound contrast agent (UCA) formulation and that is configured to distinguish between an aqueous UCA formulation and a non-aqueous UCA formulation.

100. The apparatus of embodiment 99, wherein the apparatus provides activation of the aqueous UCA formulation for a shorter period of time than that of the non-aqueous UCA formulation.

101. The apparatus of embodiment 99 wherein the apparatus activates the aqueous UCA formulation at a first shaking intensity and the non-aqueous UCA formulation at a second shaking intensity.

102. A shaking device comprising a holder, a device for shaking the holder, wherein the holder shakes a vial containing an ultrasound contrast agent (UCA) formulation for various predetermined periods of time.

103. A shaking device according to embodiment 102, further comprising a device for automatically identifying a predetermined period of time during which the vial must be shaken to form gas-filled microspheres.

104. A shaking device according to embodiments 102 or 103, wherein the shaking device reciprocates the vial while it is in the holder.

105. A shaking device according to any one of embodiments 102-104, wherein the first predetermined time period is approximately 45 seconds.

106. A shaking device according to any one of embodiments 102-105, wherein the second predetermined time period is approximately 75 seconds.

107. A shaking device according to any one of embodiments 102-106, wherein the vial identification tool provides an RFID reader that responds to a first RFID tag by shaking the vial for a first period of time and which responds to a second RFID tag by shaking the vial for a during the second period of time, and the first and second periods of time are different.

108. A shaking device according to any one of embodiments 102-107, wherein the vial identification tool includes a microchip reader that responds to the first microchip by shaking the vial for a first time period and that responds to the second microchip by shaking the vial for a second time period.

109. A shaking device according to any one of embodiments 102-108, wherein the vial identification tool includes a barcode scanner that responds to a first barcode by shaking the vial for a first period of time, and which responds to a second barcode by shaking the vial for a second period of time.

110. A shaking device according to any one of embodiments 107-109, wherein the RFID label, microchip, or barcode is on the vial.

111. A shaking device according to any one of embodiments 102-110, wherein the UCA formulation is a non-aqueous UCA formulation.

112. A shaking device for forming gas-filled microspheres, comprising an identification device configured to identify and distinguish between a first vial and a second vial, each vial containing an ultrasound contrast agent (UCA) formulation, and an automated shaking device configured with the ability to perform shaking for only one of at least two different predetermined time periods based on the identification information

- 33 042049 bottle formations.

113. A shaking device according to embodiment 112, wherein at least two different predetermined time periods are about 45 seconds and about 75 seconds.

114. A shaking device according to embodiments 112 or 113, wherein the identification device comprises an RFID reader, a microchip reader, or a barcode scanner.

115. A shaking device for forming gas-filled microspheres, comprising a holder configured to identify and distinguish between a first vial and a second vial, each vial containing an ultrasound contrast agent (UCA) composition, and an automated device for shaking the vial in the holder in within one of two predetermined time periods based on whether the first vial or the second vial has been identified.

116. The shaking device according to embodiment 115, wherein the shaking device reciprocates the vial when it is in the holder.

117. A shaking device according to embodiments 115 or 116, wherein the holder accepts a first configuration if a first vial is present and a second configuration if a second vial is present, the first configuration indicating the presence of the first vial and the second configuration indicating the presence second vial.

118. The shaking device according to any one of embodiments 115-117, wherein the first predetermined time period is approximately 45 seconds.

119. The shaking device according to any one of embodiments 115-118, wherein the second predetermined time period is approximately 75 seconds.

120. A shaking device according to any one of embodiments 115-119, wherein the holder comprises an RFID reader, a microchip reader, or a barcode scanner.

121. The device or shaking device according to any of the above embodiments, further comprising a counter that counts the number of times the shaking device is applied, the number of times the shaking device shakes during the first time period, and/or the number of times the shaking device shakes during the second time period. .

122. A shaking device comprising a holder capable of identifying a vial containing an ultrasound contrast agent (UCA) composition and a device for shaking the holder, where the holder is configured to shake only for a predetermined period of time to form gas-filled microspheres based on vial identification information.

123. A shaking device comprising a holder, a device for shaking the holder, where the holder is configured to shake only for a predetermined period of time, and a device for identifying a vial containing an ultrasound contrast agent (UCA) composition when it is in the holder and then shaking the identified vial for a predetermined period of time to form gas-filled microspheres, where the vial identification tool includes an RFID reader, a microchip reader, or a barcode scanner.

124. A kit containing a device or shaker according to any of embodiments 97-123 with instructions for activating an ultrasound contrast agent (UCA) formulation.

125. A kit according to embodiment 124 further comprising a vial containing a formulation (UCA).

126. A kit according to embodiments 124 or 125, wherein the UCA formulation is a non-aqueous UCA formulation.

127. A permanent computer-readable medium programmed with a plurality of instructions that, when executed by at least one shaker processor, performs a method, the method comprising determining, based at least in part on identification information about the type of sample in the vial containing the contrast agent means for ultrasound examination (UCA), inserted into the holder of the shaking device, at least one performed action; and instructing the shaking device to perform the determined at least one action based at least in part on the identification information.

128. A shaking device, comprising a holder configured to identify the type of sample in a vial containing an ultrasound contrast agent (UCA) formulation inserted into the holder;

at least one storage device configured to store at least one data structure providing identification of one or more actions, you

- 34 042049 completed in relation to each of the many types of samples;

at least one processor programmed to access at least one data structure to determine one or more actions to be performed on the vial based on the identified sample type; and at least one component configured to perform one or more actions defined by at least one processor.

Examples

Example 1

A commercially available, FDA approved, aqueous formulation of UCA, DEFINITY® (Lantheus Medical Imaging, Inc.) is brought into active form (activated) by mechanical agitation (described in US Pat. No. 6,039,557, the contents of which are incorporated herein by reference and may be used in this method) PFP/lipids solution using VIALMIX®. This results in the incorporation of gas into the lipid microspheres and represents the active product (see DEFINITY® prescribing information). Optimal activation with VIALMIX® DEFINITY® consistently yields gas-filled microspheres that can be analyzed for number and size distribution using a particle meter (Malvern FPIA-3000 Sysmex) when diluted with saline solution with lower and upper limits of 1 and 80 microns.

In this experiment, DEFINITY® was activated for various time periods, DEFINITY-II was activated for 75 seconds, and the effect on average microsphere diameter and concentration was analyzed.

Vials (Nipro Glass Americas, Nipro, Cat. No. 2702, B33BA, 2 ^cc , 13 mm, Type I, Silica Cylindrical Vial) containing aqueous UCA (DEFINITY®) or non-aqueous UCA (DEFINITY-II) were activated with VIALMIX®. The formed microspheres were analyzed after resuspension for number and size distribution using a particle meter (Malvern FPIA-3000 Sysmex) when diluted with saline with a lower and upper limit of 1 and 80 microns. The activation time to obtain the optimal number of microspheres and equivalent diameter was different for the two products. Activating DEFINITY® by shaking for a longer time (75 s) than the recommended 45 s resulted in a significantly lower number of microspheres, but a similar average diameter, (see Tables 1 and 2).

Table 1

DEFINITY® with different activation times

DEFINITY® when activated 45 s DEFINITY® when activated 75 s vial number Average Diameter (µm) Quantity/ml x 10 ⁹ Average Diameter (µm) Quantity/ml x 1 10 ⁹ 1 1.36 2.56 1.49 0.86 2 1.35 2.41 1.52 0.81 3 1.39 2.64 1.46 0.89 Average 1.37 2.54 1.49 0.85

table 2

DEFINITY II

DEFINITY-II when activated 75 s vial number Average Diameter (µm) Quantity/ml x 1 10 ⁹ 1 1.55 4.95 2 1.49 5.05 3 1.48 4.74 Average 1.51 4.91

Example 2

A device that can distinguish and activate with the correct shaking period

A) vials with an aqueous composition of UCA (DEFINITY®) with a corresponding RFID tag;

C) non-aqueous UCA vials (DEFINITY-II) with the appropriate RFID tag; or

C) vials without labels or with incorrect labels,

- 35 042049 was created by modifying VIALMIX®.

An image showing the front panel of the device is shown in FIG. 2. The operation described in the following paragraph was carried out using an RFID tag reader installed in the inside of the lid of the device near the vial holder, together with an ATmega328P microcontroller to read the RFID tags on the vials in the holder and either facilitate or hinder the operation of the device on the base the presence or absence of a label with an identifiable unique identifier (UID). The RFID reader used contains the MFRC522 integrated circuit, which complies with ISO/IEC 14443A standards. The tags used are MIFARE Ultralite self-adhesive 50x15mm labels operating at 13.56MHz.

To activate a DEFINITY® (or DEFINITY-II) vial, the device is first turned on using the rocker switch on the rear panel, the lid is opened and the vial is placed in the vial holder as directed in the VIALMIX® operating instructions. An RFID tag reader is included in the VIALMIX® wiring so that the shaker can only be turned on by closing the lid along with identification of the corresponding tag by the RFID reader. When a tag with a UID associated with DEFINITY® is detected, the front panel green LED labeled DEFINITY® illuminates and pressing the start button on the front panel initiates activation within the standard 45 s period. When a tag with a UID associated with DEFINITY-II is detected, the green LED on the front panel labeled DEFINITY-II is illuminated and pressing the start button on the front panel initiates activation for a total of 75 s (standard period of 45 s followed by additional activation period of 30 s). When a conforming ISO 14443ARFID tag with an unrecognizable UID is identified, the red LED labeled as other lights up and pressing the start button on the front panel does not initiate activation. When there is no corresponding ISO 14443ARFID tag, none of the LEDs are illuminated and pressing the start button on the front panel again does not start the activation.

The above sequence was used in an experiment with a DEFINITY® vial having a conforming ISO 14443A RFID tag recognized by the microcontroller as conforming to DEFINITY®. When the vial was placed in the holder and the lid was closed, the DEFINITY® LED was illuminated and the vial was activated. Subsequent particle size distribution analysis with a Malvern FPIA-3000 Sysmex indicated that the microsphere size spectrum and total bubble concentration were within acceptable limits for activated DEFINITY® (see Table 3). Similarly, the experiment was performed with a vial of DEFINITY-II having a conforming ISO 14443A RFID tag, recognized by the microcontroller as conforming to DEFINITY-II. When the vial was placed in the holder and the lid was closed, the DEFINITY-P LED was illuminated and the vial was activated. The non-aqueous contrast medium was resuspended with 0.9% saline and then analyzed for particle number and particle size using a Malvern FPIA-3000 Sysmex. Testing has shown that both DEFINITY® and DEFINITY-P, with the appropriate RFID tag, can activate. The microsphere size was very similar to DEFINITY® and the total number of microspheres was approximately 1.8 times higher.

Table 3

Vial with DEFINITY® with RFID

DEFINITY® when activated 45 s DEFINITY-II when activated 75 s vial number Average Diameter (µm) Quantity/ml x 10 ⁹ Average Diameter (µm) Quantity/ml x 10 ⁹ 1 1.36 2.10 1.60 3.96 2 1.39 2.41 1.54 4.48 3 1.36 2.37 1.49 4.02 Average 1.37 2.29 1.54 4.15

A front view of VIALMIX® modified for RFID recognition with RFID tagged vials is shown in FIG. 2.

- 36 042049

An example of software used to provide RFID recognition.

/* * This allows the shaker to read HF (ie Mifare) RFID tags and distinguish between DEFINITY®, DEFINITY-II - DGII - and missing/invalid * RFID tag. For this demo only, the appropriate LED for either DEFINITY® or DGII will light up and allow the activation to proceed. For an unrecognizable but readable RFID tag, the red LED will light up and activation will be blocked. For a missing/unrecognized RFID tag, no LED will light up and activation will be blocked * This is derived from the MFRC522 example in the literature called Dumpinfo.ino; Cm.

* website github.com, miguelbalboa, rfid for more details and other examples.

* A typical schematic/program showing how to read data from a PICC (namely an RFID tag or card) using an MFRC522 based RFID reader on the Arduino SPI interface.

* When Arduino and MFRC522 module are connected (see pinout * below), enter this circuit into Arduino IDE, then validate/compile and upload it.

* To see the output use Tools, IDE Serial Control (press Ctrl+Shft+M).

* When you bring a PICC (namely an RFID tag or card) within the reading distance * of the MFRC522 reader/PCD, the serial output will show the ID/UID, type, and * any data blocks it can read. Note: You may see Communication Waiting* messages if the PICC is too early in read range.

* If your reader supports it, this circuit/program will read all * submitted PICCs (namely, reading multiple tags). So if you place two or more * PICCs on top of each other and present them to the reader, it will output all the data * first, then the next PICC. Note that * it may take some time as all data blocks are swapped out, so keep the PICC at * read distance until complete.

* Commonly used pinout:

* MFRC522 Arduino Arduino Arduino Arduino Arduino •A· Reader/PCD Uno Mega Nano v3 Leonardo/Migo * Signal Pro Micro Conclusion Conclusion Conclusion Conclusion Conclusion Conclusion (for shaking

devices)

- 37 042049 * RST/Reset RST 9 5 D9

Reboot/1СЗР-5 RST

* SPI ss SDA(SS) 10 53 D10 10 10 SPI MOST MOST eleven / ICSP-4 51 Dll ICSP-4 16 SPI MISO MISO 12 / ICSP-1 50 D12 ICSP-1 14 SPI SCK SCK 13 / ICSP-3 52 D13 ICSP-3

* Pins used for other functions:

* Pin D1: Not used * Pin D2: Activates green DEFINITY® LED * Pin D3: Activates green LED DEFINITY-II * Pin D4: Activates red LED Other * Pin D5: Control relay to send lid open/close signal to shaker PCB * Pin D6: Reads the lid open/close microswitch * Pin D7 Reads the START button on the front panel * Pin D8: Reads the CANCEL button on the front panel * Pins D9 - D13 are used to communicate with the RFID reader, see the table above, using the column for Arduinon Nano V3 * Pin A3: Control relay to send the Start signal to the shaker PCB * Pin A2: Control relay to send the Cancel signal to the shaker PCB */ #include <SPI.h>

#include <MFRC522.h>

#define RST_PIN 9 // #define SS_PIN 10 MFRC522 mfrc522(SS_PIN, RST _PIN); // II Creates copy 1 4FRC522 II constants - pin numbers const int DLEPin = 2; II number output green LED DEFINITY® const int DGIILEDPin - 3; // number output green LED DEFINITY- II Gen II const int RedLEDPin — 4; // number output red LED unidentified const int CoverRelayPin — 5; // number output relay const int CoverSwitchPin - 6; // number output ₍ , connected WITH cover microswitch const int STARTRelayPin = A3; // number output relay START const int CANCELRelayPin - A2; // number output relay CANCEL const int STARTButtonPin - 7; // number output START buttons const int CANCELButtonPin - 8; // number output CANCEL buttons

- 38 042049 // more uid constants for recognizable tags:

const int uidl[] const int uid2[] const int uid3[] const int uid4[] const int uid5[] const int uid6[] const int uid7[] const int uid8[] const int uid9[] const int uidl0[] {0x4, OxEl, 0x8B, 0xD2, {0x4, 0x30, 0x8B, 0xD2,

0x11,

0x11, {0x4, 0x7B, 0x8B, 0xD2, 0x11, {0x4, 0x62, 0x8B, 0xD2, 0x11, {0x4, 0x7A, 0x8B, 0xD2, 0x11, {0x4, 0x61, 0x8B, 0xD2, 0x11, OxAD , 0x8B, 0xD2, 0x11, {0x4, 0x96, 0x8B, 0xD2, 0x11, {004, OxAC, 0x8B, 0xD2, 0x11, {0x4, 0x95, 0x8B, 0xD2, 0x11,

0x20, 0x80};

0x20, 0x80};

0x20, 0x80};

0x20, 0x80};

0x20, 0x80};

0x20, 0x80};

0x20, 0x80);

0x20, 0x80};

0x20, 0x80};

0x20, 0x80};

//label #1 //label #2 //label #3 //label #4 //label #5 //label #6 //label #7 //label #8 //label ##9 //label # 10 //tag number of recognizable elements //Variables int TagNumFound = 0; unsigned long Timed; int CancelCondx=0;

//------------------------------------------------ --------------void setup() {

II Variables:

mfrc522.PCD_Init() ; // Init MFRC522

ShowReaderDetails(); II Show data of PCD reader - MFRC522 cards

II installation of digital and analog pins as output or input, when need : pinMode(DLEDPin, OUTPUT); pinMode(DGIILEDPin, OUTPUT); pinMode(RedLEDPin, OUTPUT); pinMode(CoverRelayPin, OUTPUT); pinMode(CoverSwitchPin, INPUT); pinMode(STARTRelayPin, OUTPUT); pinMode(STARTButtonPin, INPUT); pinMode(CANCELRelayPin, OUTPUT); pinMode(CANCELButtonPin, INPUT);

//------------------------------------------------ ---------------//--------------------------------- ------------------------------void loop ( ) { byte Bytel;

ClearLights();

digitalWrite(CoverRelayPin,LOW);

CancelCondx=0;

//wait until the lid is open, turning off the relay again and again....

while (digitalRead( CoverSwitchPin)==HIGH) { digitalWrite(CoverRelayPin,LOW);

}

- 39 042049 // as soon as the cover is closed, RFID reading starts // Searching for new cards if ( ! mfrc522.PICC IsNewCardPresent () ) { return;

)

II Selecting one of the cards if ( ! mf rc522.PICC ReadCardSerial()) { return;

} //get second byte uid RFID

Bytel=GetBytel(mfrc522.uid.uidByte, mfrc522.uid.size);

//get the detected tag number (1-10), based on this second UID byte

TagNumFound = Get tag number(Bytel) ;

II Performing the appropriate action for the label found:

// For tags 8 and 9 (DEFINITY®) or 3 and 10 (DGII), turn on the corresponding green LED // and turn on the relay // for all others, keep the relay off and turn on the red LED switch (TagNumFound) { case 0:

UnrecognizedTag();

break;

case 1:

UnrecognizedTag();

break;

case 2 :

UnrecognizedTag();

break;

case 3:

DEFINITYGenllTag();

break;

case 4 :

UnrecognizedTag();

break;

case 5:

UnrecognizedTag();

break;

case 6:

UnrecognizedTag();

break;

case 7 :

UnrecognizedTag();

break;

case 8 :

DEFINITYTag();

break;

case 9:

DEFINITY®Tag();

break;

case 10 :

DEFINITYGenllTag();

break;

} //wait until cover is closed while (digitalRead (CoverSwitchPin)==LOW) {

- 40 042049 //then the relay immediately opens when the cover is opened digitalWrite(CoverRelayPin,LOW);

ClearLights();

II Output debugging information about the card; PICO HaltA() is automatically called //mfrc522. PICO DumpToSerial ( &(mfrc522.uid) ) ;

//then return and repeat this again.... } AND------------------------------------ --------------------------------void ClearLights ( ) { digitalWrite(RedLEDPin, LOW); digitalWrite(DGIILEDPin, LOW); digitalWrite(DLEPin, LOW);

} //---------------------------------------------- ---------------void UnrecognizedTag() { digitalWrite(RedLEDPin, HIGH);

digitalWrite(CoverRelayPin, LOW); return;

} //---------------------------------------------- ---------------void DEFINITY®Tag() { digitalWrite(DLEPin, HIGH); //turn on the green LED DEFINITY® //Firstly, wait for the START signal, then continue if the lid is closed do { //wait for the START button to be pressed - LOW for BUEEOPRPZ=Pressed while (digitalRead(STARTButtonPin)==HIGH) { } } while (digitalRead( CoverSwitchPin)==HIGH) ; //start shaking only if cover switch is closed - LOW=3aKpbiT digitalWrite(CoverRelayPin,HIGH) ;

//Second, send a START signal to the shaking device (hold it for 100ms) digitalWrite(STARTRelayPin, HIGH); delay(100) ; digitalWrite(STARTRelayPin, LOW);

//Third, while shaking is in progress, maintain the test of the CANCEL button and the lid switch, and hold for the required time.

// Exit if the lid is open or CANCEL is pressed // just to make sure the wait is a little longer than 45s Time0 = millis();

while (millis()-TimeO <45500){ if (digitalRead (CANCELButtonPin)==LOW) { digitalWrite(CANCELRelayPin, HIGH);

delay(100); digitalWrite(CANCELRelayPin, LOW); break;

-41 042049 if (digitalRead (CoverSwitchPin)==HIGH) { digitalWrite(CoverRelayPin, LOW); break;

} } II then finished with return;

} //---------------------------------------------- ---------------void DEFINITYGenllTag() { digitalWrite(DGIILEDPin, HIGH); //turn on green LED DGII

II //Firstly, wait for the START signal, then continue if the cover is closed do { //wait for the START button to be pressed - LOW for bubbinPinz=Pressed while (digitalRead(STARTButtonPin)==HIGH) { } } while (digitalRead(CoverSwitchPin) ==HIGH); //start shaking only if cover switch is closed - LOW=3aKpbiT digitalWrite(CoverRelayPin,HIGH);

//Second, send START signal to uc VIALMIX® (hold it for 100ms) digitalWrite(STARTRelayPin, HIGH); delay(100); digitalWrite(STARTRelayPin, LOW);

//Third, while shaking is in progress, maintain the test of the CANCEL button and the lid switch, and hold for the required time.

// Exit if the lid is open or CANCEL is pressed // just to make sure the wait is a little longer than 45s Time0 = millis();

while (millis()-TimeO <45500){ if (digitalRead (CANCELButtonPin)==LOW) { digitalWrite(CANCELRelayPin, HIGH);

delay(100); digitalWrite(CANCELRelayPin, LOW); CancelCondx=l; break;

} if (digitalRead (CoverSwitchPin)==HIGH) { digitalWrite(CoverRelayPin, LOW); CancelCondx=l; break;

) } if (CancelCondx==l) return;

//additional time for DGII

- 42 042049

II switch open and closed cover digitalWrite(CoverRelayPin, LOW); delay(200); digitalWrite(CoverRelayPin, HIGH);

//pressing the START button (holding it down for 100ms) digitalWrite(STARTRelayPin, HIGH); delay(100); digitalWrite(STARTRelayPin, LOW); //wait 30 more seconds TimeO = millis () ;

while (millis()-TimeO < 30000)( if (digitalRead (CANCELButtonPin)==LOW) { digitalWrite (CANCELRelayPin, HIGH); delay (100); digitalWrite(CANCELRelayPin, LOW); break; } if (digitalRead (CoverSwitchPin) ==HIGH) { digitalWrite (CoverRelayPin, LOW); break; } } //then stop it after 30s digitalWrite (CANCELRelayPin, HIGH); delay(200); digitalWrite(CANCELRelayPin, LOW);

// toggle open and closed cover again to turn off beeper digitalWrite(CoverRelayPin, LOW); delay(500);

digitalWrite(CoverRelayPin, HIGH); return; } //---------------------------------------------- ---------------byte GetBytel(byte *buffer, byte buffersize) { return buffer[l]; } //---------------------------------------------- ---------------int Get tag number(byte FirstByte) { int tag=0; switch (FirstByte) { case 0xA6 : tag=l; break; case 0x85:

- 43 042049 tag=2;

break;

case 0x7B:

tag=3; break;

case 0x62:

tag=4;

break;

case 0x7A:

tag=5; break;

case 0x61:

tag=6; break;

case OxAD:

tag=7;

break;

case 0x96:

tag=8; break;

case OxAC:

tag=9; break;

case 0x95:

tag=l0; break;

} return tag;

} //---------------------------------------------- ---------------void ShowReaderDetails() { // Get the MFRC522 software version byte v = mfrc522.PCD ReadRegister(mfrc522.VersionReg);

Serial. print (F(MFRC522 Software Version: Ox));

Serial. print(v, hex); if(v==0x91)

Serial. print(F(=vl.0));

else if (v == 0x92)

Serial. print(F(=v2.0));

else

Serial. print(F( (unknown)));

Serial. print();

// When 0x00 or OxFF is returned, there is probably no connection if ((v == 0x00) II (v == OxFF)) {

Serial. printin (F(WARNING: Communication failure, is the MFRC522 properly connected?));

} }

Example 3

A device that can distinguish and activate with the correct shaking period A) vials with an appropriately barcoded UCA (DEFINITY®) aqueous formulation, B) vials with an appropriately barcoded UCA non-aqueous formulation (DEFINITY-II) or C) vials without a barcode or with wrong barcode was created by modifying VIALMIX®. An image showing the front panel of the device is shown in FIG. 3. The operation described in the following paragraph was carried out using a barcode scanner located near the vial holder, together with a computer to read the barcodes on the vial in the holder and either facilitate or hinder the operation of the device based on the presence or absence of a barcode.

- 44 042049 yes with identifiable identification number. The barcode scanner used was a standard keyboard simulator connected via USB. Barcodes for demonstration purposes were generated online using the barcodesinc website, generator, index.php.

To activate a DEFINITY® or DEFINITY-II vial, the device is first turned on using the rocker switch on the rear panel, the lid is opened, and the vial is placed in the vial holder as directed in the operating instructions. A barcode scanner is included in the VIALMIX® wiring so that the shaker can only be turned on by closing the lid and identifying the appropriate barcode label with the barcode scanner. When a barcode associated with DEFINITY® is detected, pressing the start button on the front panel initiates activation. When a barcode associated with DEFINITY-II is detected, pressing the start button on the front panel also initiates activation. When a barcode with an unrecognized identifier is read, pressing the start button on the front panel does not initiate activation. Similarly, when there is no barcode, pressing the start button on the front panel does not initiate activation.

A front view of VIALMIX® modified to be recognized by a linear/barcode scanner with barcoded (or labeled) vials is shown in FIG. 3.

Example 4

A device that can distinguish between A) vials containing an aqueous UCA formulation (DEFINITY®) and B) vials containing a non-aqueous UCA formulation (DEFINITY-II) was created by modifying the vial holder on the VIALMIX® shaker. The vial holder lever on the VIALMIX® device has been modified to allow for limited vial holding, shaking and activation according to acceptable product specifications while preventing insertion of a larger vial. Vial discrimination was achieved by the design of a holder tube attached to a shaker arm with a diameter that would accommodate a smaller vial (Schott, West Pharmaceuticals, #6800-0314) but not a larger commercial DEFINITY® vial (Nipro Glass Americas, Nipro, cat. no. 2702, B33BA, 2 cm ³ , 13 mm, type I, silicon cylindrical vial), e.g. An image showing the vial holder is shown in FIG. 4. A non-aqueous formulation of UCA was made and filled into a smaller Schott vial. This vial, easily inserted into the holder tube on the shaker arm by a spring on the holder base to keep the vial from moving in the holder, was fixed with a screw on the cap and activated by shaking. An aqueous formulation of UCA, DEFINITY®, was made and dosed into an existing commercial Nipro Glass Americas, Nipro, cat. No. 2702, B33BA, ² cc, 13 mm, type I, silicon cylindrical vial. The diameter of this vial prevented it from fitting into the holder tube and prevented its activation. In an additional study, DEFINITY® was made and filled into a smaller Schott vial placed in a holder tube and activated. Non-aqueous contrast agent in Nipro Glass Americas, Nipro, cat. No. 2702, B33BA, 2 cm ³ , 13 mm, type I, silicon cylindrical vial did not fit the holder tube.

An experimental study was performed using a modified shake arm tube design and a vial with a diameter of 14.5-15 mm and a height of 35.0-35.3 mm (stopper/snap cap measurements) with either DEFINITY® shaken for 45 seconds, or with DEFINITY-II shaking for 75 seconds. Subsequent particle size distribution analysis with a Malvern FPIA-3000 Sysmex indicated that either DEFINITY® or DEFINITY-II could be activated in a VIALMIX® with a tubular shake arm design if they were made in an appropriately sized vial. In accordance with previous experience after resuspension, the microsphere diameters are similar, but more microspheres are formed with DEFINITY-II (Table 4).

Table 4

With modified vial holder

DEFINITY® when activated 45 s DEFINITY-II when activated 75 s vial number Average Diameter (µm) Quantity/ml x 10 ⁹ Average Diameter (µm) Quantity/ml x 10 ⁹ 1 1.39 2.16 1.60 3.81 2 1.38 2.29 1.46 4.37 3 1.40 1.95 1.38 4.97 Average 1.39 2.13 1.48 4.38

A front view of VIALMIX® modified to hold vials of various shapes and/or sizes is shown in FIG. 4.

Example 5

A device that can be used to change the shaking speed of DEFINITY® and DEFINITY-II was developed by replacing the AC motor in the VialMix with a Brushless DC motor (Trinamic QBL4208-100-04-025) and using a controller (TMCM-1640) and a source

- 45 042049 DC Velleman Inc. 24 volts to control speed and activation time. In this experiment, vials (Nipro Glass Americas, Nipro, Cat. No. 2702, B33BA, 2 ^cc , 13 mm, Type I, Silica Cylindrical Vial) containing an aqueous UCA formulation (DEFINITY®) or containing a non-aqueous UCA formulation (DEFINITY-II ), activated for different times and at different speeds. The formed microspheres were analyzed after resuspension for number and size distribution using a particle meter (Malvern FPIA-3000 Sysmex) with lower and upper limits of 1 and 80 microns. The activation time for various shaking speeds to obtain the optimal number of microspheres and equivalent diameter was different for the two products. In general, increasing the shaking speed reduced the time (see Tables 5 and 6). The longer shaking time required for DEFINITY-II compared to DEFINITY® can be overcome by slightly increasing the shaking speed. DEFINITY® shaking time can be reduced by increasing the shaking speed.

Table 5

DEFINITY II

RPM Activation time (s) Diameter (µm) Quantity/ml χ 10 ⁹ 4900 120 1.47 5.54 5200 60 1.47 5.87 5300 45 1.61 4.90 5400 45 1.43 5.99 5500 35 1.30 5.83

Table 6

DEFINITY®

RPM Activation time (s) Diameter (µm) Quantity/ml χ 10 ⁹ 4300 45 1.45 3.39 4800 25 1.50 2.50

Equivalents.

While several embodiments of the present invention have been described and illustrated herein, a number of other methods and/or structures for performing the function and/or obtaining the results and/or one or more of the benefits described herein are readily anticipated by those of ordinary skill in the art, and each of such variations and/or modifications is considered to be within the scope of the embodiments of the present invention described herein. In general, those skilled in the art will readily appreciate that all specifications, dimensions, materials, and designs described herein are by way of example and that the actual specifications, dimensions, materials, and/or designs will depend on the particular application or applications, in respect of which the ideas of the present invention are used. Those skilled in the art will recognize, or be able to determine with no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Therefore, it should be understood that the above embodiments are by way of example only and that, within the scope of the appended claims and their equivalents, embodiments of the present invention may be practiced in a manner other than specifically described and claimed. Embodiments of the present invention are directed to each individual feature, system, article, material, kit and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits and/or methods, unless such features, systems, articles, materials, kits and/or methods are mutually incompatible, is included within the scope of the present invention.

All definitions as defined and used herein are to be understood as taking precedence over dictionary definitions, definitions in documents incorporated by reference, and/or the ordinary meaning of certain terms.

All references, patents and patent applications disclosed in this document are incorporated by reference to the subject matter for which each is cited, which in some cases may cover the entire document.

The singular form when used in the description and claims of this document, unless expressly stated otherwise, should be understood to mean at least one.

The phrase and/or, as used herein in the description and in the claims, should be understood to mean either one or both of the elements thus combined, i.e. elements that in some cases are present together and in other cases are present separately. The plurality of elements listed using and/or should be interpreted in the same way, i.e. one or more elements combined in this way. Other elements may optionally be present other than the elements specifically defined under the pointer and/or whether or not they are related to such elements as specifically defined. Thus, as a non-limiting example, reference to A and/or B when used in conjunction with from

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Claims (20)

a catch phrase, such as containing, may refer in one embodiment to only A (optionally including elements other than B); in another embodiment, only to B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements), and so on. As used herein in the specification and in the claims, or should be understood to have the same meaning as and/or as defined above. For example, when separating objects in a list, either or and/or should be understood to be inclusive, i.e. including at least one, but also including more than one of the series or list of elements, and optionally additional non-listed items. Only terms expressly indicating otherwise, such as only one of, or exactly one of, or, when used in the claims, consisting of, will refer to the inclusion of exactly one element from a series or list of elements. In general, the term or, as used herein, should only be interpreted as indicating exclusive alternatives (i.e., one or the other, but not both) when preceded by exclusivity terms such as any of two, one of, only one from or exactly one of. Consisting principally of when used in a claim shall have its usual meaning as used in the field of patent law. As used herein in the description and in the claims, the phrase at least one in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more elements in the list of elements, but not necessarily including at least one of absolutely all elements specifically specified in the list of elements and not excluding any combination of elements in the list of elements. This definition also allows that elements other than those specifically defined in the list of elements to which the phrase at least one refers may be present, whether or not they are related to such elements as specifically defined. Thus, as a non-limiting example, at least one of A and B (or equivalently at least one of A or B, or equivalently at least one of A and/or B) may refer in one embodiment to at least one, optionally including more than one A, with B absent (and optionally including elements other than B); in another embodiment, at least one, optionally including more than one B, where A is absent (and optionally including elements other than A); in another embodiment, at least one, optionally including more than one A, and at least one, optionally including more than one B (and optionally including other elements), etc. It should also be understood that, unless expressly stated otherwise, in any methods claimed herein that include more than one step or step, the order of the steps or steps of the method is not necessarily limited to the order in which the steps or steps of the method are listed. In the claims, as well as in the description above, all transitional phrases such as containing, including, carrying, having, accommodating, providing for, holding, consisting of, etc., should be understood as open, i.e. meaning inclusive without limitation. Only passphrases consisting of and consisting primarily of will be closed or semi-closed passphrases, respectively, as set out in Section 2111.03 of the Patent Examining Manual of the US Patent Office. CLAIM 1. A method of forming gas-filled microspheres, including determining the identification information of the composition of the ultrasound contrast agent (UCA) in a vial placed in the device, and the vial includes an indicator, where the device contains a detector for reading the indicator and where determining the identification information of the UCA composition includes the ability to distinguish between aqueous UCA and non-aqueous UCA based on the indicator on the vial; determining the action to be taken on the vial based on the identity of the UCA formulation; and performing an action on the vial, wherein a processor embedded in the device instructs the components of the device to perform an action, where the action performed is the activation of the UCA composition with predetermined shaking intensities and/or for a predetermined period of time to form gas-filled microspheres where the detector is an RFID reader, a barcode scanner, a color scanner, or a microchip reader. 2. The method according to claim 1, where the device is a shaking device that contains a holder, and where the determination of the action to be performed includes the selection of the shaking intensity from at least two predetermined shaking intensity values and/or the selection from at least two predetermined periods time and where The action includes activating the UCA formulation at a selected shaking intensity and/or for a selected period of time using a shaking device to form gas-filled microspheres. 3. The method according to claim 1 or 2, where at least two different predetermined time periods are selected from about 45, 60-120, or 75 seconds. 4. A method according to one of the preceding claims, wherein at least two different predetermined time periods are about 45 seconds and about 75 seconds. 5. Method according to one of the preceding claims, wherein at least two predetermined shaking intensity values are selected from at least 1000, at least 2000, at least 3000, at least 4000, at least 4500 or at least 5000 shaking movements per minute. 6. The method according to one of the preceding claims, wherein the predetermined shaking intensity values are 4530 shaking movements per minute and the activation is performed for 45 seconds. 7. The method according to one of the preceding claims, wherein the activation of the aqueous UCA formulation and the non-aqueous UCA formulation is performed with different shaking intensity and the shaking duration is constant. 8. The method according to claim 7, wherein the shaking time is approximately 45 seconds. 9. A method of using gas-filled microspheres for ultrasound imaging, comprising forming gas-filled microspheres as defined in any one of claims 1-8. 10. The method according to any one of claims 1 to 9, wherein the device further comprises at least one storage device configured to store a look-up table that stores information about the action being performed. 11. The method according to any one of claims 1 to 10, wherein the device further comprises a battery. 12. The method according to any one of claims 1-11, wherein the non-aqueous UCA formulation contains DPPA, DPPC, MPEG5000-DPPE, propylene glycol, glycerin and perfluorocarbon gas and contains less than 5% water (w/w). 13. The method according to any one of claims 1-12, wherein the aqueous UCA formulation contains DPPA, DPPC and MPEG5000-DPPE in aqueous solution together with gaseous perflutren. 14. The method according to any one of claims 1-12, wherein the aqueous UCA formulation comprises DPPA, DPPC and PEG5000-DPPE in aqueous solution along with gaseous perfluorocarbon. 15. Shaking device for the formation of gas-filled microspheres containing the holder; a detector selected from an RFID reader, a barcode scanner, a color scanner, or a microchip reader, where the detector is configured to identify and distinguish between a first vial and a second vial based on an indicator of each vial, wherein the first vial contains an aqueous formulation of ultrasonic contrast agent (UCA) , and the second vial contains a non-aqueous ultrasound contrast agent (UCA) formulation; and a processor configured to perform the following method: determining, based on the identification information of the UCA composition in the vial inserted into the holder, at least one action to be performed; issuing a command to the shaking device to perform certain at least one action based on the identification information; and an automated shaking device configured to shake at a predetermined intensity and/or for a predetermined period of time based on vial identification information. 16. The shaking device according to claim 15, wherein the device further comprises at least one storage device configured to store a lookup table that stores information about the action being performed. 17. Shaking device according to claim 15 or 16, where the device further comprises a battery. 18. A shaking device according to any one of claims 15 to 17, wherein the action performed is a predetermined shaking intensity selected from two or more predefined shaking intensity values and/or a predetermined period of time selected from two or more predetermined periods time. 19. Shaking device according to any one of claims 15 to 18, comprising (a) a plurality of processors; or (b) at least one storage device configured to store a look-up table or data structure that stores information about an action performed on particular identities. 20. The shaking device according to any one of claims 15-19, wherein the non-aqueous UCA composition contains DPPA, DPPC, MPEG5000-DPPE, propylene glycol, glycerin, and gaseous perfluorocarbon and contains -