EA040910B1 - METHODS FOR OBTAINING CONTRAST MEDIA FOR ULTRASOUND EXAMINATION - Google Patents

Description

Prior Art

Gas-encapsulated lipid microspheres are used as contrast agents in ultrasound imaging applications.

Short description

The present invention provides improved methods for the preparation of phospholipid-based ultrasound contrast agents. The present invention is based in part on the unexpected discovery that certain phospholipid-based ultrasound contrast agent formulations are sensitive to divalent metal cations. In the presence of specific levels of certain such cations, the phospholipids and possibly other components in the formulation precipitate, rendering the formulation unusable. Until now, it has not been understood that certain divalent metal cations have the ability to have such a negative effect on the composition of the ultrasound contrast agent.

Based on these results, the present invention contemplates improved methods for the synthesis of such formulations that prevent such unwanted precipitation of phospholipids, as well as formulations resulting from such methods. Also provided are methods for using these improved formulations in the synthesis of improved ultrasound contrast agents and their use in imaging subjects in need thereof.

Thus, in one aspect, provided herein is a method for preparing a suspension of phospholipids comprising providing stock DPPA, DPPC, and MPEG5000-DPPE, measuring calcium concentration in one or more or all of the stock DPPC, DPPA, and MPEG5000-DPPE, combining stock DPPA , DPPC and/or MPEG5000-DPPE with a non-aqueous solvent to form a phospholipid solution and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. In some embodiments, the method further comprises measuring the concentration of calcium in the non-aqueous solvent. In some embodiments, the pooled measured calcium concentration of the DPPA, DPPC, and/or MPEG5000-DPPE parent forms is low (i.e., the sum or pooled calcium concentration of the measured parent forms, whether or not the measured parent forms are DPPA alone, DPPC alone, MPEG5000-DPPE alone, DPPA and DPPC, DPPA and MPEG5000-DPPE, DPPC and MPEG5000-DPPE, or DPPA, DPPC and MPEG5000-DPPE, is low).

In some embodiments, the combined measured calcium concentration in the DPPA, DPPC, and/or MPEG-DPPE stock forms and the non-aqueous solvent is low (i.e., the sum or pooled calcium concentration for the measured components, whether or not these measured stocks are separately DPPA or DPPA and non-aqueous solvent, separately DPPC or DPPA and non-aqueous solvent, separately MPEG5000-DPPE or MPEG5000-DPPE and non-aqueous solvent, DPPA and DPPC or DPPA, DPPC and non-aqueous solvent, DPPA and MPEG5000-DPPE or DPPA, MPEG5000-DPPE and non-aqueous solvent, DPPC and MPEG5000-DPPE or DPPC, MPEG5000-DPPE and non-aqueous solvent, DPPA, DPPC and MPEG5000-DPPE or DPPA, DPPC, MPEG5000-DPPE and non-aqueous solvent, is low).

In some embodiments, the calcium concentrations in the original forms of DPPC, DPPA, and MPEG5000-DPPE are measured.

In some embodiments, the calcium concentrations in the original forms of DPPC, DPPA, and MPEG5000-DPPE are measured, and the combined measured calcium concentration in the original forms of DPPA, DPPC, MPEG-DPPE, and the non-aqueous solvent is low.

Thus, depending on the embodiment, only the calcium concentration in DPPA is measured, or only the calcium concentration in DPPC is measured, or only the calcium concentration in MPEG5000-DPPE is measured, or only the calcium concentrations in DPPA and DPPC are measured (and such concentrations are summed to obtain a combined measured calcium concentration), or only the calcium concentrations in DPPA and MPEG5000-DPPE are measured (and such concentrations are summed to obtain a combined measured calcium concentration), or only calcium concentrations in DPPC and MPEG5000-DPPE are measured (and such concentrations are summed to obtain a combined measured calcium concentration calcium) or measure only the calcium concentrations in DPPA, DPPC and MPEG5000-DPPE (and such concentrations are summed to give a combined measured calcium concentration), or only calcium concentrations in DPPA and non-aqueous solvent are measured (and such concentrations are summed to give a combined measured calcium concentration ), or change measure only calcium concentrations in DPPC and non-aqueous solvent (and such concentrations are summed to give a combined measured calcium concentration), or measure only calcium concentrations in MPEG5000-DPPE and non-aqueous solvent (and such concentrations are summed to give a combined measured calcium concentration), or measure only calcium concentrations in DPPA, DPPC and non-aqueous solvent (and such concentrations are summed to give a combined measured calcium concentration), or only calcium concentrations in DPPA, MPEG5000-DPPE and non-aqueous solvent are measured (and such concentrations are summed to give a combined measured calcium concentration), or measure only calcium concentrations in DPPC, MPEG5000-DPPE and non-aqueous solvent (and such concentrations are summed to give a combined measured calcium concentration), or calcium concentrations are measured in DPPA, DPPC, MPEG5000-DPPE and non-aqueous solvent (and such concentrations are summed with receipt of combined measured calcium concentration). It should be clear that each combination of components is considered when obtaining a combined measured or characteristic (as discussed below) calcium concentration. It should be understood that the terms DPPA, DPPA lipid, DPPA phospholipid, DPPA parent, DPPA lipid parent, and DPPA parent phospholipid are to be used interchangeably unless the context clearly indicates otherwise. Similar interchangeable terms are used for DPPC and MPEG5000-DPPE.

In another aspect, a variant of the above method is provided. Such a method includes providing stock DPPC and MPEG5000-DPPE, measuring the calcium concentration in one or both of the stock DPPC and MPEG5000-DPPE, combining stock DPPC and MPEG5000-DPPE with a non-aqueous solvent to form a phospholipid solution, and combining the phospholipid solution with an aqueous solvent. to form a suspension of phospholipids. In some embodiments, the method further comprises measuring the concentration of calcium in the non-aqueous solvent. The various embodiments above apply equally to this method and should be understood as such.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising providing stock forms of DPPA, DPPC and MPEG5000-DPPE, measuring the calcium concentration in one or more or all of the stock forms of DPPC, DPPA and MPEG5000-DPPE, combining stock forms of DPPA, DPPC and /or MPEG5000-DPPE, characterized by combined measured low concentration of calcium, with a non-aqueous solvent to form a solution of phospholipids and combining the solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the method further comprises measuring the calcium concentration in the non-aqueous solvent and the DPPA, DPPC, MPEG500-DPPE stocks, wherein the non-aqueous solvent has a combined measured low calcium concentration.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining the original form of MPEG5000-DPPE, the original form of DPPA, the original form of DPPC and a non-aqueous solvent, each of which is characterized by a characteristic concentration of calcium, with the formation of a solution of phospholipids, and the combined characteristic concentration calcium in the original MPEG5000-DPPE, the original DPPA, the original DPPC, and the non-aqueous solvent corresponds to a low calcium concentration, and combining the phospholipid solution with the aqueous solvent to form the phospholipid suspension.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining the original form of MPEG5000-DPPE, the original form of DPPC and a non-aqueous solvent, each of which is characterized by a characteristic calcium concentration, to form a solution of phospholipids, and the combined characteristic calcium concentration in the original form MPEG5000-DPPE, the original form of DPPC and non-aqueous solvent corresponds to a low concentration of calcium, and the combination of a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising selecting an original form of MPEG5000-DPPE, an original form of DPPA, and an original form of DPPC, one, two or all of which are characterized by a characteristic calcium concentration, where the combined characteristic calcium concentration corresponds to a low calcium concentration, combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. Components such as phospholipid precursors and/or non-aqueous solvent are also selected based on a single or combined characteristic calcium concentration.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising selecting a parent form of MPEG5000-DPPE and a parent form of DPPC, one or both of which have a characteristic calcium concentration, wherein the combined characteristic calcium concentration corresponds to a low calcium concentration, combining said parent form MPEG5000- DPPE, the original form of DPPC and a non-aqueous solvent to form a solution of phospholipids and combining the solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. Components such as phospholipid precursors and/or non-aqueous solvent are also selected based on the individual or combined characteristic calcium concentration.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, including selecting the original form of MPEG5000-DPPE, the original form of DPPA and the original form of DPPC, each of which is characterized by a characteristic concentration

- 2 040910 calcium, where the combined characteristic calcium concentration corresponds to a low calcium concentration, combining said original form MPEG5000-DPPE, original form DPPA, original form DPPC and a non-aqueous solvent to form a phospholipid solution, and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the non-aqueous solvent has an intrinsic calcium concentration, and the combined intrinsic calcium concentration of the original MPEG5000-DPPE, DPPA, and DPPC forms and the non-aqueous solvent is low.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the calcium concentration in the original form of MPEG5000DPPE, combining the original form of MPEG5000-DPPE, characterized by the measured low calcium concentration, with the original form of DPPA, the original form of DPPC and a non-aqueous solvent to form a solution of phospholipids and combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 115 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the calcium concentration in the original form of MPEG5000-DPPE, combining the original form of MPEG5000-DPPE, characterized by the measured low calcium concentration, with the original form of DPPC and a non-aqueous solvent to form a solution of phospholipids, and combining the solution phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 115 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the calcium concentration in the original form of DPPC, combining the original form of DPPC, characterized by the measured low calcium concentration, with the original form of DPPA, the original form of MPEG5000-DPPE and a non-aqueous solvent to form a solution of phospholipids and combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 90 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the calcium concentration in the original form of DPPC, combining the original form of DPPC, characterized by the measured low calcium concentration, with the original form of MPEG5000-DPPE and a non-aqueous solvent to form a solution of phospholipids, and combining the solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 90 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the calcium concentration in the original form of DPPA, combining the original form of DPPA, characterized by the measured low calcium concentration, with the original form of DPPC, the original form of MPEG5000-DPPE and a non-aqueous solvent to form a solution of phospholipids and combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 780 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising measuring the concentration of calcium in a non-aqueous solvent, combining the non-aqueous solvent having the measured low calcium concentration with the original form of DPPA, the original form of DPPC and the original form of MPEG5000-DPPE to form a solution of phospholipids, and combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the low calcium concentration is less than 0.7 ppm.

In another aspect, the present invention further provides a method for preparing a phospholipid suspension, comprising combining the original forms of MPEG5000-DPPE, DPPA and DPPC with a non-aqueous solvent to form a phospholipid solution, measuring the calcium concentration in the phospholipid solution, and combining the phospholipid solution having the measured low calcium concentration with an aqueous solvent to form a suspension of phospholipids.

In another aspect, the present invention further provides a method for preparing a phospholipid suspension, comprising selecting a stock MPEG5000-DPPE having no or low calcium concentration, combining said stock MPEG5000DPPE, stock DPPA, stock DPPC, and a non-aqueous solvent to form a phospholipid solution, and combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, the original form of MPEG5000-DPPE is further characterized by the absence or low content of divalent metal cations.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining the original form of MPEG5000-DPPE, the original form

- 3 040910

DPPA, the original form of DPPC and a non-aqueous solvent to form a phospholipid solution having no or low calcium concentration, and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

In another aspect, the present invention further provides a method for obtaining imaging of a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject in the result of ultrasound with contrast enhancement, and the suspension of phospholipids obtained using any of the above methods.

In another aspect, the present invention further provides a method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject as a result contrast-enhanced ultrasonography, wherein the phospholipid suspension is prepared by a method comprising measuring the calcium concentration in the original form of MPEG5000-DPPE, combining the original form of MPEG5000-DPPE, characterized by the measured low calcium concentration, with the original form of DPPA, the original form of DPPC and a non-aqueous solvent with forming a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

In another aspect, the present invention further provides a method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject as a result contrast-enhanced ultrasound, wherein the phospholipid suspension is obtained by a method comprising combining the original forms of MPEG5000-DPPE, DPPA and DPPC with a non-aqueous solvent to form a phospholipid solution, measuring the calcium concentration in the phospholipid solution, and combining the phospholipid solution characterized by the measured low calcium concentration, with an aqueous solvent to form a suspension of phospholipids.

In another aspect, the present invention further provides a method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject as a result contrast-enhanced ultrasonography, wherein the phospholipid suspension is prepared by a method comprising selecting a stock MPEG5000-DPPE having no or low calcium concentration, combining said stock MPEG5000-DPPE, stock DPPA, stock DPPC, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

In another aspect, the present invention further provides a method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject as a result contrast-enhanced ultrasound, wherein the phospholipid suspension is prepared by a method comprising combining the original form of MPEG5000-DPPE, the original form of DPPA, the original form of DPPC, and a non-aqueous solvent to form a phospholipid solution having no or low calcium concentration, and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising separately combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a PG-containing non-aqueous solvent, under the condition of low or no calcium, to form a phospholipid solution and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids. In some embodiments, a no or low calcium condition is less than 0.7 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising sequentially combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a PG-containing non-aqueous solvent, under the condition of low or no calcium, in an order-independent manner, to form a solution of phospholipid- 4 040910 and combining the solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, a no or low calcium condition is less than 0.7 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining, in the absence of methanol and toluene, the original forms of DPPA, DPPC and MPEG5000-DPPE to form a homogeneous mixture of phospholipids, combining a homogeneous mixture of phospholipids with a PG-containing non-aqueous solvent, under the condition of low calcium content or its absence, with the formation of a solution of phospholipids and combining the solution of phospholipids with an aqueous solvent to form a suspension of phospholipids. In some embodiments, a no or low calcium condition is less than 0.7 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a homogeneous mixture solvent to form a homogeneous mixture of phospholipids, evaporating the solvent for a homogeneous mixture to form a dried homogeneous mixture of phospholipids, combining the dried homogeneous mixture phospholipids with a PG-containing non-aqueous solvent, under the condition of low or no calcium content, to form a phospholipid solution, and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension. In some embodiments, a no or low calcium condition is less than 0.7 ppm.

In another aspect, the present invention further provides a method for preparing a suspension of phospholipids, comprising combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a homogeneous mixture solvent to form a homogeneous mixture of phospholipids, precipitating, in the absence of MTBE, a homogeneous mixture of phospholipids using a second solvent for homogeneous mixture, combining the precipitated homogeneous mixture of phospholipids with a non-aqueous solvent, under the condition of low or no calcium content, to form a phospholipid solution, and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids. In some embodiments, a no or low calcium condition is less than 0.7 ppm.

In some embodiments of any of the above methods, the method further comprises combining the phospholipid suspension with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres. In some embodiments of any of the above methods, the method further comprises administering an ultrasound contrast agent to the subject and obtaining one or more contrast-enhanced ultrasound images of the subject.

In another aspect, the present invention further provides a method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres, administering the ultrasound contrast agent to the subject, and obtaining one or more contrast images of the subject as a result ultrasound with contrast enhancement, and the suspension of phospholipids obtained using any of the above methods.

In other aspects, the present invention further provides a composition comprising a phospholipid solution containing DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent and having a low calcium concentration, as well as a composition comprising a phospholipid solution containing DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent, wherein DPPA, DPPC and MPEG5000-DPPE and the non-aqueous solvent have a combined intrinsic calcium ion content that is low. In some embodiments, the non-aqueous solvent contains propylene glycol (eg, propylene glycol may be the only non-aqueous solvent or it may be used in combination with one or more other solvents to form a non-aqueous solvent). In some embodiments, the implementation of the non-aqueous solvent contains propylene glycol and glycerin. In some embodiments, the non-aqueous solvent contains a buffer. In some embodiments, the buffer is an acetate buffer. In some embodiments, the implementation of the composition contains a gaseous perfluorocarbon. In some embodiments, the gaseous perfluorocarbon is perflutren. Thus, in some cases, the composition is provided in a container, such as a vial, and the gas is provided in the space above the product in the container. Also provided are methods of combining a solution of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres. The method may further comprise administering an ultrasound contrast agent to the subject and obtaining one or more contrast-enhanced ultrasound images of the subject.

In some embodiments of the various aspects provided herein, the non-aqueous solvent comprises (i) propylene glycol or (ii) propylene glycol and glycerin.

In some embodiments of various aspects provided herein, the non-aqueous solvent contains a buffer. In some embodiments of various aspects provided herein, the non-aqueous solvent contains an acetate buffer.

In some embodiments of the various aspects provided herein, the aqueous solvent contains a buffer. In some embodiments of the various aspects provided herein, the aqueous solvent contains a phosphate buffer.

In some embodiments of the various aspects provided herein, the original forms of DPPC, DPPA, and MPEG5000-DPPE are individually combined with a non-aqueous solvent to form a phospholipid solution.

In some embodiments of various aspects provided herein, the original forms of DPPC, DPPA, and MPEG5000-DPPE are sequentially combined with a non-aqueous solvent in an order-independent manner to form a phospholipid solution.

In some embodiments of various aspects provided herein, the original forms of DPPC, DPPA, and MPEG5000-DPPE are combined with each other to form a mixture of phospholipids, and then the mixture of phospholipids is combined with a non-aqueous solvent to form a solution of phospholipids. The mixture of phospholipids may be heterogeneous or homogeneous.

In some embodiments of various aspects provided herein, the original forms of DPPC, DPPA, and MPEG5000-DPPE are combined with each other to form a homogeneous mixture of phospholipids, and a homogeneous mixture of phospholipids is combined with a non-aqueous solvent to form a solution of phospholipids. In some embodiments of various aspects provided herein, a homogeneous mixture of phospholipids is formed using a dissolution-precipitation method in an organic solvent, including dissolving the original forms of DPPC, DPPA and MPEG5000-DPPE in a mixture of methanol and toluene, optionally concentrating the mixture of phospholipids/methanol/ toluene and then contacting the concentrated phospholipid/methanol/toluene mixture with methyl tert-butyl ether (MTBE) to precipitate the phospholipids to form a homogeneous mixture of phospholipids. In some embodiments of various aspects provided herein, a homogeneous mixture of phospholipids is formed by dissolving the original forms of DPPC, DPPA, and MPEG5000-DPPE in a homogeneous mixture solvent system other than the methanol/toluene solvent system, optionally concentrating the phospholipid/solvent mixture, and then contacting the concentrated phospholipid/solvent mixture with methyl tert-butyl ether (MTBE) to precipitate the phospholipids to form a homogeneous mixture of phospholipids. In some embodiments of the various aspects provided herein, a homogeneous mixture of phospholipids is formed by dissolving the parent forms of DPPC, DPPA, and MPEG5000-DPPE in a homogeneous mixture solvent system, such as, but not limited to, a methanol/toluene solvent system, and then lyophilizing or otherwise drying the mixture to remove the solvent to obtain a homogeneous mixture of phospholipids.

In some embodiments of the various aspects provided herein, the method further comprises placing a suspension of phospholipids in a vial and introducing gaseous perfluorocarbon into the headspace of the product in the vial.

In some embodiments of the various aspects provided herein, the method further comprises activating the phospholipid suspension with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres.

In some embodiments of the various aspects provided herein, the method further comprises administering an ultrasound contrast agent to a subject and obtaining one or more contrast-enhanced ultrasound images of the subject.

In some embodiments of the various aspects provided herein, the method further includes measuring the calcium concentration in the original form of DPPA and/or the original form of DPPC and/or a mixture of phospholipids and/or a homogeneous mixture of phospholipids.

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

Brief description of graphic materials

Fig. 1 is a photograph of four phospholipid solutions having different degrees of precipitation. The figure shows the definitions on the appearance scale: 0, +, ++ and +++, as used in the examples.

Fig. 2 is a photograph showing the appearance of the solution with successive additions of DPPC, MPEG5000-DPPE, DPPA, and calcium acetate.

Fig. 3 is a photograph showing the appearance of the titratable solutions for study 4 described in the examples.

Fig. 4 is a photograph showing the appearance of solutions made with different proportions of MPEG5000-DPPE and MPEG5000-DPPE containing high levels of calcium (Ca ⁺² ).

Fig. 5 is a graph showing the filtration flow rate for an aqueous formulation of phospholipids prepared with single phospholipids or a homogeneous mixture of phospholipids containing either high or low concentrations of calcium. Studies 19 and 23 were made with low calcium concentration components, while studies 20 and 24 contained high levels of calcium (see Table 6 for details). Each point on the graph represents the average of three consecutive filtration rate measurements with the corresponding time in each study.

Fig. 6 is a graph showing the filtration flow rate for a non-aqueous phospholipid composition prepared with single phospholipids or a homogeneous mixture of phospholipids containing either high or low concentrations of calcium. Studies 33 and 35 were made with low calcium concentration components, while studies 34 and 36 contained high levels of calcium (see Table 9 for details). Each point on the graph represents the average of three consecutive filtration rate measurements with the corresponding time in each study.

Detailed description

This document provides improved methods for the production of contrast agents for ultrasound (UCA) based on phospholipids. These improvements are based in part on the unexpected discovery that certain phospholipid formulations for use in the preparation of ultrasound contrast agents are sensitive to the presence and amount of certain divalent metal cations. Specifically, it was unexpectedly found that divalent metal cations such as calcium, at certain concentrations, when added to the phospholipid-based formulation used to make the primary ultrasound contrast agent, DEFINITY®, caused the phospholipid and possibly other components of the formulation to precipitate out of solution, while making the composition unusable. Such formulations are usually produced in large batches and thus an accidental addition of calcium, for example, will render the entire batch unusable. This may lead to performance degradation.

It has also surprisingly been found that certain phospholipids are more susceptible to precipitation caused by the presence of divalent metal cations such as calcium. Specifically, DPPA in a non-aqueous solvent such as propylene glycol is more likely to precipitate in the presence of certain concentrations of divalent metal cations such as calcium. The same sensitivity was not observed or was observed to a lesser extent with other phospholipids such as DPPC and DPPE. This different precipitation profile can easily result in a phospholipid composition, and ultimately a UCA having a different composition from the intended or desired phospholipid composition. Thus, the presence of divalent metal cations may not only reduce the overall yield of UCA (e.g., due to the non-filterability of the precipitated phospholipid formulation such as the phospholipid suspensions described herein), it may also adversely affect the distribution of phospholipids in UCA. This is a problem because it can result in UCA formulations with a completely unknown phospholipid content. It is well known in the pharmaceutical field that the composition of these UCA formulations should remain constant and consistently reproducible, and batch-to-batch variations should be avoided or minimized to the highest extent possible.

Thus, the present invention provides improved methods for preparing phospholipid formulations such as the phospholipid solutions and phospholipid suspensions described herein. These methods increase the yields of such formulations by reducing the likelihood of precipitation of phospholipids. They also provide, in a more reliable and reproducible manner, phospholipid compositions characterized by predicted phospholipid profiles and distributions. These methods benefit from the novel and unexpected results described herein and provide phospholipid formulations with the desired levels and proportions of phospholipids without resorting to sediment detection.

General information about the composition of phospholipids.

Provided herein are methods for preparing improved phospholipid solutions, phospholipid suspensions, and ultimately UCA formulations. As will be described in more detail, in some instances, UCA formulations can be formed from non-aqueous solutions of phospholipids that are combined with a gas such as perflutren. In other cases, the UCA formulation can be formed by combining a non-aqueous solution of phospholipids with an aqueous solvent to form a suspension of phospholipids, which is combined with a gas such as perflutren. These and other phospholipid-containing compositions are collectively referred to herein as phospholipid compositions. Each of

- 7 040910 specific compositions will be described in more detail below. The phospholipid formulations of the present invention may contain the three phospholipids that are used in the manufacture of FDA-approved DEFINITY® microspheres. These three phospholipids are:

(1) 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (referred to herein as DPPC);

(2) 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (referred to herein as DPPA);

(3) N-(methoxypolyethylene glycol-5000-carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (referred to herein as MPEG5000-DPPE).

Phospholipid formulations of the present invention may contain DPPC and MPEG-5000-DPPE.

In some instances, modified forms of one or more of these phospholipids may be used. For example, 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (DPPE) can be conjugated to polyethylene glycol (PEG). PEG conjugated to DPPE or other phospholipid may have a molecular weight (MW or length) selected from 1000-10000, in some non-limiting cases. More typically, PEG may have a MW of approximately 5000, in which case it is called PEG5000 and when conjugated to DPPE is called PEG5000-DPPE. PEG is typically conjugated to a phospholipid, such as DPPE, at the phospholipid end group rather than the aliphatic chain end group. PEG may contain a hydroxy or methoxy group at the end and may be referred to as HO-PEG5000 or MPEG5000, respectively.

In the case of conjugation with DPPE, the conjugate may be referred to as HO-PEG5000-DPPE or MPEG5000-DPPE as an example. The full chemical name of the latter conjugate is N-(methoxypolyethylene glycol-5000-carbamoyl)-1,2-dipalmitoyl-sn-glycero-3 phosphatidylethanolamine, monosodium salt (herein referred to as MPEG5000-DPPE).

DPPA, DPPC and MPEG5000-DPPE can be used in mole percentages of about 77-90 mole% DPPC, about 5-15 mole% DPPA and about 5-15 mole% DPPE, including MPEG5000-DPPE. Preferred ratios of each phospholipid 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).

The remainder of the present invention will refer specifically to DPPA, DPPC and MPEG5000-DPPE for convenience and brevity, but it should be understood that the ideas presented herein are intended to cover the ways in which they are used and/or compositions, which contain these or other phospholipids alone or in combination, such as, without limitation, the combination of DPPC and MPEG5000-DPPE.

The various methods provided herein include measuring the concentration of divalent metal in the components used to prepare the phospholipid formulations described herein. Particularly important are the components used to prepare the phospholipid solutions, especially since precipitation appears to be a phenomenon observed initially at the stage of formation of the phospholipid solution, and not at the stage of formation of the suspension of phospholipids. Methods that can be used to measure the concentration of divalent metal cations, such as the concentration of calcium and magnesium, are described in more detail in this document, including examples. Some methods may include measuring the concentration of divalent metal cations in only one component, such as, for example, MPEG5000-DPPE. Other methods may include measuring the concentration of divalent metal cations in two or more components, such as, for example, two or three phospholipids. In some embodiments, the implementation of the components can be combined together before the implementation of the measurement. Still other methods involve measuring the concentration of divalent metal cations in all components, including the non-aqueous solvent, used to prepare a phospholipid formulation, such as a phospholipid solution. Such a measurement can be performed before or after combining the components.

For example, the measurement of the individual components used to prepare the phospholipid solution can be performed, or it can be performed on the phospholipid solution itself.

Various other methods provided herein provide for the selection of components used to prepare phospholipid formulations, such as phospholipid solutions, based on their concentration of divalent metal cations. More specifically, the methods include selecting one or more components that are absent or low in divalent metal cations or are identified as such, including the absence or low concentration of calcium or the absence or low concentration of magnesium. Some methods may involve selecting a single component, such as MPEG5000-DPPE, that is free or low in divalent metal cations or identified as such, including low or low calcium or low or low magnesium. Some methods may involve selecting two or more or all of the components based on their combined divalent metal cation concentration. Thus, it is contemplated that DPPA, DPPC, and MPEG5000-DPPE, as well as other components such as, without limitation, the non-aqueous solvent and/or its individual components, may individually

- 8 040910 be characterized by the absence of divalent metal cations or their low concentration, but that when used together, the combined concentration of divalent metal cations will no longer satisfy the requirement of the absence of divalent metal cations or their low concentration and will cause precipitation. Thus, in these and other instances, two, three or all of the components, such as two or three phospholipids, can be selected such that their combined divalent metal cation concentration corresponds to the absence or low concentration of divalent metal cations.

Phospholipid solution.

As used herein, a phospholipid solution refers to a composition containing one or more phospholipids in a non-aqueous solvent. The phospholipid solution may contain at least DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent. The phospholipid solution may at least contain DPPC and MPEG5000-DPPE in a non-aqueous solvent.

A non-aqueous solvent, as used herein, is a solvent that causes the phospholipids to dissolve, thereby providing a solution (ie, a phospholipid solution). Preferably, the non-aqueous solvent present in the phospholipid solution is pharmaceutically acceptable, particularly as it reaches the final UCA formulation that is administered to a subject, including a human subject. In certain embodiments, the non-aqueous solvent used to prepare the phospholipid solution is not or does not contain methanol or toluene or methyl tert-butyl ether (MTBE).

The non-aqueous solvent in the phospholipid solution may be a single solvent or it may be a combination of solvents. Non-aqueous solvents include, without limitation, propylene glycol (which may be referred to herein as PG) and glycerin (which may be referred to herein as G). Both are provided as liquid source forms. In some instances, the non-aqueous solvent in the phospholipid solution may be PG alone, or it may be a mixture of PG and G (which may be referred to as PG/G). A non-aqueous solvent that contains at least PG may be referred to herein as a PG-containing non-aqueous solvent. PG/G blends include ratios ranging from 5:1 to 1:5 (w/w). In some embodiments, a PG:G w/w ratio is used. 1:1 (and in this document it is called a 1:1 mixture).

The phospholipid solution may additionally contain one or more buffers. Such buffers are buffers capable of buffering a non-aqueous solvent such as those mentioned above. Examples include, without limitation, an acetate buffer (eg, a combination of sodium acetate and acetic acid), a benzoate buffer (eg, a combination of sodium benzoate and benzoic acid), and a salicylate buffer (eg, a combination of sodium salicylate and salicylic acid). Other buffers that can be used include 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. In some embodiments, an acetate buffer is used. The buffer used in the non-aqueous solvent may be other than a phosphate buffer, meaning that it is not a phosphate buffer.

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 solvent may range from about 1 to about 100 mM, including from about 1 to about 50 mM, or from about 1 to about 20 mM, or from about 1 to about 10 mM, or from about 1 to about 5 mm, including approximately 5 mm.

Accordingly, the phospholipid solution may contain one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueous solvent that is or contains PG, and optionally a buffer such as an acetate buffer.

The phospholipid solution can be prepared in a number of ways, several of which are described in more detail below. In general, a non-aqueous solvent may be heated prior to contacting the phospholipids and, if used, the buffer may first be placed in the solvent prior to contacting the phospholipids. The solvent and then the solution can be stirred to help dissolve the phospholipids.

Importantly, the precipitation of phospholipids associated with divalent metal cations has been found to occur in a non-aqueous solvent and thus during the preparation of a phospholipid solution. Thus, as described herein, various methods include the steps of measuring the concentration of divalent metal cations in various components used to obtain a solution of phospholipids, including phospholipids, either alone or together, a non-aqueous solvent such as PG and G, a buffer such as acetate buffer, if used, and the like.

In some embodiments, the concentration of divalent metal cations in a suspension of phospholipids may be measured, instead of or in addition to measuring the concentration of cations.

- 9 040910 divalent metals in a solution of phospholipids.

Visual observation of the phospholipid solution can be done to detect the precipitate, although not necessarily. Fig. 1 is a photograph showing various phospholipid solutions having various degrees of sedimentation.

In some embodiments, the phospholipid solution is then used to prepare a phospholipid suspension, described in more detail below.

In some embodiments, the phospholipid solution is directly contacted with a gas, such as perfluorocarbon gas, to form gas-encapsulated phospholipid microspheres, without first contacting the phospholipid solution with an aqueous solvent. Namely, in some cases, gas-encapsulated phospholipid microspheres are obtained by bringing into contact and vigorous shaking (referred to as activation) of a (non-aqueous) solution of phospholipids and gas. Such microspheres may then be contacted with an aqueous solvent to form UCA.

suspension of phospholipids.

As used herein, a suspension of phospholipids refers to an aqueous formulation of phospholipids containing a solution of phospholipids and an aqueous solvent. The phospholipid suspension may contain one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE. The suspension of phospholipids will at least contain one or more phospholipids, such as one or more phospholipids, a non-aqueous solvent such as PG, and an aqueous solvent.

An aqueous solvent, as used herein, is water or contains it as its main component (by weight). The aqueous solvent may further contain one or more salts and thus may be referred to as an aqueous saline solution. It may additionally or alternatively contain a buffer and thus may be referred to as aqueous buffered saline or aqueous buffered solvent. Preferably, the aqueous solvent, whether containing salt(s) or buffer(s), is pharmaceutically acceptable because, like a solution of phospholipids, it reaches the final UCA formulation that is administered to a subject, including a human subject.

Salts that can be included in the aqueous solvent include, without limitation, sodium chloride.

Buffers that can be included in the aqueous solvent include, without limitation, phosphate buffer, 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 (tris-(hydroxymethyl)methylamine) buffer. Typically, either the non-aqueous solvent or the aqueous solvent contains a buffer, 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 aqueous solvent may range from about 1 to about 100 mM, including from about 1 to about 50 mM, or from about 10 to about 30 mM, or from about 20 to about 30 mM, or from about 20 to about 25 mm, including approximately 25 mm.

Accordingly, the phospholipid suspension may contain one or more phospholipids such as DPPA, DPPC and MPEG5000-DPPE, a non-aqueous solvent that is or contains PG, an aqueous solvent that may contain one or more salts such as sodium chloride, and optionally a buffer such as an acetate buffer or a phosphate buffer. Phospholipid suspensions can be physically characterized as phospholipids suspended, rather than dissolved, in an aqueous solvent.

A suspension of phospholipids is usually obtained by bringing into contact a solution of phospholipids, which is non-aqueous, with an aqueous solvent. The aqueous solvent may already contain any salts and/or any buffers, or alternatively they may be added after contact with the phospholipid solution. The aqueous solvent may be stirred to ensure mixing of the phospholipid solution with the aqueous solvent. The aqueous solvent may also be heated before being brought into contact with the phospholipid solution, which in some cases may also be heated.

Surprisingly, the concentration of divalent metal cations in an aqueous solvent is not as important as the concentration in the non-aqueous solution of phospholipids (and its combined components). For example, it has been surprisingly found that after a solution of phospholipids containing no precipitate is obtained, it can be combined with an aqueous solvent which has a high concentration of divalent metal cations without causing any visible precipitation of the phospholipids. Thus, it was surprisingly found that the sensitivity of a phospholipid to a high content of divalent metal cations exists only in a solution of phospholipids or in the presence of a non-aqueous solvent, but not beyond this point. Similarly, it was found that after the formation of a precipitate in

- 10 040910 solution of phospholipids, contact with an aqueous solvent, even heated, does not lead to its dissolution. This different sensitivity of phospholipids, and in particular DPPA, to high levels of divalent metal cations, such as calcium levels, has not previously been taken into account and considered as an unexpected result.

While the concentration of divalent metal cations in an aqueous solvent does not appear to cause precipitation of one or more phospholipids, it unexpectedly causes precipitation of other components, including, most importantly, phosphate, which may be present if a phosphate buffer is used in an aqueous solvent. Thus, in some cases, the methods provided herein may further include measuring the concentration of divalent metal cations in the components used to obtain aqueous suspensions of phospholipids that contain phosphate. Alternatively, the methods may include selecting individual components or combined components that are characterized, alone or in combination, by the absence or low concentration of divalent metal cations.

The phospholipid suspension can then be used to prepare gas-encapsulated phospholipid microspheres.

Gas-encapsulated phospholipid microspheres and UCA formulations containing them.

As will be appreciated, the phospholipid-based ultrasound contrast agents of the present invention are gas-encapsulated phospholipid microspheres. These microspheres can be obtained in a number of ways. For example, a solution of phospholipids can be contacted with an aqueous solvent to form a suspension of phospholipids, and a suspension of phospholipids can be contacted with a gas such as perfluorocarbon gas to form gas-encapsulated phospholipid microspheres. As another example, a non-aqueous solution of phospholipids can be contacted with a gas, such as gaseous perfluorocarbon, to form gas-encapsulated phospholipid microspheres. In either case, the phospholipid composition, whether it is a non-aqueous solution of phospholipids or an aqueous suspension of phospholipids, is combined with the gas in a manner sufficient to form gas-encapsulated phospholipid microspheres. This usually involves vigorous shaking or other mixing. Sufficient shaking or mixing is usually achieved using a device such as VIALMIX® and is usually not achieved manually.

The phospholipid solution or phospholipid suspension is provided in a container such as a vial containing a gas in the space above the product.

A gaseous perfluorocarbon, such as perflutren, is introduced into the headspace of such containers, typically by means of a gas exchange process. This given vial is then vigorously shaken to form gas-encapsulated phospholipid microspheres. This process, known as activation, is carried out by the end user or medical personnel just prior to administration to a subject.

The microspheres contain a gas, such as gaseous perfluorocarbon, including, without limitation, gaseous perflutrene, in their internal cavity. The phospholipid shell 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 µm, or less than 6 µm, or less than 3 µm, or more preferably less than 2 µm. The microspheres may have an average diameter ranging from 0.5 to 3 µm, or from 1 to 2 µm, or from 1.4 to 1.8 µm, or from 1.4 to 1.6 µm. The average diameter may be approximately 1.4 microns.

The process of obtaining gas-encapsulated phospholipid microspheres is known as activation. Compositions that contain a sufficient concentration of gas-encapsulated phospholipid microspheres may be referred to herein as activated formulations.

It will be understood that the concentration of gas microspheres that is sufficient will depend on whether the gas microspheres are prepared using a phospholipid solution (without intermediate use of an aqueous solvent) or prepared using a suspension of phospholipids. Typically, a UCA formulation administered to a subject will contain on the order of at least about 1x10 ⁷ microspheres/ml of the administered formulation, or at least 5x10 ⁷ microspheres/ml, or at least 7.5x10 ⁷ microspheres/ml, or at least 1x10 ⁸ microspheres/ml, or at least 1x10 ⁹ microspheres/ml, or approximately 5x10 ⁹ microspheres/ml. The range of microsphere concentrations may in some cases be from 1x10 ⁷ to 1x10 ¹⁰ microspheres/ml of the formulation administered, and more typically from 5x10 ⁷ to 5x10 ⁹ microspheres/ml.

Depending on how they are obtained, gas microspheres may be in a non-aqueous solvent or in an aqueous solvent. Despite this, prior to administration to a subject, they are usually diluted in an aqueous solution, which may be saline or buffered aqueous solution or buffered saline. A UCA formulation to be administered, usually intravenously, to a subject, including a human subject, may have a pH 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 still 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).

The gas is preferably substantially insoluble in the phospholipid formulations provided herein, including phospholipid solution and phospholipid suspension. 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 may be used are described in US Pat. No. 5,656,211 and incorporated herein by reference. In an important embodiment, the gas is perfluoropropane.

Divalent metal cations and methods for their measurement.

Divalent metal cations are divalent metal ions with a valence of 2. They include barium(2+), beryllium(2+), cadmium(2+), calcium(2+), chromium(2+), cobalt(2+), copper(2+), europium(2+), gadolinium(2+), germanium(2+), iron(2+), lanthanum(2+), lead(2+), magnesium(2+), manganese( 2+), mercury(2+), nickel(2+), osmium(2+), platinum(2+), ruthenium(2+), strontium(2+), tin(2+), uranium(2+ ), vanadium(2+), yttrium(2+) and zinc(2+).

In some embodiments, the divalent metal cations of interest are calcium, magnesium, and manganese. In some embodiments, the divalent metal cations of interest are calcium and magnesium, and thus only calcium and magnesium are measured, or components are selected based only on their calcium and magnesium content. In some embodiments, the divalent metal cation of interest is calcium, and thus only calcium is measured or components are selected based on their calcium content alone.

Influence of divalent metal cations.

As described herein, divalent metal cations may be present in one or more of the components used to make UCA formulations. Their presence may not be taken into account until these components are combined with a non-aqueous solvent to form a solution of phospholipids, after which, for example, precipitation of phospholipids may begin, or until these components are combined with an aqueous solvent to form a suspension of phospholipids, after which, for example, phosphate precipitation. Surprisingly, the present invention found that the original form of the MPEG5000-DPPE phospholipid contained calcium and magnesium in high enough concentrations to cause precipitation of at least the DPPA phospholipid when combined. Thus, divalent metal cations may have different effects on different phospholipids, and it may not be apparent to the user whether a phospholipid (or other component) contains such cations in sufficient concentration to cause precipitation.

The inventors of the present invention have found during the preparation of various UCA formulations that the non-aqueous solvent becomes cloudy when combined with a homogeneous mixture of phospholipids containing the phospholipids DPPA, DPPC and MPEG5000-DPPE. Additionally, it was determined that the cloudy appearance was probably caused by precipitation of the DPPA phospholipid. However, the authors were not aware of the fact that MPEG5000-DPPE contained high concentrations of calcium and magnesium ions, and that these concentrations of calcium and magnesium probably caused DPPA precipitation. Interestingly, such cations do not appear to affect the ability of MPEG5000-DPPE to remain in solution and thus the user will not take this fact into account until this phospholipid is combined with others in a non-aqueous mixture. Additional studies, described in more detail herein, showed that precipitation occurred when the original form of MPEG5000-DPPE, later characterized as having a high calcium concentration, was combined with DPPA, regardless of the order of addition or the presence of other components, such as other phospholipids such as like DPPC and DPPA. The propensity of DPPA to precipitate in a non-aqueous solvent containing PG in the presence of a sufficiently high concentration of divalent metal cations, such as a concentration of calcium and magnesium, but not in an aqueous solvent characterized by similarly high concentrations of either or both of calcium and magnesium, was even more unexpected. In other words, the calcium concentrations that caused DPPA to precipitate in the non-aqueous solvent containing PG did not cause DPPA to precipitate in the aqueous solvent, and this too was unexpected.

Absence of divalent metal cations or their low concentration.

- 12 040910

As used herein, the components are selected such that they are characterized by the absence or low concentration of divalent metal cations or are identified as such, which includes the absence or low concentration of calcium. This concentration of divalent metal cations is expressed as a w/w value. (ie, the weight of the divalent metal cation per unit weight of the base matrix or solvent in which the component of interest resides). The concentration in micrograms per gram may alternatively be referred to as parts per million or ppm.

The absence or low concentration of calcium in a given component will further depend on how much of that component is used or, in other words, how much of that component is diluted to form a phospholipid solution or phospholipid suspension.

In the simplest case, only one component is of interest and only its calcium concentration is measured, or only that component is selected based on its calcium concentration. Based on the present invention, one skilled in the art will understand and be able to determine how much calcium will be allowed in a given component to avoid precipitation in the phospholipid solution or phospholipid suspension.

As an example, the concentration of calcium in the original form of the phospholipid (which is usually provided as a solid, such as powder) is expressed as the weight of calcium per gram of phospholipid. An example is calcium weight per gram of MPEG5000-DPPE or calcium weight per gram of DPPC. If two components, such as two phospholipids, are combined, the measure may be the weight of calcium per gram of combined MPEG5000-DPPE and DPPC.

The absence of a divalent metal cation, such as calcium, refers to a concentration of such a cation that cannot be detected using methods known in the art and/or presented herein.

The absence of divalent metal cations or their low concentration in the original form of the phospholipid will depend on the specific component.

Absence or low concentration of divalent metal cations in the original form of MPEG5000-DPPE phospholipid provides less than 510 μg/g (i.e. 1 μg of divalent metal cation per 1 g of MPEG5000-DPPE) (also referred to as less than 510 ppm), including less than 345 ppm , less than 230 ppm, less than 115 ppm, less than 57.5 ppm and less than 11.5.

Absence or low concentration of divalent metal cations in the original form of DPPC phospholipid provides less than 390 μg/g (i.e. 1 μg of divalent metal cation per 1 g of DPPC) (also referred to as less than 390 ppm), including less than 270 ppm, less than 180 ppm , less than 90 ppm, less than 45 ppm and less than 9 ppm.

Absence or low concentration of divalent metal cations in the original form of DPPA phospholipid provides less than 3440 µg/g (i.e. 1 µg of divalent metal cation per 1 g of DPPA) (also called less than 3440 ppm), including less than 2340 ppm, less than 1560 ppm , less than 780 ppm, less than 390 ppm and less than 78 ppm.

The absence or low concentration of divalent metal cations in a homogeneous mixture of phospholipids provides for less than 210 µg/g (i.e. micrograms of divalent metal cation per 1 g of a homogeneous mixture of phospholipids or the combined weight of MPEG5000-DPPE, DPPC and DPPA) (also called less than 210 ppm ), including less than 150 ppm, less than 100 ppm, less than 50 ppm, less than 25 ppm and less than 5 ppm.

The absence of divalent metal cations or their low concentration in propylene glycol provides for less than 3.1 μg / g (i.e. 1 μg of divalent metal cation per 1 g of propylene glycol) (also called less than 3.1 ppm), including less than 2.1 ppm, less than 1.4 ppm, less than 0.7 ppm, less than 0.35 ppm and less than 0.07 ppm.

The absence or low concentration of divalent metal cations in a mixture of propylene glycol and glycerol 1:1 (wt./wt.) provide less than 10.4 µg/g (i.e. 1 µg of divalent metal cation per 1 g of combined propylene glycol and glycerol) ( also referred to as less than 10.4 ppm), including less than 7.8 ppm, less than 5.2 ppm, less than 2.6 ppm, less than 1.3 ppm, and less than 0.26 ppm.

The absence or low concentration of divalent metal cations in glycerin provides for less than 20.4 µg/g (i.e. 1 µg of divalent metal cation per 1 g of glycerol) (also called less than 20.4 ppm), including less than 15.3 ppm, less than 10.2 ppm, less than 5.10 ppm, less than 2.6 ppm, less than 0.51 ppm.

The absence or low concentration of divalent metal cations in a phospholipid solution that contains only propylene glycol as a non-aqueous solvent provides for less than 3.1 μg/g (i.e. 1 μg of divalent metal cation per 1 g of all components of the phospholipid solution) (also called less than 3.1 ppm), including less than 2.1 ppm, less than 1.4 ppm, less than 0.7 ppm, less than 0.35 ppm and less than 0.07 ppm. As will be understood from the composition of the phospholipid solution, the main component by weight is the non-aqueous solvent, in this particular case propylene glycol.

It should be understood that the same concentration limits apply to the concentration of calcium and con

- 13 040910 concentration of magnesium, as well as the combined concentrations of calcium and magnesium.

The present invention contemplates measuring the concentration of divalent metal cations in one or more components of a phospholipid solution, including, for example, one, two, or all three parent forms of the phospholipids, optionally together with measuring the concentration of divalent metal cations in a non-aqueous solvent such as propylene glycol and/or glycerol. , depending on the nature of the phospholipid solution. If any of these components contain a concentration of divalent metal cations in excess of these limits given above, then it is expected that once a phospholipid solution is prepared from such a component, that phospholipid solution will be prone to precipitation.

In some embodiments, it is contemplated that one component will be analyzed for its concentration of divalent metal cations, and that if such a concentration corresponds to the absence or low concentration of divalent metal cations, the component can be combined with the remaining components, even if such components have not been analyzed for their concentration of divalent metal cations. The components of the phospholipid solution include stocks provided either singly or as a homogeneous mixture containing non-aqueous solvents such as propylene glycol and glycerol and optionally buffers.

In some embodiments, it is contemplated that more than one but not all components of the phospholipid solution will have their divalent metal cation concentration measured. In some cases, if one of the components has a divalent metal cation concentration that exceeds the no or low divalent metal cation limit above, then it cannot be used to prepare a phospholipid solution (or homogeneous mixture of phospholipids). In some cases, none of the components can be characterized or identified as having a concentration of divalent metal cations that is greater than corresponding to the absence or low concentration of divalent metal cations. However, if such components are used in combination, the combined divalent metal cation concentration can be determined based on the amount each component contributes to the phospholipid solution. It is contemplated that the combined concentration of divalent metal cations may or may not exceed the concentration corresponding to the absence or low concentration of divalent metal cations as defined above for the phospholipid solution.

As discussed throughout this document, the various levels indicated herein, when referring to divalent metal cations, apply equally to calcium. The examples show that the lowest calcium concentration at which phospholipid precipitation is apparent is about 0.7 µg calcium per gram of non-aqueous solvent (see Examples 1 and 2), otherwise referred to as about 0.7 ppm. Thus, the absence or low concentration of divalent metal cations, such as the absence or low concentration of calcium, in a non-aqueous solution of phospholipids provides less than 0.7 ppm, less than 0.35 ppm, or less than 0.07 ppm. It should be understood that the concentration of divalent metal cations in the solution of phospholipids less than 0.7 ppm will also be considered as the absence of divalent metal ions or their low concentration.

The concentration of divalent metal cations in an aqueous suspension of phospholipids can be represented as the weight of the divalent metal cation per gram of the aqueous solvent. Typically, a suspension of phospholipids is formed by diluting a non-aqueous solution of phospholipids by about 20 times in an aqueous solvent. Thus, the absence or low concentration of divalent metal ions in the phospholipid suspension provides for less than 0.035 μg/g of the phospholipid suspension. Similarly, the calcium concentration in the phospholipid suspension prepared from the phospholipid solution is less than 0.035 μg/g of the phospholipid suspension.

The concentration at which divalent metal cations precipitate phospholipids may be temperature dependent. At higher temperatures, higher cation concentrations may be tolerated until precipitation is observed. At lower temperatures, lower concentrations of cations may cause the onset of precipitation.

As an example, at temperatures of approximately 55°C (for example, 50-60°C), the absence of divalent metal cations or their low concentration corresponds to a concentration of divalent metal cations of less than 0.7 μg of calcium per 1 g of phospholipid solution or non-aqueous solvent. This level may be somewhat lower if the phospholipid solution is prepared at a lower temperature. Alternatively, this level may be slightly higher if the phospholipid solution is prepared at a higher temperature.

Sources of calcium.

As shown in the examples, the phospholipid solution is surprisingly and uniquely sensitive to specific levels of calcium. This unique sensitivity has not been previously noted. Given the effect of calcium on the preparation of the phospholipid solution and thus ultimately on the UCA, it is important to measure and thus control the calcium concentration in the phospholipid solution. Calcium may be present in each of the components of the phospholipid solution, including the original

- 14 040910 forms and non-aqueous solvents as described below.

Calcium and magnesium are divalent alkaline earth metals from Group 2 of the Periodic Table. Calcium is the fifth most abundant element by mass in the earth's crust, and the Ca2 ⁺ cation is also the fifth most abundant soluble ion in seawater. It is present at various levels in tap water depending on the hardness. The total hardness of water is the sum of the mole concentrations of Ca ²⁺ and Mg ²⁺ , ranging from soft at 0-60 ppm to very hard >181.

Calcium and magnesium are also present in varying concentrations in the crude glycerol extract from biodiesels. Calcium and magnesium levels have been reported as 12 to 163 ppm and 4 to 127 ppm, respectively, depending on the biodiesel seed oil (J.C. Thompson, 2006, Applied Engineering in Agriculture. Vol. 22(2):261-265 ). The main source of glycerin comes from this by-product of biodiesel production. The crude glycerol extract can be purified by treatment with activated carbon to remove organic impurities, alkali to remove unreacted esters of glycerol, and ion exchange to remove salts. High purity glycerin (>99.5%) is obtained by multi-stage distillation; a vacuum is advisable because of the high boiling point of glycerol (290°C).

In industry, propylene glycol is produced from propylene oxide. Various manufacturers use either a non-catalytic high temperature process at 200°C (392°F) to 220°C (428°F) or a catalytic process that occurs at 150°C (302°F) to 180°C (356°F). F) in the presence of an ion exchange resin or a small amount of sulfuric acid or alkali. The final products contain 20% propylene glycol, 1.5% dipropylene glycol and small amounts of other polypropylene glycols. Additional purification provides the final industrial or USP/JP/EP/BP purity propylene glycol, which is typically 99.5% or greater. Propylene glycol can also be converted from glycerol, a by-product of biodiesel production.

The calcium and magnesium content of PH propylene glycol and glycerin is not quantified as a USP, EP, EP, or JP.

Phospholipid DPPA contains phosphate, which can be ionized at the appropriate pH. The pKa for the two hydroxyl groups of phosphate is 6.2 and 1.8 (Tatulian Ionization and Binding, 511552, Phospholipid Handbook, Ed. G. Ceve, 1993). DPPA is commercially available in various salt forms. Usually the Na salt is used, but the Ca salt is also available.

DPPC is a zwitterion and thus does not require a counterion.

MPEG5000-DPPE is a modified DPPE that has pKa's of 1.9 and 9.3 for ethanolamine hydroxyl phosphate and amine (Tatulian Ionization and Binding, 511-552, Phospholipid Handbook, Ed. G. Ceve, 1993). MPEG500-DPPE is available as the Na salt.

Methods for measuring the concentration of divalent metal cations.

Quantitative determination of the concentration of divalent metal cations can be carried out using one of several known techniques. These include atomic spectroscopy techniques such as atomic absorption spectroscopy (AAS), flame photometry or flame atomic emission spectrometry (FAES), inductively coupled plasma atomic emission spectrometry (ICP-AES), and other techniques such as inductively coupled plasma atomic emission spectroscopy. -bound plasma or complexometric titration. Spectroscopic approaches exploit the absorption or emission characteristics of alkali metal (Group I) and alkaline earth (Group II) ions as they dissociate due to the thermal energy provided by the flame source. ICP-MS is a type of mass spectrometry that can detect metals at very low concentrations. This is achieved by ionizing the sample with an inductively coupled plasma and then using a mass spectrometer to separate and quantify these ions. Some of these methods are used in the examples.

Complexometric titration is another method for detecting the concentration of divalent metal cations. This method uses complexation of EDTA (ethylenediaminetetraacetic acid) with calcium and magnesium ions to compete with a color indicator. This provides fast colorimetric quantitation. EDTA forms a complex with calcium and magnesium ions. A blue dye called Eriochrome black T (ErioT) is used as an indicator. This blue dye also forms a complex with calcium and magnesium ions, changing from blue to pink in the process. The dye-metal ion complex is less stable than the EDTA-metal ion complex. For titration, a sample solution containing calcium and magnesium ions is reacted with an excess of EDTA. An indicator is added and it remains blue until all Ca ²⁺ and Mg ²⁺ ions present are complexed with EDTA.

Back titration is carried out using a solution of magnesium chloride. This provides a complex with excess EDTA molecules to the end point where all of the excess EDTA is complexed. The remaining magnesium ions from the magnesium chloride solution then begin to complex with the ErioT indicator, immediately changing its color from blue to pink.

- 15 040910

Synthesis methods.

The present invention provides methods for preparing phospholipid solutions and phospholipid suspensions for use with gaseous perfluorocarbon to form a UCA formulation containing gas-encapsulated phospholipid microspheres. In preferred embodiments, the phospholipids are DPPA, DPPC and DPPE such as MPEG5000-DPPE.

A solution of phospholipids can be obtained in a number of ways, described below. These methods are broadly described as uniform mixing and non-uniform mixing methods. The source forms may be in solid (eg powder) form or liquid form.

Homogeneous mixing methods.

Homogeneous mixing methods refer to methods in which phospholipids are thoroughly mixed with each other to obtain a solid homogeneous mixture of phospholipids that is more uniform (and thus more homogeneous) in terms of its content of phospholipids and the distribution of phospholipids in it, and in some cases has more high purity compared to simple mixtures of phospholipids.

In this method, a homogeneous dispersion of three phospholipids is formed by dissolving or suspending them in an appropriate solvent system for a homogeneous mixture, and then separating evenly distributed phospholipids from the solvent. Separating the blend solvent from the phospholipids may include drying, lyophilization, distillation, and the like, or it may include precipitation using an additional blend solvent. Solvents for a homogeneous mixture for neutral lipids are relatively non-polar solvents such as diethyl ether or chloroform. More polar solvents for a homogeneous mixture, such as alcohol (eg, methanol and ethanol), are required for membrane-bound lipids, which are themselves more polar. Chloroform can also be used, in particular for lipids of intermediate polarity. When mixed with methanol, chloroform becomes a common solvent. Dichloromethane (or methylene dichloride) is a similar extractant, but less oxidizable. Hexane can be used for lipids with low polarity. It can be used to extract neutral lipids from water/alcohol mixtures. Petroleum ether is a mixture of various hydrocarbons with 5-8 carbon atoms and can be used instead of hexanes in some cases. Other solvents for a homogeneous mixture include, without limitation, cyclohexane and toluene.

As will be described in more detail herein, some homogeneous mixtures are formed by contacting one or more desired phospholipids (e.g., DPPA, DPPC, and MPEG5000-DPPE) in a homogeneous mixture solvent system to first dissolve those phospholipids, then optionally concentrating the given solution, then either removing the solvent for a homogeneous mixture, or deposition of a homogeneous mixture of phospholipids from this solvent. The blend solvent should not be confused with the non-aqueous solvent which is then used to dissolve the phospholipids, thereby providing a solution of the phospholipids. It should be clear that this precipitation is a desirable phenomenon and should not be confused with the undesired precipitation of phospholipids which may occur later in the formation of the phospholipid solution due to the presence of calcium, another divalent cation, or a combination of divalent cations.

Some methods for preparing solutions of phospholipids involve contacting a homogeneous mixture of phospholipids with a non-aqueous solvent. There are various methods for preparing homogeneous mixtures of phospholipids, including, without limitation, methods of dissolution-precipitation in an organic solvent (or solvent for a homogeneous mixture, as used herein) and methods of suspension in water-lyophilization.

The method of dissolution-precipitation in an organic solvent is described in detail in US patent No. 8084056 and in published international application No. WO 99/36104, the entire content of which is incorporated by reference herein. One embodiment of this method includes the following steps.

(a) Contacting the desired phospholipids (eg DPPA, DPPC and MPEG5000-DPPE or DPPC and MPEG5000-DPPE) with the first homogeneous mixture solvent system. This system is usually a combination of solvents, such as CHCl ₃ /MeOH, CH2Cl2/MeOH and toluene/MeOH. It may be expedient to heat the resulting solution to a temperature sufficient to achieve complete dissolution. This 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. Pre-concentration

- 16 040910 respectfully 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 solvent for a homogeneous mixture. The mixture is suspended preferably at a temperature corresponding to approximately ambient temperature (for example, 15-30°C). Suitable second solvents for a homogeneous mixture are solvents which cause precipitation of phospholipids. The second solvent for the homogeneous mixture 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 solvent for a homogeneous mixture. Preferably, the collected solids are washed with another portion of the second homogenous solvent (eg, MTBE). Collection can be performed 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 resulting solid is referred to herein as a homogeneous mixture of phospholipids.

Certain methods described herein use phospholipids as a homogeneous mixture of phospholipids, including a homogeneous mixture of phospholipids obtained according to any of the homogeneous mixing methods indicated above. Some methods use homogeneous mixtures of phospholipids, with the exception of the homogeneous mixture of phospholipids obtained according to the method in methanol/toluene/MTBE above, where a mixture of methanol and toluene is used as the first solvent for a homogeneous mixture, and MTBE is used as a second solvent for a homogeneous mixture. mixtures. For clarity, the above process is referred to herein as the methanol/toluene/MTBE homogeneous mixture of phospholipids process.

As used herein, a homogeneous mixture of phospholipids should be distinguished from other mixtures of phospholipids, including those formulations that are obtained by simply combining phospholipids in their solid (including powdered) forms, as described herein.

In water-suspension-lyophilization methods, phospholipids are suspended in water at elevated temperature and then concentrated by lyophilization.

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

Some homogenous mixing methods, which are not the methanol/toluene/MTBE homogeneous phospholipid mixture method above, use a homogeneous mixture solvent system other than methanol and toluene. In these methods, phospholipids are combined under methanol and toluene free conditions (also referred to as methanol and toluene free conditions) to form a homogeneous mixture of phospholipids. Thus, the condition of the absence of methanol and toluene refers to the condition under which both of these solvents are absent.

In some homogeneous mixing methods, phospholipids are combined in a homogeneous mixture solvent to form a homogeneous mixture of phospholipids, and then the solvent for a homogeneous mixture is evaporated completely, for example, either by drying or distillation to form a dried homogeneous mixture of phospholipids. This dried homogeneous mixture of phospholipids is then brought into contact with a non-aqueous solvent such as PG in the methods presented herein.

In some homogeneous mixing methods, phospholipids are combined in an aqueous solvent and then the mixture is lyophilized to form a lyophilized phospholipid composition. Other homogeneous mixing methods combine phospholipids with other solvent systems, such as, without limitation, (1) a mixture of ethanol and cyclohexane (for example, 1:1, v:v) and (2) tertiary butanol (tert-butanol or 1 ,1-dimethylethanol), instead of water. After dissolution in these various solvents, the compositions are lyophilized. Lyophilization can be carried out by freezing in an isopropanol/CO ₂ bath or an acetone/CO ₂ bath and drying on a Virtis lyophilizer until the product is dry and flaky.

Various homogenous mixing methods that are not the methanol/toluene/MTBE homogeneous mixture of phospholipids mentioned above are described in published EP 0923383 (WO 1997/040858), the entire content of which is incorporated herein by reference.

In some homogenous mixing processes, which are not the methanol/toluene/MTBE homogeneous phospholipid blending method discussed above, the phospholipids are combined in a blending solvent, which may be a mixture of toluene and methanol, with

- 17 040910 formation of a homogeneous mixture of phospholipids, and then precipitate this homogeneous mixture of phospholipids in the absence of MTBE. In these methods, precipitation occurs in the absence of MTBE.

In other methods of homogeneous mixing, DPPA, DPPC and MPEG5000-DPPE (or DPPC and MPEG5000-DPPE) can be combined in their dry, solid forms and the combination can then be vigorously and thoroughly mixed in dry form (e.g., manually mixing the powders or using a blender). silage drum mixer, rotary screw mixer, belt type mixer, extruder, Cyclomix, Henschel mixer, Lodige type mixer, Eirich type mixer, or other type of device designed for mixing pharmaceutical powders in order to obtain a homogeneous mixture of phospholipids (for example, homogeneous dispersion of phospholipids in the mixture.Reference may be made to Deveswaran et al.Research J.Pharm.and Tech.2(2):April.-June.2009) for additional methodologies to obtain a homogeneous dry product.

Non-uniform mixing methods.

In contrast to homogeneous mixing methods, certain non-homogeneous mixing methods involve simple mixing of solid phospholipids and this generally results in a less homogeneous (and thus less homogeneously dispersed or more heterogeneously dispersed) mixture. These latter mixtures are referred to herein as mixtures of phospholipids (or heterogeneously mixed mixtures of phospholipids) to distinguish them from homogeneous mixtures of phospholipids.

Some methods for preparing solutions of phospholipids involve simply contacting the phospholipids in their solid form with a non-aqueous solvent. Phospholipids can be brought into contact with a non-aqueous solvent simultaneously or sequentially. If sequential, any order of addition can be used. The phospholipids may be added individually to the non-aqueous solvent, or they may first be combined together in any combination and then added to the non-aqueous solvent. Thus, it is contemplated that phospholipids can be added to the non-aqueous solvent separately and simultaneously, separately and sequentially, combined and simultaneously, and partially combined and sequentially. An example of the latter is the case where two phospholipids are combined together in solid form and then brought into contact with a non-aqueous solvent before or after contacting the remaining phospholipid with a non-aqueous solvent.

Thus, as an example, the phospholipids DPPA, DPPC and MPEG5000-DPPE (or DPPC and MPEG5000-DPPE) can be added separately to the non-aqueous solvent. Such addition individually may be sequential or simultaneous addition. If the addition is sequential, the order of addition may be any order, although in some cases DPPA may be added second or last as it is the least abundant and less soluble and the presence of one of the other phospholipids may facilitate its dissolution. In some cases, whether the phospholipids are provided individually, or as a simple mixture, or as a homogeneous mixture of phospholipids, they are then dissolved in a non-aqueous solvent containing PG or a PG/G mixture, as described above, to form a solution of phospholipids . The phospholipid solution may be combined with the gas, or it may be combined with an aqueous solvent to form a suspension of phospholipids, which in turn is brought into contact with the gas.

In other cases, a homogeneous mixture of phospholipids can be obtained by combining phospholipids, for example, in water, or in a mixture of ethanol and cyclohexane (for example, 1:1, v:v), or in tertiary butanol (tert-butanol or 1,1-dimethylethanol ), and this mixture is then lyophilized, and the dried product is resuspended in an aqueous solvent. In these cases, the final resuspended product may be combined with a gas such as perflutren. The present invention contemplates that any or all of the components used in a given preparation can be analyzed for their divalent metal cation concentration, and such individual or combined divalent metal cation concentration can be quantified and used to select and/or produce UCA.

Methods for obtaining a contrast agent for ultrasound, including activation.

Gas-encapsulated phospholipid microspheres are formed by combining and vigorously shaking a solution of phospholipids or suspensions of phospholipids with a gas such as gaseous perfluorocarbon. This process is referred to in this document as activation. The UCA composition thus formed contains at least phospholipids, a non-aqueous solvent such as PG, and gas, and thus activation at least results in gas-filled phospholipid microspheres. Phospholipids may be present in an aqueous solution, as is the case with DEFINITY®, or they may be present in a non-aqueous solution, as is the case with new UCA formulations, including, for example, DEFINTTY-II, described in more detail herein. Thus, in some cases, activation involves shaking an aqueous suspension of phospholipids in the presence of a gas, such as gaseous perfluorocarbon (eg, perflutren). In other cases, activation involves shaking a solution of phospholipids in the presence of a gas, gaseous perfluorocarbon (eg, perflutren). It should be understood that perflutren, gaseous perflutren and octafluoropropane are used interchangeably herein.

- 18 040910

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. The shaking must be done with sufficient force or intensity to ensure the formation of foam 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 shaking will vary depending on the type of UCA composition to be activated. For example, in some cases the (aqueous) suspension of phospholipids can be shaken at a lower shaking intensity than the (non-aqueous) solution of phospholipids. The present invention contemplates that in such cases, the shaking time (or duration, as these terms are used interchangeably herein) can be constant.

DEFINITY® can be activated using VIALMIX® as described below. Activation of DEFINLTY®, which involves vigorous shaking of the (aqueous) suspension of phospholipids in the presence of perflutren, lasts approximately 45 seconds with VIALMIX®. 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 also be activated with VIALMIX®. Activation of DEFINITY-II, which involves vigorous shaking of a (non-aqueous) solution of phospholipids in the presence of perflutren, lasts approximately 60-120 seconds. 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 solution of phospholipids, 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 shaking mechanisms or devices 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. VIALMIX® for example, target shaking is performed at 4530 rpm, and 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 rpm or more. 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,

- 19 040910 Shaking at 4530 rpm for 45 seconds will be achieved in 3398 full revolutions on VIALMIX®. Shaking at 3000 rpm will take 68 seconds to reach the same rpm. 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 centipoise 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 glycerol 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-like state into the liquid-crystalline state of the phospholipid used. By the gel-to-liquid crystal phase transition temperature is meant the temperature at which a phospholipid 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 phospholipids 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 skilled in the art will appreciate that phospholipids or phospholipid microspheres can 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 accomplished by inserting a syringe needle or needleless element (e.g. PINSYNC®) into a container, including foam if necessary, and withdrawing a predetermined amount of liquid into the syringe barrel by retracting the plunger, or by adding an aqueous liquid, mixing and aspirating a predetermined 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.

Methods for applying an ultrasound contrast agent to obtain imaging of a subject.

Also provided herein are methods of using gas-encapsulated phospholipid microspheres and formulations thereof. Gas microspheres and formulations thereof can be used in vivo with human or non-human subjects, or they can be used in vitro. They may be used for diagnostic or therapeutic purposes, or both for diagnostic and therapeutic purposes.

When applied to human subjects, the gas-encapsulated phospholipid microspheres and their formulations can be applied directly (as is) or can be further diluted in solution, including in 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, circulatory 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,

- 20 040910 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 may be that amount of gas-encapsulated phospholipid microspheres that allows imaging of a subject or region of a subject's body.

Examples

1. Examples of ways.

1.1. Phospholipids, and homogeneous mixtures of phospholipids, and reagents.

The phospholipids were used either as individual powders combined together as powders or as a mixture or homogeneously mixed together by dissolving and drying (details are described below). The measured content of individual phospholipids was used to estimate the final concentrations of calcium or magnesium in a non-aqueous concentrate or aqueous preparation, if direct measurements were not made in the mixture. All studies used low calcium solvents.

1.1.1. Homogeneous mixture of phospholipids.

One homogeneous mixture of phospholipids (LB) was obtained by dissolving DPPC, DPPA, MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) in toluene/methanol, concentrated under vacuum and heating, and then suspended by adding methyl tert-butyl ether (MTBE). The solid material was collected, washed with MTBE and dried (according to US patent No. 8084056). Alternatively, DPPC, DPPA and MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) were solubilized at 55°C in methanol. The methanol was then evaporated and the solids were recovered as a homogeneous mixture of phospholipids. Similarly, DPPC, DPPA and MPEG5000-DPPE (0.401:0.045:0.304 [wt:wt:wt]) were combined together as solid powders and the powders were mixed together with a spatula.

1.1.1.1. Method for determining the residual solvent for a homogeneous mixture of phospholipids.

Residual solvent in a homogeneous mixture of phospholipids was determined by FID using GC in the headspace of the product. The sample was weighed, transferred to separate 20 cm ³ headspace vials and dissolved in N-methylpyrrolidone. A number of residual solvent standards were prepared in N-methylpyrrolidone. Standards and samples were analyzed by FID using headspace GC. The concentration of each residual solvent was calculated from the calibration curve for that solvent.

1.1.2. Calcium measurements.

Calcium levels were determined in a single lipid, a homogeneous mixture of lipids, glycerin and propylene glycol using either ICP-MS or AA. Magnesium and other metal ions were also measured using these methods in some samples.

1.1.1.2. ICP-MS method (inductively coupled plasma mass spectrometry).

Samples were obtained by weighing in a pre-cleaned quartz digester. Added known additives, and then mixed with nitric acid and hydrochloric acid. The samples were digested in a closed type microwave system to digest the samples. After cooling, an internal standard solution was added and diluted and analyzed by ICP-MS using He collision mode.

1.1.1.3. Method AA (atomic absorption spectroscopy).

Samples were prepared by weighing in a dry, metal-free digester and dissolved in nitric acid and hydrochloric acid and refluxed with H ₂ O ₂ . The sample solution was washed with water and filtered. A number of standards were used to calibrate the AA, and then the absorbance of the samples was read from the calibration curve. The results for a single lipid, a homogeneous mixture of phospholipids and solvent compositions are presented in table. 1.

Table 1

The level of Ca ⁺² and Mg ⁺² in a single lipid, a homogeneous mixture of lipids and a solvent

materials Ca ⁺² (ppm) ^b Mg ⁺² (ppm) ^b Homogeneous mixture of phospholipids, lot 1 ^with Not detected Not detected Homogeneous mixture of phospholipids, lot 2 ^cd 370 54

- 21 040910

MPEG5000-DPPE (High Ca ⁺² ) Batch 1 980 150 MPEG5000-DPPE (High Ca ⁺² ) Batch 2 520 110 MPEG5000-DPPE (low concentration Ca ⁺² ) 4 Undefined DPPC Batch 1 Not detected Not detected DPPC Batch 2 7 Undefined DPPA 19 Undefined propylene glycol Not detected Undefined Glycerol 0.7 Undefined

^a Determination by ICP-MS.

^b ppm = parts per million, and are equivalent to µg/g.

^c The homogeneous mixture of phospholipids consists of DPPC, DPPA and MPEG5000DPPE (0.401:0.045:0.304 [wt:wt:wt]).

^d Uniform lipid blend, batch 2, made using MPEG5000-DPPE (high Ca ⁺² ), batch 1.

1.2. Getting the water composition.

1.2.1. Non-aqueous phospholipid concentrate.

Phospholipid concentrates were prepared by adding individual lipids (DPPC, DPPA and MPEG5000-DPPE low Ca ⁺² or MPEG5000-DPPE high Ca ⁺² , or combinations) in any order, adding a homogeneous mixture of phospholipids (LB), or adding LB , containing high levels of Ca ⁺² in 25-115 ml of media, which is propylene glycol (PG), or 1:1 v/v. propylene glycol/glycerol (PG/G), or glycerin, with constant stirring at 55-70°C. In some cases, the lipid concentrate was prepared without DPPA or with the addition of calcium acetate prior to lipid addition.

1.2.2. water composition.

Aqueous formulations were prepared by adding dibasic sodium phosphate, heptahydrate; monobasic sodium phosphate, monohydrate; sodium chloride; propylene glycol; glycerol and, finally, a non-aqueous phospholipid concentrate in 400-500 ml of water with constant stirring at 55-70°C. In some cases, calcium acetate was added before or after the addition of the non-aqueous phospholipid concentrate to the stock mixture. In other cases, phosphate buffer was not included.

1.3. Obtaining a non-aqueous composition.

1.3.1. Non-aqueous composition.

Lipids alone (DPPC, DPPA and low Ca ⁺² MPEG5000-DPPE, or high Ca ⁺² MPEG5000-DPPE, or combinations thereof) in any order, LB or LB containing high Ca ⁺² levels, were added to 25-100 ml propylene glycol (PG) carrier containing 0.005 M acetate buffer (90/10, sodium acetate/glacial acetic acid) with constant stirring at 60±5°C. After dissolution, glycerin was then added to obtain a non-aqueous composition.

1.4. Addition of calcium and/or magnesium using stock solutions.

1.4.1. Initial research.

Stock solutions of calcium acetate, magnesium acetate alone and mixtures of both were prepared in propylene glycol (25.4 µg Ca ⁺² /g, 28.0 µg Mg+2/g and 14.0 µg Ca ⁺² /g with 12.7 µg Mg+2/g, respectively). Individual stock solutions were added in aliquots to a total volume of 1 ml in 33 ml containing propylene glycol homogeneous lipid mixture (15 mg/ml). Solutions were compared with propylene glycol alone and the solution showing turbidity first was recorded.

1.4.2. Additional study with a reference scale.

Stock solutions of calcium acetate, monohydrate were prepared in propylene glycol (299 Ca ^{+ 2} μg/g), propylene glycol and glycerol (299 Ca ^{+ 2} μg/g) or water (6085 Ca ^{+ 2} μg/g), and the carrier was the same when added to propylene glycol , non-aqueous phospholipid concentrate or aqueous formulation (before or after addition of non-aqueous phospholipid concentrate). The maximum amount of calcium stock solution added was <12% of the total volume.

Some non-aqueous phospholipid concentrates were titrated with calcium acetate. Appearance was assessed on a scale of 0, +, ++, +++ by visual inspection. In FIG. 1 is the scale used for the determinations and was prepared using low Ca ⁺² lipids (DPPC, DPPA and MPEG5000-DPPE; 0.401:0.045:0.304 [wt:wt:wt]) formulated in propylene glycol at 15 mg total lipids/ml.

- 22 040910

1.5. Filtration.

1.5.1. water composition.

The obtained samples of aqueous suspensions of phospholipids were kept at 55°C before filtration. The samples were placed in a 60 ml temperature controlled syringe at 55° C. with a 13 mm syringe filter attached with a 0.22 μm hydrophilic polyvinylidene fluoride (PVDF) membrane. The syringe was pressurized with nitrogen at 5 psi over the product. inch. The flow rate was determined by weighing the filtered solution over time with readings every 30 seconds. The flow rates at each time point were calculated and the average flow rate at 9-10 minutes was compared with the initial flow rate (0-1 min) and expressed as a percentage. The sample before filtration was collected along with the samples during filtration to analyze the concentration of phospholipids.

1.5.2. Non-aqueous composition.

The obtained samples of non-aqueous solutions of phospholipids were kept at 60°C before filtration. Samples were placed in a 60 ml temperature controlled syringe at 60° C. with a 25 mm syringe filter attached with a 0.2 μm hydrophilic polyethersulfone (PES) membrane. The syringe was pressurized with nitrogen at 10 psi over the product. inch. The flow rate was determined by weighing the filtered solution over time with readings every 30 seconds. The flow rates at each time point were calculated and the average flow rate at 8-9 min was compared with the initial flow rate (0-1 min) and expressed as a percentage. Flow rates in clear solutions appear to increase with time as the filter is heated. Samples were collected as above.

1.6. Phospholipid analysis.

In some cases, samples were analyzed for phospholipid content. The sample was transferred to an HPLC vial and analyzed by separation using reverse phase HPLC and Corona charged aerosol detector (CAD; HPLC With Charged Aerosol Detection for the Measurement of Different Lipid Classes, I.N. Acworth, P.H. Gamache, R. McCarthy and D. Asa , ESA Biosciences Inc., Chelmsford, MA, USA; J. Waraska and I.N. Acworth, American Biotechnology Laboratory, January 2008) and quantified against reference standards.

1.7. Receipt and testing of the product.

1.7.1. water composition.

An aliquot (1.76 ml) of the filtered aqueous formulation (see Section 1.5.1) was taken into 2 ^cm3 Wheaton vials, the headspace was replaced with gaseous perfluoropropane (PFP), the vial was sealed with a West gray butyl rubber stopper and crimped. aluminum cap.

1.7.2. Non-aqueous composition.

An aliquot (0.35 ml) of the filtered aqueous formulation (see section 1.5.2) was taken into 2 cm ³ Wheaton vials, the headspace above the product was replaced with gaseous perfluoropropane (PFP), the vial was sealed with a West gray butyl rubber stopper and crimped. aluminum cap.

1.7.3. Microsphere sizing with Sysmex.

Samples were analyzed for quantitative distribution and size distribution using a particle meter (Malvern FPIA-3000 Sysmex). Aqueous or non-aqueous samples were optimally activated with VIALMIX®, part of the activated product was diluted with saline and then transferred to the Sysmex sample container. Sysmex uses a suitable sheath and analyzes the sample using low and high power fields of view to obtain size data for a specific size range (1-80 µm in current studies).

1.7.4. Ultrasonic contrast enhancement with an activated product.

Acoustic attenuation was measured for selected samples using the Philips Sonos 5500 clinical ultrasound imaging system. After optimal activation with VIALMIX®, 10 µl samples were pipetted into a 250 ml beaker containing 200 ml of 0.9% saline at room temperature . A round, paddle-shaped magnetic stirrer with a diameter of 38 mm maintained the homogeneity of the solution and served as a sound reflector. The s3 clinical transducer of the ultrasonic system was placed on top of the beaker, immediately in solution and 4.8 cm above the top of the magnetic stirrer. 5 seconds of 120 Hz images were then acquired digitally and written to disk starting 10 seconds after sample injection.

The ultrasound system was used in IBS mode, TGC was fixed at the minimum value for all depths, and LGC was turned off. The mechanical index (MI) was 0.2 with the power setting 18 dB below maximum. The reception gain was fixed at 90 and compression at 0. For each test sample, ultrasound data were obtained before (blank) and after sample injection.

Image analysis was performed using Philips QLab version 2.0, which

- 23 040910 downloaded the files received by the ultrasound system and calculated the values in dB for the IBS mode. Regions of interest were plotted on a magnetic stirrer and dB values exported to Excel. They were then averaged over a full 5 s (approximately 360 video frames) of observation. Attenuation measurements were obtained by subtracting the mean ROI of the sample from the mean ROI of the blank sample (both in dB). This was divided by twice the distance between the ultrasonic transducer and the top of the magnetic stirrer to obtain the attenuation in dB/cm. The values were then divided by the calculated concentration of microbubbles in the beaker and expressed as dB attenuation per centimeter per million microbubbles/mL.

Example 1 Effect of adding calcium to a non-aqueous solution of phospholipids.

This example shows the effect of calcium and magnesium ions on the precipitation of phospholipids.

Example 1.1. Initial studies on the effect of adding calcium and magnesium to a non-aqueous solution.

In the initial studies, a homogeneous mixture of lipids (LB, batch 1) characterized by a low concentration of divalent metal ions (Table 1 in the examples of the method) was added to propylene glycol at 55±5°C and mixed. It was verified by visual inspection that the phospholipids were completely dissolved and the resulting solution was clear. This LB solution was titrated with calcium (25.4 µg Ca ⁺² /g), magnesium (28.0 µg Mg+2/g) or a combination (1:1) to give a solution containing 14.0 µg Ca ⁺² /g and 12.7 µg Mg+2/g) and exhibited turbidity at 3.60 µg Ca ⁺² /g, 4.23 µg Mg+2/g and 2.35 µg/g combined metal ions/g nonaqueous phospholipid solution respectively.

Example 1.2. Additional studies regarding the effect of adding calcium to a non-aqueous solution.

The experiment was carried out as follows. Powder DPPC, DPPA and MPEG5000-DPPE, characterized by a low concentration of divalent metal ions (Table 1 in the examples of the method), was added either separately (in the sequence shown in Table 2), or as a mixture or a homogeneous mixture added to a heated ( 55±5°C) and mixed propylene glycol (PG) or a 1:1 mixture of propylene glycol and glycerol (PG/G). It was verified by visual inspection that the phospholipids had completely dissolved and the resulting solution was clear (=0, see method examples, section 1.4.2, FIG. 1 for the rating scale). In FIG. 2 shows the appearance of the lipid concentrate in propylene glycol with successive additions of the stock solution with DPPC, MPEG5000-DPPE, DPPA and calcium acetate (1 ml of 299 µg Ca ⁺² per 1 ml of the stock solution was added to obtain a lipid concentrate with 11.1 µg Ca ⁺² per 1 g of solution). The lipid concentrate did not turn cloudy until calcium was added.

Solutions of phospholipids in PG or in a 1:1 mixture of PG/G were titrated with a series of small additions of calcium. After each addition, the solution was evaluated for clarity (see example methods, section 1.4.2, Fig. 1 for the rating scale), and the lowest calcium concentration, giving a score of +, ++ and +++, is shown in table. 2. In FIG. 3 shows typical titration solutions in Study 4.

table 2

Effect of Adding Calcium to a Non-Aqueous Solution of Phospholipids

Adding order lipids Observed threshold values for turbidity (µg Ca ⁺² /g) for calcium titration ^a,b Study DPP WITH MPEG5000 -DPPE DPP A Non-aqueous solvent + ++ +++ 1 ^s 1 3 2 PG 1.5 2.6 >5.7 2 ^e 2 3 1 PG 1.5 2.9 >11.1 3 ^e 3 1 2 PG 2.3 4.6 11.1 4 s 1 2 3 PG 1.8 2.9 >5.7 ^5d Homogeneous mixture phospholipids PG 1.8 5.7 11.1 6 ^e Phospholipid Blend (dry) PG 1.8 5.7 11.1 ^7f 1 3 2 PGhG Lipids are not dissolved £8 1 3 2 PGhG 2.6 I 19.2 35.8

^a Defined in Section 1.4 Methods.

^b Titrated with calcium acetate stock solutions (stock solution with 299 μg Ca ⁺² /g).

^c The final concentration of DPPA (0.9 mg/ml), DPPC (8.02 mg/ml) and MPEG5000DPPE (6.08 mg/ml) was provided by adding phospholipids separately to propylene glycol (25 ml).

^d A homogeneous mixture of phospholipids (15 mg/ml), obtained using methanol to dissolve the phospholipids at 55°C, followed by drying, was dissolved in 25 ml of propylene glycol.

^e Phospholipid blend: DPPA, DPPC and MPEG5000-DPPE powders

- 24 040910 (0.045:0.401:0.304) was mixed together and used to formulate in 25 ml of propylene glycol. The final concentration was 15 mg/ml.

^f Phospholipid solution prepared by adding individual phospholipids [DPPA (0.9 mg/ml), DPPC (8.02 mg/ml) and MPEG5000-DPPE (6.08 mg/ml)] in 25 ml 1:1 (vol. /v) propylene glycol:glycerol.

^g Phospholipid solution prepared by adding individual phospholipids [DPPA (0.225 mg/ml), DPPC (2.00 mg/ml) and MPEG5000-DPPE (1.70 mg/ml)] in 100 ml 1:1 (v/v .) propylene glycol: glycerin.

Titration with calcium provided a clear concentration-dependent precipitation in the phospholipid solution, whether the phospholipids were added (singly, as a mixture, or as a homogeneous mixture) to the propylene glycol (see Table 2). The lipids were not soluble in either glycerol alone or 25 ml of 1:1 PG/G, but gave a clear solution when added to 100 ml of 1:1 PG/G (Study 8). Calcium provided concentration-dependent precipitation in this lipid solution, which is consistent with the original results (see Table 2). In general, these titration studies showed that the lowest concentrations of calcium, magnesium and the combined concentration that precipitated were 1.5 µg Ca ⁺² /g, 4.23 µg Mg+2/g and 2.35 µg combined metal ions/g non-aqueous solution of phospholipids.

Example 2. Influence of the components of a solution of phospholipids containing calcium, when mixed.

Example 2.1. Calcium in PG.

Study 9 was performed as follows. DPPC, MPEG5000-DPPE and DPPA powder, characterized by low calcium concentration (see Table 1 in the method examples) were added separately (in the sequence shown in Table 3) to heated (55±5°C) and stirred PG, containing 11 µg/g calcium. Clarity was assessed (see section 1.4) and the solution was clear after dissolution of DPPC, turned and remained cloudy after addition of DPPA, and remained cloudy after addition of MPEG5000-DPPE. The observed turbidity was evaluated as +++ (Fig. 1, section 1.4). This was different from the clear solution obtained when these phospholipids (including DPPA) were added to PG containing a low concentration of calcium (Study Stock Solution 1). This was further emphasized by a study 12 where only the phospholipids DPPC and MPEG5000-DPPE having high Ca ⁺² levels were dissolved and the solution remained clear even in the presence of calcium.

Example 2.2. Calcium in a homogeneous lipid mixture from MPEG5000-DPPE.

Initial experiments were performed on a homogeneous mixture of phospholipids (prepared using toluene and methanol to dissolve and adding MTBE to precipitate a homogeneous mixture of lipids) containing DPPC, DPPA and MPEG5000-DPPE or low (Ca ⁺² was not detected and 1 μg Mg + 2 /g, MPEG5000-DPPE), or high (980 µg Ca ⁺² /g and 150 µg Mg+2/g, MPEG5000-DPPE, batch 1) calcium and magnesium, respectively, were added to a heated (55±5°C) and mixed propylene glycol. Two homogeneous mixtures of lipids were mixed to obtain samples with approximately 0, 1.75, 4.11 and 12.9 μg of combined Ca ^{+ 2} and Mg + 2 / g of a non-aqueous solution of phospholipids. 1.75 μg of combined Ca ⁺² and Mg+2/g non-aqueous phospholipid solution showed haze.

Additional studies 10 and 11 were performed as follows. Homogeneous mixture of phospholipids (prepared using toluene and methanol to dissolve and adding MTBE to precipitate a homogeneous mixture of phospholipids) containing DPPC, DPPA and MPEG5000-DPPE, and either high (980 ppm Ca ^{+ 2} , 150 ppm Mg ^{+ 2} , batch 1 ), or low (4 ppm Ca ⁺² ) calcium was added to heated (55±5°C) and stirred PG. Clarity was evaluated (see section 1.4) and slight haze (+; see method examples in section 1.4) was observed in a homogeneous mixture of phospholipids containing a high concentration of calcium (measured as 370 ppm Ca ⁺² and 54 ppm Mg ⁺² ). This was different from the clear solution obtained by dissolving a homogeneous mixture of low calcium phospholipids (undetectable levels of Ca ⁺² and Mg ⁺² ) (see Table 3).

- 25 040910

Table 3

Influence of the components of a phospholipid solution containing calcium when mixed

Order of phospholipids additions explored DPPC MPEG5000 DPPA non-aqueous Ca ⁺² (Mg ⁺² ) µg/g Observed level bath solvent [source] turbidity ^a 11.2 (0.0) [added to 9 ^b 1 2 3 PG PG before lipid addition] 5.8 (0.9) [High +++ ^{with 12d} 1 2 - PG concentration Ca ⁺² in 0 ^e MPEG5000- DPPE] Homogeneous mixture 0.0 (0.0) 10 phospholipids, containing PG [LB, party 0 low concentration of Ca ⁺² * 1] Homogeneous mixture 5.36 (0.8) [LB, party 2] eleven phospholipids containing a high concentration of Ca ⁺² f PG +++

^a Defined in Section 1.4 Methods.

^b The final concentration of DPPA (0.9 mg/ml), DPPC (8.02 mg/ml) and MPEG5000DPPE (6.08 mg/ml) was provided by adding phospholipids separately to propylene glycol (25 ml).

^c The solution was clear when DPPC was dissolved, remained clear after addition of MPEG5000-DPPE, turned cloudy after addition of DPPA.

^d DPPC (8.02 mg/ml) and MPEG5000-DPPE containing Ca ⁺² (6.08 mg/ml;

980 ppm Ca ⁺² and 150 ppm Mg ⁺² ); the final concentration was provided by adding phospholipids separately to propylene glycol (25 ml), DPPA was not added.

^e The solution was clear after adding DPPC and MPEG5000-DPPE.

^f A homogeneous mixture of phospholipids (15 mg/ml), obtained using toluene and methanol to dissolve the phospholipids and adding MTBE to precipitate a homogeneous mixture of phospholipids, was dissolved in 25 ml of propylene glycol.

Example 2.3. Calcium from MPEG5000-DPPE added separately.

Studies 13-17 were carried out as follows. DPPA and DPPC having a low calcium concentration (see Table 1 for process examples) were added separately (in the sequence shown in Table 4) to heated (55°C±5°C) and stirred PG. Added MPEG5000-DPPE containing various proportions of material with low and high concentrations of calcium and magnesium. Clarity was evaluated (see method examples in Section 1.4, Fig. 1) and calcium and magnesium concentration dependent precipitation was observed (see Table 4 and Fig. 4).

Table 4

Calcium and magnesium from MPEG5000-DPPE added as a separate component

Order to add NIA lipido in Percentage ⁰ Metal ion concentration (µg/g) Investigated not DPPC DPPA MPEG5000 -DPPE (low Ca ⁺² ) MPEG5000 -DPPE (High Ca ⁺²⁾ Non-water solvent Ca ⁺² Mg ⁺² Total Observed turbidity ^a 13 ^b 1 2 100 0 PG θ,ι 0.0 0.1 0 14 ^b 1 2 75 25 PG 0.7 0.1 0.8 + 15 ^b 1 2 50 50 PG 1.3 0.3 1.6 ++ 16 ^b 1 2 25 75 PG 1.9 0.4 2.3 ++ 17 ^b 1 2 0 100 PG 3.1 0.6 3.7 +++ 18 1 2 n/p ^d n/p ^d PGhG ⁶ Not added Cloudy, DPPA did not dissolve

- 26 040910 ^a Defined in section 1.4 methods.

^b The final concentration of DPPA (0.9 mg/ml), DPPC (8.02 mg/ml) and MPEG5000DPPE (6.08 mg/ml) was provided by adding phospholipids separately to propylene glycol (25 ml).

^c MPEG5000-DPPE percentages; with a low Ca ⁺² content [4 ppm] and a high Ca ⁺² content [520 ppm Ca ⁺² , 110 ppm Mg ⁺² ] relative to the total amount.

^d n/p = not performed; phospholipids [DPPC (8.02 mg/mL) and DPPA (0.9 mg/mL)] did not dissolve in the solvent system of propylene and glycol.

^e Propylene glycol and glycerin 50:50 (v/v).

Brief description of example 2.

All these studies showed that the addition of calcium, either in a non-aqueous solvent, or via a homogeneous mixture of phospholipids, or when added as MPEG5000-DPPE as a separate compound, in all cases caused precipitation. The concentration at which the effect was noticeable was similar for example 2 compared to the concentrations for example 1. The lowest concentration of calcium that provided turbidity (+) was ^0.7 µg/g only Ca ⁺² and Mg ⁺² ). This is a concentration similar to 1.5-2.6 µg/g, which is shown in example 1.

Example 3 Addition of a non-aqueous solution of phospholipids to an aqueous solvent.

Example 3.1. Effect of calcium in a non-aqueous solution of phospholipids on addition to an aqueous solvent.

A number of studies have been conducted to investigate the effect of calcium in a non-aqueous solution of phospholipids prior to transfer to an aqueous formulation. These include the steps of 1) obtaining a non-aqueous solution of phospholipids, 2) obtaining an aqueous solution, and 3) combining the solutions from steps 1 and 2.

Example 3.1.1. Obtaining a non-aqueous solution: calcium added to the non-aqueous solution after the dissolution of the phospholipids.

In accordance with example 2, the first stage in studies 19, 20 and 22 was the following. DPPC, DPPA and MPEG5000-DPPE powder, characterized by low calcium concentration (see Table 1 in the method examples), were added separately (in the sequence shown in Table 5) to heated (55±5°C, except for study 22 , in which it was heated to 70°C) and mixed propylene glycol. It was verified by visual inspection that the phospholipids had completely dissolved and the resulting solution was clear (see method examples in Section 1.4, FIG. 1). A solution of calcium acetate [CA(OAc) ₂ ] in propylene glycol was added as indicated in the table. 5, the solution was stirred and a change in appearance was observed compared to the blank solvent and a score for clarity was recorded. After the addition of calcium acetate, the solution became cloudy. These propylene glycol concentrates were transferred to the aqueous phase as described below.

Example 3.1.2. Preparation of non-aqueous solution: calcium in MPEG5000-DPPE.

The first stage of studies 21 and 25 was as follows. DPPC, DPPA (not included in study 25), and MPEG5000-DPPE powder containing calcium (980 ppm, MPEG5000-DPPE, batch 1; see Table 1 in the method examples) were added separately (in the sequence shown in Table 1). 5) in heated (55±5°C) and stirred PG. Clarity was assessed and significant turbidity was observed in Study 21 (+++; see method examples in Section 1.4, Figure 1) after addition of DPPA, and the solution remained cloudy after addition of MPEG5000-DPPE, while no turbidity was observed in Study 25, at in which DPPA was absent. These non-aqueous solutions of phospholipids were transferred into the aqueous phase as described below.

Example 3.1.3. Obtaining a non-aqueous solution: calcium in a homogeneous mixture of lipids from MPEG5000-DPPE.

In accordance with example 2, the first stage in studies 23 and 24 was carried out as follows. Homogeneous mixture of phospholipids (prepared using toluene and methanol to dissolve and adding MTBE to precipitate a homogeneous mixture of lipids) containing DPPC, DPPA and MPEG5000-DPPE, either low (4 ppm, lot 2) or high (980 ppm, MPEG5000-DPPE, batch 1) calcium, respectively, was added to heated (55±5°C) and stirred propylene glycol. Clarity was assessed and significant haze (+++; see method examples in section 1.4, Fig. 1) of a homogeneous mixture of phospholipids containing a high concentration of calcium was observed. This was different from the clear solution obtained by dissolving a homogeneous mixture of low calcium phospholipids (see Table 5). These non-aqueous solutions of phospholipids were transferred into the aqueous phase as described below.

Example 3.1.4. Obtaining a non-aqueous solution: calcium in PG before adding phospholipids.

In accordance with example 2, the first stage in studies 28 and 30 was carried out as follows. Powder DPPC, MPEG5000-DPPE and DPPA, characterized by low calcium concentration (see Table 1 in the examples of the method) were added separately (in the sequence shown in Table 5) to heated (55±5°C) and stirred PG, either containing 11 μg/g calcium or calcium was added after the addition of phospholipids, respectively. Transparency was evaluated (see method examples in

- 27 040910 section 1.4, fig. 1), and in study 28 the solution was clear after dissolving DPPC and

MPEG5000-DPPE, but became and remained hazy after adding DPPA. In study 30, the solution was clear after dissolution of DPPC, DPPA and MPEG5000-DPPE, and turned cloudy after addition of Ca ⁺² . Haze for both studies was rated as +++ (see Table 5). These non-aqueous solutions of phospholipids were transferred into the aqueous phase as described below.

Example 3.2. Obtaining an aqueous solution.

For all studies, the aqueous solution was prepared as follows. In a separate container, sodium chloride (NaCl), dibasic sodium phosphate heptahydrate (Na ₂ HPO ₄ -7H ₂ O) and monobasic sodium phosphate (NaH ₂ PO ₄ -H ₂ O) were added to the water in a container with a stirrer and stirred until dissolved. Propylene glycol and glycerin were also added, if necessary, so that the final addition of the phospholipid concentrate would be resuspended in an 8:1:1 composition of water:glycerol:propylene glycol. This stirred solution was maintained at 55±5°C (with the exception of study 22, where the aqueous solution was maintained at 70°C).

Example 3.3. Combining non-aqueous and aqueous solutions.

For all studies, the addition of the non-aqueous phospholipid concentrate to the aqueous solution was performed as follows. Warm phospholipids in propylene glycol were added and stirred at 100-150 rpm. Visual observations were recorded and the time for complete dispersion or dissolution (either clear or cloudy) was noted. These aqueous suspensions were then collected and filtered through a 0.2 µm filter at 55° C. at 5 psi product pressure. inch. The flow rate was measured and samples were collected for the measurement of phospholipids (see method examples for procedure). Samples before and after filtration were analyzed to determine the level of loss of phospholipids associated with filtration.

Table 5

Effect of a divalent metal ion in a non-aqueous solution of phospholipids on addition to an aqueous solvent

non-aqueous

phospholipids ^a i i § ! To Addition of Phospholip and DavRS Ca ⁺² (Mg ⁺² ) µg/g PG [Ca ⁺² source] Observed turbidity ^with Contains phosphate buffer Appearance after adding to aqueous solution Ca ⁺² (Mg ⁺² ) concentration in aqueous solution [µg/g water] Percentage of initial filtration rate at 9-10 minutes % phospholipide after filtration ^d DPPC DPPA MPEG5000 -DPPE 19 C,E,A 0.0 (0.0) 0 Yes Transparent 0(0) 64.6 101 100 99 20 C,E,A 13.7(0) ^e [calcium acetate added after lipids] +++ Yes Turbid 0.8 (0) 1.3; blocked filter 95 76 94 21 C,A,E 3.1 (0.7) ^f [in MPEG5000-DPPE] +++ Yes Turbid 0.2 (0.03) 9.0; blocked filter 96 78 95 22 8 C,A,E 21.4(0) ^e [calcium acetate added after lipids] +++ Yes; at 70°C Turbid 1.2 (0) 8.5; blocked nth filter 98 75 97 25 C,E 5.8 (0.9) ^h [in MPEG5000-DPPE] 0 Yes Transparent 0.3 (0.04) 82.1 100 nd 99 23 LB ¹ 0(0) 0 Yes Transparent 0(0) 92.0 99 100 99 24 LB ¹ 5.36 (0.8) [Homogeneous mixture of lipids] +++ Yes Slightly hazy 0.3 (0.04) 5.2 Blocked nth filter 98 65 96 28 C,E,A 11.2 (0.0) ^k [calcium acetate in PG, then phospholipid added] +++ Yes Turbid 0.6 (0) 42.7 101 24 100 thirty C,A,E 21.4 (0.0) ^e [calcium acetate added after lipids] +++ No Turbid 1.2 (0) 9.0 blocked nth filter 89 42 86

^a Phospholipid concentrate was prepared at 15 mg/mL by dissolving

DPPC (C), MPEG5000-DPPE (E), and DPPA (A) at 0.401:0.304:0.045 in propylene glycol in that order at 55°C.

^b Phospholipid concentrates were added to a formulation vessel containing water (800 mg); dibasic sodium phosphate, heptahydrate (2.16 mg); monobasic sodium phosphate, monohydrate (2.34 mg); sodium chloride (4.84 mg), glycerin (126 mg) and propylene glycol (51.75 mg) per 1 ml of the solution. The materials were combined at 55°C in that order.

^c Defined in the methods in section 1.4.2.

^d HPLC with CAD detection, described in Section 1.6.

^e 1 ml, 2 ml and 2 ml (studies 20, 22 and 30 respectively) of the initial

- 28 040910 solution with 299 μg of Ca ⁺² per 1 g of PG after lipid addition before transfer to an aqueous solution for formulation.

^f MPEG5000-DPPE containing Ca ⁺² (6.08 mg/ml; 520 and 110 ppm Ca ⁺² and Mg ⁺² ) was used for the experiment.

^g All formulations were performed at 70°C.

^h MPEG5000-DPPE containing Ca ⁺² (6.08 mg/ml; 980 and 150 ppm Ca ⁺² and Mg ⁺² ) was used for the experiment.

¹ Prepared using toluene and methanol to dissolve lipids and add MTBE to precipitate a homogeneous mixture of lipids. The resulting homogeneous mixture of lipids was added to 25 ml of propylene glycol (15 mg/ml). Study 23 used a homogeneous lipid mixture with low Ca ⁺² content and study 24 used a homogeneous lipid mixture containing Ca ⁺² and Mg ⁺² (370 and 54 μg/g, respectively).

^k Added 1 ml stock solution with 299 µg Ca ⁺² per 1 g PG before adding lipids.

In accordance with the previous examples, these studies showed that precipitation occurred in a non-aqueous solution of phospholipids when high concentrations of calcium or calcium and magnesium were present. This occurs regardless of whether calcium was present in the propylene glycol before the addition of phospholipids, added after the addition of phospholipids, or added with one of the components with phospholipids (or with MPG5000 DPPE, or in a homogeneous mixture of phospholipids). Once a precipitate has formed, it does not disperse when mixed with an aqueous solvent. This results in a cloudy aqueous formulation that has an initially reduced filtration rate and often clogs the 0.2 µm filter (Table 5; FIG. 5). The filtrate of the cloudy aqueous preparations was clear, but phospholipid measurements showed persistently reduced levels of DPPA. This effect was evident both for phospholipids added individually and for phospholipids added as a homogeneous mixture.

Example 3.4. Effect of adding a non-aqueous solution of phospholipids to an aqueous solvent containing calcium.

A number of studies were carried out to study the effect of calcium in aqueous solution on the preparation of a suspension of phospholipids. These include the steps of 1) obtaining a non-aqueous solution of phospholipids, 2) obtaining an aqueous solution, and 3) combining the solutions from steps 1 and 2.

Example 3.4.1. Obtaining a non-aqueous solution.

In accordance with example 1, the first stage in studies 26, 27 and 29 was carried out as follows. DPPC, DPPA and MPEG5000-DPPE powder having low calcium concentration (see Table 1 for process examples) was added separately (in the sequence shown in Table 6) to heated (55±5°C) and stirred propylene glycol. It was verified by visual inspection that the phospholipid was completely dissolved and the resulting solution was clear. These propylene glycol concentrates were transferred to the aqueous phase as described below.

Example 3.4.2. Obtaining an aqueous solution.

In a separate container, sodium chloride (NaCl), dibasic sodium phosphate heptahydrate (Na ₂ HPO ₄ -7H ₂ O) and monobasic sodium phosphate (NaH ₂ PO ₄ -H ₂ O) were added to water in a container with a stirrer, and stirred until dissolved ( for research 29, phosphate salts were excluded from the composition). Propylene glycol and glycerin were also added, if necessary, so that the final addition of the non-aqueous solution of phospholipids would be resuspended in an 8:1:1 composition of water:glycerol:propylene glycol. In some studies, a solution of calcium acetate [Ca(OAc) ₂ ] in water was added, as shown in table. 6. This aqueous solution was stirred, maintained at 55±5°C. It was determined that the addition of 48.4 mg/g calcium caused a marked flocculation in the aqueous solution in the absence of any phospholipids (see Table 6, Study A). 12.2 μg/g calcium did not precipitate in aqueous solution (see Table 6, Study B).

Example 3.4.3. Combining non-aqueous and aqueous solutions.

For all studies, the addition of the non-aqueous phospholipid concentrate to the aqueous solution was performed as follows. Warm phospholipid dissolved in propylene glycol was added and stirred at 100-150 rpm. Visual observations were recorded and the time for complete dispersion or dissolution (either clear or cloudy) was noted. For study 27, the water composition was initially transparent. Calcium was titrated and a cloudy precipitate formed at concentrations >30.4 µg/g (see Table 6). However, it was noted that the phospholipid-free aqueous solution exhibited marked precipitation at 48.4 μg/g (Study A: Table 6). In Study 27, at calcium levels where the aqueous solution itself was not affected (12.2 μg/g based on Study B, Table 6), no effect was found on the clarity of the aqueous composition of the phospholipids. This was confirmed in study 26 where calcium was added to an aqueous solution (12.2 μg/g) before being combined with a non-aqueous phospholipid concentrate. This was also continued in study 29 where the phosphate buffer was omitted from the aqueous solution. Initially, calcium was added to the aqueous solution (12.2 μg/g) prior to combining with the non-aqueous phospholipid concentrate and the formulation was clear. Additional calcium was added

- 29 040910 after adding phospholipid to the formulation, not more than 96 μg/g, and no precipitation was observed.

The aqueous formulations from Studies 26 and 29 were then collected and filtered through a 0.2 µm filter at 55° C. at 5 psi product pressure. inch. The flow rate for 10 minutes did not decrease compared to the initial flow rate; all samples were filtered and overall filtration was similar to calcium-free formulations (see studies 19, 23 and 25). Samples before and after filtration were collected and compared to determine the loss of phospholipids associated with filtration. No significant loss of phospholipid was observed (see Table 6).

Table 6

Effect of adding a non-aqueous solution of phospholipids to an aqueous solvent containing divalent metal ions

Non-aqueous lipid concentrate Water suspension ³ CQ and II Yu Q L OOO CH O r. Observed turbidity ⁰ Calcium concentration in aqueous solution (µg Ca ⁺² /g) Appearance after adding lipid concentrate to water solution Containing uP0 ₄ buffer Percentage of Initial Filtration Rate by 9- % phospholipid after filtration ^d DPPC DPPA MPEG5000 -DPPE C,E,A 0 Titration 0-48.7 µg Ca ⁺² /g Clear up to 12.2, slightly turbid at 30.4 and turbid with sediment at 36.5 µg Ca ⁺² /g water Yes and/a C,E,A 0 12.2 ^e Transparent Yes 114.3 99 101 98 C,A,E 0 12.2 ^e Transparent No 100.8 99 99 98 and/a and/a 48.4 ^e Sediment Yes and/a n/a n/a n/a and/a and/a 12.2 ^e Transparent Yes and/a n/a n/a n/a

^a All formulations were performed at 55°C. The non-aqueous phospholipid solutions were added to an aqueous formulation vessel containing water (800 mg); dibasic sodium phosphate, heptahydrate (2.16 mg/ml), monobasic sodium phosphate, monohydrate (2.34 mg/ml), sodium chloride (4.84 mg/ml), glycerin (126 mg) and propylene glycol (51.75 mg), unless otherwise noted in the footnotes.

^b A is DPPA (0.9 mg/mL), C is DPPC (8.02 mg/mL), and E is MPEG5000-DPPE (6.08 mg/mL), which were added in that order in 25 ml of propylene glycol.

^c Defined in methods in section 1.4.

^d HPLC with CAD detection, described in Section 1.6.

^e Prior to adding the lipid concentrate to the water formulation vessel, 1 ml, 1 ml, 4 ml and 1 ml of calcium acetate concentrate (6.085 mg Ca ⁺² per g water) were added for tests 26, 29, A and B, respectively.

These studies have shown that calcium does not cause precipitation of phospholipids in the aqueous formulation, even up to 96 µg/g. However, at calcium levels above 12.2 µg/g, phosphate salts begin to precipitate.

Example 4. Effect of divalent metal ions on the dissolution of phospholipids in buffered propylene glycol and with the addition of glycerol.

Example 4.1. Titration of calcium in buffered non-aqueous phospholipid concentrate.

Studies 31 and 32 were performed as follows. Low calcium DPPC, DPPA and MPEG5000-DPPE powder (see Table 1 for process examples) was added either individually (in the sequence shown in Table 7) or as a homogeneous mixture of phospholipids (prepared using toluene and methanol to dissolve and adding MTBE to precipitate the lipid mixture) into heated (55±5° C.) and stirred acetate buffered propylene glycol. It was verified by visual inspection that the lipid was completely dissolved and the resulting solution was clear (see method examples in section 1.4). A solution of calcium acetate [Ca(OAc) ₂ ] in propylene glycol was used to titrate the phospholipid solution through a series of small additions. The solution was stirred and observed changes in appearance during the titration, compared with the blank solvent after each addition and recorded the evaluation of transparency. Received the transparency index (see section 1.4, Fig. 1, for the method) based on this evaluation and the lowest concentration of calcium, which gives a score of +, ++ and +++, is shown in table. 7.

- 30 040910

Table 7

The effect of calcium on the dissolution of phospholipids in buffered propylene glycol

Order of lipid addition ³ Observed threshold values for turbidity (µg/mL Ca ⁺² ) ^b Study DPP WITH DPPA MPEG500 0-DPPE One native mixture of lipids Calcium concentration (mcg/ml Ca+2) + 31 and/a n/a n/a 1 ^s Titration ^g 5.8 11.2 22.3 32 1 3 2 n/a Titration ^s 11.3 17.0 >33.5 ^a Lipids [DPPC (4.01 mg), DPPA (0.45 mg) and MPEG5000-DPPE (3.04 mg), indicated

in this order] individually or a homogeneous mixture of lipids (7.5 mg) was added to each milliliter of propylene glycol containing sodium acetate (0.74 mg) and acetic acid (0.06 mg) at 60° C. with stirring.

^b Defined in Methods Section 1.4.

^c Prepared using toluene and methanol to dissolve lipids and add MTBE to precipitate a homogeneous mixture of lipids.

Example 4.2. Titration of calcium in buffered non-aqueous phospholipid concentrate from MPEG5000-DPPE.

Studies 33-36 were carried out as follows. DPPC, DPPA and MPEG5000-DPPE either high (980 ppm, batch 1) or low Ca ⁺² (4 ppm) were added individually (in the sequence shown in Table 8) or as a homogeneous mixture of phospholipids (obtained using toluene and methanol to dissolve and adding MTBE to precipitate the lipid mixture) into heated (55±5° C.) and stirred acetate buffered propylene glycol. Clarity was assessed (see method examples in section 1.4) and turbidity (+ or ++; see method examples, Fig. 1) of a homogeneous mixture of phospholipids containing a high concentration of calcium was observed. This was different from the clear solution obtained by dissolving a homogeneous mixture of low calcium phospholipids (see Table 8).

Example 4.3. Addition of glycerin.

Glycerol was transferred to these buffered non-aqueous solutions of phospholipids with stirring at 300 rpm. Many bubbles of gas were trapped in the stirred solution, but it became clear when the stirrer was stopped. Visual observations were recorded and noted the level of transparency (either clear or cloudy). These PG/G solutions were then collected and filtered through a 0.2 μm filter at 60° C. at 10 psig product pressure. inch. The flow rate was measured and samples were collected for the measurement of phospholipids. Samples before and after filtration were compared to determine the loss of phospholipids associated with filtration.

Table 8

Effect of Calcium on the Dissolution of Phospholipids in Buffered Propylene Glycol and Added Glycerol

Concentrate based

Propylene Glycol with added Glycerol ^and Acetate/Propylene Glycol ³

Research

Clear Cloudy

Appearance after adding glycerin

Concentration of Ca ⁺² and (Mg ⁺² ) in the product [µg/g]

Percent Initially

Clear Cloudy

filtration speed at 8-9 minutes

174.2 96.7;

blocked filter 245.2 19.8: blocked filter % phospholipid after filtration ^d

100

a Lipids [DPPC (4.01 mg), DPPA (0.45 mg) and MPEG5000-DPPE (3.04 mg), in that order] alone or a homogeneous lipid mixture (7.5 mg) were added to each milliliter of propylene glycol containing sodium acetate (0.74 mg) and acetic acid (0.06 mg), at 60°C with stirring.

^b Propylene glycol containing acetate buffer and phospholipids was diluted 1:1 (v/v) with glycerol.

^c Defined in methods in section 1.4.

^d HPLC with CAD detection, described in Section 1.6.

- 31 040910 ^e Uniform lipid mixture prepared using toluene and methanol to dissolve lipids and adding MTBE to precipitate a homogeneous lipid mixture, low Ca ⁺² (batch 1) for study 33 and high Ca ⁺² (370 Ca ⁺² and 54 ppm Mg ⁺² batch 2) for study 34.

^f MPEG5000-DPPE containing Ca ⁺² (3.04 mg/ml; 980 and 150 ppm Ca ⁺² and Mg ⁺² ;

batch 1) was used in this experiment.

These studies showed that precipitation occurred in a buffered non-aqueous solution of phospholipids in a calcium concentration dependent manner. This occurred regardless of whether the buffered non-aqueous solution of phospholipids was prepared with individual phospholipids or with a homogeneous mixture of lipids and at concentrations that were slightly different. The concentration that causes initial precipitation for the buffered solution was higher (5.8-11.3 µg/g Ca ⁺² ) than for unbuffered solutions (1.5-2.3 µg Ca ⁺² /g, see table 2, studies 1-4), indicating the effect of the buffer.

Calcium from a homogeneous mixture of lipids caused precipitation in the buffered non-aqueous solution of phospholipids, as was observed in the unbuffered solution. Once a precipitate has formed, it does not disperse when mixed with glycerin. This results in a hazy, non-aqueous formulation that has an initially reduced filtration rate and often clogs the 0.2 µm filter (Table 8, FIG. 6). The filtrate of the cloudy formulations was clear, but phospholipid measurements showed slightly reduced levels of DPPA.

Example 5 Microsphere Formation and Acoustic Detection for a Manufactured Product.

Example 5.1. Aqueous suspension of phospholipids.

Studies 37 and 38 were carried out as follows. Filtered materials from studies 19 and 23 were obtained in vials (see method examples in section 1.7.1). After VIALMIX®, post-activation samples were analyzed for size and number of microspheres (see methods in section 1.7.3) and clinical ultrasonic acoustic parameters (see methods in section 1.7.4), see Table. 9.

Table 9

Quantity, size, and acoustic activity of microspheres from an aqueous suspension of phospholipids

Study Receipt basis Average diameter of microspheres (microns) ³ N=2 Microspheres per ml (x 10 ⁹ ) ^b N=2 Mean acoustic attenuation (SD) ^s (dB/cm/ ¹⁰⁶ bubbles/mL) 37 Selected low Ca ⁺² phospholipids measured in MPEG-5000 DPPE and other components 1.38, 1.36 3.73, 2.92 8.9 (0.3) 38 Homogeneous mixture of low Ca ⁺² phospholipids measured in MPEG-5000 DPPE and other components 1.34, 1.35 3.4, 2.5 9.0(1.3)

^a Mean microsphere diameter for microspheres ranging from 1 to 80 µm.

^b Average concentration of microspheres for microspheres in the range from 1 to 80 µm.

^c See method examples for more details.

These studies show that an aqueous suspension of phospholipids can be obtained using individual phospholipids or a homogeneous mixture of phospholipids, when the components are characterized by a low concentration of calcium. Both products have a microsphere diameter within the DEFINITY® specification (see package insert for DEFINITY®) and have strong acoustic attenuation of ultrasound on a clinical ultrasound machine.

Aspects and Embodiments

Various aspects and embodiments provided by the present invention are listed below.

Item 1. A method for producing a suspension of phospholipids, including providing the original forms of DPPA, DPPC and MPEG5000-DPPE;

measurement of calcium concentration in one or more original forms of DPPC, DPPA and MPEG5000-DPPE;

combining the original forms of DPPA, DPPC and/or MPEG5000-DPPE with a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 2. The method of claim 1, further comprising measuring the concentration of calcium in the non-aqueous solvent.

- 32 040910

Item 3. The method according to claim 1, where the combined measured calcium concentration of the original forms of DPPA,

DPPC and/or MPEG-DPPE is low.

Item 4. The method according to claim 1 or 3, wherein the combined measured concentration of calcium in the original forms of DPPA, DPPC and/or MPEG-DPPE and non-aqueous solvent is low.

Item 5. The method of claim 1 wherein the calcium concentrations in the original forms of DPPC, DPPA and MPEG5000-DPPE are measured.

Item 6. The method of claim 2, wherein the calcium concentrations in the original forms of DPPC, DPPA, and MPEG5000-DPPE are measured, and the combined measured calcium concentration in the original forms of DPPA, DPPC, MPEG-DPPE, and the non-aqueous solvent is low.

Item 7. A method for producing a suspension of phospholipids, including providing the original forms of DPPA, DPPC and MPEG5000-DPPE;

measurement of calcium concentration in one or more original forms of DPPC, DPPA and MPEG5000-DPPE;

combining the original forms of DPPA, DPPC and/or MPEG5000-DPPE, characterized by a combined measured low concentration of calcium, with a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 8. The method of claim 7, wherein the calcium concentration in the non-aqueous solvent is measured, and wherein the stock forms of DPPA, DPPC, MPEG500-DPPE and the non-aqueous solvent have a combined measured low calcium concentration.

Item 9. A method for preparing a phospholipid suspension, comprising combining the original form of MPEG5000-DPPE, the original form of DPPA, the original form of DPPC and a non-aqueous solvent, each of which is characterized by a characteristic calcium concentration, to form a solution of phospholipids, and the combined characteristic calcium concentration of the original form of MPEG5000-DPPE , original form of DPPA, original form of DPPC and non-aqueous solvent corresponds to a low concentration of calcium; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 10. A method for preparing a phospholipid suspension, comprising selecting an original form of MPEG5000-DPPE, an original form of DPPA, and an original form of DPPC, one, two or all three of which are characterized by a characteristic calcium concentration, where the combined characteristic calcium concentration corresponds to a low calcium concentration;

combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 11. The method of obtaining a suspension of phospholipids, including selecting the original form of MPEG5000-DPPE, the original form of DPPA and the original form of DPPC, each of which is characterized by a characteristic calcium concentration, where the combined characteristic calcium concentration corresponds to a low calcium concentration;

combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Claim 12. The method of claim 11, wherein the non-aqueous solvent has an intrinsic calcium concentration, and wherein the combined intrinsic calcium concentration of MPEG5000-DPPE, DPPA, and DPPC stock forms and the non-aqueous solvent is low.

Item 13. A method for producing a suspension of phospholipids, including measuring the concentration of calcium in the original form of MPEG5000-DPPE;

combining the original form of MPEG5000-DPPE, characterized by the measured low calcium concentration, with the original form of DPPA, the original form of DPPC and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 14. The method according to item 11, where the non-aqueous solvent contains (i) propylene glycol or (ii) propylene glycol and glycerin.

Item 15. The method according to item 13 or 14, where the non-aqueous solvent contains a buffer.

Item 16. The method according to item 13 or 14, where the non-aqueous solvent contains an acetate buffer.

Item 17. The method according to item 13 or 14, where the aqueous solvent contains a buffer.

Item 18. The method according to item 13 or 14, where the aqueous solvent contains a phosphate buffer.

Item 19. The method according to any one of claims 13-18, where the original forms of DPPC, DPPA and MPEG5000-DPPE according to

- 33 040910 separately combined with a non-aqueous solvent to form a solution of phospholipids.

Item 20. The method of any one of claims 13-18, wherein the original forms of DPPC, DPPA, and MPEG5000-DPPE are sequentially combined with a non-aqueous solvent in an order-independent manner to form a phospholipid solution.

Item 21. The method according to any one of claims 13-18, wherein the original forms of DPPC, DPPA and MPEG5000-DPPE are combined with each other to form a mixture of phospholipids, and then the mixture of phospholipids is combined with a non-aqueous solvent to form a solution of phospholipids.

Item 22. The method according to any one of claims 13-18, wherein the original forms of DPPC, DPPA and MPEG5000-DPPE are combined with each other to form a homogeneous mixture of phospholipids, and a homogeneous mixture of phospholipids is combined with a non-aqueous solvent to form a solution of phospholipids.

Item 23. The method according to item 22, where a homogeneous mixture of phospholipids is formed using a method of dissolution-precipitation in an organic solvent, including dissolving the original forms of DPPC, DPPA and MPEG5000-DPPE in a mixture of methanol and toluene, optionally concentrating a homogeneous mixture of phospholipids/methanol/toluene and then contacting the concentrated phospholipid/methanol/toluene mixture with methyl tert-butyl ether (MTBE) to precipitate the phospholipids to form a homogeneous mixture of phospholipids.

Item 24. The method according to any one of claims 13-23, wherein the low calcium concentration is less than 115 ppm.

Item 25. The method of any one of claims 13-24, further comprising placing the suspension of phospholipids in the vial and introducing gaseous perfluorocarbon into the space above the product in the vial.

Item 26. The method of claim 25, further comprising activating the phospholipid suspension with perfluorocarbon gas to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres.

Claim 27. The method of claim 26, further comprising administering an ultrasound contrast agent to the subject and obtaining one or more contrast-enhanced ultrasound images of the subject.

Item 28. The method according to any one of claims 13 to 27, further comprising measuring the calcium concentration in the original form of DPPA and/or the original form of DPPC and/or a mixture of phospholipids and/or a homogeneous mixture of phospholipids.

Item 29. A method for producing a suspension of phospholipids, including measuring the concentration of calcium in the original form of DPPC;

combining the original form of DPPC, which has a measured low calcium concentration, with the original form of DPPA, the original form of MPEG5000-DPPE and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 30. The method of item 29, wherein the low calcium concentration is less than 90 ppm.

Item 31. The method of obtaining a suspension of phospholipids, including measuring the concentration of calcium in the original form of DPPA;

combining the original form of DPPA, which has a measured low calcium concentration, with the original form of DPPC, the original form of MPEG5000-DPPE and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 32. The method of item 31, wherein the low calcium concentration is less than 780 ppm.

Item 33. The method of obtaining a suspension of phospholipids, including measuring the concentration of calcium in a non-aqueous solvent;

combining a non-aqueous solvent having a measured low calcium concentration with the original form of DPPA, the original form of DPPC and the original form of MPEG5000-DPPE to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 34. The method of item 33, wherein the low calcium concentration is less than 0.7 ppm.

Item 35. A method for producing a suspension of phospholipids, including selecting the original form of MPEG5000-DPPE, characterized by the absence of calcium or its low concentration;

combining said MPEG5000-DPPE stock, DPPA stock, DPPC stock, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 36. The method of claim 35, wherein the original form of MPEG5000-DPPE is also characterized by the absence or low content of divalent metal cations.

Item 37. A method for preparing a phospholipid suspension, comprising combining an original form of MPEG5000-DPPE, an original form of DPPA, an original form of DPPC, and a non-aqueous solvent to form a phospholipid solution having no or low concentration of calcium; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 38. A method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent containing gas-encapsulated phospholipid microspheres;

administering an ultrasound contrast agent to a subject; and obtaining one or more contrast-enhanced ultrasound images of the subject, wherein the phospholipid suspension is obtained using the method of any one of claims 1 to 37.

Item 39. A method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent containing gas-encapsulated phospholipid microspheres;

administering an ultrasound contrast agent to a subject and obtaining one or more contrast-enhanced ultrasound images of the subject, wherein the phospholipid suspension is obtained by a method comprising measuring a calcium concentration in the original form of MPEG5000-DPPE;

combining the original form of MPEG5000-DPPE, characterized by the measured low calcium concentration, with the original form of DPPA, the original form of DPPC and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 40. A method of imaging a subject comprising combining a suspension of phospholipids with a gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres;

administering an ultrasound contrast agent to a subject; and obtaining one or more contrast-enhanced ultrasound images of the subject, wherein the phospholipid suspension is obtained by a method including selecting an initial form of MPEG5000-DPPE characterized by a lack of calcium or a low concentration thereof, combining these initial MPEG5000-DPPE form, DPPA original form, DPPC original form, and a non-aqueous solvent to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 41. A method for imaging a subject, comprising combining a suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent containing gas-encapsulated phospholipid microspheres;

administering an ultrasound contrast agent to a subject and obtaining one or more contrast-enhanced ultrasound images of the subject, wherein the phospholipid suspension is obtained by a method comprising combining stock MPEG5000-DPPE, stock DPPA, stock DPPC, and a non-aqueous solvent with the formation of a solution of phospholipids, characterized by the absence of calcium or its low concentration; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 42. A method for preparing a suspension of phospholipids, comprising combining separately the original forms of DPPA, DPPC and MPEG5000-DPPE with a propylene glycol (PG) containing non-aqueous solvent, under the condition of low or no calcium, to form a solution of phospholipids; and combining the phospholipid solution with an aqueous solvent to form a phospholipid suspension.

Item 43. A method for preparing a phospholipid suspension, comprising sequentially combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a PG-containing non-aqueous solvent, under the condition of low or no calcium, in an order-independent manner, to form a solution of phospholipids and

- 35 040910 combining a solution of phospholipids with an aqueous solvent to form a suspension of phospholipids.

Item 44. A method for producing a suspension of phospholipids, including:

combining, in the absence of methanol and toluene, the original forms of DPPA, DPPC and MPEG5000-DPPE to form a homogeneous mixture of phospholipids, combining a homogeneous mixture of phospholipids with a non-aqueous solvent containing PG, in the condition of low or no calcium, to form a solution of phospholipids and combining the solution phospholipids with an aqueous solvent to form a suspension of phospholipids.

Item 45. A method for producing a suspension of phospholipids, including combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a solvent for a homogeneous mixture to form a homogeneous mixture of phospholipids;

evaporation of the solvent for a homogeneous mixture with the formation of a dried homogeneous mixture of phospholipids;

combining the dried homogeneous mixture of phospholipids with a PG-containing non-aqueous solvent, under the condition of low or no calcium content, to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 46. The method of obtaining a suspension of phospholipids, including combining the original forms of DPPA, DPPC and MPEG5000-DPPE with a solvent for a homogeneous mixture to form a homogeneous mixture of phospholipids;

precipitation, in the absence of MTBE, a homogeneous mixture of phospholipids using a second solvent for a homogeneous mixture;

combining the precipitated homogeneous mixture of phospholipids with a non-aqueous solvent, under the condition of low or no calcium content, to form a phospholipid solution; and combining the phospholipid solution with an aqueous solvent to form a suspension of phospholipids.

Item 47. The method according to any one of claims 42-46, wherein the low calcium concentration is less than 0.7 ppm.

Item 48. The method of any one of claims 42 to 47, further comprising combining the suspension of phospholipids with gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres.

Claim 49. The method of Claim 48, further comprising administering an ultrasound contrast agent to the subject and obtaining one or more contrast-enhanced ultrasound images of the subject.

Item 50. A method of imaging a subject comprising combining a suspension of phospholipids with a gaseous perfluorocarbon to form an ultrasound contrast agent comprising gas-encapsulated phospholipid microspheres;

administering an ultrasound contrast agent to a subject; and obtaining one or more contrast-enhanced ultrasound images of the subject, wherein the phospholipid suspension is obtained using the method of any one of claims 42-47.

Item 51. Composition containing a solution of phospholipids containing DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent and characterized by a low calcium concentration.

Item 52. A composition comprising a solution of phospholipids comprising DPPA, DPPC and MPEG5000-DPPE in a non-aqueous solvent, wherein the DPPA, DPPC and MPEG5000-DPPE and the non-aqueous solvent have a combined inherent low calcium ion content.

Item 53. The composition according to item 51 or 52, where the non-aqueous solvent contains propylene glycol.

Item 54. The composition according to item 51 or 52, where the non-aqueous solvent contains propylene glycol and glycerin.

Clause 55. The composition according to any one of claims 51-54, wherein the non-aqueous solvent contains a buffer.

Item 56. The composition according to item 55, where the buffer is an acetate buffer.

Clause 57. The composition according to any one of claims 51-56, further containing a gaseous perfluorocarbon.

Item 58. The composition of claim 57, wherein the gaseous perfluorocarbon is perflutrene.

Item 59. A method of contrast imaging of a subject by ultrasound, including:

(a) activating the perfluorocarbon gas phospholipid suspension to form gas-encapsulated lipid microspheres, the phospholipid suspension comprising a phospholipid solution containing one or more phospholipids and a non-aqueous solvent, one or more of which has a low calcium concentration;

- 36 040910 (b) administering the gas-encapsulated lipid microspheres to the subject; and (c) obtaining an ultrasound image of the subject.

Item 60. The method according to item 59, where one or more phospholipids provide DPPC and

MPEG-5000-DPPE.

Item 61. The method according to item 59, where one or more phospholipids provide DPPA, DPPC and MPEG-5000-DPPE.

Item 62. The method according to item 61, where DPPA, DPPC and MPEG5000-DPPE are presented in a mol% ratio of 10 to 82 to 8 (10:82:8).

Item 63. The method of any one of claims 60-62, wherein the characteristic low calcium concentration for DPPA is less than 780 ppm, for DPPC it is less than 90 ppm, and for MPEG5000-DPPE it is less than 115 ppm.

Item 64. The method of any one of claims 59-63, wherein the non-aqueous solvent comprises (a) propylene glycol or (b) propylene glycol and glycerin.

Item 65. The method of any one of claims 59-64, wherein the characteristic low calcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Item 66. The method according to any one of claims 59-65, wherein the phospholipid solution does not contain a detectable precipitate of phospholipids.

Item 67. A method of contrast imaging of a subject by ultrasound, including:

(a) activating a suspension of phospholipids with gaseous perfluorocarbon to form gas-encapsulated lipid microspheres, wherein the phospholipid suspension contains a solution of phospholipids containing one or more phospholipids and a non-aqueous solvent, and is prepared in the absence of methanol and toluene and in the absence of methyl tert-butyl an ether, wherein one or more phospholipids and a non-aqueous solvent have a low calcium concentration, (b) administering gas-encapsulated lipid microspheres to a subject, and (c) obtaining an ultrasound image of the subject.

Item 68. The method according to item 67, where one or more phospholipids provide DPPC and MPEG-5000-DPPE.

Item 69. The method according to item 67, where one or more phospholipids provide DPPA, DPPC and MPEG-5000-DPPE.

Item 70. The method according to item 69, where DPPA, DPPC and MPEG-5000-DPPE are present in a mol% ratio of 10 to 82 to 8 (10:82:8).

Item 71. The method of any one of claims 67-70, wherein the low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm.

Item 72. The method of any one of claims 67-71, wherein the non-aqueous solvent comprises (a) propylene glycol or (b) propylene glycol and glycerin.

Item 73. The method of any one of claims 67-72, wherein the low calcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Item 74. The method according to any one of claims 67-73, wherein the phospholipid solution does not contain a detectable precipitate of phospholipids.

Item 75. A process for producing gas-encapsulated lipid microspheres, comprising:

combining one or more phospholipids and a non-aqueous solvent to form a phospholipid solution, wherein the one or more phospholipids and/or non-aqueous solvent have a characteristic low calcium concentration, combining the phospholipid solution with an aqueous solution to form a phospholipid suspension, and activating the phospholipid suspension with gaseous perfluorocarbon to form lipid microspheres with encapsulated gas.

Item 76. The method according to item 75, where one or more phospholipids provide DPPC and MPEG5000-DPPE.

Item 77. The method according to item 75, where one or more phospholipids provide DPPA, DPPC and MPEG-5000-DPPE.

Item 78. The method according to item 77, where DPPA, DPPC and MPEG-5000-DPPE are present in a mol% ratio of 10 to 82 to 8 (10:82:8).

Item 79. The method of any one of claims 75-78, wherein the characteristic low calcium concentration for DPPA is less than 780 ppm, for DPPC it is less than 90 ppm, and for MPEG5000-DPPE it is less than 115 ppm.

Item 80. The method of any one of claims 75-79, wherein the non-aqueous solvent comprises (a) propylene glycol or (b) propylene glycol and glycerin.

Item 81. The method of any one of claims 75-80, wherein the characteristic low calcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Item 82. The method of any one of claims 75-81, wherein the phospholipid solution does not contain a detectable precipitate of phospholipids.

- 37 040910

Item 83. A process for preparing gas-encapsulated lipid microspheres, comprising combining one or more phospholipids and a non-aqueous solvent, in the absence of methanol and toluene, and in the absence of methyl tert-butyl ether, to form a solution of phospholipids, wherein one or more phospholipids and/ or non-aqueous solvent is characterized by a low concentration of calcium;

combining the phospholipid solution with the aqueous solution to form a phospholipid suspension; and activating the phospholipid suspension with gaseous perfluorocarbon to form gas-encapsulated lipid microspheres.

Item 84. The method according to item 83, where one or more lipids provide (a) DPPC and MPEG-5000-DPPE, or (b) DPPA, DPPC and MPEG-5000-DPPE, and/or (c) DPPA, DPPC and MPEG-5000-DPPE in a mole % ratio of 10 to 82 to 8 (10:82:8).

Item 85. The method of claim 84, wherein the low calcium concentration for DPPA is less than 780 ppm, for DPPC is less than 90 ppm, and for MPEG5000-DPPE is less than 115 ppm.

Item 86. The method of any one of claims 83-85, wherein the non-aqueous solvent comprises (a) propylene glycol or (b) propylene glycol and glycerin.

Item 87. The method of any one of claims 83-86, wherein the low calcium concentration for the non-aqueous solvent is less than 0.7 ppm.

Item 88. The method of any one of claims 83-87, wherein the phospholipid solution does not contain a detectable precipitate of phospholipids.

Item 89. The method of any one of claims 67-74, wherein the one or more phospholipids or non-aqueous solvent have a characteristic low calcium concentration.

Item 90. The method according to item 89, where the characteristic low concentration of calcium is determined using atomic absorption spectroscopy.

Item 91. The method according to any one of claims 67-74, wherein the low calcium concentration is determined using atomic absorption spectroscopy.

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 parameters, dimensions, materials, and designs described herein are by way of example and that the actual parameters, 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 understand, 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

Claims (4)

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 of 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 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) can 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 for obtaining a phospholipid solution suitable for obtaining a suspension of phospholipids, including combining 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and K- (methoxypolyethylene glycol-5000-carbamoyl)-1,2dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (MPEG5000-DPPE) in propylene glycol and/or glycerin; (a) in the presence of none, one or two, but not all three of methanol, toluene, methyl tertiary butyl ether (MTBE), wherein the MPEG5000-DPPE has a calcium and/or magnesium concentration of less than 115 parts per million (ppm); or (b) MPEG5000-DPPE selected with a calcium and/or magnesium ion concentration of less than 115 ppm (ppm) to form a phospholipid solution having a calcium and/or magnesium concentration of less than 0.7 ppm (ppm) . 2. The method of claim 1, wherein DPPA, DPPC and MPEG5000-DPPE are present in a mole % ratio of 5-15% to 77-90% to 5-15%. 3. The method of claim 1, wherein DPPA, DPPC and MPEG5000-DPPE are present in a mole % ratio of 10% to 82% to 8% (10:82:8). 4. The method according to any one of claims 1 to 3, wherein the concentration of calcium and/or magnesium for DPPA is less than 780 ppm and the concentration of calcium and/or magnesium for DPPC is less than 90 ppm. -