Ultrasound contrast agents and process for the preparation thereof

Abstract

Injectable aqueous suspension of microbubbles filled with a biocompatible gas and a method of preparation thereof. At least 10% of the total volume of gas contained in the microbubbles is contained in microbububbles with a diameter of 1.5 μm or less. The microbubbles can be obtained by preparing an emulsion comprising an aqueous medium, a phospholipid and a water immiscible organic solvent. The emulsion is then freeze-dried and then reconstituted in an aqueous suspension of gas-filled microbubbles.

Description

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation-in-part of co-pending U.S. application, U.S. Ser. No. ______, filed Aug. 2, 2005, which is a national stage application of international application PCT / IB2004 / 000243, filed Feb. 3, 2004, which claims priority to and the benefit of European application EP03002375.8, filed Feb. 4, 2003, all of which are hereby incorporated by reference.FIELD OF THE INVENTION [0002] The present invention relates to a process for the preparation of a dry or lyophilized formulation useful for preparing a gas containing contrast agent usable in diagnostic imaging and to a process for preparing said gas containing contrast agent. [0003] The invention also includes dry formulations prepared by this process, which may be reconstituted to form contrast agent suspensions useful in diagnostic imaging. The invention further includes suspensions of gas filled microbubbles useful in diagnostic imaging prepared using dry formulations...

AI Technical Summary

Benefits of technology

[0084] Viscosity enhancers and solubility aids that may suitably be employed are for example mono- or polysaccharides, such as glucose, lactose, saccharose, and dextrans, aliphatic alcohols, such as isopropyl alcohol and butyl alcohol, polyols such as glycerol, 1,2-propanediol, and the like agents. In general however we have found that it is unnecessary to incorporate additives such as viscosity enhancers, which are commonly employed in many existing contrast agent formulations, into the contrast agents of the present invention. This is a further advantage of the present invention as the number of components administered to the body of a subject is kept to a minimum and the viscosity of the contrast agents is maintained as low as possible.

[0085] As mentioned before, the Applicant has found substantially unnecessary, to add water-insoluble structure-builders, such as cholesterol, to the emulsifying mixture. As a matter of fact, it has been observed that an amount of 0.05% (w / w with respect to the total weight of the emulsifying mixture) of cholesterol dramatically reduces the conversion yield from microdroplets into gas-filled microvesicles, further resulting in a broad-dispersion of the vesicles' size. The amount of water-insoluble compounds in the emulsifying mixture, particularly of those compounds not comprising one or two fatty acid residue in their structure, is thus preferably lower than 0.050%, more preferably lower than about 0.030% by weight with respect to the total weight of the emulsion.

[0086] Emulsions produced according to step a) may advantageously be subjected to one or more washing steps, prior to the lyophilization of step b), in order to remove excess of phospholipids in the aqueous phase (not associated to the emulsion) and separate and remove optional additives such as viscosity enhancers and solubility aids, as well as undesired material such as colloidal particles, and undersized and / or oversized emulsion droplets. Such washing may be effected in per se known manner, the emulsion being separated using techniques such as decantation, flotation, centrifugation, cross flow filtration and the like.

[0087] If washing steps are foreseen, and if a lyoprotective agent was present in the original aqueous phase prior to the generation of the emulsion, said washing steps can be performed with aqueous solutions containing one or more lyoprotective agents to replace the amount of lyoprotective agents partially removed with the washings. On the other side, if no lyoprotectant was present in the emulsified aqueous-organic mixture, the formed emulsion can be washed with a lyoprotectant-containing aqueous solution, in order to introduce the lyoprotectant into the emulsified mixture or, alternatively, the lyoprotectant can be added after the washing steps, prior to lyophilisation.

[0088] If desired, the emulsion (either as such or after the washing step) can be subjected to a ultrafiltration or microfiltration step before lyophilization, in order to further reduce the amount of large size microbubbles in the final reconstituted suspension. During microfiltration, e.g. with a 5 μm or 3 μm filter, large size microdroplets are in fact retained by the filter and separated from the rest of the small size microdroplets, thus preventing the formation of large size microbubbles upon reconstitution of the lyophilized material. Microfiltration can be accomplished according to conventional techniques such as positive filtration, vacuum filtration or in-line filtration. Membranes of filtration can be Nylon, glass fiber, cellulose, paper, polycarbonate or polyester (Nuclepore®) membranes.

[0089] According to an alternative embodiment, an additional amphiphilic compound can be added after the formation of the emulsion according to the above teachings, either with or without the washing steps. In particular, an aqueous suspension of the desired compound is added to the formed emulsion, preferably under agitation and heating (preferably at less than 80° C., e.g. 40° C.-80° C., in particular 50-70° C.), in order to add said compound to the stabilizing envelope. This alternative embodiment is particularly useful to subsequently introduce into the stabilizing layer amphiphilic compounds which may otherwise negatively affect the properties of the final product if introduced in the initial mixture of the emulsion. Examples of amphiphilic compounds which can conveniently be subsequently introduced as additional components of the stabilizing envelope after the preparation of the initial emulsion are, for instance, PEG-modified phospholipids, in particular PEG-modified phosphatidylethanolamines, such as DMPE-PEG750, DMPE-PEG1000, DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000, DMPE-PEG5000, DPPE-PEG750, DPPE-PEG1000, DPPE-PEG2000, DPPE-PEG3000, DPPE-PEG4000, DPPE-PEG5000, DSPE-PEG750, DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000, DSPE-PEG5000, DAPE-PEG750, DAPE-PEG1000, DAPE-PEG2000, DAPE-PEG3000, DAPE-PEG4000 or DAPE-PEG5000. Similarly, also PEG-modified phospholipids bearing reactive moieties or targeting ligands (e.g. containing biotin, maleimide, or maleimide-peptide) can conveniently be introduced subsequently according to this method. In addition, this technique can also be used to subsequently add to the composition of the stabilizing layer other components, such as lipopeptides or polymeric surfactants. Examples of polymeric surfactants which can be conveniently added after formation of the emulsion are, for instance, ethyleneoxide-propylenoxide block copolymers, such as Pluronic F68, Pluronic F108, Pluronic F-127 (Sigma Aldrich, Missouri, USA); Polyoxyethylated alkyl ethers such as Brij® 78 (Sigma Aldrich, Missouri, USA); Polyoxyethylene fatty acid esters such as Myrj® 53 or Myrj® 59 (Sigma Aldrich, Missouri, USA); Polyoxyethylenesorbitan fatty acid ester such as Tween® 60 (Sigma Aldrich, Missouri, USA); or Polyethylene glycol tert-octylphenyl ether such as Triton®X-100 (Sigma Aldrich, Missouri, USA).

Problems solved by technology

The simple dispersion of free gas bubbles in the aqueous medium is however of limited practical interest, since these bubbles are in general not stable enough to be useful as ultrasound contrast agents.

The Applicant has however observed that the amount of agitation energy applied for generating the gas microbubble dispersion in the phospholipid-containing aqueous medium may be excessively high, particularly when small diameter microbubbles are to be obtained (e.g. 23000 rpm for 10 minutes, for obtaining a dispersion of bubbles having a volume mean diameter of about 3 μm).

This high agitation energy may determine local overheating in the aqueous dispersion of microbubbles, which may in turn cause degradation of the phospholipids contained in the aqueous medium.

In addition, the effects of an excessively high agitation energy are in general difficult to control and may result in an uncontrollable size distribution of the final microbubbles.

In addition, the conversion step from the emulsion microparticles, obtained according to the above process, into gas microbubbles results in rather low yield.