TECHNICAL FIELD

The invention relates to a new method of manufacturing a suspension of gas-filled microvesicles by reconstituting a freeze-dried product and to the suspension obtained according to said method.

BACKGROUND OF THE INVENTION

Rapid development of contrast agents in the recent years has generated a number of different compositions and formulations, which are useful in contrast-enhanced imaging of organs and tissues of human or animal body as well as in therapeutic treatments thereof.

A class of contrast agents particularly useful for Contrast Enhanced UltraSound imaging (“CEUS” imaging) includes suspensions of gas bubbles of nano- and/or micro-metric size dispersed in an aqueous medium. The gas is typically entrapped or encapsulated in a film-layer comprising, for instance, emulsifiers, oils, thickeners or sugars. These stabilized gas bubbles (dispersed in a suitable physiological solution) are generally referred to in the art with various terminologies, depending typically from the stabilizing material employed for their preparation; these terms include, for instance, “microspheres”, “microbubbles”, “microcapsules” or “microballoons”, globally referred to here as “gas-filled microvesicles” (or “microvesicles”).

UltraSound Contrast Agents (“USCAs”) can be produced according to various manufacturing methods. One of these methods, see e.g. WO94/09829, entails the dissolution of an amphiphilic material (such as a phospholipid and/or fatty acid) and of a freeze-drying protecting compound (e.g. polyetheleneglycol) in an organic solvent; the obtained mixture is then subjected to freeze-drying, typically after being filled into vials, to remove the solvent and obtain a freeze-dried product. Another method, see e.g. WO2004/069284, entails the preparation of a microemulsion of water with a water immiscible organic solvent, said emulsion comprising an amphiphilic material and a freeze-drying protecting compound. The emulsion is then and subjected (upon distribution into vials) to a freeze-drying step to remove water and solvent.

The headspace of the vials, containing a freeze-dried solid product in powder form at the bottom thereof, is then filled with a suitable gas (e.g. a fluorinated gas) and finally sealed for storage. Before use, an aqueous suspension of microbubbles is easily prepared by introducing a suitable liquid into the vial (e.g. saline) and gently shaking the vial to dissolve the freeze-dried product.

A commercially available USCA which can be manufactured according to the above method is SonoVue® (or Lumason® in the USA), from Bracco.

The Applicant has now observed that the characteristics of a suspension of gas-filled microvesicles (particularly microbubbles) reconstituted from a freeze-dried product can be improved by introducing a final controlled thermal treatment (i.e. heating) at the end of the process for manufacturing the freeze-dried solid product.

SUMMARY OF THE INVENTION

According to an aspect, the invention relates to a method of manufacturing a freeze-dried composition suitable for the preparation of a suspension of stabilized gas-filled microvesicles, said composition comprising: (i) an amphiphilic material capable of stabilizing said gas microvesicles; and (ii) a freeze-drying protecting component; which comprises:a. preparing a liquid mixture comprising said amphiphilic material and said freeze-drying protecting component in a solvent;b. freeze-drying the liquid mixture to remove said solvent and obtain a freeze-dried product comprising said amphiphilic material and said freeze-drying protecting component; andc. heating said freeze-dried product.

Preferably, said heating step comprises heating said product at a temperature higher than 35° C., more preferably at least 38° C. The heating temperature is preferably lower than 50° C., more preferably lower than 48° C.

In certain embodiments, the heating step is performed at ambient pressure.

Preferably, the heating step lasts for at least 8 hours, more preferably for at least 12 hours.

According to another aspect, the invention relates to a freeze-dried product obtained according to the manufacturing method described above.

According to another aspect, the invention relates to a suspension of gas-filled microvesicles obtained by reconstituting a freeze-dried product prepared according to the method of manufacturing described above, said suspension being obtained by admixing said product with a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas under gentle agitation.

According to a further aspect the invention relates to method for manufacturing a suspension of gas-filled microvesicles stabilized by an amphiphilic material, which comprises:a. preparing a freeze-dried product according to the manufacturing method illustrated above; andb. reconstituting said product by admixing it with a pharmaceutically acceptable liquid carrier in the presence of a physiologically acceptable gas under gentle agitation, to obtain the suspension of gas-filled microvesicles.

