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US-12616758-B2 - Freeze-dried composition for preparing calibrated gas-filled microvesicles

US12616758B2US 12616758 B2US12616758 B2US 12616758B2US-12616758-B2

Abstract

The present invention generally relates to the field of ultrasound contrast-agents (USCA). In particular, it relates to a freeze-dried composition comprising an amphiphilic material and a mixture of freeze-drying protecting components, which may be reconstituted for preparing a suspension of gas-filled microvesicles with calibrated size, useful in diagnostic or therapeutic applications. It further relates to the method for the preparation of such freeze-dried composition.

Inventors

  • Anne Lassus
  • Samir Cherkaoui

Assignees

  • BRACCO SUISSE SA

Dates

Publication Date
20260505
Application Date
20200624
Priority Date
20190625

Claims (17)

  1. 1 . A freeze-dried composition comprising i) an amphiphilic material comprising a phospholipid and ii) a freeze-drying protecting component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, is capable of providing a suspension of calibrated gas-filled microvesicles having a geometric standard deviation (GSD) of 1.2 or lower, wherein said freeze-drying protecting component is a mixture of at least two freeze-drying protecting components comprising: (a) a polyethylene glycol, wherein said polyethylene glycol has a molecular weight from 4000 to 8000 g/mol, and (b) a polyol, wherein said polyol is sorbitol or xylitol.
  2. 2 . A suspension of gas-filled microvesicles obtained by reconstituting a freeze-dried composition according to claim 1 with a pharmaceutically acceptable solution in the presence of a biocompatible gas, wherein said suspension has a geometric standard deviation (GSD) of 1.2 or lower.
  3. 3 . The suspension of gas-filled microvesicles according to claim 2 , wherein said suspension comprises the mixture of at least two freeze-drying protecting components at a concentration between 10 and 25% (w/v %).
  4. 4 . The suspension of gas-filled microvesicles according to claim 2 , wherein said suspension is characterized by a concentration of from 2.5×10 8 to 5.5×10 8 microvesicles/mL.
  5. 5 . A method of preparing a freeze-dried composition for the preparation of a reconstituted suspension of calibrated gas-filled microvesicles, comprising the steps of: a. preparing a suspension of calibrated gas-filled microvesicles comprising i) an amphiphilic material comprising a phospholipid and ii) a mixture of at least two freeze-drying protecting components comprising: (a) a polyethylene glycol, wherein said polyethylene glycol has a molecular weight from 4000 to 8000 g/mol, and (b) a polyol, wherein said polyol is sorbitol or xylitol; and b. freeze-drying the calibrated microvesicles suspension.
  6. 6 . The method of claim 5 , wherein the method of preparation of step a. comprises a microfluidic flow-focusing technique.
  7. 7 . The method of claim 5 , wherein said mixture of freeze-drying protecting components has a total concentration comprised between 100 mg/ml and 300 mg/mL.
  8. 8 . A process for the preparation of an injectable contrast agent comprising a suspension of gas-filled microvesicles, wherein said process comprises reconstituting a freeze-dried composition as defined in claim 1 , with a pharmaceutically acceptable solution in the presence of a biocompatible gas, wherein said suspension has a geometric standard deviation (GSD) of 1.2 or lower.
  9. 9 . The method of claim 5 , wherein said mixture of freeze-drying protecting components has a total concentration comprised between 120 mg/mL and 250 mg/mL.
  10. 10 . The method of claim 5 , wherein said mixture of freeze-drying protecting components has a total concentration of 200 mg/mL.
  11. 11 . The freeze-dried composition according to claim 1 , wherein said mixture of at least two freeze-drying protecting components has a total concentration from 50 mg/mL to 100 mg/mL.
  12. 12 . The freeze-dried composition according to claim 1 , wherein said mixture of at least two freeze-drying protecting components has a total concentration of 100 mg/mL.
  13. 13 . The suspension of gas-filled microvesicles according to claim 2 , wherein said suspension is characterized by a concentration of from 2.0×10 8 to 5.5×10 8 microvesicles/mL.
  14. 14 . The method of claim 5 , wherein said mixture of freeze-drying protecting components has a total concentration from 50 mg/mL to 100 mg/mL.
  15. 15 . The method of claim 5 , wherein said mixture of freeze-drying protecting components has a total concentration of 100 mg/mL.
  16. 16 . The process according to claim 8 , wherein said suspension is characterized by a concentration of from 2.0×10 8 to 5.5×10 8 microvesicles/mL.
  17. 17 . The process according to claim 8 , wherein said suspension is characterized by a concentration of from 2.5×10 8 to 5.5×10 8 microvesicles/mL.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS This application is the national stage application of corresponding international application number PCT/EP2020/067756, filed Jun. 24, 2020, which claims priority to and the benefit of European application no. 19182226.1, filed Jun. 25, 2019, which is hereby incorporated by reference in its entirety. TECHNICAL FIELD The present invention generally relates to the field of ultrasound contrast-agents (USCA). In particular, it relates to a freeze-dried composition comprising an amphiphilic material and a mixture of freeze-drying protecting components, which may be reconstituted for preparing a suspension of gas-filled microvesicles with calibrated size, useful in diagnostic or therapeutic applications. It further relates to the method for the preparation of such freeze-dried composition. BACKGROUND OF THE INVENTION Calibrated-size microvesicles (CMV) are a new generation of gaseous microbubbles having a narrow calibrated and controlled size distribution (mean sizes between 3 and 8 μm) compared to commercially available polydisperse microbubble ultrasound contrast-agents (USCA). These calibrated-size microbubbles are designed to enhance imaging sensitivity and improve efficiency to deliver drug and gene to specific organs. Calibrated microvesicles can be produced using various techniques: decantation, mechanical filtration, centrifugation, bubble sorting and flow-focusing. Particularly, the flow-focusing technology allows the manufacturing of calibrated microvesicles (typically with a geometric standard deviation (GSD) values between 1.05 and 1.08) in a highly reproducible way at a reasonable production rate (˜60 million bubbles per minute), with acceptable concentrations of suspended microvesicles (e.g. between 3×108 CMV/mL and 4×108 CMV/mL) for subsequent uses. Ref.1 [WO2018/041906 A1—BRACCO SUISSE SA] and Ref.2 [PCT application number PCT/EP2019/055325], both in the name of the Applicant, describe a method for preparing CMV, such as gas-filled microbubbles, in particular by using microfluidic technique. Notwithstanding aqueous suspensions of calibrated microvesicles are highly stable at room temperature for few weeks, this stability may pose some constraints for a pharmaceutical product development, for which longer shelf life is generally desirable. There is thus a need to develop a long-term storage procedure. Freeze-drying, also known as lyophilization, is a complex and challenging process, widely used in the pharmaceutical industry that has the advantage of preserving pharmaceutical products under a dry form over several months. In fact, freeze-dried products display greater storage stability and can be easily shipped. Freeze-drying is also used in the field of gas filled microvesicles for preparing freeze-dried dosage forms which are then reconstituted with an aqueous solvent in the presence of a gas, to form a suspension of gas-filled microvesicles. Ref3 [US2017/080113 A1—GE Healthcare] describes the preparation of a suspension of microbubbles with adjusted sizes and subsequently lyophilization with a sucrose solution thereof. Ref4 [WO97/29782 A1—NICOMED IMAGING A/S] teaches that USCA precursors which are stable at room temperature (RT) can be prepared by lyophilization of C3F8 microbubbles in the presence of a freeze-drying stabilizer, preferably sucrose. Up to now, according to Applicant's knowledge, such technique has not been applied yet for preparing a freeze-dried composition from a suspension of calibrated microvesicles. As observed by the Applicant, one of the most challenging issue in preparing a freeze-dried composition of calibrated microvesicles is related to the need of avoiding substantial modification of the characteristics of the initial suspension of calibrated microvesicles, such as concentration, monodispersity or geometric standard deviation (GSD) and/or final mean diameter. The Applicant has now found that such initial characteristics, can be preserved to an acceptable extent after the freeze-drying process by using a suitable mixture of freeze-drying protecting components. SUMMARY OF THE INVENTION In a first aspect, the present invention relates to a freeze-dried composition comprising an amphiphilic material and a freeze-drying protecting component which, upon reconstitution with a pharmaceutically acceptable solution in the presence of a biocompatible gas, provides a suspension of calibrated gas-filled microvesicles, wherein said freeze-drying protecting component is a mixture of at least two freeze-drying protecting components and wherein said reconstituted suspension of calibrated gas-filled microvesicles have a geometric standard deviation (GSD) lower than 1.2. In a preferred embodiment of the invention, the mixture of freeze-drying protecting components comprises a polymer, preferably a hydrophilic polymer, more preferably a polyglycol, and a polyol or a saccharide. In a still more preferred embodiment, said mixture comprises a polyethy