Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Ultrasound contrast agent
Reexamination Certificate
1999-08-16
2001-07-24
Hartley, Michael G. (Department: 1619)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Ultrasound contrast agent
C600S458000
Reexamination Certificate
active
06264918
ABSTRACT:
The present invention relates to the use of hollow echogenic microcapsules in ultrasound imaging.
The fact that air bubbles in the body can be used for echocardiography has been known for some time.
WO 92/18164 discloses the spray-drying of a solution of a wall-forming material, preferably a protein such as albumin, to form microcapsules. In WO 94/08627, the pressure at which the solution is sprayed into the heated chamber is reduced, to form larger microcapsules, or the half-life of the microcapsules in the bloodstream is increased, for example by including a surfactant in the solution which is sprayed, or the microcapsules are targeted to a selected part of the body, for example by suspending them in a solution of an electrically charged compound.
Our as yet unpublished patent application PCT/GB95/02673 discloses that, by including a volatile compound in the aqueous solution which is spray-dried, microcapsules with improved properties can be formed, in higher yield, with narrower size distribution and thinner shells.
SUMMARY OF THE INVENTION
We have now found, unexpectedly, that the microcapsules thus formed and which are not then freeze-dried also have a relatively long serum half-life, which enables the microcapsules to be used in new ways.
One aspect of the invention provides a method of (a) providing microcapsules which have been formed by (i) providing a solution of an aqueously-soluble material in an aqueous solvent containing water and a liquid of greater volatility than water and (ii) spraying the said solution into a gas such that the aqueous solvent evaporates, to form hollow microcapsules suitable for use as an echogenic contrast agent, (b) injecting the microcapsules into a patient and (c) subjecting the patient to ultrasonic energy to obtain an image from ultrasound reflected or absorbed by the microcapsules, characterised in that at least 5 minutes elapses between steps (b) and (c).
Imaging can be performed within the said 5 minute period, provided that it is also performed after the said period. The period between steps (b) and (c) is preferably at least 10, 15, 20, 30, 40, 50, 60, 70, 80 or 90 minutes. After a longer period, the image quality may deteriorate.
The echocontrast agent can be detected using 2-dimensional B-mode ultrasound imaging.
The advantage of such prolonged imaging is that the patient can receive the imaging agent, an initial image can be taken, the patient can then be subjected to a physiological perturbation in order to potentially change the tissue being imaged, and then another image can be taken during or after the perturbation in order to detect any changes in the said tissue without the patient having to receive a second dose of imaging agent. The physiological perturbation can be physical exercise when the tissue being imaged is the heart.
Suitable volatile liquids include ethanol (the preferred volatile liquid) (boiling point 78.3° C.), methanol (b.p. 64.5° C.), and acetone (b.p. 56° C.). The volatile liquid needs to act as a solvent or at least a co-solvent for the wall-forming material and be miscible with water at the ratios used.
The proportion of the aqueous solution which is the volatile liquid will vary according to the identity of the volatile compound, the concentration and identity of the wall-forming material, the temperature and pressures at which the solution is to be sprayed, and the microcapsule product desired. Typically, between 0.1% and 80% v/v, preferably 1-50% v/v and most preferably 5-30% v/v, for example about 20% v/v, of the solution is the volatile liquid. Mixtures of volatile liquids may be used, in which case these percentages refer to the total content of volatile liquid.
The spray-drying may be a one step process such as to provide the desired microcapsule product immediately. Alternatively, the immediate product may be subjected to further process steps, for example heating to further cross-link and insolubilise the protein shell of the microcapsules. This constitutes a two step process.
For a product which is to be injected into the human bloodstream, for example as an echogenic contrast agent in ultrasound diagnostic procedures (which is one intended use of the product), the total process is preferably carried out under sterile conditions. Thus, the protein solution is sterile and non-pyrogenic, the gas in the chamber is first passed through a 0.2 &mgr;m filter, the spray-drier is initially autoclaved and so on. Alternatively, or as well, the final product may be sterilised, for example by exposure to ionising radiation.
The wall-forming material may be any biocompatible water-soluble material, for example any of those (usually polymers) Inown in this art as microcapsule-forming agents. Preferably, it is a protein (the term being used to include non-naturally occurring polypeptides and polyamino acids). For example, it may be collagen, gelatin or (serum) albumin, in each case (if the microcapsules are to be administered to humans) preferably of human origin (ie derived from humans or corresponding in structure to the human protein) or polylysine or polyglutamate. It may be human serum albumin (HA) derived from blood donations or from the fermentation of microorganisms (including cell lines) which have been transformed or transfected to express HA. Alternatively, simple or complex carbohydrates, simple amino acids or fatty acids can be used, for example lysine, manrutol, dextran, palmitic acid or behenic acid.
Techniques for expressing HA (which term includes analogues and fragments of human albumin, for example those of EP-A-322094, and polymers of monomeric albumin) are disclosed in, for example, EP-A-201239 and BP-A-286424. All references are included herein by reference. “Analogues and fragments” of HA include all polypeptides (i) which are capable of forming a microcapsule in the process of the invention and (ii) of which a continuous region of at least 50% (preferably at least 75%, 80%, 90% or 95%) of the amino acid sequence is at least 80% homologous (preferably at least 90%, 95% or 99% homologous) with a continuous region of at least 50% (preferably 75%, 80%, 90% or 95%) of a nature-identical human album in. HA which is produced by recombinant DNA techniques may be used. Thus, the HA may be produced by expressing an HA-encoding nucleotide sequence in yeast or in another microorgamism and purifying the product, as is known in the art. Such material lacks the fatty acids associated with serum-derived material. Preferably, the HA is substantially free of fatty acids; ie it contains less than 1% of the fatty acid level of serum-derived material. Preferably, fatty acid is undetectable in the HA.
The aqueous solution or dispersion is preferably 0.1 to 50% w/v, more preferably about 1.0-25.0% w/v or 5.0-30.0% w/v protein, particularly when the material is albumin. About 5-15%w/v is optimal. Mixtures of wall-forming materials may be used, in which case the percentages in the last two sentences refer to the total content of wall-forming material.
The preparation to be sprayed may contain substances other than the wall-forming material, water and volatile liquid. Thus, the aqueous phase may contain 1-20% by weight of water-soluble hydrophilic compounds like sugars and polymers as stabilizers, eg polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, polyglutamic acid and polysaccharides such as starch, dextran, agar, xanthan and the like.
Functional agents may be included, for example at 1.0-40.0% w/w, such as X-ray contrast agents (for example Hexabrix (ioxaglic acid), Optiray (ioversol), Omnipaque (iohexol) or Isovice (iopamidol)) or magnetic resonance imaging agents (for example colloidal iron oxide or gadolinium chelates, eg gadopentetic acid).
Similar aqueous phases can be used as the carrier liquid in which the final microcapsule product is suspended before use. Surfactants may be used (0.1-5% by weight) including most physiologically acceptable surfactants, for instance egg lecithin or soya bean lecithin, or synthetic lecithins such as saturated synthetic lecithi
Johnson Richard Alan
van der Wouw Paulus Antonius
Hartley Michael G.
Sterne Kessler Goldstein & Fox P.L.L.C.
Upperton Limited
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