Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Ultrasound contrast agent
Reexamination Certificate
2000-10-25
2003-04-15
Hartley, Michael G. (Department: 1616)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Ultrasound contrast agent
C424S009510, C424S450000, C424S489000, C424S499000
Reexamination Certificate
active
06548048
ABSTRACT:
This invention relates to diagnostic and/or therapeutically active agents comprising gas microbubbles, more particularly to such agents comprising lipopeptide-stabilised gas microbubbles. These agents if desired may incorporate moieties having affinity for sites and/or structures within the body so that diagnostic imaging and/or therapy of particular locations within the body may be enhanced. Of particular interest are diagnostic agents for use in ultrasound imaging. Novel lipopeptides constitute a further feature of the invention.
It is well known that ultrasonic imaging comprises a potentially valuable diagnostic tool, for example in studies of the vascular system, particularly in cardiography, and of tissue microvasculature. A variety of contrast agents have been proposed to enhance the acoustic images so obtained, including suspensions of solid particles, emulsified liquid droplets, gas bubbles and encapsulated gases or liquids. It is generally accepted that low density contrast agents which are easily compressible are particularly efficient in terms of the acoustic backscatter they generate, and considerable interest has therefore been shown in the preparation of gas-containing and gas-generating systems.
Initial studies involving free gas bubbles generated in vivo by intracardiac injection of physiologically acceptable substances have demonstrated the potential efficiency of such bubbles as contrast agents in echography; such techniques are severely limited in practice, however, by the short lifetime of the free bubbles. Interest has accordingly been shown in methods of stabilising gas bubbles for echocardiography and other ultrasonic studies, for example using emulsifiers, oils, thickeners or sugars, or by entraining or encapsulating the gas or a precursor thereof in a variety of systems, e.g. as porous gas-containing microparticles or as encapsulated gas microbubbles.
There is a substantial body of prior art concerning the nature of encapsulating materials and gases which may be present within microparticles, microbubbles etc. One preferred encapsulating system uses negatively charged phospholidids as wail-forming materials to stabilise gas microbubbles—see WO-A-9729783, which is hereby incorporated by reference and which contains a comprehensive review of prior art in this area. Despite a large amount of research there still remains a need for stabilised gas-filled microbubbles or microparticles which can act as ultrasound contrast agents and which are both physiologically tolerable and echogenic. Many existing contrast agents, for example, are destroyed by continuous ultrasound exposure, and thus any enhancement in contrast agent stability may reduce this problem.
It has recently been found that certain peptides with alternating hydrophobic and hydrophilic residues may spontaneously form macroscopic peptide membranes which may be useful biomaterials for medical products, for example as vehicles for slow-diffusion drug delivery, separation materials, biodegradable polymers and artificial sutures. U.S. Pat. No. 5,670,483 describes membranes formed by the peptide EAK16 derived from the protein zuotin [see also Zhang, S in
Biopolymers
(1994) 34, 663; Zhang, S in
Biomaterals
(1995) 16, 1385; and Zhang, S in
Proc. Natl. Acad. Sci
(1993) 90, 3334]. The membranes are stable in aqueous solutions and are resistant to degredation by heat, enzymatic degradation and alkaline and acidic pH; they have also been found to be non-cytotoxic. These peptides are soluble in aqueous solutions and, according to U.S. Pat. No. 5,670,483, require a sequence length of at least 12 amino acid residues, preferably more than 16 residues, in order to form membrane structures.
Fujita, K. et al. in
Advances in Biophysics
(1997) 34, 127 have described supramolecular assemblies using helical peptides. When such peptides were suspended in an aqueous medium by a sonication method, a dispersion of vesicles termed “peptosomes” was obtained. These peptosomes had a similar size distribution to classical liposomes, i.e. in the nanometer range; typically their average particle size was 75 nm. Other molecular assemblies comprising peptidic structures have been described by Imanishi, Y. et al. in
Supramol. Sci
(1996) 13, where gramicidin A/PEG conjugates were found to form peptosomes also in the nanometer size range.
It has now been found that a range of lipid-substituted peptide derivatives, referred to herein as lipopeptides, may be used in the formation of stabilised gas microbubbles suitable for use as diagnostic and/or therapeutic agents, for example in ultrasound echography. Such microbubbles have been found to exhibit good stability, for example during ultrasonication in an imaging procedure. It has also surprisingly been found that lipopetides containing as few as two amino acid residues may exhibit membrane forming properties, in contrast to the findings regarding the self-assembly peptide structures of U.S. Pat. No. 5,670,483. Such short lipopeptides may be prepared relatively easily and economically and may therefore possess substantial cost advantages over naturally occurring, semi-synthetic or synthetic phospholipids such as phosphatidylserine.
Thus according to one aspect of the present invention there is provided a diagnostic and/or therapeutically active agent, e.g. an ultrasound contrast agent, comprising encapsualted gas-filled microbubbles stabilised by membrane-forming amphiphilic lipopeptides.
Viewed from another aspect the invention provides the use of an agent as hereinbefore defined as an ultrasound contrast agent.
Viewed from yet another aspect, the invention provides a method of generating enhanced images of a human or non-human animal body which comprises administering to said body an agent as as hereinbefore defined and generating an ultrasound, magnetic resonance, X-ray, radiographic or light image of at least a part of said body.
The macroscopic membranes may be formed from individual peptide units comprising from 2 to 50 aminoacyl residues. Each peptide unit may carry one or more lipophilic hydrocarbon chains of between 5 and about 50 carbons in length.
In a preferred embodiment the number of amino acid residues in the individual lipopetide units of the invention should be the least number of residues necessary to form an effective stabilised membrane and is preferably less than 20 residues, more preferably less than 10 residues, most preferably between 2 to 8 residues. Clearly, keeping the number of residues to a minimum will both reduce cost and allow easier preparation of the lipopeptides of the invention.
Any amino acid residue may be used in the preparation of individual lipopetide units according to the invention, although the lipopeptides must be amphiphilic. In a preferred embodiment the lipopeptides will comprise residues of amino acids selected from the readily available naturally occuring essential twenty amino acids.
In one embodiment a peptide unit can comprise alternating hydrophobic and hydrophilic residues, such as alanyl and diaminopropionyl, and may comprise one or more complementary sequences and/or a targeting sequence with affinity for biological receptors. In a particularly preferred embodiment, charged amino acids such as lysine and glutamic acid are selected to provide side-chain functionalities comprising positively and/or negatively charged groups respectively at neutral pH. Although not wishing to be limited by theory, it is envisaged that these charged groups help in the stabilisation of the-outer part of the membrane by forming ion-pairs or salt bridges. The alignment of oppositely charged groups leading to membrane stability is possible only if the peptide sequences involved are complementary to one another and this forms a further aspect of the invention.
The lipid component of the lipopeptides preferably comprises an alkyl, alkenyl or alkynyl chain, especially an alkyl chain. The hydrocarbon chains preferably have between 5 and 25 carbons and most preferably are obtainable from readily available fatty acid derivatives. Suitable fa
Cuthbertson Alan
Solbakken Magne
Wolfe Henry Raphael
Amersham Health AS
Chisholm Robert F.
Hartley Michael G.
Ronning, Jr. Royal N.
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