Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing – Ultrasound contrast agent
Reexamination Certificate
2000-02-18
2003-01-21
Hartley, Michael G. (Department: 1619)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
Ultrasound contrast agent
C516S011000, C516S077000
Reexamination Certificate
active
06509004
ABSTRACT:
This invention relates to improvements in and relating to compositions, particularly pharmaceutical compositions, containing gas-containing vesicles, for example for use as contrast agents in ultrasound imaging.
The use of compositions containing gas-containing vesicles (e.g. microballoons, liposomes, etc) as echogenic contrast agents in ultrasound imaging has been widely proposed in recent years and several such compositions are available or under clinical trial.
Several different types of vesicle membrane have been proposed, e.g. synthetic polymer, protein, phospholipid, etc. The present invention is concerned with vesicles having an amphiphilic membrane forming material, for example a surfactant or a lipid, in particular a phospholipid and most particularly one or more phospholipids having negatively charged groups, especially those having an overall negative charge, such as phosphatidylserines and phosphatidic acids.
Whilst gas-containing vesicles can readily be prepared from such amphiphilic membrane forming materials, for example by shaking, sonicating or rotor-stator mixing a gas and a liquid medium containing the membrane forming material, we have found that where the resulting composition is lyophilized there is a small degree of vesicle aggregation, agglomeration, flocculation or fusion following reconstitution of the lyophilisate with aqueous media.
Viewed from one aspect therefore the invention provides a process for the preparation of a pharmaceutical composition comprising an aqueous dispersion of gas-containing vesicles the membranes whereof comprise an amphiphilic membrane forming material, said process comprising:
(i) generating a liquid dispersion of gas-containing vesicles from a mixture comprising an amphiphilic membrane forming material;
(ii) lyophilizing a liquid dispersion of said gas-containing vesicles;
(iii) reconstituting the lyophilized product of step (ii) with a sterile aqueous liquid to produce an aqueous dispersion of gas-containing vesicles; and
(iv) treating the aqueous dispersion product of step (i) or step (iii) or the lyophilized product of step (ii) to produce a substantially aggregate-free sterile aqueous dispersion of gas-containing vesicles.
Preferred amphiphilic membrane-forming materials include materials having negative charges, particularly preferably those having an overall negative charge. Yet more particularly preferred amphiphilic membrane-forming materials are those comprising serine groups, and particularly phospholipids comprising serine groups e.g. phosphatidyl serine.
The terms aggregate, agglomerate, and flocculate refer to associations of individual intact vesicles. The process of the invention reduces one or more of these effects and the term aggregate (or aggregation) is thus used herein to refer to one or more of these associations.
The treatment of step (iv) may take the form of the use in the reconstitution step (iii) of a sterile aqueous liquid meeting particular criteria or alternatively or additionally it may involve chemical and/or mechanical treatment of the dispersion in step (i) or the reconstituted aqueous dispersions.
The tendency of vesicles formed using amphiphilic membrane-forming materials having negatively charged groups to agglomerate in a suspension medium containing multivalent metal cations appears to be increasingly severe as the vesicle content within the suspension medium is reduced. Increasing the phospholipid content of the amphiphilic membrane-forming materials of the membranes of the vesicles, or increasing the amount of membrane-forming materials, or increasing the phospholipid surface area of the membrane-forming materials, appears to reduce the tendency of individual intact vesicles to agglomerate. Such increases in phospholipid content or surface area can be achieved for example by adjusting accordingly the components used in the manufacture of the vesicles, or by adjusting the manufacturing process so as to increase the amount of phospholipid not incorporated in the microbubbles.
Moreover, the time taken between the filling of the sample vials and the freezing of the samples (i.e. the floating time) has been found to be the most important factor affecting the amount of agglomeration of vesicles. For example, it has been shown that immediate freezing results in a considerable reduction in agglomerates formed and that this reduction is independent of any adjustments made to the process and composition of the vesicles. Thus, preferably the time from generating the liquid dispersion of vesicles and the subsequent flotation and fractionation of the vesicles to the freeze drying or lyophilising of the liquid dispersion is minimised.
An increase in the phospholipid content or surface area can also be achieved by adjusting the manufacturing process so as to induce a shift downwards in the particle size distribution, to increase the available surface of phospholipids by way of increasing the number concentration of microbubbles present.
Such increases in phospholipid content as discussed above have not only been shown to correlate positively with reduced aggregation of vesicles on reconstitution, but also correlate positively with a reduced sensitivity to aggregation induced by calcium ions in the reconstitution medium (i.e. an increased calcium tolerance). This correlation of increased calcium tolerance with increased phospholipid content is shown in FIG.
2
. Calcium tolerance is defined as the lowest ion concentration to produce visible or microscopic agglomeration.
Preferably, the ratio of the total molar concentration in the aqueous suspension medium of non-chelated Al, Ba, Mg, Ca and Zn (and particularly preferably also Fe) to the molar concentration of the amphiphilic membrane-forming materials having negatively charged groups is less than 1:2, particularly less than 1:2.5, more preferably less than 1:3 and most preferably less than 1.6.
Thus viewed from a further aspect the invention provides a pharmaceutical composition comprising an aqueous dispersion of gas-containing vesicles the membranes whereof comprise an amphiphilic material (e.g. a phospholipid) having negatively charged groups, wherein the ratio of the total molar concentration in the aqueous dispersion of non-chelated Al, Ba, Mg, Ca and Zn (and particularly preferably also Fe) ions to the molar concentration of the amphiphilic membrane-forming material having negatively charged groups is less than 1:2, particularly less than 1:2.5, more preferably less than 1:3 and most preferably less than 1:6.
Another means of reducing aggregate formation is to reconstitute the lyophilizate with an aqueous liquid, such as sterile water, which has a total concentration of less than 100 &mgr;M of non-chelated Al, Ba, Ca, Mg and Zn ions, preferably a total of less than 50 &mgr;M, especially less than 47 &mgr;M, more especially less than 45 &mgr;M, more especially less than 40 &mgr;M, more especially less than 35 &mgr;m, more especially less than 30 &mgr;m and most especially less than 25 &mgr;M. Particularly preferably, these total concentration limits apply for other multivalent counter ions to the amphiphilic membrane forming material, for example Fe ions. Most preferably the sum of Mg and Ca ions has a concentration of less than 50 AM, especially the sum of Mg and Ca and Al ions is less than 30 &mgr;m. Preferably, the concentration of non-chelated individual divalent ions such as Mg, Ca, Ba and Zn is less than 30 &mgr;M and that of unchelated trivalent ions such as Al is less than 30 &mgr;M.
Viewed from a further aspect therefore, the invention provides a pharmaceutical composition, preferably for parenteral administration, comprising a dispersion of gas-containing vesicles in a sterile aqueous medium, wherein the total concentration of non-chelated Al, Ba, Ca, Mq and Zn ions in the resulting aqueous dispersion is Less than 100 &mgr;M, preferably less than 50 &mgr;M, especially less than 47 &mgr;M, more especially less than 45 &mgr;M, more especially less than 40 &mgr;m, more especially less than 35 &mgr;m, even more especially less than 30
Andersen Arnfinn
Fahlvik Anne Kjersti
Henriksen Ingrid
Kasparkova Vera
Odegardstuen Liv Ingrid
Amersham Health AS
Chisholm Robert F.
Hartley Michael G.
Ronning, Jr. Royal N.
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