U.S. patent application number 13/039838 was filed with the patent office on 2011-09-29 for ultrasound contrast agents and process for the preparation thereof.
This patent application is currently assigned to BRACCO SUISSE S.A.. Invention is credited to Philippe Bussat, Christian Guillot, Michel Schneider, Feng Yan.
Application Number | 20110236320 13/039838 |
Document ID | / |
Family ID | 32842689 |
Filed Date | 2011-09-29 |
United States Patent
Application |
20110236320 |
Kind Code |
A1 |
Schneider; Michel ; et
al. |
September 29, 2011 |
Ultrasound Contrast Agents And Process For The Preparation
Thereof
Abstract
Injectable aqueous suspension of microbubbles filled with a
biocompatible gas and a method of preparation thereof. At least 10%
of the total volume of gas contained in the microbubbles is
contained in microbubbles with a diameter of 1.5 .mu.m or less. The
microbubbles can be obtained by preparing an emulsion comprising an
aqueous medium, a phospholipid and a water immiscible organic
solvent. The emulsion is then freeze-dried and then reconstituted
in an aqueous suspension of gas-filled microbubbles.
Inventors: |
Schneider; Michel; (Troinex,
CH) ; Bussat; Philippe; (La Roche sur Foron, FR)
; Yan; Feng; (Grand-Lancy, CH) ; Guillot;
Christian; (Beaumont, FR) |
Assignee: |
BRACCO SUISSE S.A.
Manno
CH
|
Family ID: |
32842689 |
Appl. No.: |
13/039838 |
Filed: |
March 3, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11202008 |
Aug 11, 2005 |
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13039838 |
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10544123 |
Aug 2, 2005 |
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PCT/IB2004/000243 |
Feb 3, 2004 |
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11202008 |
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Current U.S.
Class: |
424/9.52 ;
424/400 |
Current CPC
Class: |
A61K 49/223 20130101;
A61P 43/00 20180101 |
Class at
Publication: |
424/9.52 ;
424/400 |
International
Class: |
A61B 8/00 20060101
A61B008/00; A61K 9/00 20060101 A61K009/00; A61P 43/00 20060101
A61P043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 4, 2003 |
EP |
03002375.8 |
Claims
1. An injectable aqueous suspension of microbubbles filled with a
biocompatible gas and comprising a stabilizing layer predominantly
comprising a phospholipid, wherein at least 10% of the total volume
of gas contained in the microbubbles is contained in microbubbles
with a diameter of 1.5 .mu.m or less.
2. The aqueous suspension according to claim 1 wherein at least 25%
of the total volume of gas contained in the microbubbles is
contained in microbubbles with a diameter of 1.5 .mu.m.
3. The aqueous suspension according to claim 1 wherein at least 50%
of the total volume of gas contained in the microbubbles is
contained in microbubbles with a diameter of 1.5 .mu.m
4. The injectable suspension according to claim 1 wherein at least
70% of the total volume of gas contained in the microbubbles is
contained in microbubbles with a diameter of 1.5 .mu.m
5. The aqueous suspension according to claim 1 wherein said
microbubbles have a D.sub.V50/D.sub.N ratio of about 2.00 or
lower.
6. The aqueous suspension according to claim 1 wherein said
microbubbles have a D.sub.V50/D.sub.N ratio of about 1.80 or
lower.
7. The aqueous suspension according to any one of the preceding
claims obtainable by a method which comprises the steps of: a)
preparing an aqueous-organic emulsion comprising i) an aqueous
medium including water, ii) an organic solvent substantially
immiscible with water, iii) an emulsifying composition of
amphiphilic materials comprising more than 50% by weight of a
phospholipid and iv) a lyoprotecting agent; b) lyophilizing said
emulsified mixture, to obtain a lyophilized matrix comprising said
phospholipid; c) contacting said lyophilized matrix with a
biocompatible gas; and reconstituting said lyophilized matrix by
dissolving it in a physiologically acceptable aqueous carrier
liquid.
8. A method for diagnostic imaging comprising administering to a
subject a contrast-enhancing amount of an aqueous suspension of any
one of claims 1 to 6 and imaging at least a part of said
subject.
9. A method for diagnostic imaging comprising administering to a
subject a contrast-enhancing amount of an aqueous suspension of any
one of claims 1 to 6 and imaging at least a part of said subject
wherein said imaging comprises insonating said subject by means of
an ultrasound device generating an ultrasound wave with a
predetermined transmit frequency, from which a corresponding
resonance size of microbubbles is determined, and administering a
contrast agent comprising gas-filled microbubbles having a narrow
size distribution and a mean size close to half the resonance
size.
10. A method for preparing a lyophilized matrix which, upon contact
with an aqueous carrier liquid and a gas, is reconstitutable into a
suspension of gas-filled microbubbles stabilized predominantly by a
phospholipid, said method comprising the steps of: a) preparing an
aqueous-organic emulsion comprising i) an aqueous medium including
water, ii) an organic solvent substantially immiscible with water
comprising, dispersed therein, an emulsifying composition of
amphiphilic materials comprising more than 50% by weight of a
phospholipid and iv) a lyoprotecting agent; b) lyophilizing said
emulsified mixture, to obtain a lyophilized matrix comprising said
phospholipid.
11. The method according to claim 10 wherein the lyoprotecting
agent is dissolved in the aqueous carrier.
12. The method according to claim 10 wherein the lyoprotecting
agent is polyethylenglycol.
13. A method for preparing an injectable contrast agent comprising
a liquid aqueous suspension of gas-filled microbubbles stabilized
predominantly by a phospholipid, which comprises the steps of:
preparing a lyophilized matrix according to the method of any one
of the preceding claims 10 to 12; contacting said lyophilized
matrix with a biocompatible gas; and reconstituting said
lyophilized matrix by dissolving it into a physiologically
acceptable aqueous carrier liquid, to obtain a suspension of
gas-filled microbubbles stabilized predominantly by said
phospholipid.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application, U.S.
Ser. No. 11/202,008, filed Aug. 11, 2005, which is a
continuation-in-part of U.S. application, U.S. Ser. No. 10/544,123,
filed Aug. 2, 2005, which is a national stage application of
international application PCT/IB2004/000243, filed Feb. 3, 2004,
now expired, which claims priority to and the benefit of European
application EP03002375.8, filed Feb. 4, 2003, now abandoned, all of
which are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a process for the
preparation of a dry or lyophilized formulation useful for
preparing a gas containing contrast agent usable in diagnostic
imaging and to a process for preparing said gas containing contrast
agent.
[0003] The invention also includes dry formulations prepared by
this process, which may be reconstituted to form contrast agent
suspensions useful in diagnostic imaging. The invention further
includes suspensions of gas filled microbubbles useful in
diagnostic imaging prepared using dry formulations of the invention
as well as containers or two component kits containing the dry
formulations of the invention.
BACKGROUND OF THE INVENTION
[0004] Rapid development of ultrasound contrast agents in the
recent years has generated a number of different formulations,
which are useful in ultrasound imaging of organs and tissue of
human or animal body. These agents are designed to be used
primarily as intravenous or intra-arterial injectables in
conjunction with the use of medical echographic equipment which
employs for example, B-mode image formation (based on the spatial
distribution of backscatter tissue properties) or Doppler signal
processing (based on Continuous Wave or pulsed Doppler processing
of ultrasonic echoes to determine blood or liquid flow
parameters).
[0005] A class of injectable formulations useful as ultrasound
contrast agents includes suspensions of gas bubbles having a
diameter of few microns dispersed in an aqueous medium.
[0006] Use of suspensions of gas bubbles in carrier liquid, as
efficient ultrasound reflectors is well known in the art. The
development of microbubble suspensions as echopharmaceuticals for
enhancement of ultrasound imaging followed early observations that
rapid intravenous injections of aqueous solutions can cause
dissolved gases to come out of solution by forming bubbles. Due to
their substantial difference in acoustic impedance relative to
blood, these intravascular gas bubbles were found to be excellent
reflectors of ultrasound. The injection of suspensions of gas
bubbles in a carrier liquid into the blood stream of a living
organism strongly reinforces ultrasonic echography imaging, thus
enhancing the visualisation of internal organs. Since imaging of
organs and deep seated tissues can be crucial in establishing
medical diagnosis, a lot of effort has been devoted to the
development of stable suspensions of highly concentrated gas
bubbles which at the same time would be simple to prepare and
administer, would contain a minimum of inactive species and would
be capable of long storage and simple distribution.
[0007] The simple dispersion of free gas bubbles in the aqueous
medium is however of limited practical interest, since these
bubbles are in general not stable enough to be useful as ultrasound
contrast agents.
[0008] Interest has accordingly been shown in methods of
stabilising gas bubbles for echography and other ultrasonic
studies, for example using emulsifiers, oils, thickeners or sugars,
or by entrapping or encapsulating the gas or a precursor thereof in
a variety of systems. These stabilized gas bubbles are generally
referred to in the art as "microvesicles", and may be divided into
two main categories.
[0009] A first category of stabilized bubbles or microvesicles is
generally referred to in the art as "microbubbles" and includes
aqueous suspensions in which the bubbles of gas are bounded at the
gas/liquid interface by a very thin envelope involving a surfactant
(i.e. an amphiphilic material) disposed at the gas to liquid
interface. A second category of microvesicles is generally referred
to in the art as "microballoons" or "microcapsules" and includes
suspensions in which the bubbles of gas are surrounded by a solid
material envelope formed of natural or synthetic polymers. Examples
of microballoons and of the preparation thereof are disclosed, for
instance, in European patent application EP 0458745. Another kind
of ultrasound contrast agent includes suspensions of porous
microparticles of polymers or other solids, which carry gas bubbles
entrapped within the pores of the microparticles. The present
invention is particularly concerned with contrast agents for
diagnostic imaging including an aqueous suspension of gas
microbubbles, i.e. microvesicles which are stabilized essentially
by a layer of amphiphilic material.
[0010] Microbubble suspensions are typically prepared by contacting
powdered amphiphilic materials, e.g. freeze-dried preformed
liposomes or freeze-dried or spray-dried phospholipid suspensions,
with air or other gas and then with aqueous carrier, agitating to
generate a microbubble suspension which must then be administered
shortly after its preparation.
[0011] Examples of aqueous suspensions of gas microbubbles and
preparation thereof can be found for instance in U.S. Pat. No.
5,271,928, U.S. Pat. No. 5,445,813, U.S. Pat. No. 5,413,774, U.S.
Pat. Nos. 5,556,610, 5,597,549, U.S. Pat. No. 5,827,504.
[0012] WO97/29783 discloses an alternative process for preparing
gas microbubble suspensions, comprising generating a gas
microbubble dispersion in an appropriate phospholipid-containing
aqueous medium and thereafter subjecting the dispersion to
lyophilisation to yield a dried reconstitutable product. The so
prepared dried products are reconstitutable in aqueous media
requiring only minimal agitation. As mentioned in said document,
the size of the so generated microbubbles is consistently
reproducible and in practice is independent from the amount of
agitation energy applied during reconstitution, being determined by
the size of the microbubbles formed in the initial microbubble
dispersion. The Applicant has however observed that the amount of
agitation energy applied for generating the gas microbubble
dispersion in the phospholipid-containing aqueous medium may be
excessively high, particularly when small diameter microbubbles are
to be obtained (e.g. 23000 rpm for 10 minutes, for obtaining a
dispersion of bubbles having a volume mean diameter of about 3
.mu.m). This high agitation energy may determine local overheating
in the aqueous dispersion of microbubbles, which may in turn cause
degradation of the phospholipids contained in the aqueous medium.
In addition, the effects of an excessively high agitation energy
are in general difficult to control and may result in an
uncontrollable size distribution of the final microbubbles.
Furthermore, this process involves a continuous flow of gas into
the aqueous medium during the generation of microbubbles, thus
requiring the use of relevant amounts of gases.
[0013] WO 94/01140 discloses a further process for preparing
microvesicle suspensions reconstitutable in an aqueous medium,
which comprises lyophilizing aqueous emulsions containing
parenterally acceptable emulsifiers, non polar liquids and
lipid-soluble or water-insoluble "structure-builders". Poloxamers
and phospholipids are mentioned as parenterally acceptable
emulsifiers, while mixtures of these two are employed in the
working examples. Cholesterol is the preferred water-insoluble
structure-builder, which is employed in the working examples. The
lyophilized product is then reconstituted in water, to give aqueous
suspension of gas-filled microvesicles. The gas-filled
microvesicles resulting from the reconstitution step are thus
defined by an envelope of different materials, including
emulsifiers such as poloxamers and water-insoluble
structure-builders such as cholesterol.
[0014] The process is said to result into an emulsion with
particles' size lower than 4 .mu.m, preferably lower than 2 .mu.m,
down to 0.5 .mu.m. The Applicant has however noticed that while the
reconstitution step may finally result in microvesicles having a
numerical mean diameter of less than 2 .mu.m, the corresponding
size distribution of the microvesicles population is nevertheless
relatively broad. In addition, the conversion step from the
emulsion microparticles, obtained according to the above process,
into gas microbubbles results in rather low yield.
[0015] The Applicant has now found that a much narrower
distribution of microbubbles size can be obtained if a phospholipid
is used as the main emulsifier of the above emulsion and if the
above process is conducted in the substantial absence of the above
water-insoluble structure-builders. In addition, the substantial
absence of said water-insoluble structure-builders allows to
substantially increase the conversion yield from emulsion
microparticles into gas microbubbles. The Applicant has further
observed that the above process may result in a further narrower
size distribution of microbubbles and in an increased yield if the
phospholipid is essentially the only emulsifier present in the
emulsion.
[0016] The Applicant has also found that by applying a rather low
agitation energy to an aqueous-organic emulsion during the process
as above specified, it is possible to obtain microbubbles having a
very small diameter and reduced size distribution.
SUMMARY OF THE INVENTION
[0017] An aspect of the invention relates to an injectable contrast
agent comprising gas-filled microbubbles stabilized by a
stabilizing layer predominantly comprising a phospholipid in an
aqueous carrier liquid, wherein at least 10% of the total volume of
gas contained in the microbubbles is contained in microbubbles with
a diameter of 1.5 .mu.m or less. Preferably, at least 25%, more
preferably at least 50% and even more preferably at least 70% of
the total volume of gas is contained in microbubbles with a
diameter of less than 1.5 .mu.m.
[0018] According to a preferred embodiment, said gas-filled
microbubbles have a volume median diameter (D.sub.V50) and a number
mean diameter (D.sub.N) such that the D.sub.V50/D.sub.N ratio is of
about 2.00 or lower, more preferably of about 1.60 or lower and
even more preferably of about 1.30 or lower.
[0019] Preferably, said injectable contrast agent is in the form of
an aqueous suspension of gas-filled microbubbles. According to a
preferred embodiment, said aqueous suspension is obtained by a
method which comprises the steps of:
[0020] preparing an aqueous-organic emulsion comprising i) an
aqueous medium including water, ii) an organic solvent
substantially immiscible with water, iii) an emulsifying
composition of amphiphilic materials comprising more than 50% by
weight of a phospholipid and iv) a lyoprotecting agent;
[0021] lyophilizing said emulsified mixture, to obtain a
lyophilized matrix comprising said phospholipid;
[0022] contacting said lyophilized matrix with a biocompatible gas;
and
[0023] reconstituting said lyophilized matrix by dissolving it in a
physiologically acceptable aqueous carrier liquid.
[0024] A further aspect of the invention relates to a method for
preparing a lyophilized matrix which, upon contact with an aqueous
carrier liquid and a gas, is reconstitutable into a suspension of
gas-filled microbubbles stabilized predominantly by a phospholipid,
said method comprising the steps of:
[0025] preparing an aqueous-organic emulsion comprising i) an
aqueous medium including water, ii) an organic solvent
substantially immiscible with water comprising, dispersed therein,
an emulsifying composition of amphiphilic materials comprising more
than 50% by weight of a phospholipid and iv) a lyoprotecting
agent;
[0026] lyophilizing said emulsified mixture, to obtain a
lyophilized matrix comprising said phospholipid.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The injectable contrast agent of the present invention can
be obtained from a lyophilized matrix of a reconstitutable
suspension of gas-filled microbubbles predominantly stabilized by a
phospholipid, said matrix being obtained from a method which
comprises preparing an aqueous-organic emulsion comprising i) an
aqueous medium, ii) an organic solvent substantially immiscible
with water; iii) a phospholipid and iv) a lyoprotecting agent, and
subsequently lyophilizing said emulsion.
[0028] The aqueous medium is preferably a physiologically
acceptable carrier. The term "physiologically acceptable" includes
to any compound, material or formulation which can be administered,
in a selected amount, to a patient without negatively affecting or
substantially modifying its organism's healthy or normal
functioning (e.g. without determining any status of unacceptable
toxicity, causing any extreme or uncontrollable allergenic response
or determining any abnormal pathological condition or disease
status).
[0029] Suitable aqueous liquid carriers are water, typically
sterile, pyrogen free water (to prevent as much as possible
contamination in the intermediate lyophilized product), aqueous
solutions such as saline (which may advantageously be balanced so
that the final product for injection is not hypotonic), or aqueous
solutions of one or more tonicity adjusting substances such as
salts or sugars, sugar alcohols, glycols or other non-ionic polyol
materials (eg. glucose, sucrose, sorbitol, mannitol, glycerol,
polyethylene glycols, propylene glycols and the like).
[0030] The Organic Solvent
[0031] As used herein the term "substantially immiscible with
water" referred to the organic solvent means that, when said
solvent is admixed with water, two separate phases are formed.
Water immiscible solvent are generally also known in the art as
apolar or non-polar solvents, as opposed to polar solvents (such as
water). Water immiscible solvents are in general substantially
insoluble in water. For the purposes of the present invention,
organic solvents suitable for being emulsified with the aqueous
solvent are typically those solvents having a solubility in water
of less than about 10 g/l. Preferably, the solubility of said
solvent in water is of about 1.0 g/l or lower, more preferably
about 0.2 g/l or lower and much more preferably about 0.01 g/l or
lower. Particularly preferred solvents are those having a
solubility in water of 0.001 g/l or lower. Particularly insoluble
organic solvents (e.g. perfluorocarbons) may have a solubility down
to about 1.010.sup.-6 g/l (e.g perfluorooctane, 1.6610.sup.-6
g/l).
[0032] The organic solvent is preferably lyophilisable, i.e. said
solvent has a sufficiently high vapour pressure at the
lyophilization temperatures, e.g. between -30.degree. C. and
0.degree. C., to allow for an effective and complete
evaporation/sublimation within acceptable times, e.g. 24-48 hours.
Preferably, the vapour pressure of the organic solvent is higher
than about 0.2 kPa at 25.degree. C.
[0033] The organic solvent can be selected from a broad range of
solvents and any chemical entity that is water-immiscible and
lyophilisable, as indicated above, and being preferably liquid at
room temperature (25.degree. C.). If a solvent having a boiling
point lower than room temperature is used, the vessel containing
the emulsifying mixture can advantageously be cooled below the
boiling point of said solvent, e.g. down to 5.degree. C. or
0.degree. C. As said solvent will be completely removed during the
lyophilization step, no particular constraints exist except that it
should not contain contaminants that cannot be removed through
lyophilisation or that are not acceptable for use in an injectable
composition.
[0034] Suitable organic solvents include but are not limited to
alkanes, such as branched or, preferably, linear (C.sub.5-C.sub.10)
alkanes, e.g. pentane, hexane, heptane, octane, nonane, decane;
alkenes, such as (C.sub.5-C.sub.10) alkenes, e.g. 1-pentene,
2-pentene, 1-octene; cyclo-alkanes, such as
(C.sub.5-C.sub.8)-cycloalkanes optionally substituted with one or
two methyl groups, e.g. cyclopentane, cyclohexane, cyclooctane,
1-methyl-cyclohexane; aromatic hydrocarbons, such as benzene and
benzene derivatives substituted by one or two methyl or ethyl
groups, e.g. benzene, toluene, ethylbenzene, 1,2-dimethylbenzene,
1,3-dimethylbenzene; alkyl ethers and ketones such as di-butyl
ether and di-isopropylketone; halogenated hydrocarbons or ethers,
such as chloroform, carbon tetrachloride,
2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),
2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),
tetrachloro-1,1-difluoroethane, and particularly perfluorinated
hydrocarbons or ethers, such as perfluoropentane, perfluorohexane,
perfluoroheptane, perfluoromethylcyclohexane, perfluorooctane,
perfluorononane, perfluorobenzene and perfluorodecalin,
methylperfluorobutylether, methylperfluoroisobutylether,
ethylperfluorobutylether, ethylperfluoroisobutylether; and mixtures
thereof.
[0035] The amount of solvent is generally comprised from about 1%
to about 50% by volume with respect to the amount of water used for
the emulsion. Preferably said amount is from about 1% to about 20%,
more preferably from about 2% to about 15% and even more preferably
from about 5% to about 10%. If desired, a mixture of two or more of
the above listed organic solvents can be used, the overall amount
of organic solvent in the emulsifying mixture being within the
above range.
[0036] Lyoprotective Agent
[0037] The term lyoprotective agent or "lyoprotectant" refers to a
compound which, when included in a formulation to be lyophilized,
will protect the chemical compounds from the deleterious effects of
freezing and vacuumizing, such as those usually accompanying
lyophilization, e.g. damage, adsorption and loss from vacuum
utilized in lyophilization. In addition, after the lyophilization
step, said lyoprotective agent preferably results in a solid matrix
("bulk") which supports the lyophilized phospholipid.
[0038] The present invention is not limited to the use of a
specific lyoprotectant, and examples of suitable lyoprotectants
include, but are not limited to, carbohydrates such as the
saccharides, mono-, di- or poly-saccharides, e.g. glucose,
galactose, fructose, sucrose, trehalose, maltose, lactose, amylose,
amylopectin, cyclodextrins, dextran, inuline, soluble starch,
hydroxyethyl starch (HES), sugar alcohols e.g. mannitol, sorbitol
and polyglycols such as polyethyleneglycols. A substantial list of
agents with lyoprotective effects is given in Acta Pharm. Technol.
34(3), pp. 129-139 (1988), the content of which is incorporated
herein by reference. Said lyoprotective agents can be used
singularly or as mixtures of one or more compounds.
[0039] Preferred lyoprotectants include mannitol and
polysaccharides such as dextrans (in particular those with
molecular weights above 1500 daltons), inulin, soluble starch,
hydroxyethyl starch and polyethyleneglycols, preferably of MW from
about 1000 to about 30000 daltons, more preferably from 2000 to
8000 daltons (e.g. PEG4000).
[0040] Mixtures of mannitol or polysaccharides such as dextrans,
inulin, soluble starch, hydroxyethyl starch with saccharides such
as glucose, maltose, lactose, sucrose, trehalose and erythritol
also provide excellent results.
[0041] Likewise, the present invention is not limited to any
particular amount of lyoprotectant used. However the optimal weight
concentration of lyoprotective agents in the emulsion prior to the
lyophilisation is comprised between about 1 and about 25%,
preferably between about 2 and about 20%, and even more preferably
between about 5 and about 10%.
[0042] A higher amount can be employed if it is also necessary to
provide a desired "bulk" to the lyophilized product.
[0043] The lyoprotective agent is preferably added to the
aqueous-organic mixture before emulsification of the same and in
this case the emulsification of the aqueous-organic mixture is thus
carried out in the presence of the lyoprotective agents.
Alternatively, the lyoprotectant can be added to the
aqueous-organic mixture after the emulsification thereof. In the
first case, the lyoprotectant is preferably added to the aqueous
medium, before admixing it with the organic solvent. If desired, it
is also possible to combine the two, e.g. by adding part of the
lyoprotective agent to the aqueous phase used for the preparation
of the emulsion and part to the thus obtained emulsion. If desired,
also cryoprotective agents, such as glycerol, can further be added
to the emulsion for protecting the chemical compounds from the
deleterious effects of freezing.
[0044] Phospholipids
[0045] According to the present description and claims, the term
phospholipid is intended to encompass any amphiphilic
phospholipidic compound the molecules of which are capable of
forming a film of material (typically in the form of a
mono-molecular layer) at the gas-water boundary interface in the
final microbubbles suspension. Accordingly, these material are also
referred to in the art as "film-forming phospholipids". Similarly,
in the emulsified mixture, these amphiphilic compounds are
typically disposed at the interface between the aqueous medium and
the organic solvent substantially insoluble in water, thus
stabilizing the emulsified solvent microdroplets. The film formed
by these compounds at the gas-water or water-solvent interface can
be either continuous or discontinuous. In the latter case, the
discontinuities in the film should not however be such as to impair
the stability (e.g. pressure resistance, resistance to coalescence,
etc.) of the suspended microbubbles or of the emulsified
microdroplets, respectively.
[0046] The term "amphiphilic compound" as used herein includes
compounds having a molecule with a hydrophilic polar head portion
(e.g. a polar or ionic group), capable of interacting with an
aqueous medium, and a hydrophobic organic tail portion (e.g. a
hydrocarbon chain), capable of interacting with e.g. an organic
solvent. These compounds thus generally act as "surface active
agent", i.e. compounds which are capable of stabilizing mixtures of
otherwise generally immiscible materials, such as mixtures of two
immiscible liquids (e.g. water and oil), mixtures of liquids with
gases (e.g. gas microbubbles in water) or mixtures of liquids with
insoluble particles (e.g. metal nanoparticles in water).
[0047] Amphiphilic phospholipid compounds typically contain at
least one phosphate group and at least one, preferably two,
lipophilic long-chain hydrocarbon group.
[0048] Examples of suitable phospholipids include esters of
glycerol with one or preferably two (equal or different) residues
of fatty acids and with phosphoric acid, wherein the phosphoric
acid residue is in turn bound to a hydrophilic group, such as
choline (phosphatidylcholines--PC), serine
(phosphatidylserines--PS), glycerol (phosphatidylglycerols--PG),
ethanolamine (phosphatidylethanolamines--PE), inositol
(phosphatidylinositol), and the like groups. Esters of
phospholipids with only one residue of fatty acid are generally
referred to in the art as the "lyso" forms of the phospholipid.
Fatty acids residues present in the phospholipids are in general
long chain aliphatic acids, typically containing from 12 to 24
carbon atoms, preferably from 14 to 22; the aliphatic chain may
contain one or more unsaturations or is preferably completely
saturated. Examples of suitable fatty acids included in the
phospholipids are, for instance, lauric acid, myristic acid,
palmitic acid, stearic acid, arachidic acid, behenic acid, oleic
acid, linoleic acid, and linolenic acid. Preferably, saturated
fatty acids such as myristic acid, palmitic acid, stearic acid and
arachidic acid are employed.
[0049] Further examples of phospholipid are phosphatidic acids,
i.e. the diesters of glycerol-phosphoric acid with fatty acids;
sphingolipids such as sphingomyelins, i.e. those
phosphatidylcholine analogs where the residue of glycerol diester
with fatty acids is replaced by a ceramide chain; cardiolipins,
i.e. the esters of 1,3-diphosphatidylglycerol with a fatty acid;
glycolipids such as gangliosides GM1 (or GM2) or cerebrosides;
glucolipids; sulfatides and glycosphingolipids.
[0050] As used herein, the term phospholipids include either
naturally occurring, semisynthetic or synthetically prepared
products that can be employed either singularly or as mixtures.
[0051] Examples of naturally occurring phospholipids are natural
lecithins (phosphatidylcholine (PC) derivatives) such as,
typically, soya bean or egg yolk lecithins.
[0052] Examples of semisynthetic phospholipids are the partially or
fully hydrogenated derivatives of the naturally occurring
lecithins. Preferred phospholipids are fatty acids di-esters of
phosphatidylcholine, ethylphosphatidylcholine,
phosphatidylglycerol, phosphatidic acid, phosphatidylethanolamine,
phosphatidylserine or of sphingomyelin.
[0053] Examples of preferred phospholipids are, for instance,
dilauroyl-phosphatidylcholine (DLPC),
dimyristoyl-phosphatidylcholine (DMPC),
dipalmitoyl-phosphatidylcholine (DPPC),
diarachidoyl-phosphatidylcholine (DAPC),
distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine
(DOPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine
(Ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC),
1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),
1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),
1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),
1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),),
1-palmitoyl-2-oleylphosphatidylcholine (POPC),
1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),
dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,
diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal
salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal
salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal
salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal
salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal
salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal
salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal
salts, distearoyl phosphatidic acid (DSPA),
diarachidoylphosphatidic acid (DAPA) and its alkali metal salts,
dimyristoyl-phosphatidylethanolamine (DMPE),
dipalmitoylphosphatidylethanolamine (DPPE), distearoyl
phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine
(DOPE), diarachidoylphosphatidylethanolamine (DAPE),
dilinoleylphosphatidylethanolamine (DLPE), dimyristoyl
phosphatidylserine (DMPS), diarachidoyl phosphatidylserine (DAPS),
dipalmitoyl phosphatidylserine (DPPS), distearoylphosphatidylserine
(DSPS), dioleoylphosphatidylserine (DOPS), dipalmitoyl
sphingomyelin (DPSP), and distearoylsphingomyelin (DSSP).
[0054] The term phospholipid further includes modified
phospholipid, e.g. phospholipids where the hydrophilic group is in
turn bound to another hydrophilic group. Examples of modified
phospholipids are phosphatidylethanolamines modified with
polyethylenglycol (PEG), i.e. phosphatidylethanolamines where the
hydrophilic ethanolamine moiety is linked to a PEG molecule of
variable molecular weight e.g. from 300 to 5000 daltons, such as
DPPE-PEG or DSPE-PEG, i.e. DPPE (or DSPE) having a PEG polymer
attached thereto. For example, DPPE-PEG2000 refers to DPPE having
attached thereto a PEG polymer having a mean average molecular
weight of about 2000. As explained in detail in the following,
these PEG-modified phospholipids are preferably used in combination
with non-modified phospholipids.
[0055] Both neutral and charged phospholipids can satisfactorily be
employed in the process of the present invention, as well as
mixtures thereof. As used herein and in the prior art, the term
"charged" in relation with "phospholipids" means that the
individual phospholipid molecules have an overall net charge, be it
positive or, more frequently, negative.
[0056] Examples of phospholipids bearing an overall negative charge
are derivatives, in particular fatty acid di-esters, of
phosphatidylserine, such as DMPS, DPPS, DSPS; of phosphatidic acid,
such as DMPA, DPPA, DSPA; of phosphatidylglycerol such as DMPG,
DPPG and DSPG. Also modified phospholipids, in particular
PEG-modified phosphatidylethanolamines, such as DMPE-PEG750,
DMPE-PEG1000, DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000,
DMPE-PEG5000, DPPE-PEG750, DPPE-PEG1000, DPPE-PEG2000,
DPPE-PEG3000, DPPE-PEG4000, DPPE-PEG5000, DSPE-PEG750,
DSPE-PEG1000, DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000,
DSPE-PEG5000, DAPE-PEG750, DAPE-PEG1000, DAPE-PEG2000,
DAPE-PEG3000, DAPE-PEG4000 or DAPE-PEG5000 can be used as
negatively charged molecules. Also the lyso-form of the above cited
phospholipids, such as lysophosphatidylserine derivatives (e.g.
lyso-DMPS, -DPPS or -DSPS), lysophosphatidic acid derivatives (e.g.
lyso-DMPA, -DPPA or -DSPA) and lysophosphatidylglycerol derivatives
(e.g. lyso-DMPG, -DPPG or -DSPG), can advantageously be used as
negatively charged compound.
[0057] Examples of phospholipids bearing an overall positive charge
are derivatives of ethylphosphatidylcholine, in particular esters
of ethylphosphatidylcholine with fatty acids, such as
1,2-Distearoyl-sn-glycero-3-Ethylphosphocholine (Ethyl-DSPC or
DSEPC), 1,2-Dipalmitoyl-sn-glycero-3-Ethylphosphocholine
(Ethyl-DPPC or DPEPC).
[0058] Preferably, blends of two or more phospholipids, at least
one with a neutral charge and at least one with an overall net
charge, are employed. More preferably, blends of two or more
phospholipids, at least one with neutral and at least one with
negative charge are employed. The amount of charged phospholipid,
may vary from about 95% to about 5% by weight, with respect to the
total amount of phospholipid, preferably from 80% to 20% by weight.
The presence of at least minor amounts, such as 5% to 20% by wt.
with respect to the total weight of phospholipid, of a (negatively)
charged phospholipid may help preventing aggregation of bubbles or
emulsion droplets. It is however possible to use a single
phospholipid, neutral or charged, or a blend of two or more
phospholipids, all neutral or all with an overall net charge.
[0059] Preferred phospholipids are DAPC, DPPA, DSPA, DMPS, DPPS,
DSPS, DPPE, DSPE, DSPG, DPPG and Ethyl-DSPC. Most preferred are
DSPA, DPPS or DSPS.
[0060] Preferred mixtures of phospholipids are mixtures of DPPS
with DPPC, DSPC or DAPC (from 95/5 to 5/95 w/w), mixtures of DSPA
with DSPC or DAPC (from 95/5 to 5/95 w/w), mixtures or DSPG or DPPG
with DSPC or mixtures of DSPC with Ethyl-DSPC. Most preferred are
mixtures of DPPS/DSPC (from 50/50 to 10/90 w/w) or DSPA/DSPC (from
50/50 to 20/80 w/w).
[0061] The amount of phospholipid is generally comprised between
about 0.005 and about 1.0% by weight with respect to the total
weight of the emulsified mixture. Larger amounts might of course be
employed but considering that the end product is an injectable
contrast agent, it is preferred not to use excess of additives
unless strictly necessary to provide for a stable and suitable
product. In general, by using an amount of phospholipid larger than
that indicated as the upper limit of the above range, essentially
no or a very negligible improvement is observed in terms of bubble
population, bubble size distribution, and bubble stability.
Typically, higher amounts of phospholipid are required when higher
volumes of organic solvent are used. Thus, when the volume of
organic solvent amounts to about 50% the volume of the water phase,
an amount of about 1% w/w of phospholipid can advantageously be
added to the emulsion. Preferably the amount of phospholipid is
comprised between 0.01 and 1.0% by weight with respect to the total
weight of the emulsified mixture and more preferably between about
0.05% and 0.5% by weight.
[0062] As mentioned before, the microbubbles produced according to
the process of the invention are stabilized predominantly by a
phospholipid, as above defined. In particular, the envelope
surrounding the gas filled microbubbles is formed by more than 50%
(w/w), preferably by at least 80%, and much more preferably by at
least 90% of a phospholipid material as above defined.
Conveniently, the substantial totality of the stabilizing envelope
of the microbubbles is formed by a phospholipid.
[0063] Other amphiphilic materials can however be admixed with the
phospholipids forming the stabilizing envelope of the gas-filled
microbubbles, in amounts of less than 50% of the total weight of
the emulsifying composition.
[0064] Examples of suitable additional envelope-stabilizing
amphiphilic materials include, for instance, lysolipids; fatty
acids, such as palmitic acid, stearic acid, lauric acid, myristic
acid, arachidic acid, arachidonic acid, behenic acid, oleic acid,
linoleic acid or linolenic acid, and their respective salts with
alkali or alkali metals; lipids bearing polymers, such as chitin,
hyaluronic acid, polyvinylpyrrolidone or polyethylene glycol (PEG),
also referred as "pegylated lipids"; lipids bearing sulfonated
mono- di-, oligo- or polysaccharides; lipids with ether or
ester-linked fatty acids; polymerized lipids; diacetyl phosphate;
dicetyl phosphate; stearylamine; ceramides; polyoxyethylene fatty
acid esters (such as polyoxyethylene fatty acid stearates);
polyoxyethylene fatty alcohols; polyoxyethylene fatty alcohol
ethers; polyoxyethylated sorbitan fatty acid esters; glycerol
polyethylene glycol ricinoleate; ethoxylated soybean sterols;
ethoxylated castor oil; ethylene oxide (EO) and propylene oxide
(PO) block copolymers; sterol esters of sugar acids including
cholesterol glucuronides, lanosterol glucoronides,
7-dehydrocholesterol glucoronide, ergosterol glucoronide,
cholesterol gluconate, lanosterol gluconate, or ergosterol
gluconate; esters of sugar acids and alcohols including lauryl
glucoronide, stearoyl glucoronide, myristoyl glucoronide, lauryl
gluconate, myristoyl gluconate, or stearoyl gluconate; esters of
sugars with aliphatic acids including sucrose laurate, fructose
laurate, sucrose palmitate, sucrose stearate, glucuronic acid,
gluconic acid or polyuronic acid; esters of glycerol with
(C.sub.12-C.sub.24), preferably (C.sub.14-C.sub.22) dicarboxylic
fatty acids and their respective salts with alkali or alkali-metal
salts, such as 1,2-dipalmitoyl-sn-3-succinylglycerol or
1,3-dipalmitoyl-2-succinylglycerol; saponins including
sarsasapogenin, smilagenin, hederagenin, oleanolic acid, or
digitoxigenin; long chain (C.sub.12-C.sub.24) alcohols, including
n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol,
or n-octadecyl alcohol;
6-(5-cholesten-3.beta.-yloxy)-1-thio-.beta.-D-galactopyranoside;
digalactosyldiglyceride;
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxy-1-thio-.beta.-D-galact-
opyranoside;
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxyl-1-thio-.beta.-D-manno-
pyranoside;
12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic
acid;
N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)-methylamino)octadec-
anoyl]-2-aminopalmitic acid;
N-succinyldioleylphosphatidylethanolamine;
1-hexadecyl-2-palmitoylglycerophosphoethanolamine;
palmitoylhomocysteine; alkylammonium salts comprising at least one
(C.sub.10-C.sub.20), preferably (C.sub.14-C.sub.18), alkyl chain,
such as, for instance, stearylammonium chloride, hexadecylammonium
chloride, dimethyldioctadecylammonium bromide (DDAB),
hexadecyltrimethylammonium bromide (CTAB); tertiary or quaternary
ammonium salts comprising one or preferably two
(C.sub.10-C.sub.20), preferably (C.sub.14-C.sub.18), acyl ester
residue, such as, for instance,
1,2-distearoyl-3-trimethylammonium-propane (DSTAP),
1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),
1,2-oleoyl-3-trimethylammonium-propane (DOTAP),
1,2-distearoyl-3-dimethylammonium-propane (DSDAP): and mixtures or
combinations thereof.
[0065] Small amounts of fatty acids and lyso forms of the
phospholipids may also form as degradation products of the original
phospholipid products, e.g. as a consequence of heating the
emulsion.
[0066] Preferred additional envelope-stabilizing amphiphilic
materials are those compounds comprising one or two fatty acid
residues in their molecule, in particular one or two linear
(C.sub.10-C.sub.20)-acyl, preferably (C.sub.14-C.sub.18)-acyl
chains, such as, for instance, the above listed fatty acids, their
respective salts and derivatives.
[0067] Particularly preferred additional envelope-stabilizing
amphiphilic materials are those compounds capable of conferring an
overall net charge to the stabilizing envelope, i.e. compounds
bearing an overall positive or negative net charge. Examples of
suitable negatively of positively charged compounds are, for
instance, lyso-phospholipids, i.e. the lyso-form of the above cited
phospholipids, such as lysophosphatidylserine derivatives (e.g.
lyso-DMPS, -DPPS or -DSPS), lysophosphatidic acid derivatives (e.g.
lyso-DMPA, -DPPA or -DSPA) and lysophosphatidylglycerol derivatives
(e.g. lyso-DMPG, -DPPG or -DSPG); bile acid salts such as cholic
acid salts, deoxycholic acid salts or glycocholic acid salts;
(C.sub.12-C.sub.24), preferably (C.sub.14-C.sub.22) fatty acid
salts such as, for instance, palmitic acid salt, stearic acid salt,
1,2-dipalmitoyl-sn-3-succinylglycerol salt or
1,3-dipalmitoyl-2-succinylglycerol salt; alkylammonium salts with a
halogen counter ion (e.g. chlorine or bromine) comprising at least
one (C.sub.10-C.sub.20) alkyl chain, preferably (C.sub.14-C.sub.18)
alkyl chain, such as, for instance stearylammonium chloride,
hexadecylammonium chloride, dimethyldioctadecylammonium bromide
(DDAB), hexadecyltrimethylammonium bromide (CTAB); tertiary or
quaternary ammonium salts with a halogen counter ion (e.g. chlorine
or bromine) comprising one or preferably two (C.sub.10-C.sub.20)
acyl chain, preferably (C.sub.14-C.sub.18) acyl ester residue, such
as, for instance, 1,2-distearoyl-3-trimethylammonium-propane
(DSTAP), 1,2-dipalmitoyl-3-trimethylammonium-propane (DPTAP),
1,2-oleoyl-3-trimethylammonium-propane (DOTAP),
1,2-distearoyl-3-dimethylammonium-propane (DSDAP).
[0068] If it is desired to obtain "targeted" ultrasound contrast
agents, i.e. contrast agents containing microbubbles that could
selectively bind to a specific site after in vitro or in vivo
administration, according to the process of the present invention
it is also possible to start directly from a phospholipid at least
part of which has been modified by the introduction of a suitably
selected targeting ligand or alternatively, and preferably,
starting from phospholipid at least part of which contain a
possibly protected reactive group capable of being coupled at a
later stage with the suitably selected targeting ligand containing
a complementary reactive function (e.g. avidin-biotin link).
[0069] Therefore, in this specific context, the term "phospholipid"
is intended to encompass both modified and unmodified
phospholipids, thus including phospholipids modified by linking a
targeting ligand or a protective reactive group to the amphiphilic
molecule of the phospholipid.
[0070] The term "targeting ligand" includes within its meaning any
compound, moiety or residue having, or being capable to promote, a
targeting activity of the microbubbles of the invention towards any
biological or pathological site within a living body. Materials or
substances which may serve as targeting ligands include, for
example, but are not limited to proteins, including antibodies,
antibody fragments, receptor molecules, receptor binding molecules,
glycoproteins and lectins; peptides, including oligopeptides and
polypeptides; peptidomimetics; saccharides, including mono and
polysaccharides; vitamins; steroids, steroid analogs, hormones,
cofactors, bioactive agents and genetic material, including
nucleosides, nucleotides and polynucleotides. Targets to which
targeting ligand may be associated include tissues such as, for
instance, myocardial tissue (including myocardial cells and
cardiomyocites), membranous tissues (including endothelium and
epithelium), laminae, connective tissue (including interstitial
tissue) or tumors; blood clots; and receptors such as, for
instance, cell-surface receptors for peptide hormones,
neurotransmitters, antigens, complement fragments, and
immunoglobulins and cytoplasmic receptors for steroid hormones.
[0071] Examples of suitable targets and targeting ligands are
disclosed, for instance, in U.S. Pat. No. 6,139,819, which is
herein incorporated by reference.
[0072] In one preferred embodiment the targeting ligands can be
bound to the amphiphilic molecules forming the stabilizing envelope
through a covalent bond.
[0073] In such a case the specific reactive moiety that needs to be
present in the phospholipid or lipid molecule when a targeting
amphiphilic molecule is desired, will depend on the particular
targeting ligand to be coupled thereto. As an example, if the
targeting ligand can be linked to the amphiphilic molecule through
an amino group, suitable reactive moieties for the amphiphilic
molecule may be isothiocyanate groups (that will form a thiourea
bond), reactive esters (to form an amide bond), aldehyde groups
(for the formation of an imine bond to be reduced to an alkylamine
bond), etc.; if the targeting ligand can be linked to the
amphiphilic molecule through a thiol group, suitable complementary
reactive moieties for the amphiphilic molecule include haloacetyl
derivatives or maleimides (to form a thioether bond); and if the
targeting ligand can be linked to the amphiphilic molecule through
a carboxylic group, suitable reactive moieties for the amphiphilic
molecule might be amines and hydrazides (to form amide or
alkylamide bonds). The reactive moiety can be linked either
directly to the phospholipid molecule or to a modifying moiety
(e.g. PEG) linked to the phospholipid.
[0074] As indicated above, in a preferred embodiment, when a
contrast agent containing targeted microbubbles is desired, at
least part of the starting phospholipid will contain a suitable
reactive moiety and the targeting ligand containing the
complementary functionality will be linked thereto either at any
step before the lyophilization, by adding the targeting ligand
containing the complementary functionality into the phase
containing the functionalised phospholipids/lipids, either before,
during or after the generation of the emulsion, or just before the
reconstitution step. In this latter case it would be possible to
fully exploit the flexibility of the system as the microbubbles
containing at least part of the film-forming phospholipids, or of
the associated lipids, suitably functionalised, might then be bound
to any desired targeting ligand, sharing the same reactive
complementary group.
[0075] Not necessarily however the targeting ligand needs to be
bound to the amphiphilic molecules through a covalent bond. The
targeting ligands may also be suitably associated to the
microbubbles via physical and/or electrostatic types of
interactions. As an example, a functional moiety having a high
affinity and selectivity for a complementary moiety can be
introduced into the phospholipid molecule, while the complementary
moiety will be linked to the targeting ligand. For instance, an
avidin (or streptavidin) moiety (having high affinity for biotin)
can be covalently linked to a microbubble stabilizing phospholipid
while the complementary biotin moiety can be incorporated into a
suitable targeting ligand, e.g. a peptide or an antibody. The
biotin-labelled targeting ligand will thus be associated to the
avidin-labelled microbubble by means of the avidin-biotin coupling
system. According to an alternative embodiment, a biotin-containing
phospholipid can be used as a compound to form the stabilizing
envelope of a microbubble; biotin-containing phospholipid
incorporated in the stabilizing envelope is then reacted first with
avidin (or neutravidin) and then with a biotin-containing ligand.
Examples of biotin/avidin labelling of phospholipids and peptides
are also disclosed in the above cited U.S. Pat. No. 6,139,819.
Alternatively, van der Waal's interactions, electrostatic
interactions and other association processes may associate or bind
the targeting ligand to the amphiphilic molecules.
[0076] Examples of suitable specific targets to which the
microbubbles of the invention can be directed are, for instance,
fibrin, the .alpha..sub.v.beta..sub.3 receptor or the GPIIbIIIa
receptor on activated platelets. Fibrin and platelets are in fact
generally present in "thrombi", i.e. coagula which may form in the
blood stream and cause a vascular obstruction. Suitable binding
peptides are disclosed, for instance, in the above cited U.S. Pat.
No. 6,139,819. Further binding peptides specific for
fibrin-targeting are disclosed, for instance, in International
patent application WO 02/055544, which is herein incorporated by
reference.
[0077] Other examples of important targets include receptors in
vulnerable plaques and tumor specific receptors, such as kinase
domain region (KDR) and VEGF (vascular endothelial growth
factor)/KDR complex. Binding peptides suitable for KDR or VEGF/KDR
complex are disclosed, for instance, in International Patent
application WO 03/74005 and WO 03/084574, both herein incorporated
by reference.
[0078] Process
[0079] The emulsifying step a) of the process of the present
invention can be carried out by submitting the aqueous medium and
the core solvent in the presence of at least one phospholipid to
any appropriate emulsion-generating technique known in the art,
such as, for instance, sonication, shaking, high pressure
homogenization, micromixing, membrane emulsification, high speed
stirring or high shear mixing, e.g. using a rotor-stator
homogenizer. For instance, a rotor-stator homogenizer is employed,
such as Polytron.RTM. PT3000. The agitation speed of the
rotor-stator homogenizer can be selected depending from the
components of the emulsion, the volume of the emulsion and of the
diameter of the vessel containing the emulsion and the desired
final diameter of the microdroplets of solvent in the emulsion. In
general, it has been observed that, when using a rotor-stator
homogenizer having a probe of about 3 cm diameter immersed in a
50-80 ml mixture contained in 3.5-5 cm diameter beaker, an
agitation speed of about 8000 rpm is typically sufficient to obtain
microdroplets having a mean numerical diameter sufficiently reduced
to result, after lyophilization and reconstitution of the
lyophilized matrix, in gas-filled microbubbles having a diameter of
less than about 1.8 .mu.m. By increasing the agitation speed at
about 12000 rpm, it is in general possible to obtain gas-filled
microbubbles having a number mean diameter of less than about 1.5
.mu.m, while with an agitation speed of about 14000-15000 rpm,
gas-filled microbubbles having a number mean diameter of about 1.0
.mu.m or less can generally be obtained. In general it has been
observed that by increasing the agitation speed above about 18000
rpm, slight further reduction of microbubbles size is obtained.
[0080] Alternatively, a micromixing technique can also be employed
for emulsifying the mixture. As known, a micromixer typically
contain at least two inlets and at least one outlet. The organic
solvent is thus introduced into the mixer through a first inlet (at
a flow rate of e.g. 0.05-5 ml/min), while the aqueous phase is
introduced through the second inlet (e.g. at a flow rate of 2-100
ml/min). The outlet of the micromixer is then connected to the
vessel containing the aqueous, so that the aqueous phase drawn from
said vessel at subsequent instants and introduced into the
micromixer contains increasing amounts of emulsified solvent. When
the whole volume of solvent has been added, the emulsion from the
container can be kept under recirculation through the micromixer
for a further predetermined period of time, e.g. 5-120 minutes, to
allow completion of the emulsion.
[0081] Depending on the emulsion technique, the organic solvent can
be introduced gradually during the emulsification step or at once
before starting the emulsification step. Alternatively the aqueous
medium may be gradually added to the water immiscible solvent
during the emulsification step or at once before starting the
emulsification step. The phospholipid can be either dispersed in
the aqueous medium or in the organic solvent, before admixing the
two, or it may be separately added the aqueous-organic mixture
before or during the emulsification step. Preferably, the
phospholipid is dispersed in the organic solvent (preferably
cyclooctane).
[0082] The emulsification of step a) is conveniently carried out at
room temperature, e.g. at a temperature of 22.degree.
C..+-.5.degree. C., or at higher temperatures, for instance
50.degree. C.-60.degree. C. (e.g. in the case of core solvents with
high boiling points) or at lower temperature, for instance
0.degree. C.-10.degree. C. (e.g. in the case of core solvents with
boiling points close to room temperature). The temperature is
preferably kept below the boiling temperature of the organic
solvent, preferably at least 5.degree. C. below said temperature,
more preferably at least 10.degree. C. below. As prolonged exposure
of the mixture at high temperatures (e.g. 90.degree. C. or more)
may cause possible degradations of phospholipids, with consequent
formation of the respective lyso-derivatives, it is in general
preferred to avoid such prolonged heating at high temperatures.
[0083] If necessary, the aqueous medium containing the
phospholipids can be subjected to controlled heating, in order to
facilitate the dispersion thereof. For instance, the phospholipid
containing aqueous suspension can be heated at about 60-70.degree.
C. for about 15 minutes and then allowed to cool at the temperature
at which the emulsion step is then carried out.
[0084] As previously mentioned, additional amphiphilic materials,
such as those previously listed, can also be introduced into the
emulsifying mixture containing the phospholipid. The amount of said
additional amphiphilic compounds is preferably not higher than
about 50% by weight with respect to the total weight of amphiphilic
material, more preferably not higher than 20% by weight, down to an
amount of e.g. about 0.1%.
[0085] The aqueous medium may, if desired, further contain one or
more excipients.
[0086] As used herein, the term "excipient" refers to any additive
useful in the present invention, such as those additives employed
to increase the stability of the emulsion or of the lyophilisate
intermediate and/or to provide for pharmaceutically acceptable and
stable final compositions.
[0087] Exemplary excipients in this regard are, for instance,
viscosity enhancers and/or solubility aids for the
phospholipids.
[0088] Viscosity enhancers and solubility aids that may suitably be
employed are for example mono- or polysaccharides, such as glucose,
lactose, saccharose, and dextrans, aliphatic alcohols, such as
isopropyl alcohol and butyl alcohol, polyols such as glycerol,
1,2-propanediol, and the like agents. In general however we have
found that it is unnecessary to incorporate additives such as
viscosity enhancers, which are commonly employed in many existing
contrast agent formulations, into the contrast agents of the
present invention. This is a further advantage of the present
invention as the number of components administered to the body of a
subject is kept to a minimum and the viscosity of the contrast
agents is maintained as low as possible.
[0089] As mentioned before, the Applicant has found substantially
unnecessary, to add water-insoluble structure-builders, such as
cholesterol, to the emulsifying mixture. As a matter of fact, it
has been observed that an amount of 0.05% (w/w with respect to the
total weight of the emulsifying mixture) of cholesterol
dramatically reduces the conversion yield from microdroplets into
gas-filled microvesicles, further resulting in a broad-dispersion
of the vesicles' size. The amount of water-insoluble compounds in
the emulsifying mixture, particularly of those compounds not
comprising one or two fatty acid residue in their structure, is
thus preferably lower than 0.050%, more preferably lower than about
0.030% by weight with respect to the total weight of the
emulsion.
[0090] Emulsions produced according to step a) may advantageously
be subjected to one or more washing steps, prior to the
lyophilization of step b), in order to remove excess of
phospholipids in the aqueous phase (not associated to the emulsion)
and separate and remove optional additives such as viscosity
enhancers and solubility aids, as well as undesired material such
as colloidal particles, and undersized and/or oversized emulsion
droplets. Such washing may be effected in per se known manner, the
emulsion being separated using techniques such as decantation,
flotation, centrifugation, cross flow filtration and the like.
[0091] If washing steps are foreseen, and if a lyoprotective agent
was present in the original aqueous phase prior to the generation
of the emulsion, said washing steps can be performed with aqueous
solutions containing one or more lyoprotective agents to replace
the amount of lyoprotective agents partially removed with the
washings. On the other side, if no lyoprotectant was present in the
emulsified aqueous-organic mixture, the formed emulsion can be
washed with a lyoprotectant-containing aqueous solution, in order
to introduce the lyoprotectant into the emulsified mixture or,
alternatively, the lyoprotectant can be added after the washing
steps, prior to lyophilisation.
[0092] If desired, the emulsion (either as such or after the
washing step) can be subjected to a ultrafiltration or
microfiltration step before lyophilization, in order to further
reduce the amount of large size microbubbles in the final
reconstituted suspension. During microfiltration, e.g. with a 5
.mu.m or 3 .mu.m filter, large size microdroplets are in fact
retained by the filter and separated from the rest of the small
size microdroplets, thus preventing the formation of large size
microbubbles upon reconstitution of the lyophilized material.
Microfiltration can be accomplished according to conventional
techniques such as positive filtration, vacuum filtration or
in-line filtration. Membranes of filtration can be Nylon, glass
fiber, cellulose, paper, polycarbonate or polyester
(Nuclepore.RTM.) membranes.
[0093] According to an alternative embodiment, an additional
amphiphilic compound can be added after the formation of the
emulsion according to the above teachings, either with or without
the washing steps. In particular, an aqueous suspension of the
desired compound is added to the formed emulsion, preferably under
agitation and heating (preferably at less than 80.degree. C., e.g.
40.degree. C.-80.degree. C., in particular 50-70.degree. C.), in
order to add said compound to the stabilizing envelope. This
alternative embodiment is particularly useful to subsequently
introduce into the stabilizing layer amphiphilic compounds which
may otherwise negatively affect the properties of the final product
if introduced in the initial mixture of the emulsion. Examples of
amphiphilic compounds which can conveniently be subsequently
introduced as additional components of the stabilizing envelope
after the preparation of the initial emulsion are, for instance,
PEG-modified phospholipids, in particular PEG-modified
phosphatidylethanolamines, such as DMPE-PEG750, DMPE-PEG1000,
DMPE-PEG2000, DMPE-PEG3000, DMPE-PEG4000, DMPE-PEG5000,
DPPE-PEG750, DPPE-PEG1000, DPPE-PEG2000, DPPE-PEG3000,
DPPE-PEG4000, DPPE-PEG5000, DSPE-PEG750, DSPE-PEG1000,
DSPE-PEG2000, DSPE-PEG3000, DSPE-PEG4000, DSPE-PEG5000,
DAPE-PEG750, DAPE-PEG1000, DAPE-PEG2000, DAPE-PEG3000, DAPE-PEG4000
or DAPE-PEG5000. Similarly, also PEG-modified phospholipids bearing
reactive moieties or targeting ligands (e.g. containing biotin,
maleimide, or maleimide-peptide) can conveniently be introduced
subsequently according to this method. In addition, this technique
can also be used to subsequently add to the composition of the
stabilizing layer other components, such as lipopeptides or
polymeric surfactants. Examples of polymeric surfactants which can
be conveniently added after formation of the emulsion are, for
instance, ethyleneoxide-propylenoxide block copolymers, such as
Pluronic F68, Pluronic F108, Pluronic F-127 (Sigma Aldrich,
Missouri, USA); Polyoxyethylated alkyl ethers such as Brij.RTM. 78
(Sigma Aldrich, Missouri, USA); Polyoxyethylene fatty acid esters
such as Myrj.RTM. 53 or Myrj.RTM. 59 (Sigma Aldrich, Missouri,
USA); Polyoxyethylenesorbitan fatty acid ester such as Tween.RTM.
60 (Sigma Aldrich, Missouri, USA); or Polyethylene glycol
tert-octylphenyl ether such as Triton.RTM.X-100 (Sigma Aldrich,
Missouri, USA).
[0094] The Applicant has in fact observed that the use of a mixture
containing limited amounts (e.g. less than 10% by weight) of a PEG
modified phospholipid (e.g. DSPE-PEG or DPPE-PEG) together with a
film forming phospholipid (e.g. DPPS or a 50:50 mixture of
DAPC/DPPS) for preparing an emulsion according to the process of
the invention, may determine a substantial broadening of the size
distribution in the final product, with respect to the size
distribution of microbubbles obtained from an emulsion containing
only the film forming phospholipid. On the other side, if an
emulsion containing only the film forming phospholipid is first
prepared and then an aqueous suspension of the PEG modified
phospholipid is subsequently added to the obtained emulsion (e.g.
under agitation for 1 hour, at a temperature of about 60.degree.
C.), it has been observed that a rather high amount (typically more
than 30% by weight) of the PEG modified phospholipid can be
incorporated into the stabilizing envelope, without substantially
affecting the size distribution of the final product.
[0095] According to a preferred embodiment, the emulsion is
subjected to a controlled additional heating treatment before the
lyophilization step. The additional heating of the emulsion is
preferably performed into a sealed container. The heat treatment
can vary from about 15 minutes to about 90 minutes, at temperatures
comprised from about 60.degree. C. to about 125.degree. C.,
preferably from about 80.degree. C. to about 120.degree. C. In
general, the higher the temperature, the shortest the time of the
thermal treatment. During the heating, the emulsion can optionally
be kept under agitation.
[0096] As observed by the Applicant, while this additional thermal
treatment may result in a partial degradation of the phospholipids
(e.g. with a content of about 5-20% w/w of lysolipids in the final
product, when the emulsion is heated at about 100-120.degree. C.
for about 30 min), it has nevertheless the great advantage of
allowing a substantial narrowing of the size distribution and an
increase of the total number of microbubbles in the final
suspension, independently from the working conditions of the
initial emulsification step (e.g. type of organic solvent,
emulsifying technique, optional washing steps, etc.).
[0097] The thermally treated emulsion can then be directly
subjected to lyophilization, typically without the need of further
washing steps.
[0098] Lyophilization of the emulsion according to step b) may be
carried out by initially freezing the emulsion and thereafter
lyophilizing the frozen emulsion, by per se generally known methods
and devices. Since the dried, lyophilized, product will normally be
reconstituted by addition of a carrier liquid prior to
administration, the emulsion may advantageously be filled into
sealable vials prior to lyophilization so as to give vials each
containing an appropriate amount, e.g. a single dosage unit, of
lyophilized dried product for reconstitution into an injectable
form. By lyophilizing the emulsion in individual vials rather than
in bulk, handling of the delicate honeycomb-like structure of the
lyophilized product and the risk of at least partially degrading
this structure are avoided.
[0099] Following lyophilization, the vacuum can be removed in the
lyophilizer by introducing the desired gas to form the microbubbles
in the final formulation of the contrast agent. This will allow to
fill the headspace of the vials with the desired gas and then seal
the vials with an appropriate closure. Alternatively, the vial can
be kept under vacuum and sealed, while the gas is added at a later
stage, e.g. just before administration, for instance when the gas
is a radioactive or a hyperpolarized gas.
[0100] The so obtained lyophilized product in the presence of the
suitable gas can thus be stably stored for several months before
being reconstituted by dissolving it into an aqueous carrier
liquid, to obtain a suspension of gas-filled microbubbles.
[0101] Any biocompatible gas, gas precursor or mixture thereof may
be employed to fill the above microvesicles, the gas being selected
depending on the chosen modality.
[0102] The gas may comprise, for example, air; nitrogen; oxygen;
carbon dioxide; hydrogen; nitrous oxide; a noble or inert gas such
as helium, argon, xenon or krypton; a radioactive gas such as
Xe.sup.133 or Kr.sup.81; a hyperpolarized noble gas such as
hyperpolarized helium, hyperpolarized xenon or hyperpolarized neon;
a low molecular weight hydrocarbon (e.g. containing up to 7 carbon
atoms), for example an alkane such as methane, ethane, propane,
butane, isobutane, pentane or isopentane, a cycloalkane such as
cyclobutane or cyclopentane, an alkene such as propene, butene or
isobutene, or an alkyne such as acetylene; an ether; a ketone; an
ester; halogenated gases, preferably fluorinated gases, such as or
halogenated, fluorinated or perfluorinated low molecular weight
hydrocarbons (e.g. containing up to 7 carbon atoms); or a mixture
of any of the foregoing. Where a halogenated hydrocarbon is used,
preferably at least some, more preferably all, of the halogen atoms
in said compound are fluorine atoms.
[0103] Fluorinated gases are preferred, in particular
perfluorinated gases, especially in the field of ultrasound
imaging. Fluorinated gases include materials which contain at least
one fluorine atom such as, for instance fluorinated hydrocarbons
(organic compounds containing one or more carbon atoms and
fluorine); sulfur hexafluoride; fluorinated, preferably
perfluorinated, ketones such as perfluoroacetone; and fluorinated,
preferably perfluorinated, ethers such as perfluorodiethyl ether.
Preferred compounds are perfluorinated gases, such as SF.sub.6 or
perfluorocarbons (perfluorinated hydrocarbons), i.e. hydrocarbons
where all the hydrogen atoms are replaced by fluorine atoms, which
are known to form particularly stable microbubble suspensions, as
disclosed, for instance, in EP 0554 213, which is herein
incorporated by reference.
[0104] The term perfluorocarbon includes saturated, unsaturated,
and cyclic perfluorocarbons. Examples of biocompatible,
physiologically acceptable perfluorocarbons are: perfluoroalkanes,
such as perfluoromethane, perfluoroethane, perfluoropropanes,
perfluorobutanes (e.g. perfluoro-n-butane, optionally in admixture
with other isomers such as perfluoro-isobutane), perfluoropentanes,
perfluorohexanes or perfluoroheptanes; perfluoroalkenes, such as
perfluoropropene, perfluorobutenes (e.g. perfluorobut-2ene) or
perfluorobutadiene; perfluoroalkynes (e.g. perfluorobut-2-yne); and
perfluorocycloalkanes (e.g. perfluorocyclobutane,
perfluoromethylcyclobutane, perfluorodimethylcyclobutanes,
perfluorotrimethylcyclobutanes, perfluorocyclopentane,
perfluoromethylcyclopentane, perfluorodimethylcyclopentanes,
perfluorocyclohexane, perfluoromethylcyclohexane and
perfluorocycloheptane). Preferred saturated perfluorocarbons have
the formula C.sub.nF.sub.n+2, where n is from 1 to 12, preferably
from 2 to 10, most preferably from 3 to 8 and even more preferably
from 3 to 6. Suitable perfluorocarbons include, for example,
CF.sub.4, C.sub.2F.sub.6, C.sub.3F.sub.8, C.sub.4F.sub.8,
C.sub.4F.sub.10, C.sub.5F.sub.12, C.sub.6F.sub.12, C.sub.6F.sub.14,
C.sub.7F.sub.14, C.sub.7F.sub.16, C.sub.8F.sub.18, and
C.sub.9F.sub.20.
[0105] Particularly preferred gases are SF.sub.6 or
perfluorocarbons selected from CF.sub.4, C.sub.2F.sub.6,
C.sub.3F.sub.8, C.sub.4F.sub.8, C.sub.4F.sub.10 or mixtures
thereof; SF.sub.6, C.sub.3F.sub.8 or C.sub.4F.sub.10 are
particularly preferred.
[0106] It may also be advantageous to use a mixture of any of the
above gases in any ratio. For instance, the mixture may comprise a
conventional gas, such as nitrogen, air or carbon dioxide and a gas
forming a stable microbubble suspension, such as sulfur
hexafluoride or a perfluorocarbon as indicated above. Examples of
suitable gas mixtures can be found, for instance, in WO 94/09829,
which is herein incorporated by reference. The following
combinations are particularly preferred: a mixture of gases (A) and
(B) in which the gas (B) is a fluorinated gas, preferably selected
from SF.sub.6, CF.sub.4, C.sub.2F.sub.6, C.sub.3F.sub.6,
C.sub.3F.sub.8, C.sub.4F.sub.6, C.sub.4F.sub.8, C.sub.4F.sub.10,
C.sub.5F.sub.10, C.sub.5F.sub.12 or mixtures thereof, and (A) is
selected from air, oxygen, nitrogen, carbon dioxide or mixtures
thereof. The amount of gas (B) can represent from about 0.5% to
about 95% v/v of the total mixture, preferably from about 5% to
80%.
[0107] In some instances it may be desirable to include a precursor
to a gaseous substance (i.e. a material that is capable of being
converted to a gas in vivo). Preferably the gaseous precursor and
the gas derived therefrom are physiologically acceptable. The
gaseous precursor may be pH-activated, photo-activated, temperature
activated, etc. For example, certain perfluorocarbons may be used
as temperature activated gaseous precursors. These
perfluorocarbons, such as perfluoropentane or perfluorohexane, have
a liquid/gas phase transition temperature above room temperature
(or the temperature at which the agents are produced and/or stored)
but below body temperature; thus, they undergo a liquid/gas phase
transition and are converted to a gas within the human body.
Furthermore, the term "gas" as used herein includes mixtures in
vapor form at the normal human body temperature of 37.degree. C.
Compounds which at the temperature of 37.degree. C. are liquid may
thus also be used in limited amounts in admixture with other
gaseous compounds, to obtain a mixture which is in a vapor phase at
37.degree. C.
[0108] For ultrasonic echography, the biocompatible gas or gas
mixture is preferably selected from air, nitrogen, carbon dioxide,
helium, krypton, xenon, argon, methane, halogenated hydrocarbons
(including fluorinated gases such as perfluorocarbons and sulfur
hexafluoride) or mixtures thereof. Advantageously, perfluorocarbons
(in particular C.sub.4F.sub.10 or C.sub.3F.sub.8) or SF.sub.6 can
be used, optionally in admixture with air or nitrogen.
[0109] For the use in MRI the microbubbles will preferably contain
a hyperpolarized noble gas such as hyperpolarized neon,
hyperpolarized helium, hyperpolarized xenon, or mixtures thereof,
optionally in admixture with air, CO.sub.2, oxygen, nitrogen,
helium, xenon, or any of the halogenated hydrocarbons as defined
above.
[0110] For use in scintigraphy, the microbubbles according to the
invention will preferably contain radioactive gases such as
Xe.sup.133 or Kr.sup.81 or mixtures thereof, optionally in
admixture with air, CO.sub.2, oxygen, nitrogen, helium, kripton or
any of the halogenated hydrocarbons as defined above.
[0111] The lyophilized composition in contact with the gas can then
be very easily reconstituted by the addition of an appropriate
sterile aqueous injectable and physiologically acceptable carrier
liquid such as sterile pyrogen-free water for injection, an aqueous
solution such as saline (which may advantageously be balanced so
that the final product for injection is not hypotonic), or an
aqueous solution of one or more tonicity-adjusting substances such
as salts (e.g. of plasma cations with physiologically tolerable
counterions), or sugars, sugar alcohols, glycols and other
non-ionic polyol materials (e.g. glucose, sucrose, sorbitol,
mannitol, glycerol, polyethylene glycols, propylene glycols and the
like), requiring only minimal agitation such as may, for example,
be provided by gentle hand-shaking.
[0112] As observed by the Applicant, the so obtained reconstituted
microbubbles have generally a number mean diameter which is
slightly lower than the number mean diameter measured for the
microdroplets of the emulsion. The mean number diameter of the
microbubbles is in general from about 60% to about 90% of the mean
number diameter of the emulsion's microdroplets. In most cases, a
mean number diameter of the microbubbles of about 70-75% of the
mean number diameter of the microdroplets has been observed.
[0113] Where the dried product is contained in a vial, this is
conveniently sealed with a septum through which the carrier liquid
may be injected using an optionally pre-filled syringe;
alternatively the dried product and carrier liquid may be supplied
together in a dual chamber device such as a dual chamber syringe.
It may be advantageous to mix or gently shake the product following
reconstitution. However, as noted above, in the stabilized contrast
agents according to the invention the size of the gas microbubbles
may be substantially independent of the amount of agitation energy
applied to the reconstituted dried product. Accordingly no more
than gentle hand-shaking may be required to give reproducible
products with consistent microbubble size.
[0114] The microbubble suspensions generated upon reconstitution in
water or an aqueous solution may be stable for at least 12 hours,
thus permitting considerable flexibility as to when the dried
product is reconstituted prior to injection.
[0115] Unless it contains a hyperpolarized gas, known to require
special storage conditions, the lyophilised residue may be stored
and transported without need of temperature control of its
environment and in particular it may be supplied to hospitals and
physicians for on site formulation into a ready-to-use
administrable suspension without requiring such users to have
special storage facilities.
[0116] Preferably in such a case it can be supplied in the form of
a two component kit.
[0117] Said two component kit can include two separate containers
or a dual-chamber container. In the former case preferably the
container is a conventional septum-sealed vial, wherein the vial
containing the lyophilized residue of step b) is sealed with a
septum through which the carrier liquid may be injected using an
optionally prefilled syringe. In such a case the syringe used as
the container of the second component is also used then for
injecting the contrast agent. In the latter case, preferably the
dual-chamber container is a dual-chamber syringe and once the
lyophilisate has been reconstituted and then suitably mixed or
gently shaken, the container can be used directly for injecting the
contrast agent. In both cases means for directing or permitting
application of sufficient bubble forming energy into the contents
of the container are provided. However, as noted above, in the
stabilised contrast agents according to the invention the size of
the gas microbubbles is substantially independent of the amount of
agitation energy applied to the reconstituted dried product.
Accordingly no more than gentle hand shaking is generally required
to give reproducible products with consistent microbubble size.
[0118] It can be appreciated by one ordinary skilled in the art
that other two-chamber reconstitution systems capable of combining
the dried powder with the aqueous solution in a sterile manner are
also within the scope of the present invention. In such systems, it
is particularly advantageous if the aqueous phase can be interposed
between the water-insoluble gas and the environment, to increase
shelf life of the product. Where a material necessary for forming
the contrast agent is not already present in the container (e.g. a
targeting ligand to be linked to the phospholipid during
reconstitution), it can be packaged with the other components of
the kit, preferably in a form or container adapted to facilitate
ready combination with the other components of the kit.
[0119] No specific containers, vial or connection systems are
required; the present invention may use conventional containers,
vials and adapters. The only requirement is a good seal between the
stopper and the container. The quality of the seal, therefore,
becomes a matter of primary concern; any degradation of seal
integrity could allow undesirables substances to enter the vial. In
addition to assuring sterility, vacuum retention is essential for
products stoppered at ambient or reduced pressures to assure safe
and proper reconstitution. As to the stopper, it may be a compound
or multicomponent formulation based on an elastomer, such as
poly(isobutylene) or butyl rubber.
[0120] The contrast agents obtainable by the process of the present
invention may be used in a variety of diagnostic imaging
techniques, including in particular ultrasound and Magnetic
Resonance. Possible other diagnostic imaging applications include
scintigraphy, light imaging, and X-ray imaging, including X-ray
phase contrast imaging.
[0121] Their use in diagnostic ultrasound imaging and in MR
imaging, e.g. as susceptibility contrast agents and as
hyperpolarized gas bubbles, constitute preferred features of the
invention. A variety of imaging techniques may be employed in
ultrasound applications, for example including fundamental and
harmonic B-mode imaging, pulse or phase inversion imaging and
fundamental and harmonic Doppler imaging; if desired
three-dimensional imaging techniques may be used.
[0122] In vivo ultrasound tests in rabbits, dogs and pigs have
shown that contrast agents according to the invention may produce
an increase in backscattered signal intensity from the myocardium
of 15-25 dB following intravenous injection of doses as low as
0.001 ml/kg body weight. Signals may be observed at even lower
doses using more sensitive techniques such as color Doppler or
power pulse inversion. At these low doses, attenuation in
blood-filled compartments such as the heart chambers has been found
to be sufficiently low to permit visualization of regions of
interest in the myocardial vasculature. Tests have also shown such
intravenously injected contrast agents to be distributed throughout
the whole blood pool, thereby enhancing the echogenicity of all
vascularised tissues, and to be recirculated. They have also been
found useful as general Doppler signal enhancement aids, and may
additionally be useful in ultrasound-computed tomography and in
physiologically triggered or intermittent imaging.
[0123] For ultrasound applications such as echocardiography, in
order to permit free passage through the pulmonary system and to
achieve resonance with the preferred imaging frequencies of about
0.1-15 MHz, microbubbles having an average size of 0.1-10 .mu.m,
e.g. 0.5-7 .mu.m are generally employed. As described above,
contrast agents according to the invention may be produced with a
very narrow size distribution for the microbubble dispersion within
the range preferred for echocardiography, thereby greatly enhancing
their echogenicity as well as their safety in vivo, and rendering
the contrast agents of particular advantage in applications such as
blood pressure measurements, blood flow tracing and ultrasound
tomography.
[0124] In ultrasound applications the contrast agents of the
invention may, for example, be administered in doses such that the
amount of phospholipid injected is in the range 0.1-200 .mu.g/kg
body weight, typically 10-200 .mu.g/kg in the absence of a washing
step for the emulsion and 0.1-30 .mu.g/kg if the emulsion has been
washed prior to lyophilisation. It will be appreciated that the use
of such low levels of phospholipid is of substantial advantage in
minimising possible toxic side effects. Furthermore, the low levels
of phospholipids present in effective doses may permit dosage
increases to prolong observation times without adverse effects.
[0125] By suitably selecting the components of the mixture and in
particular the amount of agitation energy applied during the
emulsion of the aqueous-organic mixture, it is possible to obtain
gas-filled microbubbles with the desired numerical mean diameter
and size distribution.
[0126] In particular, by exploiting the process according to the
present invention it is possible to obtain contrast agents
comprising phospholipid-stabilized small-sized gas microbubbles
characterized by having relatively small mean dimensions and a
particularly useful narrow and controlled size distribution.
[0127] As known by those skilled in the art, the dimensions of
micro/nano particles and their respective size distribution can be
characterized by a number of parameters, the most frequently used
being the mean diameter in number D.sub.N, the median diameter in
number D.sub.N50, the mean diameter in volume D.sub.V and the
median diameter in volume D.sub.V50. While diameters in number
provide an indication of the mean number dimension of the
particles, the diameter in volume provides information on how the
total volume of the particles is distributed among the whole
population. As the presence of very few large volume particles in a
population of otherwise small volume particles may cause the
corresponding D.sub.V value to be shifted towards high values, it
is sometimes more convenient to use the D.sub.V50 value for
evaluating the distribution of a particles' population. D.sub.V50
is a calculated value indicating that half of the total of
particles' internal volume is present in particles having a
diameter lower than D.sub.V50; this allows to reduce the effects of
accidentally formed large volume particles in the evaluation of the
size distribution. Clearly, mono-sized particles show identical
D.sub.N, D.sub.N50, D.sub.V and D.sub.V50 values. On the other
side, an increasing broadening of particles' distribution will
result in a larger difference between these various values with a
corresponding variation of the respective ratio thereof (e.g.
increase of D.sub.V/D.sub.N ratio). For example, particles
populations containing primarily small particles (e.g. particles
with a diameter around 2 .mu.m) with nevertheless a small
percentage of large particles (for instance particles with a
diameter above 8 .mu.m) show higher D.sub.V or D.sub.V50 values as
compared to the D.sub.N value, with correspondingly higher
D.sub.V/D.sub.N or D.sub.V50/D.sub.N ratios. In addition, the
microbubbles preparations can be characterized by the amount of gas
contained in microbubbles below a predetermined diameter.
[0128] According to an aspect of the present invention, gas-filled
microbubbles compositions are provided wherein at least 10% of the
total volume of gas contained in the microbubbles of the
composition is contained in microvesicles with a diameter of 1.5
.mu.m or less. Preferably, said amount of gas contained in
microvesicles with diameter of 1.5 .mu.m or less is at least 25%,
more preferably at least 50% and even more preferably at least 70%.
In some embodiments of the invention, said amount is even higher
than 90%. The D.sub.V50/D.sub.N ratio of the microbubbles
composition is preferably of about 2.10 or lower, more preferably
of about 1.80 or lower, much more preferably of about 1.50 or
lower. Microbubbles with lower values of the D.sub.V50/D.sub.N
ratio, e.g. 1.20, and even lower, e.g. 1.05, can easily be
obtained.
[0129] Viewed from another aspect, the process of the present
invention allows to prepare microbubbles having a mean diameter in
number (D.sub.N) of less than 1.70 .mu.m and a median diameter in
volume (D.sub.V50) such that the D.sub.V50/D.sub.N ratio is of
about 2.30 or lower, preferably lower than 2.10. Preferably said
D.sub.N value is of 1.60 .mu.m or lower, more preferably of 1.50
.mu.m or lower, much more preferably of 1.30 .mu.m or lower.
Microbubbles with lower values of D.sub.N, e.g. of about 1 .mu.m,
or even lower, e.g. 0.85 .mu.m and down to 0.80 .mu.m, can easily
be obtained with the process of the invention.
[0130] The concentration of microbubbles in the reconstituted
suspension is in general of at least 1.times.10.sup.8 particles per
milliliter, preferably of at least 1.times.10.sup.9 particles per
milliliter.
[0131] The above values of gas amount, D.sub.V50, D.sub.N and
number of microbubbles refer to a measurement made by using a
Coulter Counter Mark II apparatus fitted with a 30 .mu.m aperture,
with a measuring range of 0.7 to 20 .mu.m.
[0132] This specific category of contrast agents are particularly
valuable in ultrasound imaging, in particular for imaging
techniques relying on non-linear scattering of microbubbles, as
explained below.
[0133] Most recent ultrasound contrast-imaging methods exploit the
nonlinear scattering characteristics of ultrasound contrast agents.
From the literature (e.g. Eatock et al., Journal of the Acoustical
Society of America, vol. 77(5), pp 1692-1701, 1985) it is known
that nonlinear scattering is significant only for microbubbles
which are smaller than, or close to, resonance size. In particular,
microbubbles with dimensions of half the resonance size can
conveniently be employed. "Half the resonance size" is the size of
a microbubble with a resonance frequency that equals twice the
centre frequency of the transmitted ultrasound wave (which for
particular applications may be of up to about 60 MHz). When imaging
a volume containing a microbubble-based ultrasound contrast agent,
the detectability of the microbubble echoes against tissue echoes
is enhanced by the level of nonlinear scattering by the
microbubbles, and decreased by the attenuation caused by the
microbubbles located between the probe and the region of interest.
Attenuation along the transmit path reduces the ultrasound-energy
available for generating nonlinear bubble-response; attenuation
along the receive path removes echo-energy able to reach the
ultrasound probe. In the case of a suspension comprising a wide
range of microbubble sizes, the microbubbles at resonance size, and
larger than resonance size, mainly contribute to transmit-receive
attenuation, without contributing in an efficient way to the
nonlinear echo signals. Therefore, the overall acoustic response
for nonlinear imaging greatly benefits from the use of a calibrated
set of microbubbles having a narrow size distribution and a mean
size close to half the resonance size. Preferably, microbubbles
preparations having a size distribution corresponding to a
D.sub.V50/D.sub.N ratio of about 2.30 or lower, more preferably of
2.10 or lower and much more preferably of 2.00 or lower are
employed. Preferably, the mean size of the employed microbubbles is
of about .+-.10% of half the resonance size, more preferably of
about .+-.5% of half the resonance size.
[0134] A yet still further aspect of the present invention thus
relates to a method of diagnostic imaging which comprises
administering to a subject a contrast-enhancing amount of a
contrast agent comprising gas-filled microbubbles with the size and
size distribution as above specified and imaging at least a part of
said subject. In particular, said diagnostic imaging includes
insonating said subject by means of an ultrasound device generating
an ultrasound wave with a predetermined transmit frequency, from
which a corresponding resonance size of microbubbles is determined,
and administering a contrast agent comprising gas-filled
microbubbles having a narrow size distribution and a mean size
close to half the resonance size. Preferably, the narrow size
distribution and a mean size of the microbubbles are as above
defined. For instance, a HDI 5000 ultrasound machine from Philips
(e.g. in pulse inversion mode, with L7-4 probe and Mechanical Index
of 0.07), can be used in the diagnostic imaging method. According
to this method, said subject is a vertebrate and said contrast
agent is introduced into the vasculature or into a body cavity of
said vertebrate. Said contrast agent can be supplied as a kit, such
as those previously described, comprising the lyophilized product
in contact with the gas and an aqueous medium for
reconstitution.
Embodiments of the Invention Include
[0135] 1. Methods for preparing a lyophilized matrix which, upon
contact with an aqueous carrier liquid and a gas, is
reconstitutable into a suspension of gas-filled microbubbles
stabilized predominantly by a phospholipid, said method comprising
the steps of:
[0136] a) preparing an aqueous-organic emulsion comprising i) an
aqueous medium including water, ii) an organic solvent
substantially immiscible with water; iii) an emulsifying
composition of amphiphilic materials comprising more than 50% by
weight of a phospholipid and iv) a lyoprotecting agent;
[0137] b) lyophilizing said emulsified mixture, to obtain a
lyophilized matrix comprising said phospholipid. [0138] 2. Methods
for preparing an injectable contrast agent comprising a liquid
aqueous suspension of gas-filled microbubbles stabilized
predominantly by a phospholipid, which comprises the steps of:
[0139] a) preparing an aqueous-organic emulsion comprising i) an
aqueous medium including water, ii) an organic solvent
substantially immiscible with water; iii) an emulsifying
composition of amphiphilic materials comprising more than 50% by
weight of a phospholipid and iv) a lyoprotecting agent;
[0140] b) lyophilizing said emulsion, to obtain a lyophilized
matrix comprising said phospholipid;
[0141] c) contacting said lyophilized matrix with a biocompatible
gas;
[0142] d) reconstituting said lyophilized matrix by dissolving it
into a physiologically acceptable aqueous carrier liquid, to obtain
a suspension of gas-filled microbubbles stabilized predominantly by
said phospholipid. [0143] 3. Methods according to embodiments 1 or
2 wherein the step a) of preparing the emulsion comprises the
following steps:
[0144] a1) preparing a suspension by dispersing the emulsifying
composition and the lyoprotective agent in the aqueous medium;
[0145] a2) admixing the obtained suspension with the organic
solvent;
[0146] a3) submitting the mixture to controlled agitation, to
obtain an emulsion. [0147] 4. Methods according to any of the
preceding embodiments, wherein the organic solvent has a solubility
in water of less than 10 g/l. [0148] 5. Methods according to any of
the preceding embodiments, wherein the organic solvent has a
solubility in water of 1.0 g/l or lower. [0149] 6. Methods
according to any of the preceding embodiments, wherein the organic
solvent has a solubility in water of 0.2 g/l or lower. [0150] 7.
Methods according to any of the preceding embodiments, wherein the
organic solvent has a solubility in water of about 0.01 g/l or
lower. [0151] 8. Methods according to any of the preceding
embodiments, wherein the organic solvent has a solubility in water
of 0.001 g/l or lower. [0152] 9. Methods according to embodiment 1,
wherein the organic solvent is selected among branched or linear
alkanes, alkenes, cyclo-alkanes, aromatic hydrocarbons, alkyl
ethers, ketones, halogenated hydrocarbons, perfluorinated
hydrocarbons and mixtures thereof. [0153] 10. Methods according to
embodiment 9 wherein the solvent is selected among pentane, hexane,
heptane, octane, nonane, decane, 1-pentene, 2-pentene, 1-octene,
cyclopentane, cyclohexane, cyclooctane, 1-methyl-cyclohexane,
benzene, toluene, ethylbenzene, 1,2-dimethylbenzene,
1,3-dimethylbenzene, di-butyl ether and di-isopropylketone,
chloroform, carbon tetrachloride,
2-chloro-1-(difluoromethoxy)-1,1,2-trifluoroethane (enflurane),
2-chloro-2-(difluoromethoxy)-1,1,1-trifluoroethane (isoflurane),
tetrachloro-1,1-difluoroethane, perfluoropentane, perfluorohexane,
perfluoroheptane, perfluorononane, perfluorobenzene,
perfluorodecalin, methylperfluorobutylether,
methylperfluoroisobutylether, ethylperfluorobutylether,
ethylperfluoroisobutylether and mixtures thereof [0154] 11. Methods
according to any of the preceding embodiments, wherein the amount
of organic solvent is from about 1% to about 50% by volume with
respect to the amount water. [0155] 12. Methods according to any of
the preceding embodiments wherein the lyoprotecting agent is
selected among carbohydrates, sugar alcohols, polyglycols and
mixtures thereof. [0156] 13. Methods according to embodiment 12
wherein the lyoprotecting agent is selected among glucose,
galactose, fructose, sucrose, trehalose, maltose, lactose, amylose,
amylopectin, cyclodextrins, dextran, inuline, soluble starch,
hydroxyethyl starch (HES), erythritol, mannitol, sorbitol,
polyethyleneglycols and mixtures thereof. [0157] 14. Methods
according to embodiment 12 or 13 wherein the amount of
lyoprotecting agent from about 1% to about 25% by weight with
respect to the weight of water. [0158] 15. Methods according to any
of embodiments 1, 2 or 3 wherein the phospholipid is selected among
dilauroyl-phosphatidylcholine (DLPC),
dimyristoyl-phosphatidylcholine (DMPC),
dipalmitoyl-phosphatidylcholine (DPPC),
diarachidoyl-phosphatidylcholine (DAPC),
distearoyl-phosphatidylcholine (DSPC), dioleoyl-phosphatidylcholine
(DOPC), 1,2 Distearoyl-sn-glycero-3-Ethylphosphocholine
(Ethyl-DSPC), dipentadecanoyl-phosphatidylcholine (DPDPC),
1-myristoyl-2-palmitoyl-phosphatidylcholine (MPPC),
1-palmitoyl-2-myristoyl-phosphatidylcholine (PMPC),
1-palmitoyl-2-stearoyl-phosphatidylcholine (PSPC),
1-stearoyl-2-palmitoyl-phosphatidylcholine (SPPC),),
1-palmitoyl-2-oleylphosphatidylcholine (POPC),
1-oleyl-2-palmitoyl-phosphatidylcholine (OPPC),
dilauroyl-phosphatidylglycerol (DLPG) and its alkali metal salts,
diarachidoylphosphatidyl-glycerol (DAPG) and its alkali metal
salts, dimyristoylphosphatidylglycerol (DMPG) and its alkali metal
salts, dipalmitoylphosphatidylglycerol (DPPG) and its alkali metal
salts, distearoylphosphatidylglycerol (DSPG) and its alkali metal
salts, dioleoyl-phosphatidylglycerol (DOPG) and its alkali metal
salts, dimyristoyl phosphatidic acid (DMPA) and its alkali metal
salts, dipalmitoyl phosphatidic acid (DPPA) and its alkali metal
salts, distearoyl phosphatidic acid (DSPA),
diarachidoylphosphatidic acid (DAPA) and its alkali metal salts,
dimyristoyl-phosphatidylethanolamine (DMPE),
dipalmitoylphosphatidylethanolamine (DPPE), distearoyl
phosphatidyl-ethanolamine (DSPE), dioleylphosphatidyl-ethanolamine
(DOPE), diarachidoylphosphatidylethanolamine (DAPE),
dilinoleylphosphatidylethanolamine (DLPE), polyethyleneglycol
modified dimyristoyl-phosphatidylethanolamine (DMPE-PEG),
polyethyleneglycol modified dipalmitoylphosphatidylethanolamine
(DPPE-PEG), polyethyleneglycol modified distearoyl
phosphatidyl-ethanolamine (DSPE-PEG), polyethyleneglycol modified
dioleylphosphatidyl-ethanolamine (DOPE-PEG), polyethyleneglycol
modified diarachidoylphosphatidylethanolamine (DAPE-PEG),
polyethyleneglycol modified dilinoleylphosphatidylethanolamine
(DLPE-PEG), dimyristoyl phosphatidylserine (DMPS), diarachidoyl
phosphatidylserine (DAPS), dipalmitoyl phosphatidylserine (DPPS),
distearoylphosphatidylserine (DSPS), dioleoylphosphatidylserine
(DOPS), dipalmitoyl sphingomyelin (DPSP), and
distearoylsphingomyelin (DSSP) and mixtures thereof. [0159] 16.
Methods according to embodiment 1 wherein the emulsifying
composition of amphiphilic materials comprises a phospholipid or an
amphiphilic material bearing an overall net charge. [0160] 17.
Methods according to embodiments 1, 2, 3 or 13, wherein the amount
of phospholipid is from about 0.005% to about 1.0% by weight with
respect to the total weight of the emulsified mixture. [0161] 18.
Methods according to embodiment 17 wherein the amount of
phospholipid is from 0.01% to 1.0% by weight with respect to the
total weight of the emulsified mixture. [0162] 19. Methods
according to embodiment 1, 2 or 3 wherein the phospholipid includes
a targeting ligand or a protective reactive group capable of
reacting with a targeting ligand. [0163] 20. Methods according to
any of embodiments 1, 2, 3, 15 or 16 wherein the emulsion further
contains an amphiphilic material selected from lysolipids; fatty
acids and their respective salts with alkali or alkali metals;
lipids bearing polymers; lipids bearing sulfonated mono- di-,
oligo- or polysaccharides; lipids with ether or ester-linked fatty
acids; polymerized lipids; diacetyl phosphate; dicetyl phosphate;
stearylamine; ceramides; polyoxyethylene fatty acid esters;
polyoxyethylene fatty alcohols; polyoxyethylene fatty alcohol
ethers; polyoxyethylated sorbitan fatty acid esters; glycerol
polyethylene glycol ricinoleate; ethoxylated soybean sterols;
ethoxylated castor oil; ethylene oxide (EO) and propylene oxide
(PO) block copolymers; sterol esters of sugar acids; esters of
sugars with aliphatic acids; esters of glycerol with
(C.sub.12-C.sub.24) dicarboxylic fatty acids and their respective
salts with alkali or alkali-metal salts; saponins; long chain
(C.sub.12-C.sub.24) alcohols;
6-(5-cholesten-3.beta.-yloxy)-1-thio-.beta.-D-galactopyranoside;
digalactosyldiglyceride;
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxy-1-thio-.beta.-D-galact-
opyranoside;
6-(5-cholesten-3.beta.-yloxy)hexyl-6-amino-6-deoxyl-1-thio-.beta.-D-manno-
pyranoside;
12-(((7'-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic
acid;
N-[12-(((7'-diethylaminocoumarin-3-yl)carbonyl)-methylamino)octadec-
anoyl]-2-aminopalmitic acid;
N-succinyldioleylphosphatidylethanolamine;
1-hexadecyl-2-palmitoylglycerophosphoethanolamine;
palmitoylhomocysteine; alkylammonium salts comprising at least one
(C.sub.10-C.sub.20) alkyl chain; tertiary or quaternary ammonium
salts comprising at least one (C.sub.10-C.sub.20) acyl chain linked
to the N-atom through a (C.sub.3-C.sub.6) alkylene bridge: and
mixtures or combinations thereof. [0164] 21. Methods according to
embodiment 1 or 2 wherein the aqueous-organic emulsion of step a)
is subjected to a washing step before the lyophilizing step b).
[0165] 22. Methods according to embodiment 1 or 2 wherein the
aqueous-organic emulsion of step a) is subjected to a
microfiltration step before the lyophilizing step b). [0166] 23.
Methods according to embodiment 1 or 2 which further comprises
adding an aqueous suspension comprising a further amphiphilic
compound to the aqueous-organic emulsion obtained according to step
a), before the lyophilization step b), thus obtaining a second
aqueous-organic emulsion comprising said further amphiphilic
compound. [0167] 24. Methods according to embodiment 23 which
further comprises heating the mixture of said aqueous suspension
and of said aqueous-organic emulsion. [0168] 25. Methods according
to embodiment 23 wherein said mixture is heated at a temperature of
from about 40.degree. C. to about 80.degree. C. [0169] 26. Methods
according to embodiment 23 wherein said amphiphilic compound is a
PEG-modified phospholipid, a PEG-modified phospholipid bearing a
reactive moiety or a PEG-modified phospholipid bearing a targeting
ligand [0170] 27. Methods according to embodiments 1, 2 or 23 which
further comprises, before the lyophilization step b), subjecting
the aqueous-organic emulsion to a controlled heating. [0171] 28.
Methods according to embodiment 27, wherein said controlled heating
is effected at a temperature of from about 60.degree. C. to
125.degree. C. [0172] 29. Methods according to embodiment 28,
wherein said controlled heating is effected at a temperature of
from about 80.degree. C. to 120.degree. C. [0173] 30. Methods
according to embodiment 28, wherein said emulsion is contained in a
sealed vial. [0174] 31. Methods according to embodiment 2 or 3
wherein the biocompatible gas is selected among air; nitrogen;
oxygen; carbon dioxide; hydrogen; nitrous oxide; inert gases; a low
molecular weight hydrocarbon, including a (C.sub.1-C.sub.7) alkane,
a (C.sub.4-C.sub.7) cycloalkane, a (C.sub.2-C.sub.7) alkene and a
(C.sub.2-C.sub.7) alkyne; an ether; a ketone; an ester; a
halogenated (C.sub.1-C.sub.7) hydrocarbon, ketone or ether; or a
mixture of any of the foregoing. [0175] 32. Methods according to
embodiment 31 wherein the halogenated hydrocarbon gas is selected
among bromochlorodifluoro-methane, chlorodifluoromethane,
dichlorodifluoro-methane, bromotrifluoromethane,
chlorotrifluoromethane, chloropentafluoroethane,
dichlorotetrafluoroethane and mixtures thereof. [0176] 33. Methods
according to embodiment 31 wherein the halogenated hydrocarbon gas
is a perfluorinated hydrocarbon. [0177] 34. Methods according to
embodiment 33 wherein the perfluorinated hydrocarbon gas is
perfluoromethane, perfluoroethane, a perfluoropropane, a
perfluorobutane, a perfluoropentane, a perfluorohexane, a
perfluoroheptane; perfluoropropene, a perfluorobutene,
perfluorobutadiene, perfluorobut-2-yne, perfluorocyclobutane,
perfluoromethylcyclobutane, a perfluorodimethylcyclobutane, a
perfluorotrimethylcyclo-butane, perfluorocyclopentane,
perfluoromethylcyclopentane, a perfluorodimethylcyclo-pentane,
perfluorocyclohexane, perfluoromethylcyclohexane,
perfluoromethylcyclohexane and mixtures thereof. [0178] 35.
Injectable aqueous suspensions of microbubbles filled with a
biocompatible gas and comprising a stabilizing layer predominantly
comprising a phospholipid, wherein said microbubbles have a number
mean diameter (D.sub.N) of less than 1.70 .mu.m and a volume median
diameter (D.sub.V50) such that the D.sub.V50/D.sub.N ratio is of
about 2.00 or lower. [0179] 36. Aqueous suspensions according to
embodiment 35 wherein said microbubbles have a D.sub.N value of
1.60 .mu.m or lower, preferably of 1.50 .mu.m or lower, more
preferably of 1.30 .mu.m or lower. [0180] 37. Aqueous suspensions
according to embodiment 35 wherein said microbubbles have a
D.sub.V50/D.sub.N ratio of about 1.80 or lower, preferably of about
1.60 or lower, more preferably of about 1.50 or lower. [0181] 38.
Contrast agents for use in diagnostic imaging comprising an aqueous
suspension according to any of embodiments 35 to 37. [0182] 39.
Methods for diagnostic imaging comprising administering to a
subject a contrast-enhancing amount of an aqueous suspension
according to any of embodiments 35 to 37 and imaging at least a
part of said subject. [0183] 40. Methods according to embodiment 39
which include insonating said subject by means of an ultrasound
device generating an ultrasound wave with a predetermined transmit
frequency, from which a corresponding resonance size of
microbubbles is determined, and administering a contrast agent
comprising gas-filled microbubbles having a narrow size
distribution and a mean size close to half the resonance size.
[0184] The following non-limitative examples are given for better
illustrating the invention.
EXAMPLES
[0185] The following materials have been employed in the following
examples.
Phospholipids:
[0186] DPPS Dipalmitoylphosphatidylserine (Genzyme) IUPAC:
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine [0187] DPPG
Dipalmitoylphosphatidylglycerol sodium salt (Genzyme) IUPAC:
1,2-Dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] [0188] DSPA
Distearoyl phosphatidic acid sodium salt (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3-phosphate [0189] DSPG
Distearoylphosphatidylglycerol sodium salt (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3-phosphoserine) [0190] DSPC
Distearoylphosphatidylcholine (Genzyme) IUPAC:
1,2-Distearoyl-sn-glycero-3-phosphocholine [0191] DSEPC
Distearoylethylphosphatidylcholine (Avanti Polar Lipids) IUPAC:
1,2-Distearoyl-sn-glycero-3-Ethylphosphocholine [0192] DAPC
Diarachidoylphosphatidylcholine (Avanti polar Lipids) IUPAC:
1,2-Diarachidoyl-sn-glycero-3-phosphocholine [0193] DSTAP
1,2-Distearoyl-3-trimethylammonium-propane chloride (Avanti Polar
Lipids) [0194] DSPE-PEG2000 Distearoylphosphatidylethanolamine
modified with PEG2000, sodium salt (Nektar Therapeutics) [0195]
DSPE-PEG5000 Distearoylphosphatidylethanolamine modified with
PEG5000, sodium salt (Nektar Therapeutics) [0196] DSPE-PEG2000-
Distearoylphosphatidylethanolamine modified with PEG2000-maleimide
maleimid (Avanti Polar lipids) [0197] SATA
N-Succinimidyl-5-acetylthioacetate (Pierce) [0198] RGD-4C
H-Ala-Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys-Gly-NH.sub.2 (AnaSpec
Inc.)
Solvents:
Perfluoro-n-hexane (C.sub.6F.sub.14), by Fluka
[0199] perfluoromethylcyclohexane (CF.sub.3-cyclo-C.sub.6F.sub.11),
by Fluka perfluoro-n-heptane (C.sub.7F.sub.16), by Fluka
perfluoro-n-nonane (C.sub.9F.sub.20), by Aldrich perfluorodecalin,
by Aldrich
Cyclohexane, by Fluka
Cyclooctane, by Fluka
[0200] n-Decane, by Fluka n-Octane, by Fluka meta xylene, by Fluka
Diisopropyl cetone, by Fluka
CCl.sub.4, by Fluka
Lyoprotectants:
Mannose, by Fluka
Glucose, by Fluka
Sorbitol, by Fluka
Mannitol, by Fluka
Maltose, by Fluka
Dextran 6000, by Fluka
Dextran 15000, by Fluka
Dextran 40000, by Fluka
Inulin, by Fluka
Characterization of Microdroplets and Microbubbles.
[0201] The size distribution of the emulsions microdroplets has
been determined:
[0202] a) by means of a Coulter counter (Counter Mark II apparatus
fitted with a 30 .mu.m aperture with a measuring range of 0.7 to 20
.mu.m), when the emulsion has been submitted to a washing step; 10
.mu.l of emulsion were diluted in 100 ml of saline at room
temperature and allowed to equilibrate for 3 minutes prior to
measurement;
[0203] b) by means of a laser light scattering particle sizer
(Malvern Mastersizer, dilution 200.times., focal length 45 mm,
standard presentation), if the emulsion has not been subjected to a
washing step.
[0204] The size distributions, volume concentrations and number of
the microbubbles (after lyophilisation and reconstitution with an
aqueous phase) were determined by using a Coulter Counter Mark II
apparatus fitted with a 30 .mu.m aperture with a measuring range of
0.7 to 20 .mu.m. 50 .mu.l of microbubble samples were diluted in
100 ml of saline at room temperature and allowed to equilibrate for
3 minutes prior to measurement.
[0205] The amounts of phospholipids in the final preparations
(emulsion of microbubbles suspension) were determined by HPLC-MS
analysis, with the following set up: Agilent 1100 LC chromatograph,
MN CC 125/2 mm--5 C8 column from Maherey Nagel, Agilent MSD G1946D
detector.
Lyophilization
[0206] The lyophilization methodology and apparatus, where not
otherwise specified, were as follows. The emulsion (optionally
after the washing step, if present) is first frozen at -45.degree.
C. for 5 minutes and then freeze-dried (lyophilized) at room
temperature at a pressure of 0.2 mbar, by using a Christ-Alpha 2-4
freeze-drier.
Example 1
Preparations 1a-1n
[0207] 10 mg of DPPS are added to about 10 ml of an 10% (w/w)
mannitol aqueous solution; the suspension is heated at 65.degree.
C. for 15 minutes and then cooled at room temperature (22.degree.
C.). Perfluoroheptane (8% v/v) is added to this aqueous phase and
emulsified in a beaker of about 4 cm diameter by using a high speed
homogenizer (Polytron T3000, probe diameter of 3 cm) for 1 minute
at the speed indicated in table 1. The resulting median diameter in
volume (D.sub.V50) and a mean diameter in number (D.sub.N) of
microdroplets of the emulsion are shown in table 1. The emulsion is
then centrifuged (800-1200 rpm for 10 minutes, Sigma centrifuge
3K10) to eliminate the excess of the phospholipid and the separated
pellets (microdroplets) were recovered and re-suspended in the same
initial volume of a 10% mannitol aqueous solution.
[0208] The washed emulsion is then collected into a 100 ml balloon
for lyophilization, frozen and then freeze-dried according to the
above standard procedure. The lyophilized is then exposed to an
atmosphere containing 35% of perfluoro-n-butane and 65% of nitrogen
and then dispersed in a volume of water twice than the initial one
by gentle hand shaking. The microbubble suspension obtained after
reconstitution with distilled water is analyzed using a Coulter
counter. The concentration of microbubbles in the obtained
suspensions was of about 1.times.10.sup.9 particles per ml. The
respective microbubbles median diameter in volume (D.sub.V50), mean
diameter in volume (D.sub.V), mean diameter in number (D.sub.N) an
the amount of microbubbles with diameter larger than 3 .mu.m
(percentage over the total number of microbubbles) are given in
table 1. When more than one example has been performed at the same
agitation speed, the values indicated in table 1 are referred to
the mean calculated value of each parameter.
TABLE-US-00001 TABLE 1 EMULSION Gas-filled microbubbles Agitation
D.sub.N D.sub.V50 D.sub.N D.sub.V50/ >3 .mu.m <1.5 .mu.m Ex.
(rpm) D.sub.V50 (.mu.m) (.mu.m) (.mu.m) D.sub.V (.mu.m) (.mu.m)
D.sub.N part. % vol. % 1a 8000 4.58 1.77 2.92 3.33 1.51 1.93 5.44
10.9 1b 9000 4.66 1.94 3.19 3.45 1.53 2.08 6.61 10.0 1c 10000 3.04
1.74 2.16 2.53 1.33 1.62 1.88 22.6 1d 11000 3.05 1.80 2.17 3.33
1.32 1.65 1.55 23.7 1e 12000 2.84 1.69 1.86 2.17 1.24 1.50 0.93
33.1 1f 12500 2.79 1.68 1.75 2.05 1.22 1.44 0.65 35.7 1g 14000 2.20
1.52 1.39 2.45 1.08 1.29 0.23 58.0 1h 14500 2.00 1.38 1.19 1.39
1.01 1.19 0.06 73.3 1i 15000 1.88 1.39 1.22 2.20 1.01 1.21 0.06
70.3 1j 15500 2.19 1.48 1.24 1.46 1.02 1.22 0.11 68.7 1k 16000 1.83
1.32 1.27 3.08 0.99 1.28 0.10 65.2 1l 17000 1.40 1.12 0.91 1.03
0.87 1.05 0.01 95.7
Example 2
Preparations 2a-2j
[0209] The same procedure adopted for example 1 is followed, with
the only difference that the phospholipid is a mixture of DPPS (20%
w/w) and DSPC (80% w/w), the total amount of phospholipid remaining
unchanged. The results are summarized in table 2.
TABLE-US-00002 TABLE 2 EMULSION Gas-filled microbubbles Agitation
D.sub.N D.sub.V50 D.sub.N D.sub.V50/ >3 .mu.m <1.5 .mu.m Ex.
(rpm) D.sub.V50 (.mu.m) (.mu.m) (.mu.m) D.sub.V (.mu.m) (.mu.m)
D.sub.N part. % vol. % 2a 6000 8.75 3.07 7.55 9.05 2.27 3.33 21.81
1.2 2b 10000 3.54 1.90 3.00 3.71 1.47 2.04 5.05 11.7 2c 12000 3.04
1.83 2.45 3.73 1.32 1.85 2.15 19.8 2d 12500 2.85 1.76 2.21 3.24
1.27 1.74 1.57 24.4 2e 13000 2.98 1.83 2.25 3.04 1.28 1.76 1.76
23.5 2f 13500 2.91 2.05 1.88 2.46 1.20 1.57 0.87 33.8 2g 14000 2.45
1.67 1.82 2.66 1.16 1.57 0.57 36.5 2h 14500 2.18 1.55 1.58 3.04
1.09 1.44 0.38 46.5 2i 15000 1.94 1.42 1.34 1.96 1.04 1.28 0.31
61.5 2j 16000 1.81 1.38 1.35 2.30 1.03 1.31 0.14 59.0
Example 3
Preparation 3a-3p
[0210] The same procedure adopted for examples 2 is followed, with
the only difference that the DPPS/DSPC weight ratio is varied, as
reported in table 3. The results are summarized in table 3.
TABLE-US-00003 TABLE 3 EMULSION Gas-filled microbubbles DPPS/DSPC
Agitation D.sub.V50 D.sub.N D.sub.V50 D.sub.N >3 .mu.m <1.5
.mu.m Ex. ratio (rpm) (.mu.m) (.mu.m) (.mu.m) (.mu.m)
D.sub.V50/D.sub.N part. % vol. % 3a 80/20 12000 2.44 1.54 1.68 1.19
1.41 0.48 39.4 3b 75/25 12000 2.53 1.66 1.73 1.18 1.47 0.62 38.3 3c
60/40 11000 3.53 1.86 2.75 1.45 1.90 4.00 13.6 3d 60/40 12000 2.62
1.60 1.78 1.21 1.47 0.72 35.4 3e 60/40 14000 2.36 1.60 1.59 1.13
1.41 0.36 44.7 3f 50/50 12000 2.81 1.68 2.28 1.30 1.75 2.05 22.6 3g
40/60 11000 3.00 1.72 2.44 1.32 1.85 2.31 19.2 3h 40/60 12000 2.88
1.75 2.07 1.27 1.63 1.45 25.8 3i 40/60 13000 2.61 1.69 1.76 1.16
1.52 0.57 37.6 3j 40/60 14000 2.06 1.43 1.41 1.07 1.31 0.23 43.8 3k
40/60 14500 2.39 1.67 1.64 1.15 1.43 0.49 46.5 3l 30/70 11000 3.12
1.75 2.64 1.37 1.93 2.76 16.3 3m 30/70 12000 3.08 1.81 2.38 1.34
1.78 2.45 19.7 3n 25/75 11000 3.15 1.85 2.46 1.31 1.88 2.15 20.7 3o
10/90 11000 3.72 2.26 3.14 1.47 2.13 4.60 12.1 3p 5/95 11000 4.53
2.23 4.08 1.54 2.65 6.35 7.4
Example 4
[0211] The same procedure adopted for example 2 is followed, with
the only difference that mixtures of DSPA and DPPS with different
weight ratios were prepared. The results are summarized in table
4.
TABLE-US-00004 TABLE 4 Emulsion Gas-filled microbubbles DSPA/DPPS
Agitation D.sub.V50 D.sub.N D.sub.V50 D.sub.N >3 .mu.m <1.5
.mu.m Ex. Ratio (rpm) (.mu.m) (.mu.m) (.mu.m) (.mu.m)
D.sub.V50/D.sub.N part. % Vol. % 4a 25/75 12000 2.61 1.63 1.94 1.24
1.56 1.07 30.4 4b 50/50 11000 2.81 1.86 2.35 1.39 1.69 2.67 18.4 4c
50/50 12000 2.35 1.57 1.84 1.19 1.55 0.74 34.4 4d 75/25 12000 2.50
1.65 2.11 1.27 1.66 1.45 25.6
Example 5
Preparations 5a-5i
[0212] The same procedure adopted for example 1 is followed, with
the only difference that a 1/1 (w/w) phospholipid mixture of DPPG
and DSPC has been employed (total concentration 1.0 mg/ml) in
admixture with 10% w/w (with respect to the total weight of
phospholipid) of palmitic acid. The results are summarized in table
5.
TABLE-US-00005 TABLE 5 EMULSION Gas-filled microbubbles Agitation
D.sub.V50 D.sub.N D.sub.V50 D.sub.N D.sub.V50/ >3 .mu.m <1.5
.mu.m Ex (rpm) (.mu.m) (.mu.m) (.mu.m) (.mu.m) D.sub.N part. % Vol.
% 5a 6000 10.02 2.64 6.87 2.07 3.32 18.00 1.8 5b 8000 5.31 2.49
3.73 1.62 2.30 7.97 7.5 5c 9000 5.04 2.69 3.20 1.55 2.06 6.22 9.5
5d 10000 3.82 2.02 2.85 1.38 2.07 2.65 16.4 5e 10500 3.36 1.96 2.51
1.32 1.89 2.44 20.0 5f 11000 3.22 1.87 2.31 1.28 1.81 1.41 23.3 5g
12000 2.69 1.61 1.74 1.14 1.53 0.52 39.2 5h 13000 2.28 1.56 1.56
1.07 1.46 0.23 47.3 5i 14000 2.00 1.44 1.30 1.00 1.30 0.26 32.7
Example 6
[0213] The same procedure adopted for example 1 is followed, with
the only difference that DSEPC is used as phospholipid and
perfluorohexane is used as the organic solvent. The applied
rotation speed is of 11000 rpm. Dimensions, size distribution and
percentage of microbubbles with diameter larger than 3 .mu.m were
as follows.
TABLE-US-00006 D.sub.V50 (.mu.m) D.sub.N (.mu.m) D.sub.V50/D.sub.N
>3 .mu.m (%) 1.65 1.11 1.49 0.30
Example 7
Preparations 7a-7i
[0214] Distilled water (10 ml) containing DPPS (10 mg) as
phospholipid is heated at 70.degree. C. for 15 minutes and then
cooled at room temperature. 0.8 ml of an organic solvent as
specified in the following table 6 were emulsified in this aqueous
phase using a high speed homogenizer (Polytron T3000) at 10000 rpm
for 1 minute. The emulsion is added to 10 ml of a 15% dextran 15000
solution, frozen and lyophilized (0.2 mbar, 24 hours). After
lyophilisation, air is introduced in the lyophilizer. The
microbubble suspension obtained after reconstitution with distilled
water is analyzed using a Coulter counter. Table 6 summarizes the
results in terms of dimensions and size distribution of
microbubbles.
TABLE-US-00007 TABLE 6 Ex. Solvent D.sub.V50 (.mu.m) D.sub.N
(.mu.m) D.sub.V50/D.sub.N 7a C.sub.6F.sub.14 2.77 1.44 1.92 7b
CF.sub.3-cyclo-C.sub.6F.sub.11 2.24 1.30 1.72 7c C.sub.7F.sub.16
2.48 1.40 1.77 7d C.sub.9F.sub.20 2.46 1.36 1.81 7e
perfluorodecalin 3.76 1.52 2.47 7f Cyclohexane 2.61 1.41 1.85 7g
Cyclooctane 2.43 1.35 1.80 7h Decane 2.01 1.12 1.79 7i Octane 2.87
0.96 2.99 7j meta xylene 2.45 1.21 2.02 7k Diisopropyl cetone 1.83
1.05 1.74 7l CCl.sub.4 1.90 1.27 1.50
Example 8
[0215] The above example 7 is repeated with the same methodology,
by using perfluoro hexane as the organic solvent and different
lyoprotecting agents at different concentrations as outlined in
table 7. Table 7 summarizes the results in terms of dimensions and
size distribution of microbubbles.
TABLE-US-00008 TABLE 7 Lyoprotectant and D.sub.V50 Ex.
concentration (w/w) (.mu.m) D.sub.N (.mu.m) D.sub.V50/D.sub.N 8a
Mannose 5% 4.35 1.90 2.29 8b Glucose 5% 2.59 0.96 2.70 8c Sorbitol
5% 3.84 1.40 2.74 8d Mannitol 10% 2.22 1.22 1.82 8e Mannitol 5%
2.24 1.21 1.85 8f Mannitol 4% 2.54 1.45 1.75 8g Maltose 5% 3.42
0.99 3.45 8h Dextran 6000 7.5% 3.30 1.48 2.23 8j Dextran 15000 5%
2.55 1.31 1.95 8k Dextran 15000 7.5% 2.77 1.44 1.92 8i Dextran
40000 7.5% 2.54 1.32 2.29 8l Inulin 5% 3.58 1.43 2.70
Example 9
Preparations 9a-9e
[0216] Example 1 is repeated by emulsifying the mixture at a speed
of 10000 rpm. In addition, the same example is repeated by adding
different amounts of Pluronic F68 (a poloxamer corresponding to
Poloxamer 188) into the aqueous phase prior to emulsification, as
outlined in table 8. Table 8 shows the results of the comparative
experiment, in terms of size distribution and conversion yield of
the microbubbles. Conversion yield is given as the percentage
number of gas-filled microbubbles formed upon reconstitution of the
lyophilized matrix with respect to the number of microdroplets
measured in the emulsion.
TABLE-US-00009 TABLE 8 Conversion Example Pluronic* (mg/ml)
D.sub.V50 D.sub.N D.sub.V50/D.sub.N yield (%) 9a 0 2.42 1.38 1.75
28.0 9b 0.25 4.64 1.97 2.36 18.8 9c 0.5 13.85 1.38 10.04 7.3 9d 1.0
12.59 1.49 8.45 3.2 9e 2.0 15.80 1.23 12.85 0.5 *Concentration
referred to the volume of aqueous phase
[0217] The above results show that with a concentration of
poloxamer corresponding to half the concentration of the
phospholipid (i.e. about 33% of the total amount of surfactants in
the mixture), both conversion yields and size distribution of
microbubbles are negatively affected.
Example 10
Preparations 10a-10d
[0218] Example 9 is repeated, but instead of adding Pluronic F68 to
the aqueous phase, different amounts of cholesterol (from Fluka)
were added to the organic phase, prior to emulsification, as
outlined in table 9. Table 9 shows the results of the comparative
experiment, in terms of size distribution and conversion yield
(from the microdroplets of the emulsion) of the microbubbles.
TABLE-US-00010 TABLE 9 Cholesterol* Conversion Example (mg/ml)
D.sub.V50 D.sub.N D.sub.V50/D.sub.N yield (%) 10a 0 2.42 1.38 1.75
28.0 10b 0.10 3.79 1.31 2.89 17.8 10c 0.25 1.35 1.05 1.28 5.7 10d
0.50 14.02 1.70 8.25 0.8 *Concentration referred to the volume of
the aqueous phase
[0219] The above results show that with a concentration of 0.050%
(w/w) of cholesterol in the aqueous phase, both conversion yield
and size distribution of microbubbles are highly negatively
affected. A concentration of 0.025%, while it may provide
acceptable dimensions and size distribution of microbubbles, still
results in a rather low conversion yield.
Example 11
[0220] Distilled water (30 ml) containing 60 mg of DPPS and 3 g of
mannitol is heated to 70.degree. C. during 15 minutes then cooled
to room temperature.
[0221] Perfluoroheptane is emulsified in this aqueous phase using a
high speed homogenizer (Polytron.RTM., 12500 rpm, 1 minute).
[0222] The resulting emulsion, showing a median diameter in volume
(D.sub.V50) of 2.3 .mu.m and a mean diameter in number (D.sub.N) of
2.0 .mu.m, is washed once by centrifugation, resuspended in 30 ml
of a 10% solution of mannitol in distilled water and then divided
in three portions (3.times.10 ml).
[0223] The first portion (A) is used as such for the subsequent
lyophilization step. The second portion (B) is collected into a
syringe and hand-injected through a 5 .mu.m Nuclepore.RTM. filter
(47 mm--Polycarbonate). The third portion (C) is filtered through a
3 .mu.m Nuclepore.RTM. filter (47 mm--Polycarbonate) with the same
method.
[0224] The emulsions were frozen in 100 ml balloon (-45.degree. C.
for 5 minutes) then freeze dried (0.2 mBar, for 72 hours).
[0225] Atmospheric pressure is restored by introducing a 35/65
mixture of C.sub.4F.sub.10 and air. The respective lyophilisates
were dispersed in distilled water (10 ml). The so obtained
microbubbles suspensions are analysed using a Coulter counter and
the results are reported in the following table
TABLE-US-00011 D.sub.V50 D.sub.N D.sub.V50/D.sub.N <1.5 .mu.m
Part A 1.71 1.12 1.53 40.2 Part B 1.65 1.12 1.47 42.3 Part C 1.57
1.09 1.44 46.3
[0226] As shown by the above results, the additional filtration
step allows to further reduce the dimension of the microbubbles and
to reduce the respective size distribution.
Example 12
[0227] Example 1 has been repeated, by using 10 mg of a 7/3 (w/w)
mixture of DSPC/DSTAP, at an agitation speed of 11000 rpm.
Characterization of emulsion droplets and microbubbles were as
follows:
TABLE-US-00012 Emulsion droplets Gas-filled microbubbles D.sub.V50
D.sub.N D.sub.V50 D.sub.N >3 .mu.m <1.5 .mu.m 2.36 1.48 2.10
1.12 0.63 32.7
Example 13
[0228] The preparation of example 1 is repeated, by emulsifying the
mixture at a speed of 10000 rpm (example 13a).
[0229] The same preparation is repeated, by further adding about
0.9 mg of DSPE-PEG2000 (about 8.3% of the total amount of dispersed
phospholipids) to the initial aqueous suspension (example 13b).
[0230] No washing by centrifugation is performed on either the two
preparations. Table 10 shows the characterization of the two
preparations, both of the emulsion and of the microbubbles
suspension.
TABLE-US-00013 TABLE 10 Emulsion Microbubbles D.sub.V50 D.sub.N
D.sub.V50 D.sub.N D.sub.V50/ Conversion Example (.mu.m) (.mu.m)
(.mu.m) (.mu.m) D.sub.N Yield (%) 13a 3.19 1.66 2.66 1.33 2.00 29.5
13b 4.32 1.43 5.81 1.18 4.92 18.8
[0231] The above results show that with a concentration of DSPE-PEG
of less than 10% by weight (with respect to the total amount of
phospholipids), both conversion yields and size distribution of
microbubbles are negatively affected.
Example 14
[0232] The preparation of example 11 is repeated, by replacing DPPS
with the same amount of a 1:1 (w/w) mixture of DAPC/DPPS.
[0233] The resulting emulsion is divided in three portions of 10
ml, without washing it by centrifugation.
[0234] Aqueous suspensions of DSPE-PEG2000 and of DSPE-PEG5000 are
separately prepared by dispersing 25 mg of the respective DSPE-PEG
in 5 ml of a 10% mannitol solution under sonication (3 mm
sonication probe, Branson 250 sonifier, output 30%, for 5 min).
[0235] An aliquot of 2.5 ml of a 10% mannitol solution is then
added to a first portion of the emulsion (example 14a)
[0236] An aliquot of 2.5 ml of the prepared DSPE-PEG2000 suspension
is added to a second portion of the emulsion (example 14b)
[0237] An aliquot of 2.5 ml of the prepared DSPE-PEG5000 suspension
is added to a third portion of the emulsion (example 14c)
[0238] The three mixtures are heated at 60.degree. C. under
stirring for one hour. After cooling at room temperature, the size
of microdroplets are determined by means of Malvern Mastersizer.
Results are reported in table 11.
[0239] The emulsions are then freeze dried according to the
procedure of example 11. Atmospheric pressure is restored by
introducing a 35/65 mixture of C.sub.4F.sub.10 and air. The
respective lyophilisates were dispersed in distilled water (10 ml).
The so obtained microbubbles suspensions were analysed using a
Coulter counter (see table 11).
[0240] Microbubble suspensions are then washed twice with distilled
water by centrifugation (180 g/10 min) and lyophilized again
according to the above procedure. The amount of DSPE-PEG in the
dried composition is determined by means of HPLC-MS. Results are
given in the following table 11.
TABLE-US-00014 TABLE 11 Emulsion Microbubbles D.sub.V50 DSPE-PEG
Example (.mu.m) D.sub.N (.mu.m) D.sub.V50 (.mu.m) D.sub.N (.mu.m)
(% w/w) 14a 2.6 2.3 1.9 1.1 0.0 14b 2.5 2.3 3.4 1.3 35.5 14c 2.5
2.3 2.2 1.2 37.9
[0241] As inferable from the above results, the subsequent addition
of a DSPE-PEG suspension to the formed emulsion allows introducing
relatively high amounts of DSPE-PEG in the composition of the
stabilizing layer (in this case more than 30% of the total weight
of the phospholipids forming the stabilizing envelope), without
negatively affecting the final properties of the microbubbles.
[0242] Similar results can be obtained with other PEG-modified
phospholipids, in particular DSPE-PEG2000-Biotin or
DSPE-PEG2000-Maleimide, and with peptide bearing phospholipids, in
particular DSPE-PEG2000-maleimide-SATA-RGD4C. This latter peptide
bearing phospholipid can be prepared according to known techniques,
by reacting the RGD-4C peptide with SATA, deprotecting the thiol
group of SATA and reacting the deprotected RGD4C-SATA with
DSPE-PEG2000-maleimide. The preparation method described in
"Development of EGF-conjugated liposomes for targeted delivery of
boronated DNA-binding agents", by Bohl Kullberg et al.,
Bioconjugate chemistry 2002, 13, 737-743, (describing the insertion
of a EGF protein in a DSPE-PEG-maleimide molecule), can be
conveniently used.
Example 15
[0243] 20 mg of a 80/20 (w/w) DSPC/DSPA mixture are dissolved in
1.6 ml of cyclooctane at 80.degree. C. and the suspension is added
to 20 ml of distilled water containing 10% (w/w) of PEG4000
(Fluka). The mixture is emulsified by using a high speed
homogenizer (PolytronT3000) for 1 minute at 8000 rpm.
[0244] DSPE-PEG1000 (0.29 .mu.mole) and
DSPE-PEG2000-maleimide-SATA-RGD4C (0.29 .mu.mole) are dissolved in
EtOH/water 9/1 v/v; after evaporation, the obtained lipid film is
dried overnight at 25.degree. C. and 0.2 mBar and resuspended in
320 .mu.l of water at 60.degree. C.
[0245] The obtained solution is added to the emulsion previously
prepared and the resulting emulsion is heated under stirring at
80.degree. C. for 1 hour, followed by cooling at to room
temperature. The emulsion is then centrifuged (1300 g/10 min) to
eliminate the excess of phospholipid and the floating microdroplets
are recovered and resuspended in 40 ml of PEG4000 10% solution.
[0246] The resulting emulsion is sampled in DINER vials (1 ml per
vial) and the vials are frozen at -50.degree. C. for 2 hours
(Christ Epsilon lyophilizer), then freeze-dried at -25.degree. C.
and 0.2 mBar for 12 hours, with a final drying step at 30.degree.
C. and 0.05 mBar for 6 hours.
[0247] The lyophilized product is then exposed to an atmosphere
containing 35% of perfluoro-n-butane and 65% of nitrogen and the
vials are sealed.
[0248] The product is finally dispersed in a volume of water twice
that of the initial volume by gentle hand shaking.
[0249] Similar results are obtained when the DSPC/DSPA mixture is
replaced by a 80/20 DSPC/Stearic acid mixture.
Example 16
[0250] 10 mg of a 1:1 (w/w) DPPS/DSPC mixture are added to about 10
ml of a 10% (w/w) mannitol aqueous solution.
[0251] The mixture is heated at 70.degree. C. for 15 minutes and
then cooled at room temperature (22.degree. C.). Cyclooctane is
added at a flow rate 0.2 mL/min through an inlet of a micromixer
(standard slit Interdigidital micro Mixer, housing SS 316Ti with
nickel-on-copper inlay, 40 .mu.m.times.300 .mu.m, Institut fur
Microtechnik Mainz GmbH) to the aqueous phase circulating at 20
ml/min at room temperature, for a total amount of 7.4% (v/v) of
organic solvent. Upon completion of the addition of the organic
solvent, the emulsion is recirculated in the micromixer for
additional 20 minutes.
[0252] The emulsion is then divided into five aliquots of 2 ml each
and it is introduced into five vials DIN8R. Four vials are sealed
and heated for 30 minutes at temperatures of 60, 80, 100 and
120.degree. C., respectively, as indicated in table 12, while the
fifth is not heated.
[0253] The emulsions are then cooled to room temperature and the
content of the five vials is subjected to lyophilization according
to the following procedure. 1 ml of each emulsion is collected into
a DIN8R vial and frozen at -5.degree. C.; the temperature is
lowered to -45.degree. C. during 1 hour and the emulsion is then
freeze-dried at -25.degree. C. and 0.2 mbar during 12 hours
(Telstar Lyobeta35 lyophilizer), with a final drying step at
30.degree. C. and 0.2 mbar for 5 hours.
[0254] The lyophilized product is then exposed to an atmosphere
containing 35% of perfluoro-n-butane and 65% of nitrogen and then
dispersed in a volume of water twice than the initial one by gentle
hand shaking. Table 12 shows the result of the characterization of
the final suspension of microbubbles.
TABLE-US-00015 TABLE 12 Number of .mu.bubbles Heating D.sub.V50
D.sub.N D.sub.V50/D.sub.N per ml of emulsion no heating 10.45 1.63
6.41 5.34 .times. 10.sup.7 60.degree. C. 4.85 1.32 3.67 7.83
.times. 10.sup.7 80.degree. C. 5.34 1.29 4.14 8.51 .times. 10.sup.7
100.degree. C. 6.96 1.66 4.19 4.92 .times. 10.sup.8 120.degree. C.
3.05 1.50 2.03 8.69 .times. 10.sup.8
[0255] From the above results, it appears that by subjecting the
formed emulsion to a thermal treatment allows to narrow the size
distribution of the final microbubble suspension, while also
increasing the total number of microbubbles. In particular, by
increasing the heating temperature above 100.degree. C., it is
possible to obtain a relatively narrow size distribution of
microbubbles also in the absence of any washing step of the
emulsion, as well as an increase of the total number of
microbubbles in the suspension.
Example 17
[0256] 20 mg of 80/20 (w/w) DSPC/DSPA phospholipid mixture are
dissolved in 1.6 ml of cyclooctane at 80.degree. C. and the
suspension is added to 20 ml of distilled water containing 10%
(w/w) of PEG4000 (Fluka).
[0257] The mixture is emulsified by using a high speed homogenizer
(PolytronT3000) for 1 minute at 8000 rpm.
[0258] The resulting emulsion is heated under stirring at
80.degree. C. for 1 hour then cooled to room temperature.
Afterwards it is centrifuged (1300 rpm for 10 min) to eliminate the
excess of phospholipid and the floating microdroplets are recovered
and resuspended in 40 ml of PEG4000 10% solution.
[0259] The resulting emulsion is sampled in DINER vials (1 ml per
vial) and the vials are frozen at -50.degree. C. for 2 hours
(Christ Epsilon lyophilizer), then freeze-dried at -25.degree. C.
and 0.2 mBar for 12 hours, with a final drying step at 30.degree.
C. and 0.05 mBar for 6 hours.
[0260] The lyophilized product is then exposed to an atmosphere
containing 35% of perfluoro-n-butane and 65% of nitrogen and the
vials are sealed.
[0261] The product is finally dispersed in a volume of water twice
that of the initial volume by gentle hand shaking.
[0262] Table 13 shows the characterization of the emulsion and of
the microbubble suspension.
TABLE-US-00016 TABLE 13 Emulsion Microbubbles D.sub.V50 (.mu.m)
D.sub.N (.mu.m) D.sub.V50 (.mu.m) D.sub.N (.mu.m) D.sub.V50/D.sub.N
<1.5 .mu.m 2.89 1.66 2.51 1.27 1.98 22.1
Example 18
[0263] Distilled water (10 ml) containing 10 mg of DPPS and 1 g of
mannitol is heated to 70.degree. C. during 15 minutes then cooled
to room temperature. DPPE-MPB
(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl-
)butyramide]Na salt--Avanti Polar Lipids) is added (4.8% by
weight--0.5 mg). This phospholipid is dispersed in the aqueous
phase using a ultrasound bath (Branson 1210--3 minutes).
[0264] Perfluoroheptane (0.8 ml from Fluka) is emulsified in this
aqueous phase (cooled with a ice bath) using a high speed
homogenizer (Polytron.RTM. T3000, 15000 rpm, 1 minute).
[0265] The resulting emulsion showed a median diameter in volume
(D.sub.V50) of 2.3 .mu.m and a mean diameter in number (D.sub.N) of
2.1 .mu.m as determined with a Malvern Mastersizer.
[0266] The emulsion is washed twice by centrifugation then
resuspended in 9.5 ml of a 10% solution of mannitol in distilled
water. The washed emulsion is frozen (-45.degree. C., 5 minutes)
then freeze dried (under 0.2 mBar, for 24 hours).
[0267] Atmospheric pressure is restored by introducing a 35/65
mixture of C.sub.4F.sub.10 and air. The lyophilisate is dispersed
in distilled water (20 ml), microbubbles were washed once by
centrifugation and then redispersed in 4 ml of an EDTA containing
phosphate buffered saline (molar composition: 10 mM phosphate, 2.7
mM KCl, 137 mM NaCl, 10 mM EDTA), containing 3.4 mg of
thioacetylated avidin, 400 .mu.l of a hydroxylamine solution (13.92
mg in PBS 50 mM, pH: 7.5) were added to deprotect the thiol group
of the thioacetylated avidin.
[0268] The suspension is stirred by inversion on a disk rotator
(Fisher Scientific) for 2 hours. Then 150 .mu.l of NaOH 1N were
added.
[0269] The so obtained avidin-labelled microbubbles were washed
twice with PBS by centrifugation (10000 rpm, 10 minutes, Sigma
centrifuge 3K10). The microbubbles suspension obtained is analysed
using a Coulter counter showing a D.sub.V50 diameter of 1.6 .mu.m
and a D.sub.N of 1.2 .mu.m.
[0270] The efficacy of targeted microbubbles composition was tested
both in vitro and in vivo.
[0271] In Vitro Experiment:
[0272] To test the effective bonding of acetylated avidin to the
surface of the microbubbles, two sets of fibrin containing wells
were prepared. In the first set, only a fibrin surface is present.
In the second set, the fibrin is pre-treated with a biotin-labelled
antifibrin peptide (DX-278, disclosed in WO 02/055544). Microbubble
suspensions prepared as above were added to the wells
(5.times.10.sup.8 microbubbles/well). After 2 hours of incubation
(upside down) and several washings, the fibrin surfaces in the two
set of wells were observed by means of an optical microscope. While
essentially no microbubble could be observed in the wells without
the biotinylated antifibrin peptide, a massive coverage of
microbubbles was observed in the biotinylated antifibrin peptide
containing wells.
[0273] In Vivo Experiment:
[0274] A thrombus is formed in the abdominal aorta of two rabbits
by the FeCl.sub.3 method (Lockyer et al., 1999, Journal of
Cardiovascular Pharmacology, vol 33, pp 718-725).
[0275] Echo imaging is performed with an HDI 5000 ultrasound
machine (Philips), pulse inversion mode, L7-4 probe, MI: 0.07.
[0276] A biotinylated antibody (CD41 specific for the GPIIB/IIIA
receptor of activated platelets) is then injected intravenously to
the two rabbit.
[0277] After 30 minutes, the microbubble suspension comprising
avidin-labelled microbubbles is injected intravenously
(1.times.10.sup.9 microbubbles/ml) in the first rabbit. Fifteen
minutes after the injection, a strong opacification of the thrombus
is observed for the suspension. This opacification is still visible
after at least one hour from the injection.
[0278] The same amount of the microbubble suspension without
avidin-labelled microbubbles is injected intravenously in the
second rabbit. Only a light opacification of the thrombus is
observed.
* * * * *