U.S. patent application number 10/734730 was filed with the patent office on 2005-01-06 for diagnostic/therapeutic agents.
This patent application is currently assigned to Amersham Health AS. Invention is credited to Bryn, Klaus, Cuthbertson, Alan, Godal, Aslak, Gogstad, Geir, Hellebust, Halldis, Hoff, Lars, Hogset, Anders, Klaveness, Jo, Lovhaug, Dagfinn, Naevestad, Anne, Rongved, Pal, Solbakken, Magne, Tolleshaug, Helge.
Application Number | 20050002865 10/734730 |
Document ID | / |
Family ID | 46149778 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050002865 |
Kind Code |
A1 |
Klaveness, Jo ; et
al. |
January 6, 2005 |
Diagnostic/therapeutic agents
Abstract
Targetable diagnostic and/or therapeutically active agents, e.g.
ultrasound contrast agents, comprising a suspension in an aqueous
carrier liquid of a reporter comprising gas-containing or
gas-generating material, said agent being capable of forming at
least two types of binding pairs with a target.
Inventors: |
Klaveness, Jo; (Oslo,
NO) ; Rongved, Pal; (Oslo, NO) ; Hogset,
Anders; (Oslo, NO) ; Tolleshaug, Helge; (Oslo,
NO) ; Cuthbertson, Alan; (Oslo, NO) ; Godal,
Aslak; (Oslo, NO) ; Hoff, Lars; (Oslo, NO)
; Gogstad, Geir; (Oslo, NO) ; Bryn, Klaus;
(Oslo, NO) ; Naevestad, Anne; (Oslo, NO) ;
Lovhaug, Dagfinn; (Oslo, NO) ; Hellebust,
Halldis; (Oslo, NO) ; Solbakken, Magne; (Oslo,
NO) |
Correspondence
Address: |
Li CAI
Amersham Health, Inc.
101 Carnegie Center
Princeton
NJ
08540-6231
US
|
Assignee: |
Amersham Health AS
Oslo
NO
|
Family ID: |
46149778 |
Appl. No.: |
10/734730 |
Filed: |
December 15, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10734730 |
Dec 15, 2003 |
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09925715 |
Aug 10, 2001 |
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6680047 |
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09925715 |
Aug 10, 2001 |
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08959206 |
Oct 28, 1997 |
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6331289 |
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60049263 |
Jun 7, 1997 |
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60049264 |
Jun 6, 1997 |
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60049266 |
Jun 7, 1997 |
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Current U.S.
Class: |
424/9.52 |
Current CPC
Class: |
A61K 47/6925 20170801;
A61K 49/0047 20130101; A61K 51/065 20130101; A61K 51/1227 20130101;
A61K 47/62 20170801; Y10S 977/93 20130101; A61K 51/1255 20130101;
A61K 49/0058 20130101; Y10S 977/928 20130101; A61K 49/0056
20130101; A61K 49/0054 20130101; A61K 49/0043 20130101; A61K 47/542
20170801; A61K 49/223 20130101; A61K 49/0091 20130101; A61K 49/0002
20130101; Y10S 977/929 20130101 |
Class at
Publication: |
424/009.52 |
International
Class: |
A61K 049/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 1996 |
GB |
9622366.4 |
Oct 28, 1996 |
GB |
9622369.8 |
Feb 4, 1997 |
GB |
9702195.0 |
Apr 24, 1997 |
GB |
9708265.5 |
Jun 6, 1997 |
GB |
9711837.6 |
Jun 6, 1997 |
GB |
9711839.2 |
Claims
1. A targetable diagnostic and/or therapeutically active agent
comprising a suspension in an aqueous carrier liquid of a reporter
comprising gas-containing or gas-generating material, said agent
being capable of forming at least two types of binding pairs with a
target.
2. An agent as claimed in claim 1 wherein the gas comprises air,
nitrogen, oxygen, carbon dioxide, hydrogen, an inert gas, a sulphur
fluoride, selenium, hexafluoride, a low molecular weight
hydrocarbon, a ketone, an ester, a halogenated low molecular weight
hydrocarbon or a mixture of any of the foregoing.
3. An agent as claimed in claim 2 wherein the gas comprises a
perfluorinated ketone, perfluorinated ether or perfluorocarbon.
4. An agent as claimed in claim 2 wherein the gas comprises sulphur
hexafluoride or a perfluoropropane, perfluorobutane or
perfluoropentane.
5. An agent as claimed in claim 1 comprising gas microbubbles
stabilised by a coalescence resistant surface membrane, a
filmogenic protein, a polymer material, a non-polymeric and
non-polymerisable wall-forming material or a surfactant.
6. An agent as claimed in claim 5 wherein said surfactant comprises
at least one phospholipid.
7. An agent as claimed in claim 6 wherein at least 750 of the said
surfactant material comprises phospholipid molecules individually
bearing net overall charge.
8. An agent as claimed in claim 7 wherein at least 750 of the
film-forming surfactant material comprises one or more
phospholipids selected from phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and cardiolipins.
9. An agent as claimed in claim 8 wherein at least 800 of said
phospholipids comprise phosphatidylserines.
10. An agent as claimed in claim 1 wherein said gas-containing or
gas-generating material is conjugated to at least two vectors or to
one vector capable of binding to at least two binding sites.
11. An agent as claimed in claim 1 wherein said gas-containing or
gas-generating material is conjugated to one or more targeting
vectors having specificity for one or more cellular surface
receptors and further comprises moieties capable of binding to a
receptor system so as to induce a therapeutic response.
12. An agent as claimed in claim 1 wherein the vector or vectors
are selected from antibodies; cell adhesion molecules; cell
adhesion molecule receptors; cytokines; growth factors; peptide
hormones and pieces thereof; non-bioactive binders of receptors for
cell adhesion molecules, cytokines, growth factors and peptide
hormones; oligonucleotides and modified oligonucleotides;
DNA-binding drugs; protease substrates/inhibitors; molecules
generated from combinatorial libraries; small bioactive molecules;
and proteins and peptides which bind to cell-surface
proteoglycans.
13. An agent as claimed in claim 1 wherein the vector or vectors
have affinity for targets at a level such that the agent interacts
with but does not fixedly bind to said targets.
14. An agent as claimed in claim 13 wherein the vector or vectors
are selected from ligands for cell adhesion proteins and cell
adhesion proteins which have corresponding ligands on endothelial
cell surfaces.
15. An agent as claimed in claim 1 wherein the vector or vectors
are sited such that they are not readily exposed to the target.
16. An agent as claimed in claim 1 wherein the vector or vectors
are coupled or linked to the reporter by means of avidin-biotin
and/or streptavidin-biotin interactions.
17. An agent as claimed in claim 1 wherein the vector or vectors
may be covalently or non-covalently coupled or linked to the
reporter.
18. An agent as claimed in claim 1 wherein the vector is coupled or
linked to the reporter by means of electrostatic charge
interaction.
19. An agent as claimed in claim 1 which further contains moieties
which are radioactive or are effective as X-ray contrast agents,
light imaging probes or spin labels.
20. An agent as claimed in claim 1 further comprising a therapeutic
compound.
21. An agent as claimed in claim 20 wherein said therapeutic
compound is an antineoplastic agent, blood product, biological
response modifier, antifungal agent, hormone or hormone analogue,
vitamin, enzyme, antiallergic agent, tissue factor inhibitor,
platelet inhibitor, coagulation protein target inhibitor, fibrin
formation inhibitor, fibrinolysis promoter, antiangiogenic,
circulatory drug, metabolic potentiator, antitubercular, antiviral,
vasodilator, antibiotic, antiinflammatory, antiprotozoan,
antirheumatic, narcotic, opiate, cardiac glycoside, neuromuscular
blocker, sedative, local anaesthetic, general anaesthetic or
genetic material.
22. An agent as claimed in claim 20 wherein said therapeutic
compound is covalently coupled or linked to the reporter through
disulphide groups.
23. An agent as claimed in claim 20 wherein a lipophilic or
lipophilically-derivatised therapeutic compound is linked to the
reporter through hydrophobic interactions.
24. A combined formulation comprising: i) a first administrable
composition comprising a pre-targeting vector having affinity for a
selected target; and ii) a second administrable composition
comprising an agent as claimed in any of the preceding claims, said
agent comprising a vector having affinity for said pre-targeting
vector.
25. A combined formulation as claimed in claim 24 wherein said
pre-targeting vector comprises a monoclonal antibody.
26. A combined formulation comprising: i) a first administrable
composition comprising an agent an agent as claimed in claim 1, and
ii) a second administrable composition comprising a substance
capable of displacing or releasing said agent from its target.
27. A combined formulation comprising: i) a first administrable
composition comprising an agent as claimed in claim 22, and ii) a
second administrable composition comprising a reducing agent
capable of reductively cleaving the disulphide groups coupling or
linking the therapeutic compound and reporter in the agent of said
first administrable composition.
28. A process for the preparation of a targetable diagnostic and/or
therapeutically active agent as defined in claim 1 which comprises
coupling or linking at least one vector to a reporter comprising
gas-containing or gas-generating material: such that said agent is
capable of forming at least two types of binding pairs with a
target.
29. A process as claimed in claim 28 wherein a therapeutic compound
is also combined with the reporter.
30. (canceled).
31. A method of generating enhanced images of a human or non-human
animal body which comprises administering to said body an agent as
claimed in claim 1 and generating an ultrasound, magnetic
resonance, X-ray, radiographic or light image of at least a part of
said body.
32. A method as claimed in claim 31 which comprises the steps: i)
administering to said body a pre-targeting vector having affinity
for a selected target; and thereafter ii) administering an agent,
said agent comprising a vector having affinity for said
pre-targeting vector.
33. A method as claimed in claim 32 wherein said pre-targeting
vector comprises a monoclonal antibody.
34. A method as claimed in claim 31 which comprises the steps: i)
administering to said body an agent; and thereafter ii)
administering a substance capable of displacing or releasing said
agent from its target.
35. A method as claimed in claim 31 wherein said agent further
comprises a therapeutic compound.
36. A method as claimed in claim 35 wherein said therapeutic
compound is covalently coupled or linked to the reporter through
disulphide groups, and a composition comprising a reducing agent
capable of reductively cleaving said disulphide groups is
subsequently administered.
37. A method for in vitro investigation of targeting by an agent as
defined in claim 1 wherein cells expressing a target are fixedly
positioned in a flow chamber, a suspension of said agent in a
carrier liquid is passed through said chamber, and binding of said
agent to said cells is examined.
38. A method as claimed in claim 37 wherein the flow rate of
carrier liquid is controlled to simulate shear rates encountered in
vivo.
Description
[0001] This invention relates to diagnostic and/or therapeutically
active agents, more particularly to diagnostic and/or
therapeutically active agents incorporating moieties having
affinity for sites and/or structures within the body so that
diagnostic imaging and/or therapy of particular locations within
the body may be enhanced. Of particular interest are diagnostic
agents for use in ultrasound imaging, which are hereinafter
referred to as targeted ultrasound contrast agents.
[0002] It is well known that ultrasonic imaging comprises a
potentially valuable diagnostic tool, for example in studies of the
vascular system, particularly in cardiography, and of tissue
microvasculature. A variety of contrast agents has been proposed to
enhance the acoustic images so obtained, including suspensions of
solid particles, emulsified liquid droplets, gas bubbles and
encapsulated gases or liquids. It is generally accepted that low
density contrast agents which are easily compressible are
particularly efficient in terms of the acoustic backscatter they
generate, and considerable interest has therefore been shown in the
preparation of gas-containing and gas-generating systems.
[0003] Gas-containing contrast media are also known to be effective
in magnetic resonance (MR) imaging, e.g. as susceptibility contrast
agents which will act to reduce MR signal intensity.
Oxygen-containing contrast media also represent potentially useful
paramagnetic MR contrast agents.
[0004] Furthermore, in the field of x-ray imaging it has been
observed that gases such as carbon dioxide may be used as negative
oral contrast agents or intravascular contrast agents.
[0005] The use of radioactive gases, e.g. radioactive isotopes of
inert gases such as xenon, has also been proposed in scintigraphy,
for example for blood pool imaging.
[0006] Targeted ultrasound contrast agents may be regarded as
comprising (i) a reporter moiety capable of interacting with
ultrasound irradiation to generate a detectable signal; (ii) one or
more vectors having affinity for particular target sites and/or
structures within the body, e.g. for specific cells or areas of
pathology; and (iii) one or more linkers connecting said reporter
and vector(s), in the event that these are not directly joined.
[0007] The molecules and/or structure to which the contrast agent
is intended to bind will hereinafter be referred to as the target.
In order to obtain specific imaging of a selected region/structure
in the body the target must be present and available in this
region/structure. Ideally it will be expressed only in the region
of interest, but usually will also be present at other locations in
the body, creating possible background problems. The target may
either be a defined molecular species (i.e. a target molecule) or
an unknown molecule or more complex structure (i.e. a target
structure) which is present in the area to be imaged, and is able
to bind specifically or selectively to a given vector molecule.
[0008] The vector is attached to the reporter moiety in order to
bind these moieties to the region/structure to be imaged. The
vector may bind specifically to a chosen target, or it may bind
only selectively, having affinty also for a limited number of other
molecules/structures, again creating possible background
problems.
[0009] There is a limited body of prior art relating to targeted
ultrasound contrast agents. Thus, for example, U.S. Pat No.
5,531,980 is directed to systems in which the reporter comprises an
aqueous suspension of air or gas microbubbles stabilised by one or
more film-forming surfactants present at least partially in
lamellar or laminar form, said surfactant(s) being bound to one or
more vectors comprising "bioactive species designed for specific
targeting purposes". It is stated that the microbubbles are not
directly encapsulated by surfactant material but rather that this
is incorporated in liquid-filled liposomes which stabilise the
microbubbles. It will be appreciated that lamellar or laminar
surfactant material such as phospholipids present in such liposomes
will inevitably be present in the form of one or more lipid
bilayers with the lipophilic tails "back-to-back" and the
hydrophilic heads both inside and outside (see e.g. Schneider, M.
on "Liposomes as drug carriers: 10 years of research" in Drug
targeting, Nyon, Switzerland, 3-5 Oct. 1984, Buri, P. and Gumma, A.
(Ed), Elsevier, Amsterdam 1984).
[0010] EP-A-0727225 describes targeted ultrasound contrast agents
in which the reporter comprises a chemical having a sufficient
vapour pressure such that a proportion of it is a gas at the body
temperature of the subject. This chemical is associated with a
surfactant or albumin carrier which includes a protein-, peptide-
or carbohydrate-based cell adhesion molecule ligand as vector. The
receptor moieties in such contrast agents correspond to the phase
shift colloid systems described in WO-A-9416739; it is now
recognised that administration of such phase shift colloids may
lead to generation of microbubbles which grow uncontrollably,
possibly to the extent where they cause potentially dangerous
embolisation of, for example, the myocardial vasculature and brain
(see e.g. Schwarz, Advances in Echo-Contrast [1994(3)], pp
48-49).
[0011] WO-A-9320802 proposes that tissue-specific ultrasonic image
enhancement may be achieved using acoustically reflective
oligolamellar liposomes conjugated to tissue-specific ligands such
as antibodies, peptides, lectins etc. The liposomes are
deliberately chosen to be devoid of gas and so will not have the
advantageous echogenic properties of gas-based ultrasound contrast
agents. Further references to this technology, e.g. in targeting to
fibrin, thrombi and atherosclerotic areas are found in publications
by Alkanonyuksel, H. et al. in J. Pharm. Sci. (1996) 85(5),
486-490; J. Am. Coll. Cardiol. (1996) 27(2) Suppl A, 298A; and
Circulation, 68 Sci. Sessions, Anaheim 13-16 Nov. 1995.
[0012] There is also a number of publications concerning ultrasound
contrast agents which refer in passing to possible use of
monoclonal antibodies as vectors without giving significant
practical detail and/or to reporters comprising materials which may
be taken up by the reticuloendothelial system and thereby permit
image enhancement of organs such as the liver--see, for example
WO-A-9300933, WO-A-9401140, WO-A-9408627, WO-A-9428874, U.S. Pat
No. 5,088,499, U.S. Pat No. 5,348,016 and U.S. Pat No. 5,469,854.
In general these prior art targeted contrast agents are intended to
enhance contrast at specific sites in the body, for example tumour
cells, by using one vector to bind strongly to one target, in order
to achieve concentration at the target cells. In contrast to this
principle of using one vector to bind with high affinity to one
target, the present invention is based in part on the finding that
diagnostic and/or therapeutically active agents with more
favourable properties may be obtained by use of multiple kinds of
vector-target interactions (e.g. involving agents associated with a
plurality of different vectors and/or with one or more vectors
having affinity for different targets on the same or different cell
types). In this way, binding of gas-containing and gas-generating
diagnostic and/or therapeutic agents may, for example, be obtained
by forming multiple binding pairs between one vector with
specificity for more than one receptor or between more than one
vector with affinity for one or more types of target, with either
low or high affinities. Such multiple binding of the
vector-conjugated agent to one or more target molecules/structures
may result in advantageous targeting properties, for example by
enhancing target specificity and/or by distinguishing interactions
at a desired target area from background interactions with lower
levels of molecules/structures similar to target expressed
elsewhere in the body.
[0013] It is well known to use one vector binding with high
affinity to one target. The present invention, however, is based on
the finding that the desired binding of gas-containing and
gas-generating diagnostic and/or therapeutic agents may be obtained
by forming multiple binding pairs with low affinity between one
type of vector and one type of target, or by forming multiple
binding pairs between one or more types of vectors and one or more
types of target, with either low or high affinities. Thus multiple
binding of the vector conjugated agent to one or more target
molecules/structures may have advantageous targeting properties,
for example in enhancing target specificity and/or in
distinguishing interactions at a desired target area from
background interactions with lower levels of molecules/structures
similar to target expressed elsewhere in the body.
[0014] Thus according to one aspect of the present invention there
is provided a targetable diagnostic and/or therapeutically active
agent, e.g. an ultrasound contrast agent, comprising a suspension
in an aqueous carrier liquid, e.g. an injectable carrier liquid, of
a reporter comprising gas-containing or gas-generating material
characterised in that said agent is capable of forming at least two
types of binding pairs, e.g. being conjugated to at least two
vectors or to one vector capable of binding to at least two binding
sites.
[0015] One advantageous embodiment of the invention is based on the
additional finding that limited adhesion to targets is a highly
useful property of diagnostic and/or therapeutically active agents,
which property may be achieved using vectors giving temporary
retention rather than fixed adhesion to a target. Thus such agents,
rather than being fixedly retained at specific sites, may for
example effectively exhibit a form of retarded flow along the
vascular endothelium by virtue of their transient interactions with
endothelial cells. Such agents may thus become concentrated on the
walls of blood vessels, in the case of ultrasound contrast agents
providing enhanced echogenicity thereof relative to the bulk of the
bloodstream, which is devoid of anatomical features. They therefore
may permit enhanced imaging of the capillary system, including the
microvasculature, and so may facilitate distinction between normal
and inadequately perfused tissue, e.g. in the heart, and may also
be useful in visualising structures such as Kupffer cells, thrombi
and atherosclerotic lesions or for visualising neo-vascularized and
inflamed tissue areas. The present invention is well suited to
imaging changes occurring in normal blood vessels which are
situated in areas of tissue necrosis.
[0016] It will be appreciated that binding affinities are dependent
on numbers of interactions as well as their strength. The density
of vector molecules at the surface of the reporter units may
therefore be selected so as appropriately to adjust the degree of
interactions between particular agents and targets.
[0017] The term multiple-specificity is also used to describe an
injectable carrier liquid, of gas-containing or gas-generating
material composed of one or more vectors with a specificity for one
or more cellular surface receptors while at the same time
comprising a second element with specificity for a substrate or
receptor system binding to which induces a therapeutic response.
Thus included within the scope of the present invention are
multiple-specific imaging agents comprising a targeting vector,
such as the anti-fibrin antibody described by Lanza et al.
(Circulation, (1996) 94 (12),pp 3334), annexin V atherosclerotic
plaque binding peptides such as YRALVDTLK, or any other vector
known to associate with fibrin clots, in combination with a drug or
enzyme with fibrinolytic activity such as streptokinase,
plasminogen activator (tPA), urokinase (uPA) or prourokinase
(scuPA) resulting in a localised therapeutic antithrombotic effect.
This invention is also extended to include vectors with increased
specificity for tumour cells in combination with vectors or drug
molecules functioning as chemotherapeutic agents capable of
inhibiting tumour growth.
[0018] It is well known that many, if not all, target molecules are
not expressed exclusively at target sites; a common situation is
that such molecules are over-expressed by target cells or at a
target structure but are also expressed at lower levels elsewhere
in the body. The use of reporters carrying a multiplicity of
vectors with relatively low affinity for the target may be
advantageous in this situation, since the reporter will then tend
to concentrate in regions of high target density which permit
multiple (and therefore strong) binding to the reporter (e.g. a
gas-containing agent incorporating the vectors folic acid and
glutathione for multiple-specific binding to folic acid receptors
and glutathione-S-trasferase receptors respectively which are
over-expressed as tumour cells). Areas of low target density, on
the other hand, will not provide sufficient interaction with such
low affinity vectors to bind the target. In such embodiments of the
invention, low affinity vectors may be regarded as having an
association constant Kafor interaction with a target molecule or
structure of less than 10.sup.8M.sup.-1, e.g. less than 10.sup.7
M.sup.-1, preferably less than 10.sup.6M.sup.-1. A further
embodiment of this invention is thus based on the finding that the
desired binding of gas-containing and gas-generating diagnostic
and/or therapeutic agents may be obtained by forming binding pairs
with low affinity between more than one type of vector and one or
more type of target. Multiple vectors may therfore be used to
increase specificity, so that the reporter will bind only to target
cells or structures expressing a particular combination of target
molecules.
[0019] It may also be useful to select a plurality of vectors which
bind to different parts, e.g. epitopes, of a target structure in
order to give increased binding strength. This may be particularly
advantageous when the target density is low.
[0020] Products comprising two or more vectors with different
specificities, i.e. which bind to different target molecules on
different cells, may advantageously be used as "general purpose"
agents for detection of a range of diseases, e.g. different forms
of cancer. Thus, for example, the use of such agents may enable
detection of metastases, which are often heterogeneous with respect
to expression of target molecules (i.e. antigens).
[0021] Within the context of the present invention, the reporter
unit will usually remain attached to the vectors. In another type
of targeting procedure, sometimes called pre-targeting, the vector
(often, a monoclonal antibody) is administered alone; subsequently,
the reporter is administered, coupled to a moiety which is capable
of specifically binding the vector molecule (when the vector is an
antibody, the reporter may be coupled to an immunoglobulin-binding
molecule, such as protein A or an anti-immunoglobulin antibody). An
advantage of this protocol is that time may be allowed for
elimination of the vector molecules that do not bind their targets,
substantially reducing the background problems that are connected
with the presence of an excess of reporter-vector conjugate. Within
the context of the present invention, pre-targeting with one
specific vector might be envisaged, followed by reporter units that
are coupled to another vector and a moiety which binds the first
vector.
[0022] Within the context of the present invention, in some cases
and in particular for the assessment of blood perfusion rates in
defined areas, for example in myocardium, it is of interest to
measure the rate at which ultrasound contrast agents bound to the
target are displaced or released from the target. This can be
achieved in a controlled fashion by subsequent administration of a
vector or other agent able to displace or release the contrast
agent from the target.
[0023] Vectors useful in accordance with the invention include
ligands for cell adhesion proteins, as well as cell adhesion
proteins themselves where these have corresponding ligands on
endothelial cell surfaces. Examples of cell adhesion proteins
include integrins, most of which bind the Arg-Gly-Asp (RGD) amino
acid sequence. If desired, the vector may be targeted to specific
cell adhesion proteins expressed mainly on activated endothelial
cells such as are found at or close to sites of inflammation or
other pathological responses. Other vectors which may be used
include proteins and peptides that bind to cell-surface
proteoglycans, which are complexes of proteins and sulphated
polysaccarides found on most cells, including endothelial cells.
Such proteoglycans contribute to the negative surface charge of all
nucleated cells from vertebrate animals; this charge may also be
exploited in accordance with the invention by using positively
charged vectors, e.g. comprising cationic lipids, which will
interact electrostatically with the endothelial surface.
[0024] A further aspect of the present invention is for example
where a vector or vectors is attached to the reporter or included
non-covalently into the reporter in a manner where the said vector
or vectors is not readily exposed to the targets or receptors.
Increased tissue specificity may therefore be achieved by applying
an additional process to expose the vectors, e.g. the agent is
exposed after administration to external ultrasound to change the
diffusibility of the moieties containing the vectors.
[0025] The reporter may be in any convenient form, for example
being any appropriate gas-containing or gas-generating ultrasound
contrast agent formulation. Representative examples of such
formulations include microbubbles of gas stabilised (e.g. at least
partially encapsulated) by a coalescence-resistant surface membrane
(for example gelatin, e.g. as described in WO-A-8002365), a
filmogenic protein (for example an albumin such as human serum
albumin, e.g. as described in U.S. Pat No. 4,718,433, U.S. Pat No.
4,774,958, U.S. Pat No. 4,844,882, EP-A-0359246, WO-A-9112823,
WO-A-9205806, WO-A-9217213, WO-A-9406477 or WO-A-9501187), a
polymer material (for example a synthetic biodegradable polymer as
described in EP-A-0398935, an elastic interfacial synthetic polymer
membrane as described in EP-A-0458745, a microparticulate
biodegradable polyaldehyde as described in EP-A-0441468, a
microparticulate N-dicarboxylic acid derivative of a polyamino
acid--polycyclic imide as described in EP-A-0458079, or a
biodegradable polymer as described in WO-A-9317718 or
WO-A-9607434), a non-polymeric and non-polymerisable wall-forming
material (for example as described in WO-A-9521631), or a
surfactant (for example a polyoxyethylene-polyoxypropylene block
copolymer surfactant such as a Pluronic, a polymer surfactant as
described in WO-A-9506518, or a film-forming surfactant such as a
phospholipid, e.g. as described in WO-A-9211873, WO-A-9217212,
WO-A-9222247, WO-A-9428780 or WO-A-9503835).
[0026] Other useful gas-containing contrast agent formulations
include gas-containing solid systems, for example microparticles
(especially aggregates of microparticles) having gas contained
therewithin or otherwise associated therewith (for example being
adsorbed on the surface thereof and/or contained within voids,
cavities or pores therein, e.g. as described in EP-A-0122624,
EP-A-0123235, EP-A-0365467, WO-A-9221382, WO-A-9300930,
WO-A-9313802, WO-A-9313808 or WO-A-9313809). It will be appreciated
that the echogenicity of such microparticulate contrast agents may
derive directly from the contained/associated gas and/or from gas
(e.g. microbubbles) liberated from the solid material (e.g. upon
dissolution of the microparticulate structure).
[0027] The disclosures of all of the above-described documents
relating to gas-containing contrast agent formulations are
incorporated herein by reference.
[0028] Gas microbubbles and other gas-containing materials such as
microparticles preferably have an initial average size not
exceeding 10 .mu.m (e.g. of 7 .mu.m or less) in order to permit
their free passage through the pulmonary system following
administration, e.g. by intravenous injection.
[0029] Where phospholipid-containing compositions are employed in
accordance with the invention, e.g. in the form of
phospholipid-stabilised gas microbubbles, representative examples
of useful phospholipids include lecithins (i.e.
phosphatidylcholines), for example natural lecithins such as egg
yolk lecithin or soya bean lecithin and synthetic or semisynthetic
lecithins such as dimyristoylphosphatidylc- holine,
dipalmitoylphosphatidylcholine or distearoylphosphatidylcholine;
phosphatidic acids; phosphatidylethanolamines; phosphatidylserines;
phosphatidylglycerols; phosphatidylinositols; cardiolipins;
sphingomyelins; fluorinated analogues of any of the foregoing;
mixtures of any of the foregoing and mixtures with other lipids
such as cholesterol. The use of phospholipids predominantly (e.g.
at least 75%) comprising molecules individually bearing net overall
charge, e.g. negative charge, for example as in naturally occurring
(e.g. soya bean or egg yolk derived), semisynthetic (e.g. partially
or fully hydrogenated) and synthetic phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and/or cardiolipins, may be particularly advantageous.
[0030] Other exemplary lipids which may be used to prepare
gas-containing contrast agents include fatty acids, stearic acid,
palmitic acid, 2-n-hexadecylstearic acid, oleic acid and other acid
containing lipid structures. These lipid structures are considered
particularly interesting when coupled by amide bond formation to
amino acids containing one or more amino groups. The resulting
lipid modified amino acids (e.g. dipalmitoyllysine,
distearoyl-2,3-diaminopropionic acid) are considered useful
precursors for the attachment of functionalised spacer elements
featuring coupling sites for conjugation of one or more vector
molecules.
[0031] A further extension of this invention relates to the
synthesis of lipopeptide structures comprising a lipid reporter
attached to a linker portion (e.g. PEG, polyamino acid, alkylhalide
etc) the said linker being suitably functionalised for coupling to
one or more vector molecules. A particular preference is the
inclusion of a positively charged linker element (e.g. two or more
lysine residues) for anchoring of the reporter element in the
microbubble through electrostatic interaction with the negatively
charged membrane. Multiple-specific targeting is achievable by
mixing and `doping` of phospholipid gas-containing structures with
one or more targeted lipopeptide sequences. Multiple-specificity
can also be achieved by assembling more than one vector on a
branched lysine core structure such as those described by Tam et.
al. (Proc. Natl. Acad. Sci. USA, 1989, 86, 9084) or by
incorporating multiple vectors in a linear sequence.
Multiple-specificity can also be achieved using lipopeptides or
phospholipids comprising combinatorial libraries synthesised by
chemical synthesis as described by Lowe (Combinatorial Chemistry,
Chemical Society Reviews, 1995, 309-317).
[0032] Also within the scope of this invention are functionalised
microbubbles carrying one or more reactive groups for non-specific
reaction with receptor molecules located on cell surfaces.
Microbubbles comprising a thiol moiety,for example, can bind to
cell surface receptors via disulphide exchange reactions. The
reversible nature of this covalent bond means that bubble flow can
be controlled by altering the redox environment. Similarly
`activated` microbubbles of membranes comprising active esters such
as N-hydroxysuccinimide esters can be used to modify amino groups
found on a multiplicity of cell surface molecules.
[0033] Representative examples of gas-containing microparticulate
materials which may be useful in accordance with the invention
include carbohydrates (for example hexoses such as glucose,
fructose or galactose; disaccharides such as sucrose, lactose or
maltose; pentoses such as arabinose, xylose or ribose; .alpha.-,
.beta.- and .gamma.-cyclodextrins; polysaccharides such as starch,
hydroxyethyl starch, amylose, amylopectin, glycogen, inulin,
pulullan, dextran, carboxymethyl dextran, deXtran phosphate,
ketodextran, aminoethyldextran, alginates, chitin, chitosan,
hyaluronic acid or heparin; and sugar alcohols, including alditols
such as mannitol or sorbitol), inorganic salts (e.g. sodium
chloride), organic salts (e.g. sodium citrate, sodium acetate or
sodium tartrate), X-ray contrast agents (e.g. any of the
commercially available carboxylic acid and non-ionic amide contrast
agents typically containing at least one 2,4,6-triiodophenyl group
having substituents such as carboxyl, carbamoyl, N-alkylcarbamoyl,
N-hydroxyalkylcarbamoyl, acylamino, N-alkylacylamino or
acylaminomethyl at the 3- and/or 5-positions, as in metrizoic acid,
diatrizoic acid, iothalamic acid, ioxaglic acid, iohexol, iopentol,
iopamidol, iodixanol, iopromide, metrizamide, iodipamide, meglumine
iodipamide, meglumine acetrizoate and meglumine diatrizoate), and
polypeptides and proteins (e.g. gelatin or albumin such as human
serum albumin).
[0034] Any biocompatible gas may be present in the reporter of
contrast agents according to the invention, the term "gas" as used
herein including any substances (including mixtures) substantially
or completely in gaseous (including vapour) form at the normal
human body temperature of 37.degree. C. The gas may thus, for
example, comprise air; nitrogen; oxygen; carbon dioxide; hydrogen;
an inert gas such as helium, argon, xenon or krypton; a sulphur
fluoride such as sulphur hexafluoride, disulphur decafluoride or
trifluoromethylsulphur pentafluoride; selenium hexafluoride; an
optionally halogenated silane such as methylsilane or
dimethylsilane; a low molecular weight hydrocarbon (e.g. containing
up to 7 carbon atoms), for example an alkane such as methane,
ethane, a propane, a butane or a pentane, a cycloalkane such as
cyclopropane, cyclobutane or cyclopentane, an alkene such as
ethylene, propene, propadiene or a butene, or an alkyne such as
acetylene or propyne; an ether such as dimethyl ether; a ketone; an
ester; a halogenated low molecular weight hydrocarbon (e.g.
containing up to 7 carbon atoms); or a mixture of any of the
foregoing. Advantageously at least some of the halogen atoms in
halogenated gases are fluorine atoms; thus biocompatible
halogenated hydrocarbon gases may, for example, be selected from
bromochlorodifluoromethane, chlorodifluoromethane,
dichlorodifluoromethane, bromotrifluoromethane,
chlorotrifluoromethane, chloropentafluoroethane,
dichlorotetrafluoroethane, chlorotrifluoroethylene, fluoroethylene,
ethylfluoride, 1,1-difluoroethane and perfluorocarbons, e.g.
perfluoroalkanes such as perfluoromethane, perfluoroethane,
perfluoropropanes, perfluorobutanes (e.g. perfluoro-n-butane,
optionally in admixture with other isomers such as
perfluoro-iso-butane), perfluoropentanes, perfluorohexanes and
perfluoroheptanes; perfluoroalkenes such as perfluoropropene,
perfluorobutenes-(e.g. perfluorobut-2-ene) and perfluorobutadiene;
perfluoroalkynes such as perfluorobut-2-yne; and
perfluorocycloalkanes such as perfluorocyclobutane,
perfluoromethylcyclobutane, perfluorodimethylcyclobutanes,
perfluorotrimethyl-cyclobutanes, perfluorocyclopentane,
perfluoromethyl-cyclopentane, perfluorodimethylcyclopentanes,
perfluorocyclohexane, perfluoromethylcyclohexane and
perfluorocycloheptane. Other halogenated gases include methyl
chloride, fluorinated (e.g. perfluorinated) ketones such as
perfluoroacetone and fluorinated (e.g. perfluorinated) ethers such
as perfluorodiethyl ether. The use of perfluorinated gases, for
example sulphur hexafluoride and perfluorocarbons such as
perfluoropropane, perfluorobutanes and perfluoropentanes, may be
particularly advantageous in view of the recognised high stability
in the bloodstream of microbubbles containing such gases.
[0035] The reporter may be made by any convenient process, for
example by making gas-containing or gas-generating formulations.
Representative examples include the preparation of a suspension of
gas microbubbles by contacting a surfactant with gas and mixing
them in the presence of an aqueous carrier, as described in WO
9115244; or by atomising a solution or dispersion of a wall-forming
material in the presence of a gas in order to obtain hollow
microcapsules, as described in EP 512693A1; preparation of solid
microspheres by a double emulsion process, as described in U.S.
Pat. No. 5,648,095; or a process for forming hollow microcapsules
by spray-drying as described in EP 681843A2; or preparing
gas-filled liposomes by shaking an aqueous solution comprising a
lipid in the presence of a gas as described in U.S. Pat. No.
5,469,854.
[0036] A suitable process for attachment of the desired vector to
the reporter comprises a surface modification of the preformed
reporter with a suitable linker employing reactive groups on the
surface of both the reporter and vector. It may be particularly
advantageous physically to mix the reporter material with the
vector-containing substance at any step of the process. Such a
process will result in incorporation or an attachment of the vector
to the reporter. An optional process step may remove the excess of
vector not bound to the reporter by washing the gas-containing
particles following separation, by for example, floatation. A
preferred aspect is the use of lipopeptide structures incorporating
functional groups such as thiol, maleimide biotin etc. which can be
premixed if desired with other reporter molecules before formation
of gas-containing agents. The attachment of vector molecules may be
carried out using the linker reagents listed below.
[0037] Linking of a reporter unit to the desired vectors may be
achieved by covalent or non-covalent means, usually involving
interaction with one or more functional groups located on the
reporter and/or vectors. Examples of chemically reactive functional
groups which may be employed for this purpose include amino,
hydroxyl, sulfhydryl, carboxyl, and carbonyl groups, as well as
carbohydrate groups, vicinal diols, thioethers, 2-aminoalcohols,
2-aminothiols, guanidinyl, imidazolyl and phenolic groups.
[0038] Covalent coupling of reporter and vectors may therefore be
effected using linking agents containing reactive moieties capable
of reaction with such functional groups. Examples of reactive
moieties capable of reaction with sulfhydryl groups include
.alpha.-haloacetyl compounds of the type X--CH.sub.2CO-- (where
X=Br, Cl or I), which show particular reactivity for sulfhydryl
groups but which can also be used to modify imidazolyl, thioether,
phenol and amino groups as described by Gurd, F. R. N. in Methods
Enzymol. (1967) 11, 532. N-Maleimide derivatives are also
considered selective towards sulfhydryl groups, but may additionaly
be useful in coupling to amino groups under certain conditions.
N-maleimides may be incorporated into linking systems for
reporter-vector conjugation as described by Kitagawa, T. et al. in
Chem. Pharm. Bull. (1981) 29, 1130 or used as polymer crosslinkers
for bubble stabilisation as described by Kovacic, P. et al. in J.
Am. Chem. Soc. (1959) 81, 1887. Reagents such as 2-iminothiolane,
e.g. as described by Traut, R. et al. in Biochemistry (1973) 12,
3266, which introduce a thiol group through conversion of an amino
group, may be considered as sulfhydryl reagents if linking occurs
through the formation of disulphide bridges. Thus reagents which
introduce reactive disulphide bonds into either the reporter or the
vectors may be useful, since linking may be brought about by
disulphide exchange between the vector and reporter; examples of
such reagents include Ellman's reagent (DTNB),
4,4'-dithiodipyridine, methyl-3-nitro-2-pyridyl disulphide and
methyl-2-pyridyl disulphide (described by Kimura, T. et al. in
Analyt. Biochem. (1982) 122, 271).
[0039] Examples of reactive moieties capable of reaction with amino
groups include alkylating and acylating agents. Representative
alkylating agents include:
[0040] i) .alpha.-haloacetyl compounds, which show specificity
towards amino groups in the absence of reactive thiol groups and
are of the type X--CH.sub.2CO-- (where X=Cl, Br or I), e.g. as
described by Wong, Y-H. H. in Biochemistry (1979) 24, 5337;
[0041] ii) N-maleimide derivatives, which may react with amino
groups either through a Michael type reaction or through acylation
by addition to the ring carbonyl group as described by Smyth, D. G.
et al. in J. Am. Chem. Soc. (1960) 82, 4600 and Biochem. J. (1964)
91, 589;
[0042] iii) aryl halides such as reactive nitrohaloaromatic
compounds;
[0043] iv) alkyl halides as described by McKenzie, J. A. et al. in
J. Protein Chem. (1988) 7, 581;
[0044] v) aldehydes and ketones capable of Schiff's base formation
with amino groups, the adducts formed usually being stabilised
through reduction to give a stable amine;
[0045] vi) epoxide derivatives such as epichlorohydrin and
bisoxiranes,which may react with amino, sulfhydryl or phenolic
hydroxyl groups;
[0046] vii) chlorine-containing derivatives of s-triazines, which
are very reactive towards nucleophiles such as amino, sufhydryl and
hydroxy groups;
[0047] viii) aziridines based on s-triazine compounds detailed
above, e.g. as described by Ross, W. C. J. in Adv. Cancer Res.
(1954) 2, 1, which react with nucleophiles such as amino groups by
ring opening;
[0048] ix) squaric acid diethyl esters as described by Tietze, L.
F. in Chem. Ber. (1991) 124, 1215; and
[0049] x) .alpha.-haloalkyl ethers, which are more reactive
alkylating agents than normal alkyl halides because of the
activation caused by the ether oxygen atom, e.g. as described by
Benneche, T. et al. in Eur. J. Med. Chem. (1993) 28, 463.
[0050] Representative amino-reactive acylating agents include:
[0051] i) isocyanates and isothiocyanates, particularly aromatic
derivatives, Which form stable urea and thiourea derivatives
respectively and have been used for protein crosslinking as
described by Schick, A. F. et al. in J. Biol. Chem. (1961) 236,
2477;
[0052] ii) sulfonyl chlorides, which have been described by Herzig,
D. J. et al. in Biopolymers (1964) 2, 349 and which may be useful
for the introduction of a fluorescent reporter group into the
linker;
[0053] iii) Acid halides;
[0054] iv) Active esters such as nitrophenylesters or
N-hydroxysuccinimidyl esters;
[0055] v) acid anhydrides such as mixed, symmetrical or
N-carboxyanhydrides;
[0056] vi) other useful reagents for amide bond formation as
described by Bodansky, M. et al. in `Principles of Peptide
Synthesis` (1984) Springer-Verlag;
[0057] vii) acylazides, e.g. wherein the azide group is generated
from a preformed hydrazide derivative using sodium nitrite, e.g. as
described by Wetz, K. et al. in Anal. Biochem. (1974) 58, 347;
[0058] viii) azlactones attached to polymers such as
bis-acrylamide, e.g. as described by Rasmussen, J. K. in Reactive
Polymers (1991) 16, 199; and
[0059] ix) Imidoesters, which form stable amidines on reaction with
amino groups, e.g. as described by Hunter, M. J. and Ludwig, M. L.
in J. Am. Chem. Soc. (1962) 84, 3491.
[0060] Carbonyl groups such as aldehyde functions may be reacted
with weak protein bases at a pH such that nucleophilic protein
side-chain functions are protonated. Weak bases include
1,2-aminothiols such as those found in N-terminal cysteine
residues, which selectively form stable 5-membered thiazolidine
rings with aldehyde groups, e.g. as described by Ratner, S. et al.
in J. Am. Chem. Soc. (1937) 59, 200. Other weak bases such as
phenyl hydrazones may be used, e.g. as described by Heitzman, H. et
al. in Proc. Natl. Acad. Sci. USA (1974) 71, 3537.
[0061] Aldehydes and ketones may also be reacted with amines to
form Schiff's bases, which may advantageously be stabilised through
reductive amination. Alkoxylamino moieties readily react with
ketones and aldehydes to produce stable alkoxamines, e.g. as
described by Webb, R. et al. in Bioconjugate Chem. (1990) 1,
96.
[0062] Examples of reactive moieties capable of reaction with
carboxyl groups include diazo compounds such as diazoacetate esters
and diazoacetamides, which react with high specificity to generate
ester groups, e.g. as described by Herriot R. M. in Adv. Protein
Chem. (1947) 3, 169. Carboxylic acid modifying reagents such as
carbodiimides, which react through O-acylurea formation followed by
amide bond formation, may also usefully be employed; linking may be
facilitated through addition of an amine or may result in direct
vector-receptor coupling. Useful water soluble carbodiimides
include 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbo- diimide (CMC)
and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), e.g. as
described by Zot, H. G. and Puett, D. in J. Biol. Chem. (1989) 264,
15552. Other useful carboxylic acid modifying reagents include
isoxazolium derivatives such as Wo6dwards reagent K; chloroformates
such as p-nitrophenylchloroformate; carbonyldiimidazoles such as
1,1'-carbonyldiimidazole; and N-carbalkoxydihydroquinolines such as
N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.
[0063] Other potentially useful reactive moieties include vicinal
diones such as p-phenylenediglyoxal, which may be used to react
with guanidinyl groups, e.g. as described by Wagner et al. in
Nucleic acid Res. (1978) 5, 4065; and diazonium salts, which may
undergo electrophilic substitution reactions, e.g. as described by
Ishizaka, K. and Ishizaka T. in J. Immunol. (1960) 85, 163.
Bis-diazonium compounds are readily prepared by treatment of aryl
diamines with sodium nitrite in acidic solutions. It will be
appreciated that functional groups in the reporter and/or vector
may if desired be converted to other functional groups prior to
reaction, e.g. to confer additional reactivity or selectivity.
Examples of methods useful for this purpose include conversion of
amines to carboxylic acids using reagents such as dicarboxylic
anhydrides; conversion of amines to thiols using reagents such as
N-acetylhomocysteine thiolactone, S-acetylmercaptosuccinic
anhydride, 2-iminothiolane or thiol-containing succinimidyl
derivatives; conversion of thiols to carboxylic acids using
reagents such as a-haloacetates; conversion of thiols to amines
using reagents such as ethylenimine or 2-bromoethylamine;
conversion of carboxylic acids to amines using reagents such as
carbodiimides followed by diamines; and conversion of alcohols to
thiols using reagents such as tosyl chloride followed by
transesterification with thioacetate and hydrolysis to the thiol
with sodium acetate.
[0064] Vector-receptor coupling may also be effected using enzymes
as zero-length crosslinking agents; thus, for example,
transglutaminase, peroxidase and xanthine oxidase have been used to
produce crosslinked products. Reverse proteolysis may also be used
for crosslinking through amide bond formation.
[0065] Non-covalent vector-receptor coupling may, for example, be
effected by electrostatic charge interactions e.g. between a
polylysinyl-functionalised reporter and a
polyglutamyl-functionalised vector, through chelation in the form
of stable metal complexes or through high affinity binding
interaction such as avidin/biotin binding. Polylysine, coated
non-covalently to the negatively charged membrane surface can also
increase non-specifically the affinity of a microbubble for a cell
through charge interactions.
[0066] Alternatively, vectors may be coupled to a protein or
peptide sequence known to bind phospholipids. In many instances, a
single molecule of phospholipid may attach to a protein such as a
translocase, while other proteins may attach to surfaces consisting
mainly of phospholipid head groups and so may be used to attach
vectors to phospholipid microspheres; one example of such a protein
is .beta.2-glycoprotein I (Chonn, A., Semple, S. C. and Cullis, P.
R., Journal of Biological Chemistry (1995) 270, 25845-25849).
Phosphatidylserine-binding proteins have been described, e.g. by
Igarashi, K. et al. in Journal of Biological Chemistry 270(49),
29075-29078. Annexins are a class of phospholipid-binding proteins,
many of which bind particularly avidly to phosphatidyl-serine
(reviewed in Raynal, P. and H. B. Pollard. Annexins: the problem of
assessing the biological role for a gene family of multifunctional
calcium- and phospholipid-binding proteins". Biochim. Biophys. Acta
1197: 63-93). A conjugate of a vector with such a
phosphatidylserine-binding protein may therefore be used to attach
the vector to phosphatidylserine-encapsulated microbubbles. When
the amino acid sequence of a binding protein is known, the
phospholipid-binding portion may be synthesised or isolated and
used for conjugation with a vector, thus avoiding the biological
activity which may be located elsewhere in the molecule.
[0067] It is also possible to obtain molecules that bind
specifically to the surface (or in the "membrane") of microspheres
by direct screening of molecular libraries for microsphere-binding
molecules. For example, phage libraries displaying small peptides
could be used for such selection. The selection may be made by
simply mixing the microspheres and the phage display library and
eluting the phages binding to the floating microspheres. If
desired, the selection can be done under "physiological conditions"
(e.g. in blood) to eliminate peptides which cross-react with blood
components. An advantage of this type of selection procedure is
that only binding molecules that do not destabilize the
microspheres should be selected, since only binding molecules
attached to intact floating microspheres will rise to the top. It
may also be possible to introduce some kind of "stress" during the
selection procedure (e.g. pressure) to ensure that destabilizing
binding moieties are not selected. Furthermore the selection could
be done under shear conditions e.g. by first letting the phages
react with the microspheres and then letting the microspheres pass
through a surface coated with anti-phage antibodies under flow
conditions. In this way it may be possible to select binders which
may resist shear conditions present in vivo. Binding moieties
identified in this way may be coupled (chemically via peptide
synthesis, or at the DNA-level using recombinant vectors) to a
vector molecule, constituting a general tool for attaching any
vector molecule to the microspheres.
[0068] A vector which comprises or is coupled to a peptide or
lipopeptide linker which contains a element capable of mediating
membrane insertion may also be useful. One example is described by
Leenhouts, J. M. et al. in Febs Letters (1995) 370(3), 189-192.
Non-bioactive molecules consisting of known membrane insertion
anchor/signal groups may also be used as vectors for certain
applications, an example being the H1 hydrophobic segment from the
Na, K-ATPase .alpha.-subunit described by Xie, Y. and Morimoto, T.
in J. Biol. Chem. (1995) 270(20), 11985-11991. The anchor group may
also be fatty acid(s) or cholesterol.
[0069] Coupling may also be effected using avidin or streptavidin,
which have four high affinity binding sites for biotin. Avidin may
therefore be used to conjugate vector to reporter if both vector
and reporter are biotinylated. Examples are described by Bayer, E.
A. and Wilchek, M. in Methods Biochem. Anal. (1980) 26, 1. This
method may also be extended to include linking of reporter to
reporter, a process which may encourage bubble association and
consequent potentially increased echogenicity.
[0070] Non-covalent coupling may also utilise the bifunctional
nature of bispecific immunoglobulins. These molecules can
specifically bind two antigens, thus linking them. For example,
either bispecific IgG or chemically engineered bispecific F(ab)'2
fragments may be used as linking agents. Heterobifunctional
bispecific antibodies have also been reported for linking two
different antigens, e.g. as described by Bode, C. et al. in J.
Biol. Chem. (1989) 264, 944 and by Staerz, U. D. et al. in Proc.
Natl. Acad. Sci. USA (1986) 83, 1453. Similarly, any reporter
and/or vector containing two or more antigenic determinants (e.g.
as described by Chen, Aa et al. in Am. J. Pathol. (1988) 130, 216)
may crosslink antibody molecules and lead to formation of
multi-bubble cross-linked assemblies of potentially increased
echogenicity.
[0071] So-called zero-length linking agents, which induce direct
covalent joining of two reactive chemical groups without
introducing additional linking material (e.g. as in amide bond
formation induced using carbodiimides or enzymatically) may, if
desired, be used, as may agents such as biotin/avidin systems which
induce non-covalent reporter-vector linking and agents which induce
hydrophobic or electrostatic interactions.
[0072] Most commonly, however, the linking agent will comprise two
or more reactive moieties, e.g. as described above, connected by a
spacer element. The presence of such a spacer permits bifunctional
linkers to react with specific functional groups within a molecule
or between two different molecules, resulting in a bond between
these two components and introducing extrinsic linker-derived
material into the reporter-vector conjugate. The reactive moieties
in a linking agent may be the same (homobifunctional agents) or
different (heterobifunctional agents or, where several dissimilar
reactive moieties are present, heteromultifunctional agents),
providing a diversity of potential reagents that may bring about
covalent bonding between any chemical species, either
intramolecularly or intermolecularly.
[0073] The nature of extrinsic material introduced by the linking
agent may have a critical bearing on the targeting ability and
general stability of the ultimate product. Thus it may be desirable
to introduce labile linkages, e.g. containing spacer arms which are
biodegradable or chemically sensitive or which incorporate
enzymatic cleavage sites. Alternatively the spacer may include
polymeric components, e.g. to act as surfactants and enhance bubble
stability. The spacer may also contain reactive moieties, e.g. as
described above to enhance surface crosslinking, or it may contain
a tracer element such as a fluorescent probe, spin label or
radioactive material.
[0074] Spacer elements may typically consist of aliphatic chains
which effectively separate the reactive moieties of the linker by
distances of between 5 and 30 .ANG.. They may also comprise
macromolecular structures such as poly(ethylene glycols). Such
polymeric structures, hereinafter referred to as PEGS, are simple,
neutral polyethers which have been given much attention in
biotechnical and biomedical applications (see e.g. Milton Harris,
J. (ed) "Poly(ethylene glycol) chemistry, biotechnical and
biomedical applications" Plenum Press, New York, 1992). PEGs are
soluble in most solvents, including water, and are highly hydrated
in aqueous environments, with two or three water molecules bound to
each ethylene glycol segment; this has the effect of preventing
adsorption either of other polymers or of proteins onto
PEG-modified surfaces. PEGs are known to be nontoxic and not to
harm active proteins or cells, whilst covalently linked PEGs are
known to be non-immunogenic and non-antigenic. Furthermore, PEGs
may readily be modified and bound to other molecules with only
little effect on their chemistry. Their advantageous solubility and
biological properties are apparent from the many possible uses of
PEGs and copolymers thereof, including block copolymers such as
PEG-polyurethanes and PEG-polypropylenes.
[0075] Appropriate molecular weights for PEG spacers used in
accordance with the invention may, for example, be between 120
Daltons and 20 kDaltons.
[0076] The major mechanism for uptake of particles by the cells of
the reticuloendothelial system (RES) is opsonisation by plasma
proteins in blood; these mark foreign particles which are then
taken up by the RES. The biological properties of PEG spacer
elements used in accordance with the invention may serve to
increase contrast agent circulation time in a similar manner to
that observed for PEGylated liposomes (see e.g. Klibanov, A. L. et
al. in FEBS Letters (1990) 268, 235-237 and Blume, G. and Cevc, G.
in Biochim. Biophys. Acta (1990) 1029, 91-97).
[0077] Other potentially useful protein modifications which can be
made to vectors include partial or complete deglycosidation by
neuraminidase, endoglycosydases or periodate, since deglycosidation
often results in less uptake by liver, spleen, macrophages etc.,
whereas neo-glycosylation of proteins often results in increased
uptake by the liver and macrophages); preparation of truncated
forms by proteolytic cleavage, leading to reduced size and shorter
half life in circulation; and cationisation, e.g. as described by
Kumagi et al. in J. Biol. Chem. (1987) 262, 15214-15219; Triguero
et al. in Proc. Natl. Acad. Sci. USA (1989) 86, 4761-4765;
Pardridge et al. in J. Pharmacol. Exp. Therap. (1989) 251, 821-826
and Pardridge and Boado, Febs Lett. (1991) 288, 30-32.
[0078] Increased coupling efficiency to areas of interest may also
be achieved using antibodies bound to the terminii of PEG spacers
(see e.g. Maruyama, K. et al. in Biochim. Biophys. Acta (1995)
1234, 74-80 and Hansen, C. B. et al. in Biochim. Biophys. Acta
(1995) 1239, 133-144).
[0079] In some instances it is considered advantageous to include a
PEG component as a stabiliser in conjunction with a vector or
vectors or directly to the reporter in the same molecule where the
PEG does not serve as a spacer.
[0080] Other representative spacer elements include structural-type
polysaccharides such as polygalacturonic acid, glycosaminoglycans,
heparinoids, cellulose and marine polysaccharides such as
alginates, chitosans and carrageenans; storage-type polysaccharides
such as starch, glycogen, dextran and aminodextrans; polyamino
acids and methyl and ethyl esters thereof, as in homo- and
co-polymers of lysine, glutamic acid and aspartic acid; and
polypeptides, oligonucleotides and oligosaccharides, which may or
may not contain enzyme cleavage sites.
[0081] In general, spacer elements may contain cleavable groups
such as vicinal glycol, azo, sulfone, ester, thioester or
disulphide groups. Spacers containing biodegradable methylene
diester or diamide groups of formula
--(Z).sub.m.Y.X.C(R.sup.1R.sup.2).X.Y.(Z).sub.n--
[0082] [where X and Z are selected from --O--, --S--, and --NR--
(where R is hydrogen or an organic group); each Y is a carbonyl,
thiocarbonyl, sulphonyl, phosphoryl or similar acid-forming group:
m and n are each zero or 1; and R.sup.1 and R.sup.2are each
hydrogen, an organic group or a group --X.Y.(Z).sub.m--, or
together form a divalent organic group] may also be useful; as
discussed in, for example, WO-A-9217436 such groups are readily
biodegraded in the presence of esterases, e.g. in vivo, but are
stable in the absence of such enzymes. They may therefore
advantageously be linked to therapeutic agents to permit slow
release thereof.
[0083] Poly[N-(2-hydroxyethyl)methacrylamides] are potentially
useful spacer materials by virtue of their low degree of
interaction with cells and tissues (see e.g. Volfov, I., Rhov, B.
and V. R. and Vetvicka, P. in J. Bioact. Comp. Polymers (1992) 7,
175-190). Work on a similar polymer consisting mainly of the
closely related 2-hydroxypropyl derivative showed that it was
endocytosed by the mononuclear phagocyte system only to a rather
low extent (see Goddard, P., Williamson, I., Bron, J., Hutchkinson,
L. E., Nicholls, J. and Petrak, K. in J. Bioct. Compat. Polym.
(1991) 6, 4-24. ).
[0084] Other potentially useful poymeric spacer materials
include:
[0085] i) copolymers of methyl methacrylate with methacrylic acid;
these may be erodible (see Lee, P. I. in Pharm. Res. (1993) 10,
980) and the carboxylate substituents may cause a higher degree of
swelling than with neutral polymers;
[0086] ii) block copolymers of polymethacrylates with biodegradable
polyesters (see e.g. San Roman, J. and Guillen-Garcia, P. in
Biomaterials (1991) 12, 236-241);
[0087] iii) cyanoacrylates, i.e. polymers of esters of
2-cyanoacrylic acid--these are biodegradable and have been used in
the form of nanoparticles for selective drug delivery (see
Forestier, F., Gerrier, P., Chaumard, C., Quero, A. M., Couvreur,
P. and Labarre, C. in J. Antimicrob. Chemoter. (1992) 30,
173-179);
[0088] iv) polyvinyl alcohols, which are water-soluble and
generally regarded as biocompatible (see e.g. Langer, R. in J.
Control. Release (1991) 16, 53-60);
[0089] v) copolymers of vinyl methyl ether with maleic anhydride,
which have been stated to be bioerodible (see Finne, U., Hannus, M.
and Urtti, A. in Int. J. Pharm. (1992) 78. 237-241);
[0090] vi) polyvinylpyrrolidones, e.g. with molecular weight less
than about 25,000, which are rapidly filtered by the kidneys (see
Hespe, W., Meier, A. M. and Blankwater, Y. M. in
Arzeim.-Forsch./Drug Res. (1977) 27, 1158-1162);
[0091] vii) polymers and copolymers of short-chain aliphatic
hydroxyacids such as glycolic, lactic, butyric, valeric and caproic
acids (see e.g. Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9),
including copolymers which incorporate aromatic hydroxyacids in
order to increase their degradation rate (see Imasaki, K., Yoshida,
M., Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka,
H. and Nagai. T. in Int. J. Pharm. (1992) 81, 31-38);
[0092] viii) polyesters consisting of alternating units of ethylene
glycol and terephthalic acid, e.g. Dacron.sup.R, which are
non-degradable but highly biocompatible;
[0093] ix) block copolymers comprising biodegradable segments of
aliphatic hydroxyacid polymers (see e.g. Younes, H., Nataf, P. R.,
Cohn, D., Appelbaum, Y. J., Pizov, G. and Uretzky, G. in Biomater.
Artif. Cells Artif. Organs (1988) 16, 705-719), for instance in
conjunction with polyurethanes (see Kobayashi, H., Hyon, S. H. and
Ikada, Y. in "Water-curable and biodegradable prepolymers"--J.
Biomed. Mater. Res. (1991) 25, 1481-1494);
[0094] x) polyurethanes, which are known to be well-tolerated in
implants, and which may be combined with flexible "soft" segments,
e.g. comprising poly(tetra methylene glycol), poly(propylene
glycol) or poly(ethylene glycol)) and aromatic "hard" segments,
e.g. comprising 4,4'-methylenebis(phenylene isocyanate) (see e.g.
Ratner, B. D., Johnston, A. B. and Lenk, T. J. in J. Biomed. Mater.
Res: Applied Biomaterials (1987) 21, 59-90; Sa Da Costa, V. et al.
in J. Coll. Interface Sci. (1981) 80, 445-452 and Affrossman, S. et
al. in Clinical Materials (1991) 8, 25-31);
[0095] xi) poly(1,4-dioxan-2-ones), which may be regarded as
biodegradable esters in view of their hydrolysable ester linkages
(see e.g. Song, C. X., Cui, X. M. and Schindler, A. in Med. Biol.
Eng. Comput. (1993) 31, S147-150), and which may include glycolide
units to improve their absorbability (see Bezwada, R. S., Shalaby,
S. W. and Newman, H. D. J. in Agricultural and synthetic polymers:
Biodegradability and utilization (1990) (ed Glass, J. E. and Swift,
G.), 167-174--ACS symposium Series, #433, Washington D.C.,
U.S.A.--American Chemical Society);
[0096] xii) polyanhydrides such as copolymers of sebacic acid
(octanedioic acid) with bis(4-carboxy-phenoxy)propane, which have
been shown in rabbit studies (see Brem, H., Kader, A., Epstein, J.
I., Tamargo, R. J., Domb, A., Langer, R. and Leong, K. W. in Sel.
Cancer Ther. (1989) 5, 55-65) and rat studies (see Tamargo, R. J.,
Epstein, J. I., Reinhard, C. S., Chasin, M. and Brem, H. in J.
Biomed. Mater. Res. (1989) 23, 253-266) to be useful for controlled
release of drugs in the brain without evident toxic effects;
[0097] xiii) biodegradable polymers containing ortho-ester groups,
which have been employed for controlled release in vivo (see Maa,
Y. F. and Heller, J. in J. Control. Release (1990) 14, 21-28);
and
[0098] xiv) polyphosphazenes, which are inorganic polymers
consisting of alternate phosphorus and nitrogen atoms (see Crommen,
J. H., Vandorpe, J. and Schacht, E. H. in J. Control. Release
(1993) 24, 167-180).
[0099] The following tables list linking agents and agents for
protein modification which may be useful in preparing targetable
contrast agents in accordance with the invention.
[0100] Heterobifunctional Linking Agents
1 Linking agent Reactivity 1 Reactivity 2 Comments ABH carbohydrate
photoreactive ANB-NOS --NH.sub.2 photoreactive APDP(1) --SH
photoreactive iodinable disulphide linker APG --NH.sub.2
photoreactive reacts selectively with Arg at pH 7-8 ASIB(1) --SH
photoreactive iodinable ASBA(1) --COOH photoreactive iodinable EDC
--NH.sub.2 --COOH zero-length linker GMBS --NH.sub.2 --SH
sulfo-GMBS --NH.sub.2 --SH water-soluble HSAB --NH.sub.2
photoreactive sulfo-HSAB --NH.sub.2 photoreactive water-soluble MBS
--NH.sub.2 --SH sulfo-MBS --NH.sub.2 --SH water-soluble
M.sub.2C.sub.2H carbohydrate --SH MPBH carbohydrate --SH NHS-ASA(1)
--NH.sub.2 photoreactive iodinable sulfo-NHS- --NH.sub.2
photoreactive water-soluble, ASA(1) iodinable sulfo-NHS-LC-
--NH.sub.2 photoreactive water-soluble, ASA(1) iodinable PDPH
carbohydrate --SH disulphide linker PNP-DTP --NH.sub.2
photoreactive SADP --NH.sub.2 photoreactive disulphide linker
sulfo-SADP --NH.sub.2 photoreactive water-soluble disulphide linker
SAED --NH.sub.2 photoreactive disulphide linker SAND --NH.sub.2
photoreactive water-soluble disulphide linker SANPAH --NH.sub.2
photoreactive sulfo-SANPAH --NH.sub.2 photoreactive water-soluble
SASD(1) --NH.sub.2 photoreactive water-soluble iodinable disulphide
linker SIAB --NH.sub.2 --SH sulfo-SIAB --NH.sub.2 --SH
water-soluble SMCC --NH.sub.2 --SH sulfo-SMCC --NH.sub.2 --SH
water-soluble SMPB --NH.sub.2 --SH sulfo-SMPB --NH.sub.2 --SH
water-soluble SMPT --NH.sub.2 --SH sulfo-LC-SMPT --NH.sub.2 --SH
water-soluble SPDP --NH.sub.2 --SH sulfo-SPDP --NH.sub.2 --SH
water-soluble sulfo-LC-SPDP --NH.sub.2 --SH water-soluble
sulfo-SAMCA(2) --NH.sub.2 photoreactive sulfo-SAPB --NH.sub.2
photoreactive water-soluble Notes: (1) = iodinable; (2) =
fluorescent
[0101] Homobifunctional Linking Agents
2 Linking agent Reactivity Comments 0 --NH.sub.2 BMH --SH BASED (1)
photoreactive iodinable disulphide linker BSCOES --NH.sub.2
sulfo-BSCOES --NH.sub.2 water-soluble DFDNB --NH.sub.2 DMA
--NH.sub.2 DMP --NH.sub.2 DMS --NH.sub.2 DPDPB --SH disulphide
linker DSG --NH.sub.2 DSP --NH.sub.2 disulphide linker DSS
--NH.sub.2 DST --NH.sub.2 sulfo-DST --NH.sub.2 water-soluble DTBP
--NH.sub.2 disulphide linker DTSSP --NH.sub.2 disulphide linker EGS
--NH.sub.2 sulfo-EGS --NH.sub.2 water-soluble SPBP --NH.sub.2
[0102] Biotinylation Agents
3 Agent Reactivity Comments biotin-BMCC --SH biotin-DPPE*
preparation of biotinylated liposomes biotin-LC-DPPE* preparation
of biotinylated liposomes biotin-HPDP --SH disulphide linker
biotin-hydrazide carbohydrate biotin-LC-hydrazide carbohydrate
iodoacetyl-LC-biotin --NH.sub.2 NHS-iminobiotin --NH.sub.2 reduced
affinity for avidin NHS-SS-biotin --NH.sub.2 disulphide linker
photoactivatable biotin nucleic acids sulfo-NHS-biotin --NH.sub.2
water-soluble sulfo-NHS-LC-biotin --NH.sub.2 Notes: DPPE =
dipalmitoylphosphatidylethanolamine; LC = long chain
[0103] Agents for Protein Modification
4 Agent Reactivity Function Ellman's reagent --SH
quantifies/detects/protects DTT --S.S-- reduction 2-mercaptoethanol
--S.S-- reduction 2-mercaptylamine --S.S-- reduction Traut's
reagent --NH.sub.2 introduces --SH SATA --NH.sub.2 introduces
protected --SH AMCA-NHS --NH.sub.2 fluorescent labelling
AMCA-hydrazide carbohydrate fluorescent labelling AMCA-HPDP --S.S--
fluorescent labelling SBF-chloride --S.S-- fluorescent detection of
--SH N-ethylmaleimide --S.S-- blocks --SH NHS-acetate --NH.sub.2
blocks and acetylates --NH.sub.2 citraconic anhydride --NH.sub.2
reversibly blocks and introduces negative charges DTPA --NH.sub.2
introduces chelator BNPS-skatole tryptophan cleaves tryptophan
residue Bolton-Hunter --NH.sub.2 introduces iodinable group
[0104] Linking agents used in accordance with the invention will in
general bring about linking of vector to reporter or reporter to
reporter with some degree of specificity, and may also be used to
attach one or more therapeutically active agents.
[0105] Ultrasound imaging modalities which may be used in
accordance with the invention include two- and three-dimensional
imaging techniques such as B-mode imaging (for example using the
time-varying amplitude of the signal envelope generated from the
fundamental frequency of the emitted ultrasound pulse, from
sub-harmonics or higher harmonics thereof or from sum or difference
frequencies derived from the emitted pulse and such harmonics,
images generated from the fundamental frequency or the second
harmonic thereof being preferred), colour Doppler imaging and
Doppler amplitude imaging, and combinations of the two latter with
any of the modalities (techniques) above. Surprisingly, the second
harmonic signals from the targeted monolayer microspheres were
found to be excellent when used in accordance with the present
invention. To reduce the effects of movement, successive images of
tissues such as the heart or kidney may be collected with the aid
of suitable synchronisation techniques (e.g. gating to the ECG or
respiratory movement of the subject). Measurement of changes in
resonance frequency or frequency absorption which accompany
arrested or retarded microbubbles may also usefully be made to
detect the contrast agent.
[0106] The present invention provides a tool for therapeutic drug
delivery in combination with vector-mediated direction of the
product to the desired site. By "therapeutic" or "drug" is meant an
agent having a beneficial effect on a specific disease in a living
human or non-human animal. Whilst combinations of drugs and
ultrasound contrast agents have been proposed in, for example,
WO-A-9428873 and WO-A-9507072, these products lack vectors having
affinity for particular sites and thereby show comparitively poor
specific retention at desired sites prior to or during drug
release.
[0107] Therapeutic compounds used in accordance with the present
invention may be encapsulated in the interior of the microbubbles
or attached to or incorporated in the encapsulating walls. Thus,
the therapeutic compound may be linked to a part of the wall, for
example through covalent or ionic bonds, or may be physically mixed
into the encapsulating material, particularly if the drug has
similar polarity or solubility to the membrane material, so as to
prevent it from leaking out of the product before it is intended to
act in the body. The release of the drug may be initiated merely by
wetting contact with blood following administration or as a
consequence of other internal or external influences, e.g.
dissolution processes catalyzed by enzymes or the use of of
ultrasound. The destruction of gas-containing microparticles using
external ultrasound is a well known phenomenon in respect of
ultrasound contrast agents, e.g. as described in WO-A-9325241; the
rate of release may be varied depending on the type of therapeutic
application, using a specific amount of ultrasound energy from the
transducer.
[0108] The therapeutic agent may be covalently linked to the
encapsulating membrane surface using a suitable linking agent, e.g.
as described herein. Thus, for example, one may initially prepare a
phospholipid or lipopeptide derivative to which the drug is bonded
through a biodegradable or selectively cleavable linker followed by
incorporation of the material into the microbubble. Alternatively
lipidated drug molecules which do not require processing to
liberate an active drug are incorporated directly into the
membrane. The active lipidated-drug can be released by increasing
the strength of the ultrasound beam.
[0109] Exemplary drug delivery systems suitable for use in the
present compositions include any known therapeutic drugs or active
analogues thereof containing thiol groups which are coupled to
thiol containing microbubbles under oxidative conditions yielding
disulphide bridges. In combination with a vector or vectors the
drug/vector modified microbubbles are allowed to accumulate in the
target tissue. Administration of a reducing agent such as reduced
glutathione then liberates the drug molecule from the targeted
microbubble in the vicinity of the target cell increasing the local
concentration of the drug and enhancing therapeutic effect. The
product may also be prepared without the therapeutic if desired.
The drug may then be coupled to or coated on the microbubbles prior
to use. Thus, for example, a therapeutic could be added to a
suspension of microbubbles in aqueous media and shaken in order to
attach or adhere the therapeutic to the microbubbles.
[0110] Other drug delivery systems include vector modified
phospholipid membranes doped with lipopeptide structures comprising
a poly-L-lysine or poly-D-lysine chain in combination with a
targeting vector. Applied to gene therapy/antisense technologies
with particular emphasis on receptor-mediated drug delivery the
microbubble carrier is condensed with DNA or RNA via elecrostatic
interaction with the polycation. This method has the advantage that
the vector or vectors used for targeted delivery are not directly
attached to the polysine carrier moiety. The polylysine chain is
also anchored more tightly in the microbubble membrane due to the
presence of the lipid chains. The use of ultrasound to increase the
effectiveness of delivery is also considered useful.
[0111] Alternatively free polylysine chains are firstly modified
with drug or vector molecules then condensed onto the negative
surface of targeted microbubbles.
[0112] Representative and non-limiting examples of drugs useful in
accordance with the invention include antineoplastic agents such as
vincristine, vinblastine, vindesine, busulfan, chlorambucil,
spiroplatin, cisplatin, carboplatin, methotrexate, adriamycin,
mitomycin, bleomycin, cytosine arabinoside, arabinosyl adenine,
mercaptopurine, mitotane, procarbazine, dactinomycin (antinomycin
D), daunorubicin, doxorubicin hydrochloride, taxol, plicamycin,
aminoglutethimide, estramustine, flutamide, leuprolide, megestrol
acetate, tamoxifen, testolactone, trilostane, amsacrine (m-AMSA),
asparaginase (L-asparaginase), etoposide, interferon a-2a and 2b,
blood products such as hematoporphyrins or derivatives of the
foregoing; biological response modifiers such as muramylpeptides;
antifungal agents such as ketoconazole, nystatin, griseofulvin,
flucytosine, miconazole or amphotericin B; hormones or hormone
analogues such as growth hormone, melanocyte stimulating hormone,
estradiol, beclomethasone dipropionate, betamethasone, cortisone
acetate, dexamethasone, flunisolide, hydrocortisone,
methylprednisolone, paramethasone acetate, prednisolone,
prednisone, triamcinolone or fludrocortisone acetate; vitamins such
as cyanocobalamin or retinoids; enzymes such as alkaline
phosphatase or manganese superoxide dismutase; antiallergic agents
such as amelexanox; inhibitors of tissue factor such as monoclonal
antibodies and Fab fragments thereof, synthetic peptides,
nonpeptides and compounds downregulating tissue factor expression;
inhibitors of platelets such as, GPIa, GPIb and GPIIb-IIIa, ADP
receptors, thrombin receptors, von Willebrand factor,
prostaglandins, aspirin, ticlopidin, clopigogrel and reopro;
inhibitors of coagulation protein targets such as: FIIa FVa, FVIIa,
FVIIIA, FIXa, tissue factor, hepatins, hirudin, hirulog,
argatroban, DEGR-rFVIIa and annexin V; inhibitors of fibrin
formation and promoters of fibrionolysis such as t-PA, urokinase,
Plamin, Streptokinase, rt-Plasminogen Activator and
rStaphylokinase; antiangiogenic factors such as medroxyprogesteron,
pentosan polysulphate, suramin, taxol, thalidomide, angiostatin,
interferon-alpha, metalloproteinase inhibitors, platelet factor 4,
somatostatin, thromobospondin; circulatory drugs such as
propranolol; metabolic potentiators such as glutathione;
antituberculars such as p-aminosalicylic acid, isoniazid,
capreomycin sulfate, cyclosexine, ethambutol, ethionamide,
pyrazinamide, rifampin or streptomycin sulphate; antivirals such as
acyclovir, amantadine, azidothymidine, ribavirin or vidarabine;
blood vessel dilating agents such as diltiazem, nifedipine,
verapamil, erythritol tetranitrate, isosorbide dinitrate,
nitroglycerin or pentaerythritol tetranitrate; antibiotics such as
dapsone, chloramphenicol, neomycin, cefaclor, cefadroxil,
cephalexin, cephradine, erythromycin, clindamycin, lincomycin,
amoxicillin, ampicillin, bacampicillin, carbenicillin,
dicloxacillin, cyclacillin, picloxacillin, hetacillin, methicillin,
nafcillin, penicillin, polymyxin or tetracycline;
antiinflammatories such as diflunisal, ibuprofen, indomethacin,
meclefenamate, mefenamic acid, naproxen, phenylbutazone, piroxicam,
tolmetin, aspirin or salicylates; antiprotozoans such as
chloroquine, metronidazole, quinine or meglumine antimonate;
antirheumatics such as penicillamine; narcotics such as paregoric;
opiates such as codeine, morphine or opium; cardiac glycosides such
as deslaneside, digitoxin, digoxin, digitalin or digitalis;
neuromuscular blockers such as atracurium mesylate, gallamine
triethiodide, hexafluorenium bromide, metocurine iodide,
pancuronium bromide, succinylcholine chloride, tubocurarine
chloride or vecuronium bromide; sedatives such as amobarbital,
amobarbital sodium, apropbarbital, butabarbital sodium, chloral
hydrate, ethchlorvynol, ethinamate, flurazepam hydrochloride,
glutethimide, methotrimeprazine hydrochloride, methyprylon,
midazolam hydrochloride, paraldehyde, pentobarbital, secobarbital
sodium, talbutal, temazepam or triazolam; local anaesthetics such
as bupivacaine, chloroprocaine, etidocaine, lidocaine, mepivacaine,
procaine or tetracaine; general anaesthetics such as droperidol,
etomidate, fentanyl citrate with droperidol, ketamine
hydrochloride, methohexital sodium or thiopental and
pharmaceutically acceptable salts (e.g. acid addition salts such as
the hydrochloride or hydrobromide or base salts such as sodium,
calcium or magnesium salts) or derivatives (e.g. acetates) thereof.
Other examples of therapeutics include genetic material such as
nucleic acids, RNA, and DNA of natural or synthetic origin,
including recombinant RNA and DNA. DNA encoding certain proteins
may be used in the treatment of many different types of diseases.
For example, tumor necrosis factor or interleukin-2 genes may be
provided to treat advanced cancers; thymidine kinase genes may be
provided to treat ovarian cancer or brain tumors; interleukin-2
genes may be provided to treat neuroblastoma, malignant melanoma or
kidney cancer; and interleukin-4 genes may be provided to treat
cancer.
[0113] Lipophilic derivatives of drugs linked to the microbubble
wall through hydrophobic interactions may exhibit therapeutic
effects as part of the microbubble or after release from the
microbubble, e.g. by use of ultrasound. If the drug does not
possess the desired physical properties, a lipophilic group may be
introduced for anchoring the drug to the membrane. Preferably the
lipophilic group should be introduced in a way that does not
influence the in vivo potency of the molecule, or the lipophilic
group may be cleaved releasing the active drug. Lipophilic groups
may be introduced by various chemical means depending on functional
groups available in the drug molecule. Covalent coupling may be
effected using functional groups in the drug molecule capable of
reacting with appropriately functionalised lipophilic compounds.
Examples of lipophilic moieties include branched and unbranched
alkyl chains, cyclic compounds, aromatic residues and fused
aromatic and non-aromatic cyclic systems. In some instances the
lipophilic moiety will consist of a suitably functionalised
steroid, like cholesterol and related compounds. Examples of
functional groups particularly suitable for derivatisation include
nucleophilic groups like amino, hydroxy and sulfhydryl groups.
Suitable processes for lipophilic derivatisation of any drug
containing a sulfhydryl group, like captopril, may include direct
alkylation, e.g. reaction with an alkyl halide under basic
conditions and thiol ester formation by reaction with an activated
carboxylic acid. Representative examples of derivatisation of any
drug having carboxylic functions, like atenolol and chlorambucil,
include amide and ester formation by coupling of amines and
alcohols, respectively, possesing requested physical properties. A
preferred aspect is attachment of cholesterol to a therapeutic
compound by forming a degradable ester bond.
[0114] A preferred application of the present invention relates to
angiogenesis, which is the formation of new blood vessels by
branching from existing vessels. The primary stimulus for this
process may be inadequate supply of nutrients and oxygen (hypoxia)
to cells in a tissue. The cells may respond by secreting
angiogenetic factors, of which there are many; one example is
vascular endothelial growth factor. These factors initiate the
secretion of proteolytic enzymes which break down the proteins of
the basement membrane, as well as inhibitors which limit the action
of these potentially harmful enzymes. The combined effect of loss
of attachment and signals from the receptors for angiogenetic
factors is to cause the endothelial cells to move, multiply, and
rearrange themselves, and finally to synthetise a basement membrane
around the new vessels.
[0115] Tumors must initiate angiogenesis when they reach millimeter
size in order to keep up their rate of growth. As angiogenesis is
accompanied by characteristic changes in the endothelial cells and
their environment, this process is a promising target for
therapeutic intervention. The transformations accompanying
angiogenesis are also very promising for diagnosis, a preferred
example being malignant disease, but the concept also shows great
promise in inflammation and a variety of inflammation-related
diseases. These factors are also involved in re-vascularisation of
infarcted parts of the myocardium, which occurs if a stenosis is
released within a short time.
[0116] A number of known receptors/targets associated with
angiogenesis are given in subsequent tables. Using the targeting
principles described in the present disclosure, angiogenesis mav be
detected by the majority of the imaging modalities in use in
medicine. Contrast-enhanced ultrasound may possess additional
advantages, the contrast medium being microspheres which are
restricted to the interior of blood vessels. Even if the target
antigens are found on many cell types, the microspheres will attach
exclusively to endothelial cells.
[0117] So-called prodrugs may also be used in agents according to
the invention. Thus drugs may be derivatised to alter their
physicochemical properties and to adapt them for inclusion into the
reporter; such derivatised drugs may be regarded as prodrugs and
are usually inactive until cleavage of the derivatising group
regenerates the active form of the drug.
[0118] By targeting a gas-filled microbubble containing a
prodrug-activating enzyme to areas of pathology one may image
targeting of the enzyme, making it possible to visualise when the
micobubbles are targeted properly to the area of pathology and at
the same time have disappeared from non-target areas. In this way
one can determine the optimal time for injection of prodrug into
individual patients.
[0119] Another alternative is to incorporate the prodrug, the
prodrug-activating enzyme and the vector in the same microbubble in
a system where the prodrug will only be activated after some
external stimulus. Such a stimulus may, for example, be a
tumour-specific protease as described above, or bursting of the
bubbles by external ultrasound after the desired targeting has been
achieved.
[0120] Therapeutics may easily be delivered in accordance with the
invention to diseased or necrotic areas including the heart and
vasculature in general, and to the liver, spleen and kidneys and
other regions such as the lymph system, body cavities or
gastrointestinal system.
[0121] Products according to the present invention may be used for
targeted therapeutic delivery either in vivo or in vitro. In the
latter context the products may be useful in in vitro systems such
as kits for diagnosis of different diseases or characterisation of
different components in blood or tissue samples. Similar techniques
to those used to attach certain blood components or cells to
polymer particles(e.g. monodisperse magnetic particles) in vitro to
separate them from a sample may be used in the present
invention,using the low density of the reporter units in agents of
the present invention to effect separation of the gas-containing
material by floatation and repeated washing.
[0122] Vectors which may be usefully employed in generating
multiple-specific targetable contrast agents according to the
invention include the following:
[0123] i) Antibodies, which can be used as vectors for a very wide
range of targets, and which have advantageous properties such as
very high specificity, high affinity (if desired), the possiblity
of modifying affinity according to need etc. Whether or not
antibodies will be bioactive will depend on the specific
vector/target combination. Both conventional and genetically
engineered antibodies may be employed, the latter permitting
engineering of antibodies to particular needs, e.g. as regards
affinity and specificity. The use of human antibodies may be
preferred to avoid possible immune reactions against the vector
molecule. A further useful class of antibodies comprises so-called
bispecific antibodies, i.e. antibodies having specificity for two
different target molecules in one antibody molecule. Such
antibodies may, for example, be useful in promoting formation of
bubble clusters and may also be used for various therapeutic
purposes, e.g. for carrying toxic moieties to the target. Various
aspects of bispecific antibodies are described by McGuinness, B. T.
et al. in Nat. Biotechnol. (1996) 14, 1149-1154; by George, A. J.
et al. in J. Immunol. (1994) 152, 1802-1811; by Bonardi et al. in
Cancer Res. (1993) 53, 3015-3021; and by French, R. R. et al. in
Cancer Res. (1991) 51, 2353-2361.
[0124] ii) Cell adhesion molecules, their receptors, cytokines,
growth factors, peptide hormones and pieces thereof. Such vectors
rely on normal biological protein-protein interactions with target
molecule receptors, and so in many cases will generate a biological
response on binding with the targets and thus be bioactive; this
may be a relatively insignificant concern with vectors which target
proteoglycans.
[0125] iii) Non-peptide agonists/antagonists or non-bioactive
binders of receptors for cell adhesion molecules, cytokines, growth
factors and peptide hormones. This category may include
non-bioactive vectors which will be neither agonists nor antagonist
but which may nonetheless exhibit valuable targeting ability.
[0126] iv) Oligonucleotides and modified oligonucleotides which
bind DNA or RNA through Watson-Crick or other types of
base-pairing. DNA is usually only present in extracelluar space as
a consequence of cell damage, so that such oligonucleotides, which
will usually be non-bioactive, may be useful in, for example,
targeting of necrotic regions, which are associated with many
different pathological conditions. Oligonucleotides may also be
designed to bind to specific DNA- or RNA-binding proteins, for
example transcription factors which are very often highly
overexpressed or activated in tumour cells or in activated immune
or endothelial cells. Combinatorial libraries may be used to select
oligonucleotides which bind specifically to possible target
molecules (from proteins to caffeine) and which therefore may be
employed as vectors for targeting.
[0127] v) DNA-binding drugs may behave similarly to
oligonuclotides, but may exhibit biological acitvity and/or toxic
effects if taken up by cells.
[0128] vi) Various small molecules, including bioactive compounds
known to bind to biological receptors of various kinds. Such
vectors or their targets may be used to generate non-bioactive
compounds binding to the same targets.
[0129] vii) Vector molecules may be selected from combinatorial
libraries without necessarily knowing the exact molecular target,
by functionally selecting (in vitro, ex vivo or in vivo) for
molecules binding to the region/structure to be imaged.
[0130] viii) Various small molecules, including bioactive compounds
known to bind to biological receptors of various kinds. Such
vectors or their targets may be used for generate non-bioactive
compounds binding to the same targets.
[0131] ix) Proteins or peptides which bind to glucosamino-glycan
side chains e.g. haparan sulphate, including
glucosoaminoglycan-binding portions of larger molecules, since
binding to such glucosoaminoglycans side chains does not result in
a biological response. Proteoglycans are not found on red blood
cells, thus eliminating undesirable adsorption to these cells.
[0132] Other peptide vectors and lipopeptides thereof of particular
interest for targeted ultrasound imaging are listed below:
Atherosclerotic plaque binding peptides such as YRALVDTLK,
YAKFRETLEDTRDRMY and RALVDTEFKVKQEAGAK; Thrombus binding peptides
such as NDGDFEEIPEEYLQ and GPRG; Platelet binding peptides such as
PLYKKIIKKLLES; and cholecystokinin, .alpha.-melanocyte-stimulating
hormone, heat stable enterotoxin 1, vasoactive intestinal peptide,
synthetic alpha-M2 peptide from the third heavy chain
complementarity-determining region and analogues thereof for tumor
targeting.
[0133] The following tables identify various receptors which may be
targeted by particular types of vectors and consequent areas of use
for targetable ultrasound contrast agents according to the
invention which contain such vectors.
[0134] Protein and Peptide Vectors--Antibodies
5 Vector type Receptor Comments/areas of use Ref antibodies CD34
vascular diseases in general, (general) normal vessel wall (e.g
myocardium), activated endothelium, immune cells antibodies ICAM-1
vascular diseases in general, (general) normal vessel wall (e.g
myocardium), activated endothelium, immune cells antibodies ICAM-2
vascular diseases in general, (general) normal vessel wall (e.g
myocardium), activated endothelium, immune cells antibodies ICAM-3
vascular diseases in general, (general) normal vessel wall (e.g
myocardium), activated endothelium, immune cells antibodies
E-selectin vascular diseases in general, (general) normal vessel
wall (e.g myocardium), activated endothelium, immune cells
antibodies P-selectin vascular diseases in general, (general)
normal vessel wall (e.g myocardium), activated endothelium, immune
cells antibodies PECAM vascular diseases in general, (general)
normal vessel wall (e.g myocardium), activated endothelium, immune
cells antibodies Integrins, vascular diseases in general, (general)
e.g. VLA-1, normal vessel wall (e.g VLA-2, VLA-3, myocardium),
activated VLA-4, VLA-5, endothelium, immune cells VLA-6,
.beta..sub.1.alpha..sub.7, .beta..sub.1.alpha..sub.8,
.beta..sub.1.alpha..sub.V, LFA-1, Mac-1, CD41a, etc. antibodies
GlyCAM Vessel wall in lymph nodes (general) (quite specific for
lymph nodes) antibodies MadCam 1 Vessel wall in lymph nodes
(general) (quite specific for lymph nodes) antibodies fibrin
Thrombi (general) antibodies Tissue Activated endothelium,
(general) Factor tumours antibodies Myosin Necrosis, myocardial
(general) infaction antibodies CEA Tumours (general)
(carcinoembryonal antigen) antibodies Mucins Tumours (general)
antibodies Multiple Tumours (general) drug resistance protein
antibodies Prostate Prostate cancer (general) specific antigen
antibodies Cathepsin Tumours (proteases of various (general) B
kinds are often more or less specifically overexpressed in a
variety of tumours - Cathepsin B is such a protease) antibodies
Transferrin Tumors, (general) receptor vessel wall MoAb 9.2.27
Tumours Antigen upregulated on cell growth VAP-1 Adhesion molecule
Band 3 Upregulated during phagocytic protein activity CD44 tumor
cells .beta.2- general microglobulin MHC class I general antibody
integrin tumors, angiogenisis c .alpha.v.beta.3 antibodies CD44
tumour cells a antibodies .beta.2- general b microglobulin
antibodies MHC class 1 general b a. ) Heider, K. H., M. Sproll, S.
Susani, E. Patzelt, P. Beaumier, E. Ostermann, H. Ahorn, and G. R.
Adolf. 1996. "Characterization of a high-affinity monoclonal
antibody specific for CD44v6 as candidate for immunotherapy of
squamous cell carcinomas". Cancer Immunology Immunotherapy 43:
245-253. b). I. Roitt, J. Brostoff, and D. Male. 1985. Immunology,
London: Gower Medical Publishing, p. 4.7 c.) Stromblad, S., and D.
A. Cheresh. 1996. "Integrins, angiogenesis and vascular cell
survival". Chemistry & Biology 3: 881-885.
[0135] Protein and Peptide Vectors--Cell Adhesion Molecules
etc.
6 Vector type Receptor Comments/areas of use Ref L-selectin CD34
vascular diseases in MadCAM1 general, normal vessel wall GlyCam 1
(e.g myocardium), activated endothelium, Lymph nodes Other
selectins carbohydrate vascular diseases in ligands general, normal
vessel wall (sialyl Lewis x) (e.g myocardium), activated heparan
sulfate endothelium RGD-peptides integrins angiogenisis PECAM
PECAM, Endothelium, and other Cells in immune system Integrins,
Laminin, Endothelium, e.g. VLA-1, VLA-2, collagen, Vessel wall
VLA-3, VLA-4, fibronectin, etc. VLA-5, VLA-6, VCAM-1,
.beta..sub.1.alpha..sub.7, .beta..sub.1.alpha..sub.8,
thrombospondin, .beta..sub.1.alpha..sub.V, LFA-1, Mac-1,
vitronectin CD41a, etc. etc. Integrin Integrins, Cells in immune
system receptors, e.g. VLA-1, vessel wall e.g. Laminin, VLA-2,
VLA-3, etc. collagen, VLA-4, VLA-5, fibronectin, VLA-6,
.beta..sub.1.alpha..sub.7, VCAM-1, .beta..sub.1.alpha..sub.8,
.beta..sub.1.alpha..sub.V, thrombospondin, LFA-1, Mac-1,
vitronectin CD41a, etc. etc. Nerve cell proteoglycans angiogenesis
c adhesion N-CAM molecule (N-CAM) (homophilic) RGD-peptides
integrins
[0136] Vectors Comprising Cytokines/Growth Factors/Peptide Hormones
and Fragments Thereof
7 Vector type Receptor Comments/areas of use Ref Epidermal growth
EGF-receptor or Tumours factor related receptors Nerve growth
NGF-receptor Tumours factor Somatostatin ST-receptor Tumours
Endothelin Endothelin- Vessel wall receptor Interleukin-1
IL-1-receptor Inflammation, activated cells of different kinds
Interleukin-2 IL-2-receptor Inflammation, activated cells of
different kinds Chemokines (ca. Chemokine Inflammation 20 different
receptors, cytokines partly proteoglycans sharing receptors) Tumour
necrosis TNF-receptors Inflammation factor Parathyroid
PTH-receptors Bone diseases hormone Kidney diseases Bone
BMP-receptors Bone Diseases Morphogenetic Protein Calcitonin
CT-receptors Bone diseases Colony Corresponding Endothelium
stimulating specific factors (G-CSF, receptors, GM-CSF, M-CSF,
proteoglycans IL-3) Insulin like IGF-I receptor Tumours, growth
factor I other growing tissues Atrial ANF-receptors Kidney,
Natriuretic vessel wall Factor Vasopressin Vasopressin Kidney,
receptor vessel wall VEGF VEGF-receptor Endothelium, regions of
angiogenesis Fibroblast FGF-receptors, Endothelium growth factors
Proteoglycans Angiogenesis Schwann cell proteoglycans growth factor
specific receptors
[0137] Miscellaneous Protein and Peptide Vectors
8 Vector type Receptor Comments/areas of use Ref Streptavidin
Kidney Kidney diseases Bacterial Fibronectin Vessel wall
fibronectin- binding proteins Fc-part of Fc-receptors Monocytes
antibodies macrophages liver Transferrin transferrin- Tumours
receptor vessel walls Streptokinase/ thrombi thrombi tissue
plasminogen activator Plasminogen, Fibrin Thrombi, plasmin tumours
Mast cell proteoglycans proteinases Elastase proteoglycans
Lipoprotein proteoglycans lipase Coagulation proteoglycans enzymes
Extracellular proteoglycans superoxide dismutase Heparin cofactor
proteoglycans II Retinal survival proteoglycans factor specific
receptors Heparin-binding proteoglycans brain mitogen specific
receptors Apolipoprotein, proteoglycans e.g. specific
apolipoprotein B receptors (e.g., LDL receptor) Apolipoprotein E
LDL receptor proteoglycans Adhesion- proteoglycans promoting
proteins, e.g. Purpurin Viral coat proteoglycans proteins, e.g.
HIV, Herpes Microbial "Antigen 85" fibronectin, collagen, adhesins
complex of fibrinogen, vitronectin, mycobacteria heparan sulfate
.beta.-amyloid proteoglycans .beta.-amyloid accumulates in
precursor Alzheimer's disease Tenascin, heparan sulfate, e.g.
tenascin C integrins
[0138] Vectors Comprising Non-peptide Agonists/Antagonists of
Cytokines/Growth Factors/Peptide Hormones/Cell Adhesion
Molecules
9 Vector type Receptor Comments/areas of use Ref Endothelin
Endothelin Vessel wall antagonist receptor Desmopressin Vasopressin
Kidney (vasopressin receptor Vessel wall analogue) Demoxytocin
Oxytocin Reproductive organs, (oxytocin Receptor Mammary glands,
analogue) Brain Angiotensin II Angiotensin II Vessel wall receptor
receptors brain antagonists adrenal gland CV-11974, TCV-116
non-peptide RGD- integrins Cells in immune system analogues vessel
wall etc.
[0139] Vectors Comprising Anti-angiogenic Factors
10 Vector type Target Comments/areas of use Ref Angiostatin EC of
tumors plasminogen fragment K cartilage-derived EC of tumors J
inhibitor .beta.-Cyclodextrin tumors, C tetradecasulfate
inflammation fumagillin and analogs tumors, E inflammation
Interferon-.alpha. EC of tumors K Interferon-.gamma. EC of tumors E
interleukin-12 EC of tumors E linomide tumors, A inflammation
medroxyprogesterone EC of tumors K metalloproteinase EC of tumors K
inhibitors pentosan polysulfate EC of tumors K platelet factor 4 EC
of tumors M Somatostatin EC of tumors K Suramin EC of tumors K
Taxol EC of tumors K thalidomide EC of tumors K Thrombospondin EC
of tumors K
[0140] Vectors Comprising Angiogenic Factors
11 Comments/areas Vector type Target of use Ref acidic fibroblast
EC of tumors K growth factor adenosine EC of tumors K Angiogenin EC
of tumors K Angiotensin II EC of tumors K basement membrane tumors
e.g., tenascin, M components collagen IV basic fibroblast EC of
tumors K growth factor Bradykinin EC of tumors K Calcitonin gene-
EC of tumors K related peptide epidermal growth EC of tumors K
factor Fibrin tumors K Fibrinogen tumors K Heparin EC of tumors K
histamine EC of tumors K hyaluronic acid EC of tumors K or
fragments thereof Interleukin-1.alpha. EC of tumors K laminin,
laminin EC of tumors K fragments nicotinamide EC of tumors K
platelet acti- EC of tumors K vating factor Platelet-derived EC of
tumors K endothelial growth factor prostaglandins EC of tumors K
E1, E2 spermine EC of tumors K spermine EC of tumors K Substance P
EC of tumors K transforming EC of tumors K growth factor-.alpha.
transforming EC of tumors K growth factor-.beta. Tumor necrosis
factor-.alpha. EC of tumors K vascular endo- EC of tumors K thelial
growth factor/vascular permeability factor vitronectin A
[0141] Vector Molecules Other than Recognized Angiogenetic Factors
with Known Affinity for Receptors Associated with Angiogenesis
12 Vector type Target Comments/areas of use Ref angiopoietin
tumors, B inflammation .alpha..sub.2-antiplasmin tumors,
inflammation combinatorial tumors, for instance: compounds
libraries, compounds inflammation that bind to basement from
membrane after degradation endoglin tumors, D inflammation
endosialin tumors, D inflammation endostatin [collagen tumors, M
fragment] inflammation Factor VII related tumors, D antigen
inflammation fibrinopeptides tumors, ZC inflammation fibroblast
growth tumors, E factor, basic inflammation hepatocyte growth
tumors, I factor inflammation insulin-like growth tumors, R factor
inflammation interleukins tumors, e.g.,: IL-8 I inflammation
leukemia inhibitory tumors, A factor inflammation metalloproteinase
tumors, e.g., batimastat E inhibitors inflammation Monoclonal
antibodies tumors, for instance: to inflammation angiogenetic
factors or their receptors, or to components of the fibrinolytic
system peptides, for instance tumors, B, Q cyclic RGD.sub.DFV
inflammation placental growth factor tumors, J inflammation
placental tumors, E proliferin-related inflammation protein
plasminogen tumors, M inflammation plasminogen activators tumors, D
inflammation plasminogen activator tumors, U, V inhibitors
inflammation platelet activating tumors, inhibitors of angiogenesis
A factor antagonists inflammation platelet-derived growth tumors, E
factor inflammation pleiotropin tumors, ZA inflammation proliferin
tumors, E inflammation proliferin related tumors, E protein
inflammation selectins tumors, e.g., E-selectin D inflammation
SPARC tumors, M inflammation snake venoms tumors, Q
(RGD-containing) inflammation Tissue inhibitor of tumors, eg,,
TIMP-2 U metalloproteinases inflammation thrombin tumors, H
inflammation thrombin-receptor-activating tumors, H
tetradecapeptide inflammation thymidine phosphorylase tumors, D
inflammation tumor growth factor tumors, ZA inflammation
[0142] Receptors/Targets Associated with Angiogenesis
13 Vector type Target Comments/areas of use Ref biglycan tumors,
dermatan sulfate X inflammation proteoglycan CD34 tumors, L
inflammation CD44 tumors, F inflammation collagen type I, IV,
tumors, A VI, VIII inflammation decorin tumors, dermatan sulfate Y
inflammation proteoglycan dermatan sulfate tumors, X proteoglycans
inflammation endothelin tumors, G inflammation endothelin receptors
tumors, G inflammation fibronectin tumors P Flk-1/KDR, Flt-4
tumors, VEGF receptor D inflammation FLT-1 (fms-like tumors, VEGF-A
receptor O tyrosine kinase) inflammation heparan sulfate tumors, P
inflammation hepatocyte growth tumors, I factor receptor (c-met)
inflammation insulin-like growth tumors, R factor/mannose-6-
inflammation phosphate receptor integrins: Tumors, D, .beta..sub.3
and .beta..sub.5, inflammation P integrin
.alpha..sub.v.beta..sub.3, integrin .alpha..sub.6.beta..sub.1,
laminin receptor integrins .alpha..sub.6, integrins .beta..sub.1,
integrin .alpha..sub.2.beta..sub.1, integrin
.alpha..sub.v.beta..sub.3, integrin .alpha..sub.5 subunit of the
fibronectin receptor integrin .alpha..sub.V.beta..sub.5, fibrin
receptors. Intercellular adhesion tumors, P molecule-1 and -2
inflammation Jagged gene product tumors, T inflammation Ly-6
tumors, a lymphocyte activation N inflammation protein matrix
tumors, D metalloproteinases inflammation MHC class II tumors,
inflammation Notch gene product tumors, T inflammation Osteopontin
tumors Z PECAM tumors, alias CD31 P inflammation plasminogen
activator tumors, ZC receptor inflammation platelet-derived growth
tumors, E factor receptors inflammation Selectins: E-, P- tumors, D
inflammation Sialyl Lewis-X tumors, blood group antigen M
inflammation stress proteins: tumors, molecular chaperones glucose
regulated, heat inflammation shock families and others syndecan
tumors, T inflammation thrombospondin tumors, M inflammation TIE
receptors tumors, tyrosine kinases with Ig- E inflammation and
EGF-Iike domains tissue factor tumors, Z inflammation tissue
inhibitor of tumors, e.g., TIMP-2 U metalloproteinases inflammation
transforming growth tumors, E factor receptor inflammation
urokinase-type tumors, D plasminogen activator inflammation
receptor Vascular cellular tumors, D adhesion molecule inflammation
(VCAM) Vascular endothelial tumors, growth factor related
inflammation protein Vascular endothelial tumors, K growth factor-A
inflammation receptor von Willebrand factor- tumors, L related
antigen inflammation
[0143] Oligonucleotide Vectors
14 Vector type Receptor Comments/areas of use Ref Oligonucleotides
DNA made Tumours complementary to available by Myocardial
infarction repeated necrosis All other diseases that sequences,
e.g. involves necrosis genes for ribosomal RNA, Alu-sequences
Oligonucleotides DNA made Tumours complementary to available by
disease-specific necrosis in a mutations (e.g. region of the
mutated relevant disease oncogenes). Oligonucleotides DNA of
infective Viral or bacterial complementary to agent infections DNA
of infecting agent. Triple or As in above As in above examples
quadruple-helix examples forming Oligonucleotides Oligonucleotides
DNA-binding Tumours with recognition protein, e.g. Activated
endothelium sequence for transcription Activated immune cells
DNA-or RNA- factors (often binding proteins overexpressed/
activated in tumours or activated endothelium/ immune cells
[0144] Modified Oligonucleotide Vectors
15 Vector type Receptor Comments/areas of use Ref Phosphorothioate
As for As for unmodified oligos oligos unmodified oligos
2'-O-methyl As for As for unmodified oligos substituted unmodified
oligos oligos circular oligos As for As for unmodified oligos
unmodified oligos oligos As for As for unmodified oligos containing
unmodified hairpin oligos structure to decrease degradation oligos
with As for As for unmodified oligos terminal unmodified
phosphorothioate oligos 2'-fluoro oligos As for As for unmodified
oligos unmodified oligos 2'-amino oligos As for As for unmodified
oligos unmodified oligos DNA-binding As for Increased binding
affinity drugs conjugated unmodified as compared to pure oligos to
oligos (for oligos examples, see below) Peptide Nucleic As for
Increased binding affinity Acids (PNAs, unmodified and stability
compared to oligonucleotidss oligos standard oligos. with a peptide
backbone)
[0145] Nucleoside and Nucleotide Vectors
16 Vector type Receptor Comments/areas of use Ref Adenosine or
Adenosine Vessel wall analogues receptors Heart ADP, UDP, UTP
Various Many tissues, e.g. brain, and others nucleotide spinal
cord, kidney, spleen receptors
[0146] Receptors Comprising DNA-binding Drugs
17 Vector type Receptor Comments/areas of use Ref acridine DNA made
Tumours, derivatives available by Myocardial infarction and
distamycin necrosis all other diseases involving netropsin necrosis
or other processes actinomycin D liberating DNA from cells
echinomycin bleomycin etc.
[0147] Receptors Comprising Protease Substrates
18 Vector type Receptor Comments/areas of use Ref Peptidic or non-
Cathepsin B Tumours, a variety of which peptidic may more or less
specifically substrates overexpress proteases of various kinds,
e.g. Cathepsin B
[0148] Receptors Comprising Protease Inhibitors
19 Vector type Receptor Comments/areas of use Ref Peptidic or non-
Cathepsin B Tumours, a variety of which peptidic may more or less
specifically inhibitors overexpress proteases of e.g. N-acetyl-
various kinds, e.g. Leu-Leu- Cathepsin B norleucinal bestatin
Aminopeptidases Tumours, ([(2S,3R)-3- e.g. on cell surfaces
Amino-2-hydroxy- 4-phenyl- butanoyl]-L- leucine hydrochloride)
Pefabloc (4-(2- Serine proteases Tumours, aminoethyl)- vessel wall
benzenesulfonyl etc. fluoride hydrochloride) Commercially
Angiotensin Endothelial cells available converting inhibitors
enzyme e.g. kaptopril enalapril ricionopril Low specificity
Coagulation Vessel wall injury, non-peptidic factors tumours,
compounds etc. Protease nexins proteoglycans (extracellular
protease inhibitors) Antithrombin proteoglycans, Coagulation
actors
[0149] Vectors from Combinatorial Libraries
20 Vector type Receptor Comments/areas of use Ref Antibodies with
Any of above Any diseased or normal structure targets - or may
structure of interest, e.g. determined be unknown when thrombi,
tumours or walls of during make functional myocardial vessels
generation selection of process vector binding to chosen diseased
structure Peptides with Any of above Any diseased or normal
sequence targets - or may structure of interest, e.g. determined be
unknown when thrombi, tumours or walls of during make functional
myocardial vessels generation selection of process vector binding
to chosen diseased structure Oligonucleotides Any of above Any
diseased or normal with sequence targets - or may structure of
interest, e.g. determined be unknown when thrombi, tumours or walls
of during make functional myocardial vessels generation selection
of process vector binding to chosen diseased structure
Modifications of Any of above Any diseased or normal oligos
obtained targets - or may structure of interest, e.g. as above be
unknown when thrombi, tumours or walls of make functional
myocardial vessels selection of vector binding to chosen diseased
structure Other chemicals Any of above Any diseased or normal with
structure targets - or may structure of interest, e.g. determined
be unknown when thrombi, tumours or walls of during make functional
myocardial vessels generation selection of process vector binding
to chosen diseased structure
[0150] Carbohydrate Vectors
21 Vector type Receptor Comments/areas of use Ref neo- macrophages
general activation/ glycoproteins inflammation oligosaccharides
Asialo- liver with terminal glycoprotein galactose receptor
Hyaluronan aggrecan (a proteoglycan) "link proteins" cell-surface
receptors: CD44 Mannose Blood brain barrier, Brain tumours and
other diseases causing changes in BBB Bacterial Blood brain
barrier, glycopeptides Brain tumours and other diseases causing
changes in BBB
[0151] Lipid Vectors
22 Vector type Receptor Comments/areas of use Ref LDL-like lipids
LDL-receptor Atherosclerosis
[0152] Small Molecule Vectors
23 Vector type Receptor Comments/areas of use Ref Adrenalin
Corresponding receptors Betablockers Adrenergic beta- Myocardium
for beta-1 receptors blockers Alpha-blockers Adrenergic Vessel wall
alpha-receptors benzodiazepines serotonin- Serotonin- analogues
receptors anti-histamines Histamine- Vessel wall receptors
Acetyl-choline ACh-receptors receptor antagonists verapamil
Ca.sup.2+-channel Heart muscle blocker nifedipin Ca.sup.2+-channel
Heart muscle blocker Amiloride Na.sup.+/H.sup.+-exchanger Blocks
this exchanges in kidney and is generally upregulated in cells
stimulated by growth factors. Digitalis Na.sup.+/K.sup.+-ATP-ases
myocardium glycosides peripheral vasculature, central nervous
system Thromboxae/ Thromboxane/ Vessel wall, Prostaglandin
prostaglandin Endothelium receptor receptors antagonists or
agonists Glutathione Glutathione- Lung, receptors Brain
Leukotriene- receptors Biotin biotin transport protein on cell
surface Folate folate transport Tumours protein on cell surface
Riboflavin riboflavin transport protein on cell surface
Methotrexate folate transport protein on cell surface chlorambucil
general transport mechanisms
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[0184] Representative examples of drugs useful in accordance with
the invention include: abamectin, abundiazole, acaprazine,
acabrose, acebrochol, aceburic acid, acebutolol, acecainide,
acecarbromal, aceclidine, ac clof nac, acedapsone, acediasulfone,
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phosphanilate, chlorindanol, chlorisondamine chloride,
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chloronaphazine, chloroazodin, chlorobutanol, chlorocresol,
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cholic acid, choline chloride, choline glycerophosphate,
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cicloprolol, ciclosidomine, ciclotizolam, ciclotropium bromide,
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cintazone, cintriamide, cinperone, ciprafamide, ciprafazone,
ciprefadol, ciprocinonide, ciprofibrate, ciprofloxacin, cipropride,
ciproquazone, ciprostene, ciramadol, cirazoline, cisapride,
cisconazole, cismadinone, cisplatin, cistinexine, citalopram,
citatepine, citenamide, citenazone, citicoline, citiolone,
clamidoxic acid, clamoxyquin, clanfenur, clanobutin, clantifen,
clarithromycin, clavulanic acid, clazolam, clazolimine, clazuril,
clebopride, clefamide, clemastine, clemeprol, clemizole,
clenbuterol, clenpirin, cletoquine, clibucaine, clidafidine,
clidanac, clidinum bromide, climazolam, climbazole, climiqualine,
clindamycin, clindamycin palmitate, clindamycin phosphate,
clinofibrate, clinolamide, cliquinol, clioxanide, clipoxamine,
cliprofen, clobazam, clobenoside, clobenzepam, clobenzorex,
clobenztropine, clobetasol propionate, clobetasone butyrate,
clobutinol, clobuzarit, clocanfamide, clocapramine, clociguanil,
clocinizine, clocortolone acetate, clocortolone pivalate,
clocoumarol, clodacaine, clodanolene, clodazon, clodoxopone,
clodronic acid, clofazimine, clofenamic acid, clofenamide,
clofenciclan, clofenetamine, clofenoxyde, clofenvinfos,
clofeverine, clofexamide, clofezone, clofibrate, clofibric acid,
clofibride, clofilium phosphate, cloflucarban, clofoctol, cloforex,
clofurac, clogestone acetate, cloguanamil, clomacran, clomegestone
acetate, clometacin, clometherone, clomethiazole, clometocillin,
clomifenoxide, clominorex, clomiphene, clomipramine,
clomocycline,
clomoxir, clonazepam, clonazoline, clonidine, clonitazene,
clonitrate, clonixeril, clonixin, clopamide, clopenthixol,
cloperastine, cloperidone, clopidogrel, clopidol, clopimozide,
clopipazan, clopirac, cloponone, cloprednol, cloprostenol,
cloprothiazole, cloquinate, cloquinozine, cloracetadol, cloranolol,
clorazepate, clorethate, clorexolone, clorgiline, cloricromen,
cloridarol, clorindanic acid, clorindione, clormecaine,
cloroperone, clorophene, cloroqualone, clorotepine, clorprenaline,
clorsulon, clortermine, closantel, closiramine, clostebol,
clothiapine, clothixamide, clotiazepam, cloticasone propionate,
clotioxone, clotrimazole, clovoxamine, cloxacepride, cloxacillin,
cloxacillin benzathine, cloxazolam, cloxestradiol, cloximate,
cloxotestosterone, cloxypendyl, cloxyquin, clozapine, cobamide,
cocaine, cocarboxylase, codeine, codoxime, cofisatin, cogazocine,
colchicine, colestolone, colfenamate, colforasin, colterol,
conessine, conorphone, copper gluconate, cormethasone acetate,
corticosterone, cortisone acetate, cortisuzol, cortivazol,
cortodoxone, cotarnine chloride, cotinine, cotriptyline, coumaphos,
coumazoline, coumermycin, coumetarol, creatinolfosfate, crisnatol,
croconazole, cromakalim, cromitrile, cromolyn, cropropamide,
crospovidone, crotamiton, crotetamide, crotoniazide, crufomate,
cuprimyxin, cuproxoline, cyacetacide, cyamemazine, cyanocobalamine,
cyclacillin, cyclandelate, cyclarbamate, cyclazocine, cyclazodone,
cyclexanone, cyclindole, cycliramine, cyclizine, cyclobarbital,
cyclobendazole, cyclobenzaprine, cyclobutoic acid, cyclobutyrol,
cyclofenil, cycloguanil, cloheximide, cycloleucine, cyclomenol,
cyclomethicone, cyclomethycaine, cyclopentamine, cyclopenthiazide,
cyclopentolate, cyclopenazine, cyclophosphamide, cyclopregnol,
cyclopyrronium bromide, cycloserine, cyclosporine, cyclothiazide,
cyclovalone, cycotiamine, cycrimine, cyheptamid, cyheptropine,
cynarine, cypenamine, cypothrin, cyprazepam, cyprenophine,
cyprodenate, cyproheptadine, cyprolidol, cyproquinate, cyproterone
acetate, cyproximide, cystine, cytarabine, dacarbazine, dacemazine,
dacisteine, dacinomycin, dacuronium bromide, dagapamil,
dalbraminol, daledalin, daltroban, dametralast, damotepine,
danazol, danitracen, danosteine, danthron, dantrolene, dapiprazole,
dapsone, daptomycin, darenzepine, darodipine, datelliptium
chloride, dunorubicin, dazadrol, dazepinil, dazidamine, dazmegrel,
dazolicine, dazopride, dazoquinast, dacoxiben, deanol aceglumate,
deanol acetaminobenzoate, deazauridine, deboxamet, debrisoquin,
decamethonium bromide, decimemide, decitropine, declaben,
declenperone, decloxizine, decominol, decoquinate, deditonium
bromide, deferoxamine, deflazacort, defosfamide, dehydroacetic
acid, dehydroemetine, dehydro-7-methyltestosterone, delanterone,
delapril, delergotrile, delfantrine, delmadinone acetate,
delmetacin, delmopinol, delorazepam, deloxone, delprostenate,
dembrexine, demecarium bromide, demeclocycline, demecolcine,
demecycline, demegestone, demelverine, demexiptiline, democonazole,
demoxepam, denaverine, denbufylline, denipride, denopamine,
denpidazone, denzimol, deoxyspergualin, depramine, deprodone,
deprostil, deptropine, derpanicate, desacetylcolchicine tartrate,
desaspidin, desiclovir, descinolone acetonide, deserpidine,
desipramine, deslanoside, desmethylcolchicine,
desmethylmisonidazole, desmethylmoramide, desocriptine,
desogestrel, desomorphine, desonide, desoximetasone,
desoxycorticosterone acetate, desoxycorticosterone pivalate,
desoxypyridoxine, detajmium bitartrate, detanosal, deterenol,
detomidine, detorubicin, detrothronine, devapamil, dexamethasone,
dexamethasone, acefurate, dexamethasone acetate, dexamethasone
dipropionate, dexamethasone phosphate, dexamisole,
dexbrompheniramine, dexchlorpheniramine, dexclamol, dexetimide,
dexetozolin, dexfenfluramine, deximafen, dexindoprofen,
dexivacaine, dexlofexidine, dexmedetomidine, dexoxadrol,
dexpanthenol, dexpropranolol, dexproxibutene, dexecoverine,
dextilidine, dextroamphetamine, dextrofemine, dextromethorphan,
dextromoramide, dextrorphan, dextrothyroxine, dezaguanine,
dezocine, diacerein, diacetamate, diacetolol, diacetylmorphine,
diamfenetide, diaminomethylphenazinium chloride, diamocaine,
diampromide, diamthazole, dianhydrogalactitol, diapamide,
diarbarone, diathymosulfone, diatrizoic acid, diaveridine,
diazepam, diaziquone, diazoacetylglycine hydrazide, diazouracil,
diazoxide, dibekacin, dibemethine, dibenamine, dibenzepin,
dibrompropamidine, dibromsalan, dibrospidium chloride, dibucaine,
dibuprol, dibupyrone, dibusadol, dicarbine, dicarfen, dichlorallyl
lawsone, dichlorisone acetate, dichlormezanone,
dichlorofluormethane, dichloromethotrexate, dichlorophen,
dichlorophenarsine, dichlorotetrafluoroethane, dichloroxylenol,
dichlorphenamide, dichlorvos, diciferron, dicirenone, diclazuril,
diclofenac, diclofensine, diclofurime, diclometide, diclonixin,
dicloxacillin, dicobalt edetate, dicolinium iodide, dicresulene,
dicumarol, dicyclomine, didemnin, dideoxycytidine, didrovaltrate,
dieldrin, dienestrol, dienogest, diethadione, diethazine,
diethylpropion, diethylstilbestrol, diethylstilbestrol diphosphate,
diethylstilbestrol dipropionate, diethylthiambutene,
diethyltoluamide, dietifen, difebarbamate, difemerine, difemetorex,
difenamizole, difencloxazine, difenoximide, difenoxin, difetarsone,
difeterol, diflorasone diacetate, difloxacin, difluanine,
diflucortolone, diflurcortolone pivalate, diflumidone, diflunisal,
difluprednate, diftalone, digalloyl trioleate, digitoxin, digoxin,
dihexyverine, dihydralazine, dihydroazacytidine, dihydroergotamine,
dihydrolenperone, dihydrostreptomycin, dihydrotachysterol,
dihydroxyfluoroprogestrone, diisopromine, diisopropanolamine,
dilazep, dilevalol, dilmefone, diloxanide, diltiazem,
dimabefylline, dimecamine, dimecolonium iodide, dimecrotic acid,
dimefadane, dimefline, dimelazine, dimemorfan, dimenhydrinate,
dimenoxadol, dimeheptanol, dimepranol, dimepregnen, dimeprozan,
dimercaprol, dimesna, dimesone, dimetacrine, dimetamfetamine,
dimethadione, dimethaminostyrylquinoline, dimethazan, dimethindene,
dimethiodal, dimethisoquin, dimethisterone, dimetholizine,
dimethoxanate, dimethylhydroxytestosterone,
dimethylnorandrostadienone, dimethylnortestosterone,
dimethylstilbestrol, dimethyl, dimethylthiambutene,
dimethyltubocurarinium chloride, dimetipirium bromide, dimetofrine,
dimetridazole, diminazene, dimoxamine, dimoxaprost, dimoxyline,
dimpylate, dinaline, dinazafoqe, diniprofylline, dinitolmide,
dinoprost, dinoprostone, dinsed, diosmin, dioxadilol, dioxadrol,
dioxamate, dioxaphetyl butyrate, dioxethedrin, dioxifedrine,
dioxybenzone, dipenine bromide, diperodon, diphemanil
methylsulfate, diphenadione, diphenan, diphenhydramine, diphendiol,
diphenoxylate, diphenylpraline, diphoxazide, dipipanone,
dipipoverine, dipiverin, diprafenone, diprenorphine, diprobutine,
diprofene, diprogulic acid, diproleandomycin, diproqualone,
diproteverine, diprotriozate, diproxadol, dipyridamole,
dipyrithione, dipyrocetyl, dipyrone, dirithromycin, disobutamide,
disofenin, disogluside, disopyramide, disoxaril, distigmine
bromide, disulergine, disulfamide, disulfiram, disuprazole,
ditazole, ditercalinium chloride, dithiazanine iodide, ditiocarb,
ditiomustine, ditolamide, ditophal, divabuterol, dixanthogen,
dizatrifone, dizocilpine, dobupride, dobutamine, docarpamine,
doconazole, docusate, doliracetam, domazoline, domiodol, domiphen
bromide, domipizone, domoprednate, domoxin, domperidone, don,
donetidine, dopamantine, dopamine, dopexamine, dopropidil,
doqualast, dorastine, doreptide, dosergoside, dotarizine,
dotefonium bromide, dothiepin, doxacurium chloride, doxaminol,
doxapram, doxaprost, doxazosin, doxefazepam, doxenitoin, doxepin,
doxibetasol, doxifluridine, doxofylline, doxorubicin, doxpicomine,
doxycycline, doxylamine, dramedilol, draquinolol, deazidox,
dribendazole, drindene, drobuline, drocinonide, droclidinium
bromide, drocode, drofenine, droloxifene, drometrizole,
dromostanolone, dromostanolone propionate, dronabinol, dropempine,
droperidol, droprenilamine, dropropizine, drotaverine, drotebanol,
droxacin, droxicainide, droxicam, droxidopa, droxypropine,
dulofibrate, dulozafone, duometacin, duoperone, dupracetam,
durapatite, dyclonine, dydrogesterone, dymanthine, dyphylline,
ebastine, ebrotidine, ebselen, ecastolol, echinomycin,
echothiophate iodide, ecipramidil, eclanamine, eclazolast,
econazole, ectylurea, edelfosine, edetic acid, edetol, edifolone,
edogestrone, edoxudine, edrophonicum chloride, efaroxan, efetozole,
eflornithine, efloxate, efrotomycin, elantrine, elanzepine,
elderfield's pyrimidine mustard, elfazepam, ellagic acid,
elliptinium acetate, elmustine, elnadipine, eltenac, eltoprazine,
elucaine, elziverine, embramine, embutramide, emepronium bromide,
emetine, emiglitate, emilium tosylate, emopanil, emorfazone,
emylcamate, enalapril, enalaprilat, enbucrilate, encainide,
enciprazine, enclomiphene, encyprate, endomide, endralazine,
endrysone, enefexine, enestebol, enfenamic acid, enflurane,
eniclobrate, enilconazole, enilospirone, enisoprost, enocitabine,
enolicam, enoxacin, enoxamast, enoximone, enoxolone, eniprazole,
eniproline, enprazepine, enprofylline, enpromate, enprostil,
enrofloxacin, entsufon sodium, enviomycin, enviradene, epalretat,
epanolol, eperisone, ephedrine, epicainide, epicillin, epicriptine,
epiestriol, epimestrol, epinastine, epinephrine, epinephryl borate,
epipropidine, epirizole, epiroprim, epirubicin, epithiazide,
epitiostanol, epoprostenol, epostane, eprazinone, eprovafen,
eproxindine, eprozinol, epsiprantel, eptaloprost, eptazocine,
equilin, erdosteine, ergocalciferol, ergoloid mesylates,
ergonovine, ergosterol, ergotamine, ericolol, erizepine,
erocainide, erythrityl tetranitrate, erythromycin, erythromycin
acistrate, erythromycin ethylsuccinate, erythromycin propionate,
erythrosine, esaprazole, esculamine, eseridine, esflurbiprofen,
esmolol, esorubicin, esproquin, estazolam, estradiol, estradiol
benzoate, estradiol cypionate, estradiol dipropionate, estradiol
enanthate, estradiol undecylate, estradiol valerate, estramustine,
estramustine phosphate, estrapronicate, estrazinol, estriol,
estrofurate, estrone, estrone hydrogen sulfate, estropipate,
esuprone, etabenzarone, etacepride, etafedrine, etafenone,
etamestrol, etamiline, etamiphyllin, etamocycline, etanidazole,
etanterol, etaqualone, etasuline, etazepine, etazolate, etebenecid,
eterobarb, etersalate, ethacridine, ethacrynic acid, ethambutol,
ethamivan, ethamsylate, ethanolamine oleate, ethaverine,
ethchlorvynol, ethenzamide, ethazide, ethidium chloride,
ethinamate, ethinyl estradiol, ethiofos, ethionamide, ethsterone,
ethoheptazine, ethomoxane, ethonam, ethopropazine, ethosuximide,
ethotoin, ethoxazene, ethoxazorutoside, ethoxzolamide,
ethyybenztropine, ethyl biscoumacetate, ethyl carfluzepate, ethyl
cartrizoate, ethyl dibunate, ethyl dirazepate, ethylenediamine,
ethylestrenol, ethylhydrocupreine, ethyl loflazepate,
ethylmethylthiambutene, ethylmorphine, 9-ethyl-6-mercaptopurine,
ethyl nitrite, ethylnorepinephrine, ethylparaben, ethylphenacemide,
ethylstibamine, ethynerone, ethynodiol diacetate, ethypicone,
etibendazole, eticlopride, eticyclidine, etidocaine, etidronic
acid, etifelmine, etifenin, etifoxine, etilamfetamine, etilefrine,
etilefrin pivalate, etintidine, etiochlanolone, etipirium iodide,
etiproston, etiracetam, etiroxate, etisazole, etisomicin,
etisulergine, etizolam, etocarlide, etocrylene, etodolac,
etodroxzine, etofamide, etofenamate, etofenprox, etofibrate,
etoformin, etofuradine, etofylline, etoglucid, etolorex,
etolotifen, etoloxamine, etomidate, etomidoline, etomoxir,
etonitazene, etoperidone, etoposide, etoprindole, etoprine,
etorphine, etosalamide, etoxadrol, etoxeridine, etozolin,
etrabamine, etretinate, etryptamine, etymemazine, eucalyptol,
eucatropine, eugenol, euprocin, evandamine, Evans blue, exalamide,
exametazine, exaprolol, exepanol, exifone, exiproben, falintolol,
falipamil, famiraprinium chloride, famotidine, famotine,
famiprofazone, fanetizole, fantridone, fazadinium bromide,
fazaribine, febantel, febarbamate, februpol, febuverine, feclemine,
feclobuzone, fedrilate, felbamate, felbinac, felipyrine,
felodipine, femoxetine, fenabutene, fenacetinol, fenaclon,
fenadiazole, fenaptic acid, fenalamide, fenalcomine, fenamifuril,
penamole, fenaperone, fenbendazole, fenbencillin, fenbufen,
fenbutrazate, fencamfamine, fencibutirol, fenclexonium
metilsulfate, fenclofen4c, fenclonine, fenclorac, fenlozic acid,
fendiline, fendosal, feneritrol, fenestrel, fenethazine,
fenethylline, fenetradil, fenflumizole, fenfluramine, fenfluthrin,
fengabine, fenharmane, fenimide, feniodium chloride, fenipentol,
fenirofibrate, fenisorex, fenmetozole, fenmetramide, fenobam,
fenocinol, fenoctimine, fenofibrate, fenoldopam, fenoprofen,
fenoterol, fenoverine, fenoxazoline, fenoxedil, fenozolone,
fenpentadiol, fenperate, fenipalone, fenipramide, feniprane,
fenpiverinium bromide, fenprinast, fenproporex, fenprostalene,
fenquizone, fenretinide, fenspiride, fentanyl, fentiazac,
fenticlor, fenticonazole, fentonium bromide, fenyripol, fepentolic
acid, fepitrizol, fepradinol, feprazone, fepromide, feprosidnine,
ferriclate calcium, ferrotrenine, ferrous fumarate, ferrous
gluconate, fetoxylate, fexicaine, fexinidazole, fezatione,
fezolamine, fiacitabine, fibracillin, filenadol, filipin, fifexide,
flamenol, flavamine, flavodic acid, flavodil, flavoneactic acid,
flavoxate, flazalone, flecainide, flerobuterol, fleroxacin,
flesinoxan, flestolol, fletazepam, floctafenine, flomoxef,
flopropione, florantyrone, flordipine, floredil, florfenicol,
florifenine, flosequinan, flotrenizine, floverine, floxacillin,
floxacrine, floxuridine, fluacizine, flualamide, fluanisone,
fluazacort, flubanilate, flubendazole, flubepride, flucabril,
flucetorex, flucindole, fluciprazine, flucloronide, fluconazole,
flucrylate, flucytosine, fludalanine, fludarabine phosphate,
fludazonium chloride, fludiazepam, fludorex, fludoxopone,
fludrocortisone acetate, flufenamic acid, flufenisal, flufosal,
flufylline, fluindarol, fluindione, flumazenil, flumecinol,
flumedroxone-17-acetate, flumequine, flumeridone, flumethasone,
flumethasone pivalate, flumethiazide, flumetramide, flumexadol,
flumezapine, fluminorex, flumizole, flumoxonide, flunamine,
flunarizine, flunidazole, flunisolide, flunisolide acetate,
flunitrazepan, flunixin, flunoprost, flunoxaprofen, fluocinolone
acetonide, fluocinonide, flourcortin butyrate, fluocortolone,
fluocortolone caproate, fluorescein, fluoresone, fluoroadenosine,
3-fluoroandrostanol, fluorodopane, fluorohydroxyandrosterone,
fluorometholone, fluorometholone acetate, fluorosalan,
6-fluorotestosterone propionate, fluorouracil,
9-fluoroxotestenololactone- , 9-fluoroxotestololacetone,
fluotracen, fluoxetine, fluoxymesterone, fluparoxan, flupentixol,
fluperamide, fluperlapine, fluperolone acetate, fluphenazine,
fluphenazine enanthate, flupimazine, flupirtine, flupranone,
fluprazine, fluprednidene, fluprednisolone, fluprednisolone
valerate, fluprofen, fluprofylline, fluproquazone, fluprostenol,
fluquazone, fluradoline, flurandrenoline, flurantel, flurazepam,
flurbiprofen, fluretofen, flurithromycin, flurocitabine,
flurofamide, flurogestone acetate, flurothyl, fluroxene,
flusoxolol, fluspiperone, fluspirilene, flutamide, flutazolam,
flutemazepam, flutiazin, fluticasone propionate, flutizenol,
flutonidine, flutoprazepam, flutroline, flutropium bromide,
fluvoxamine, fluzinamide, fluzoperine, folescutol, folic acid,
fomidacillin, fominoben, fomocaine, fonazine, fopirtoline,
forfenimex, formebolone, formetorex, formintrazole, formocortal,
formoterol, fosarilate, fosazepam, foscarnet, foscolic acid,
fosenazide, fosfocreatine, fosfomycin, fosfonet, fosfosal,
fosinapril, fosmenic acid, fosmidomycin, forpirate, fostedil,
fostriecin, fotemustine, fotreamine, frabuprofen, frentizole,
fronepidil, froxiprost, ftaxilide, ftivazide, ftorafur,
ftormetazine, ftorpropazine, fubrogonium iodide, fuchsin,
fumagillin, fumoxcillin, fuprazole, furacrinic acid, furafylline,
furalazine, furaltadone, furaprofen, furazabol, furazolidone,
furazolium chloride, furbucillin, furcloprofen, furegrelate,
furethidine, furfenorex, furidarone, furmethoxadone, furobufen,
furodazole, furofenac, furomazine, furosemide, furostilbestrol,
fursalan, fursuItiamine, furterene, furtrethonium iodide, fusidic
acid, fuzlocillin, gabapentin, gabexate, gaboxadol, galantamine,
gallamine triethodide, gallopamil, galosemide, galtifenin,
gampexine, gamolenic acid, ganciclovir, ganglefene, gapicomine,
gapromidine, gefarnate, gemazocine, gemcadiol, gemeprost,
gemfibrozil, gentamicin, gentian violet, gepefrine, gepirone,
geroquinol, gestaclone, gestadienol, gestodene, gestonorone
caproate, gestrinone, giparmen, gitaloxin, gitoformate, glafenine,
glaziovine, gliamilide, glibornuride, glibutimine, glicaramide,
glicetanile, geroquinol, gestaclone, gestadienol, gestodene,
gestonorone caproate, gestrinone, giparmen, gitaloxin, gitoformate,
glafenine, glaziovine, gliamilide, glibornuride, glibutimine,
glicaramide, glicetanile,
gliclazide, glicondamide, glidazamide, gliflumide, glimepiride,
glipentide, glipizide, gliquidone, glisamuride, glisindamide,
glisolamide, glisoxepide, gloxazone, gloximonam, glucametacin,
glucosamine, glucosulfamide, glucosulfone, glucurolactone,
glucuronamide, glunicate, glutamic acid, glutaral, glutarimide,
glutaurine, glutethimide, glyburide, glybuthiazol, glybuzole,
glyceryl monostearate, glycidyl methacrylate, glycine,
glyclopyramide, glybiarsol, glycopyrrolate, glycyclamide,
glyhexamide, glymidine, glyoctamide, glypinamide, glyprothiazol,
glysobuzole, gold thiomalate, gold sodium thiosulfate, granisetron,
griseofulvin, guabenxan, guacetisal, guafecainol, guaiactamine,
guaiapate, guaietolin, guaifenesin, guaimesal, guaisteine,
guaithylline, guamecycline, guanabenz, guanacline, guanadrel,
guanazodine, guanazole, guanclofine, guancydine, guanethidine,
guanfacine, guanisoquin, guanoclor, guanoctine, guanoxabenz,
guanoxan, guanoxyfen, hadacidin, halazepam, halazone, halcinonide,
halethazole, halocortolone, halofantrine, halofenate, halofuginone,
halometasone, halonamine, halopemide, halopenium chloride,
haloperidol, haloperidol decanoate, haloperidone acetate,
haloprogesterone, haloprogin, halothane, haloxazolam, haloxon,
halquinols, hedaquinium chloride, hepronicate, heptabarbital,
heptaminol, heptaverine, heptolamide, hepzidine, hetacillin,
hetaflur, heteronium bromide, hexachlorophene, hexacyclonate,
hexacyprone, hexadiline, hexadimethrine bromide, hexafluorenium
bromide, hexamethonium bromide, hexamidine, hexapradol, hexaprofen,
hexapropymate, hexasonium iodide, hexacarbacholine bromide,
hexedine, hexestrol, hexetidine, hexobarbital, hexobendine,
hexocyclium methylsulfate, hexoprenaline, hexopyrronium bromide,
hexylcaine, hexylene glycol, hexylresorcinol, histamine,
histapyrrodine, homarylamine, homatropine, homatropine
methylbromide, homidium bromide, homochlorcyclizine, homofenazine,
homoharringtonine, homopipramol, homosalate, homotestosterone
propionate, homprenorphine, hopantenic acid, hoquizil, hycanthone,
hydracarbazine, hydralazine, hydrargaphen, hydrobentizide,
hydrochlorthiazide, hydrocodone, hydrocortamate, hydrocortisone,
hydrocortisone aceponate, hydrocortisone acetate, hydrocortisone
butyrate, hydrocortisone cypionate, hydrocortisone-phosphate,
hydrocortisone succinate, hydrocortisone valerate,
hydroflumethiazide, hydromadinone, hydromorphinol, hydromorphone,
hydroquinone, hydroxindasate, hydroxindasol, hydroxyoxocobalamin,
hydroxy amphetamine, hydroxychloroquine,
hydroxydimethandrostadienone, hydroxydione succinate,
hydroxymethylandrostanone, 10-hydroxynorehisterone,
hydroxypethidine, hydroxyphenamate, hydroxyprocaine,
hydroxyprogeserone, hydroxyprogesterone caproate, hydroxypyridine
tartrate, hydroxystilbamidine, 7-hydroxytestololacetone,
hydroxytestosterone propionate, hydroxytetracaine, hydroxytoluic
acid, hydroxyurea, hydroxyzine, hymecromone, hyoscyamine,
hypericin, ibacitabine, ibafloxacin, ibazocine, ibopamine,
ibrotamide, ibudilast, ibufenac, ibuprofen, ibuprofen piconol,
ibuproxam, ibuterol, ibuverine, icazepam, icosipiramide, icotidine,
idarubicin, idaverine, idazoxan, idebenone, idenast, idoxuridine,
idralfidine, idrocilamide, idropranolol, ifenprodil, ifosfamide,
ifoxetine, ilmofosine, iloprost, imafen, imanixil, imazodan,
imcarbofos, imexon, imiclopazine, imidazole salicylate,
imidazopyrazole, imidecyl iodine, imidocarb, imidoline, imidurea,
imiloxan, iminophendimide, imipenem, imipramine, imipraminoxide,
imirestat, imolamine, imoxiterol, impacarzine, impromidine,
improsulfan, imuracetam, inaperisone, indacrinone, indalpine,
indanazoline, indanidine, indanorex, indapamide, indatraline,
indacainide, indeloxazine, indenolol, indicine-N-oxide,
indigotindisulfonic acid, indobufen, indocate, indocyanine green,
indolapril, indolidan, indomethacin, indopanolol, indopine,
indoprofen, indoramin, indorenate, indoxole, indriline, inicarone,
inocoterone, inosine, inosine dialdehyde, inositol niacinate,
inproquone, intrazole, intriptyline, iobenzamic acid, iobutic acid,
iocarmic acid, iocetamic acid, iodamide, iodecimol, iodetryl,
iodipamide, iodixanol, iodoalphionic acid, iodol, iodophthalein,
iodoquinol, iodothiouracil, iodoxamic acid, ioglicic acid,
ioglucol, ioglucomide, ioglunide, ioglycamic acid, iogulamide,
iohexol, iodlidonic acid, iolixanic acid, iomeglamic acid,
iomeprol, iomorinic acid, iopamidol, iopanoic acid, iopentol,
iophendylate, iophenoxic acid, ioprocemic acid, iopromide, iopronic
acid, iopydol, iopydone, iosarcol, iosefamic acid, ioseric acid,
iosimide, iosulamide, iosumetic acid, iotasul, iotetric acid,
iothalamic acid, iotranic acid, iotrizoic acid, iotrolan, iotroxic
acid, ioversol, ioxabrolic acid, ioxaglic acid, ioxitalamic acid,
ioxotrizoic acid, iozomic acid, ipexidine, ipodic acid,
ipragratine, ipramidil, ipratropium bromide, iprazochrome,
ipriflavone, iprindole, iprocinodine, iproclozide, iprocrolol,
iprofenin, iproheptine, iproniazid, iproidazole, iproplatin,
iprotiazem, iproxamine, iprozilamine, ipsalazide, ipsapirone,
iquindamine, irindalone, irloxacin, irolapride, irsogladine,
isamfazone, isamoltan, isamoxole, isaxonine, isbogrel, isepamicin,
isoaminile, isobromindione, isobucaine, isobutamben, isocarboxazid,
isoconazole, isocromil, isoetharine, isofezolac, isoflupredone
acetate, isoflurane, isoflurophate, isoleucine, isomazole,
isomerol, isometamidium, isomethadone, isomethept ne, isomylamine,
isoniazid, isonixin, isoprazone, isoprednidene, isoprofen,
isoprofamide iodide, isopropicillin, isopropyl myristate, isopropyl
palmitate, isoproterenol, isosorbide, isosorbide dinitrate,
isosorbide mononitrate, isospalglumic acid, isosulfan blue,
isosulpride, isothipendyl, isotic, isotiquimide, isotretinoin,
isoxaprolol, isoxepac, isoxicam, isoxsuprine, isradipine,
itanoxone, itazigrel, itraconazole, itrocainide, ivermectin bib,
ivoqualine, josamycin, kainic acid, kalafungin, kanamycin,
kebuzone, keracyanin, ketamine, ketanserin, ketazocine, ketazolam,
kethoxal, ketipramine, ketobemidone, ketocaine, ketocainol,
ketoconazole, ketoprofen, ketorfanol, ketorolac, ketotifen,
ketotrexate, khellin, khelloside, kitasamycin, labetalol,
lacidipine, lactalfate, lactose, lactulose, lamotrigine, lamtidine,
lanatoside, lapachol, lapinone, lapyrium chloride, la, salocid,
laudexium methyl sulfate, lauralkonium chloride, laureth,
laurixamine, laurocapram, lauroguadine, laurolinium acetate, lauryl
isoquinolinium, lefetamine, leflunomide, leiopyrrole, lemidosul,
lenampicillin, leniquinsin, lenperone, leptacline, lergotrile,
letimide, letosteine, leucine, leucinocaine, leucocianidol,
leucovorin, levacecarnine, levallorphan, levamfetamine, levamisole,
levdropropizine, levisoprenaline, levlofexidine, levobunolol,
levocabastine, levocarnitine, levodopa, levofacetoperane,
levofenfluramine, levofuraltadone, levoglutamide, levomenol,
levomethadone, levomethadyl acetate, levomethorphan,
levometiomeprazine, levomopranol, levomoramide, levonantradol,
levonordeprin, levonorgestrel, levophenacyl morphan,
levopropoxyphene, levopropylcillin, levopropylhexedrine,
levoprotiline, levorin, levorphanol, levothyroxine, levoxadrol,
lexofenac, libecillide, libenzapril, lidamidine, lidocaine,
lidofenin, lidoflazine, lifibrate, lilopristone, limaprost,
lincomycin, lindane, linsidomine, liothyronine, liroldine,
lisinopril, lisuride, lithium carbonate, lithium citrate, litracen,
lividomycin, lixazinone, lobeline, lobendazole, lobenzarit,
lobuprofen, locicortone, lodaxaprine, lodacezarlodinixil,
lodiperone, lodoxamide, lodoxamide ethyl, lofemizole, lofendazam,
lofentanil, lofepramine, lofexidine, loflucarban, lombazole,
lomefloxacin, lometraline, lomevactone, lomifylline, lomofungin,
lomustine, lonapalene, lonaprofen, lonazolac, lonidamine,
loperamide, loperamide oxide, lopirazepam, loprazolam, loprodiol,
lorajmine, lorapride, loratadine, lorazepam, lorbamate, lorcainide,
lorcinadol, lorglumide, lormetazepam, lortalamine, lorzafone,
losindole, losulazine, lotifazole, lotrifen, lotucaine, lovastatin,
loxanast, loxapine, loxiglumide, loxoprofen, loxtidine, lozilurea,
lucanthone, lucartamide, lucimycin, lufuradom, lupitidine,
luprostiol, luxabendazole, lyapolate sodium, lycetamine, lydimycin,
lymecycline, lynestrenol, lysergide, lysine, mabuterol,
maduramicin, mafenide, mafoprazine, mafosfamide, magnesium citrate,
magnesium, gluconate, magnesium salicylate, malathion, malethamer,
malic acid, malotilate, manidipine, manganese gluconate, mannitol,
mannitol hexanitrate, mannomustine, mannosulfan, manozodil,
maprotiline, maridomycin, mariptiline, maroxepin, maytansine,
mazaticol, mazindol, mazipredone, mebanazine, mebendazole,
mebenoside, mebeverine, mebezonium iodide, mebhydrolin, mebiquine,
mebolazine, mebrofenin, mebutamate, mebutizide, mecamylamine,
mecarbinate, mecetronium ethylsulfate, mechlorethamine, meciadanol,
mecinarone, meclizine, meclocycline, meclocycline sulfosalicylate,
meclofenamic acid, meclofenoxate, meclonazepam, mecloqualone,
mecloralurea, meclorisone dibutyrate, mecloxamine, mecobalamin,
mecrylate, mecysteine, medazepam, medazomide, medetomidine,
medibazine, medifoxamine, medorinone, medorubicin, medrogestone,
medronic acid, medroxalol, medroxyprogestrone, medroxyprog strone
acetate, medrylamine, medrysone, mefeclorazine, mefenamic acid,
mefenidil, mefenidramium metilsulfate, mefenorex, mefeserpine,
mefexamide, mefloquine, mefruside, megalomicin, megestrol acetate,
meglitinide, megucycline, meglumine, meglutol, meladrazine,
melarsonyl, melarsoprol, melengestrol acetate, meletimide,
melinamide, melitracen, melizame, meloxicam, melperone, melphalan,
memantine, memotine, menabitan, menadiol, menadiol diphosphate,
menadiol disulfate, menadione, menadione sodium bisulfite,
menatetrenone, menbutone, menfegol, menglytate, menitrazepam,
menoctone, menogaril, menthol, meobentine, meparfynol, mepazine,
mepenzolate bromide, meperidine, mephenesin, mephenoxalone,
mephentermine, mephenyton, mephobarbital, mepindolol, mepiprazole,
mepiroxol, mepitiostane, mepivacaine, mepixanox, mepramidil,
meprednisone, meprobamate, meproscillarin, meproxitol, meprylcaine,
meptazinol, mequidox, mequinol, mequitazine, meralein, meralluride,
merbarone, merbromin, mercaptamine, mercaptomerin, mercaptopurine,
mercuderamide, mercufenol chloride, mercumatilin, mercurobutol,
mergocriptine, merophan, mersalyl, mesabolone, mesalamine,
meseclazone, mesna, mesocarb, meso-hexestrol, mesoridazine,
mesipirenone, mestanolone, mesterolone, mestranol, mesudipine,
mesulergine, mesulfamide, mesulfen, mesuprine, metabromsalan,
metacetamol, metaclazepam, metaglycodol, metahexamide,
metamelfalan, metamfazone, metamfepramone, metampicillin,
metanixin, metapramine, metaproterenol, metaraminol, metaterol,
metaxalone, metazamide, metazide, metazocine, metbufen,
meteneprost, metergoline, metergotamine, metescufylline,
metesculetol, metethoheptazine, metformin, methacholine chloride,
methacycline, methadone, methadyl acetate, methallenestril,
methallibure, methalthiazide, methamphetamine, methandriol,
methandrostenolone, methaniazide, methantheline bromide,
methaphenilene, methapyrilene, m thaqualone, metharbital,
methastyridone, methazolamide, methdilazine, methenamine,
methenolone acetate, methenolone enanthate, metheptazine,
methestrol, methetoin, methicillin, methimazole, methiodal sodium,
methioguanine, methiomeprazine, methionine, methisazone,
methitural, methixene, methocarbamol, methohexital, methopholine,
methoserpidine, methotrexate, methotrimeprazine, methoxamine,
methoxsalen, methoxyflurane, methoxyphedrine, methoxyphenamine,
methoxypromazine, methscopolamine bromide, methsuximide,
methylclothiazide, N-methyladrealone hcl, methyl alcohol,
methylatropine nitrate, methylbenactyzium bromide,
methylbenzethonium, methylchromone, methyldesorphine,
methyldihydromorphine, methyldopa, methyldopate, methylene blue,
methylphedrine, methylergonovine, methylformamide, methyl
nicotinate, 2-methyl-19-nortestosterone,
2-methyl-11-oxoprogestrone, methyl palmoxirate, methylparaben,
methylphendiate, methylprednisolone, methylprednisolone aceponate,
methylprednisolone acetate, methylprednisolone hemisuccinate,
methylprednisolone phosphate, methylpredn isolone suleptanate,
methyl salicylate, methylstreptonigrin, 4-methyltestosterone,
7-methyltestosterone, 17-methyltestosterone, 7-methyltesosterone
propionate, methylthionosine, 16-methylthioprogestone- ,
methylthiouracil, methynodiol diacetate, methyprylon, methysergide,
metiamide, metiapine, metiazinic acid, metibride, meticrane,
metildigoxin, metindizate, metioprim, metioxate, metipirox,
metipranolol, metiprenaline, metitepine, metizoline, metkephamid,
metochalcone, metocinium iodide, metoclopramide, metocurine iodide,
metofenazate, metogest, metolazone, metomidate, metopimazine,
metopon, metoprine, metoprolol, metoquizine, metoserpate,
metostilenol, metoxepin, metrafazoline, metralindole, metrazifone,
metrenperone, metribolone, metrifonate, metrifudil, metrizamide,
metrizoic acid, metronidazole, meturedepa, metyrapone, metyridine,
metyrosine, mevastatin, mexafylline, mexazolam, mexenone,
mexiletine, mexiprostil, mexoprofen, mexrenoate, mezacopride,
mezepine, mezilamine, mezlocillin, mianserin, mibolerone,
micinicate, miconazole, micronomicin, midaflur, midaglizole,
midalcipran, midamaline, midazogrel, midazolam, midecamycin,
midodrine, mifentidine, mifepristone, mifobate, miglitol,
mikamycin, milacemide, milenperone, milipertine, miloxacin,
milrinone, milverine, mimbane, minaprine, minaxolone, mindolilol,
mindoperone, minepentate, minocromil, minocycline, minoxidil,
mioflazine, mipimazole, mirincamycin, miristalkonium chloride,
miroprofen, mirosamicin, misonidazole, misoprostol, mitindomide,
mitobronitol, mitoclomine, mitoguazone, mitolactol, mitomycin,
mitonafide, mitopodozide, mitoquidone, mitotane, mitotenamine,
mitoxantrone, mitozolomide, mivacurium chloride, mixidine,
misoprostol, mitindomide, mitobronitol, mitoclomine, mitoguazone,
mitolactol, mitomycin, mitonafide, mitopodozide, mitoquidone,
mitotane, mitotenamine, mitoxantrone, mitozolomide, mivacurium
chloride, mixidine, mizoribine, mobecarb, mobenzoxamine, mocimycin,
mociprazine, moclobemide, moctamide, modafinil, modaline,
mofebutazone, mofloverine, mofoxime, molfarnate, molinazone,
molindone, molracetam, molsidomine, mometasone furoate, monalazone
disodium, monensin, monobenzone, monoethanolamine, monometacrine,
monophosphothiamine, monothioglycerol, monoxerutin, montirelin,
moperone, mopidamol, mopidralazine, moprolol, moquizone, morantel,
morazone, morclofone, morforex, moricizine, morinamide,
morniflumate, morocromen, moroxydine, morpheridine, morphine,
morsuximide, motapizone, motrazepam, motretinide, moveltipril,
moxadolen, moxalactam, moxaprindine, moxastine, moxaverine,
moxazocine, moxestrol, moxicoumone, moxipraquine, moxisylyte,
moxnidazole, moxonidine, mupirocin, murabutide, murocainid
muzolimine, mycophenolic acid, myfadol, myralact, myrophine,
myrtecaine, nabazenil, nabilone, nabitan, naboctate, nabumetone,
nadide, nadolol, nadoxolol, naepaine, nafamostat, nafazatrom,
nafcaproic acid, nafcillin, nafenodone, nafenopin, nafetolol,
nafimidone, nafiverine, naflocort, nafomine, nafoxadol, nafoxidine,
nafronyl, naftalofos, naftazone, naftifine, naftopidil, naftoxate,
naftypramide, nalbuphine, nalidixic acid, nalmefene, nalmexone,
nalorphine, naltrexone, naminterol, namoxyrate, nanaprocin,
nandrolone cyclotate, nandrolone decanoate, nandrolone,
phenpropionate, nanofin, nantradol, napactadine, napamezole,
naphazoline, naphthonone, naprodoxime, naproxen, naproxol, naranol,
narasin, natamycin, naxagolide, naxaprostene, nealbarbital,
nebidrazine, nebivolol, nebracetam, nedocromil, nefazodone,
neflumozide, nefopam, nelezaprine, neoarsphenamine, neocinchophen,
neomycin, neostigmine bromide, nequinate, neraminol, nerbacadol,
nesapidil, nesosteine, netilmicin, netobimin, neutramycin,
nexeridine, niacin, niacinamide, nialamide, niaprazine, nibroxane,
nicafenine, nicainoprol, nicametate, nicarbazin, nicarpidine,
nicergoline, niceritrol, niceverine, niclofolan, niclosamide,
nicoboxil, nicoclonate, nicocodine, nicocortonide, nicodicodine,
nicofibrate, nicofuranose, nicofurate, nicogrelate, nicomol,
nicomorphine, nicopholine, nicorandil, nicothiazone, nicotinyl
alcohol, nicoxamat, nictiazem, nictindole, nodroxyzone, nifedipine,
nifenalol, nifenazone, niflumic acid, nifluridide, nifuradene,
nifuraldezone, nifuralide, nifuratel, nifuratrone, nifurdazil,
knifurethazone, nifurfoline, nifurimide, nifurizone, nifurmazole,
nifurmerone, nifuroquine, nifuroxazide, nifuroxime, nifurpipone,
nifurpirinol, nifurprazine, nifurquinazole, nifursemizone,
nifursol, nifurthiazole, nifurtimox, nifurtoinol, nifurvidine,
nifurzide, niguldipine, nihydrazone, nikethamide, nileprost,
nilprazole, niludipine, nilutamide, nilvadipine, nimazone,
nimesulide, nimetazepam, nimidane, nimodipine, nimorazole,
nimustine, niometacin, niperotidine, nipradilol, niprofazone,
niridazole, nisbuterol, nisobamate, nisoldipine, nisoxetine,
nisterime acetate, nitarsone,
nitazoxanide, nithiamide, nitracrine, nitrafudam, nitralamine,
nitramisole, nitraquazone, nitrazepam, nitrefazole, nitrendipine,
nitricholine, nitrochlofene, nitrocycline, nitrodan,
nitrofurantoin, nitrofurazone, nitroglycerin, nitromersol,
nitromide, nitromifene, nitroscanate, nitrosulfathiazole,
nitroxinil, nitroxoline, nivazol, nivimeldone, nixylic acid,
nizatidine, nizofenone, noberastine, nocloprost, nocodazole,
nofecainide, nogalamycin, nolinium bromide, nomegestrol,
nomelidine, nomifensine, nonabine, nonaperone, nonapyrimine,
nonoxynol-4, nonoxynol-9, noracymethadol, norbolethone, norbudrine,
norclostebol, norcodeine, nordazepam, nordefrin, nordinone,
norepinephrine, norethandrolone, norethindrone, norethindrone
acetate, norethynodrel, noreximide, norfenefrine, norfloxacin,
norfloxacin succinil, norflurane, norgesterone, norgestimate,
norgestomet, norgestrel, norgestrienone, norletimol,
norlevorphanol, normethadone, normethandrone, normorphine,
norpipanone, nortestosterone propionate, nortetrazepam,
nortriptyline, norvinisterone, nosantine, noscapine, nosiheptide,
novobiocin, noxiptiline, noxytiolin, nuclomedone, nuclotixine,
nufenoxole, nuvenzepine, nylestriol, nylidrin, nystatin, obidoxime,
ociltide, ocrylate, octabenzone, octacaine, octafonium chloride,
octamoxin, octamylamine, octanoic acid, octapinol, octastine,
octaverine, octazamide, octenidine, octenidine saccharin,
octicizer, octimibate, octorylene, octodrine, octopamine,
octotiamine, octoxynol-9, octriptyline, octrizole, ofloxacin,
ofornine, oftasceine, olaflur, olaquindox, oleanomycin, oletimol,
oleyl alcohol, olivomycin a, olmidine, olpimedone, olsalazine,
oltipraz, olvanil, omeprazole, omidoline, omoconazole, omonasteine,
onapristone, ondansetron, ontianil, opiniazide, opipramol,
orazamide, orbutopril, orconazole, orestrate, ormetoprim,
ornidazole, ornipressin, ornithine, ornoprostil, orotic acid,
orotirelin, orpanoxin, orphenadrine, ortetamine, osalmid,
osmadizone, otilonium bromide, otimerate sodium, ouabain, oxabolone
cipionate, oxabrexine, oxaceprol, oxacillin, oxadimedine,
oxaflozane, oxaflumazine, oxagrelate, oxalinast, oxaliplatin,
oxamarin, oxametacin, oxamisole, oxamniquine, oxanamide,
oxandrolone, oxantel, oxapadol, oxapium iodide, oxapropanium
iodide, oxaprotiline, oxaprozin, oxarbazole, oxatomide, oxazafone,
oxazepam, oxazidione, oxazolam, oxazorone, oxcarbazepine,
oxdralazine, oxeladin, oxendolone, oxepinac, oxetacillin,
oxethazaine, oxetorone, oxfendazole, oxfenicine, oxibendazole,
oxibetaine, oxiconazole, oxidopamine, oxidronic acid, oxifentorex,
oxifungin, oxilorphan, oximonam, oxindanac, oxiniacic acid,
oxiperomide, oxiracetam, oxiramide, oxisopred, oxisuran,
oxitefonium bromide, oxitriptan, oxitriptyline, oxitropium bromide,
oxmetidine, oxodipine, oxogestone phenpropionate, oxolamine,
oxolinic acid, oxomemazine, oxonazine, oxophenarsine, oxoprostol,
oxpheneridine, oxprenoate potassium, oxprenolol, oxtriphylline,
oxybenzone, oxybutynin, oxychlorosene, oxycinchophen, oxyclozanide,
oxycodone, oxydipentonium chloride, oxyfedrine, oxymesterone,
oxymetazoline, oxymetholone, oxymorphone, oxypendyl, oxypertine,
oxyphenbutazone, oxyphenonium bromide, oxypurinol, oxypyrronium
bromide, oxyquinoline, oxyridazine, oxysonium iodide,
oxytetracycline, oxytiocin, ozagrel, ozolinone, pacrinolol,
pactamycin, padimate, pafenolol, palatrigine, paldimycin,
palmidrol, palmoxiric acid, pamabrom, pamaquine, pamatolol,
pamidronic acid, pancuronium bromide, panidazole, panomifene,
patenicate, panthenol, pantothenic acid, panuramine, papaverine,
papaveroline, parachlorophenol, paraflutizide, paraldehyde,
paramethadione, paramethasone acetate, paranyline, parapenzolate
bromide, parapropamol, pararosaniline, pararosaniline embonate,
paraxazone, parbendazole, parconazole, pareptide, parethoxycaine,
pargeverine, pargolol, pargyline, paridocaine, parodilol,
paromomycin, paroxetine, paroxypropione, parsalmide, partricin,
parvaquone, pasiniazid, paulomycin, paxamate, pazelliptine,
pazoxide, pcnu, pecilocin, pecocycline, pefloxacin, pelanserin,
pelretin, pelrinone, pemedolac, pemerid, pemoline, pempidine,
penamecillin, penbutolol, pendecamaine, penfluridol, penflutizide,
pengitoxin, penicillamine, penicillin procaine, penicillin,
penimepicycline, penimocycline, penirolol, penmesterol, penoctonium
bromide, penprostene, pentabamate, pentacynium chloride,
pentaerythritol tetranitrate, pentafluranol, pentagastrin,
pentagestrone, pentalamide, pentamethonium bromide,
pentamethylmelamine, pentamidine, pentamoxane, pentamustine,
pentapiperide, pentapiperium methylsulfate, pentaquine,
pentazocine, pentetate calcium trisodium, pentetic acid,
penthienate bromide, penthrichloral, pentiapine maleate,
pentifylline, pentigetide, pentisomicin, pentisomide, pentizidone,
pentobarbital, pentolinium tartrate, pentomone, pentopril,
pentorex, pentosan polysulfate sodium, pentostatin, pentoxifylline,
pentrinitrol, pentylenetrazole, peplomycin, pepstatin, peraclopone,
peradoxime, perafensine, peralopride, peraquinsin, perastine,
peratizole, perbufylline, perfluamine, perflunafene, pergolide,
perhexilene, periciazine, perimetazine, perindopril, perindoprilat,
perisoxal, perlapine, permethrin, perphenazine, persilic acid,
petrichloral, pexantel, phanquone, phenacaine, phenacemide,
phenacetin, phenacttropinium chloride, phenadoxone, phenaglycodol,
phenamazoline, phenampromide, phenarsone sulfoxylate, phenazocine,
phenazopyridine, phencarbamide, phencyclidine, phendimetrazine,
phenelzine, pheneridine, phenesterin, penethicillin, phenformin,
phenglutarimide, phenicarbazide, phenindamine, phenindione,
pheniprazine, pheniramine, phenisonone, phenmetrazine,
phenobarbital, phenobutiodil, phenolphtalein,
phenolsulfonphthalein, phenomorphan, phenoperidine, phenothiazine,
phenothrin, phenoxybenzamine, phenoxypropazine, phenprobamate,
phenprocoumon, phenpromethamine, phensuximide, phentermine,
phentolamine, phenylalanine, phenyl aminosalicylate,
phenylbutazone, phenylrphrine, phenylethyl alcohol, phenylmercuric
acetate, phenylmercuric borate, phenylmercuric chloride,
phenylmercuric nitrate, phenylmethylbarbituric acid,
phenylpropanolamine, phenylthilone, phenyltoloxamine, phenyramidol,
phenytoin, phetharbital, pholcodine, pholedrine, phosphoramide
mustard, phoxim, phthalofyne, phthalysulfacetamide,
phthalylsulfamethizole, phthalylsulfathiazole, physostigmine,
phytic acid, phytonadiol diphosphate, phytonadione, pibecarb,
pibenzimol, pibecarb, pibenzimol, piberaline, picafibrate,
picartamide, picenadol, picilorex, piclonidine, piclopastine,
picloxydine, picobenzide, picodralazine, picolamine, piconol,
picoperine, picoprazole, picotamide, picotrin diolamine, picumast,
pidolic acid, pifarnine, pifenate, pifexole, piflutixole, pifoxime,
piketoprofen, pildralazine, pilocarpine, pimoclone, pimefylline,
pimelautde, pimetacin, pimethixene, pimetine, pimetremide,
piminodine, pimobendan, pimondiazole, pimozide, pinacidil,
pinadoline, pinafide, pinaverium bromide, pinazepam, pincainide,
pindolol, pinolcaine, pinoxepin, pioglitazone, pipacycline,
pipamazine, pipaperone, pipazethate, pipebuzone, pipecuronium
bromide, pipemidic acid, pipenzolate bromid pipequaline,
piperacetazine, piperacillin, piperamide, piperazine,
piperazinedione, piperidolate, piperilate, piperocaine, piperoxan,
piperylone, pipobroman, pipoctanone, pipofezine, piposulfan,
pipotiazine palmiate, pipoxizine, pipoxolan, pipradimadol,
pipradol, pipramadol, pipratecol, piprinhydrinate, piprocurarium
iodide, piprofurol, piprozolin, piquindone, piquizil, piracetam,
pirandamine, pirarubicin, piraxelate, pirazmonam, pirazolac,
pirbenicillin, pirbuterol, pirdonium bromide, pirenoxine,
pirenperone, pirenzepine, pirepolol, piretanide, pirfenidone,
piribedil, piridicillin, piridocaine, piridoxilate, piridronic
acid, pirifibrate, pirindazole, pirinixic acid, pirinixil,
piriprost, piriqualone, pirisudanol, piritramide, piritrexim,
pirlimycin, pirlindole, pirmagrel, pirmenol, pirnabine, piroctone,
pirogliride, piroheptine, pirolate, pirolazamide, piromidic acid,
piroxantrone hcl, piroxicam, piroxicam cinnamate, piroxicillin,
piroximone, pirozadil, pirprofen, pirquinozol, pirralkonium
bromide, pirtenidine, pitenodil, pitofenone, pituxate,
pivampicillin, pivenfrine, pivopril, pivoxazepam, pizotyline,
plafibride, plaunotol, pleuromulin, plicamycin, podilfen,
podophylloxoxin, poldine methylsulfate, polidocanol, ploymyxin,
polythiazide, ponalrestat, ponfibrate, porfiromycin, poskine,
potassium guaiacolsulfonate, potassium nitrazepate, potassium
sodium tartrate, potassium sorbate, potassium thiocyanate,
practolol, prajmalium, pralidoxime chloride, pramipexole,
pramiracetam, pramiverine, pramoxime, prampine, pranolium chloride,
pranoprofen, pranosal, prasterone, pravastatin, praxadine,
prazepam, prazepine, praziquantel, prazitone, prazocillin,
prazosin, preclamol, prednazate, prednazoline, prednicarbate,
prednimustine, prednisolamate, prednisolone, prednisolone acetate,
prednisolone hemisuccinate, prednisolone phosphate, prednisolone
steaglate, prednisolone tebutate, prednisone, prednival, pr
dnylidene, prefenamate, pregnenolone, pregnenolone succinate,
premazepam, prenalterol, prenisteine, prenoverine, prenoxdiazine,
prenylamine, pretamazium iodide, pretiadil, pribecaine, pridefine,
prideperone, pridinol, prifelone, prifinium bromide, prifuroline,
prilocaine, primaperone, primaquine, primidolol, primidone,
primycin, prinomide, pristinamycin, prizidilol, proadifen,
probarbital, probenecid, probicromil, probucol, procainamide,
procaine, procarbazine, procaterol, prochlorperazine, procinolol,
procinonide, proclonol, procodazole, procyclidine, procymate,
prodeconium bromide, prodilidine, prodipine, prodblic acid,
profadol, profexalone, proflavine, proflazepam, progabide,
progesterone, proglumetacin, proglumide, proheptazine,
proligestone, proline, prolintane, prolonium iodide, promazine,
promegestone, promestriqne, promethazine, promolate, promoxolane,
pronetalol, propacetamol, propafenone, propamidine, propanidid,
propanocaine, propantheline bromide, proparacaine, propatyl
nitrate, propazolamide, propendiazole, propentofylline,
propenzolate, properidine, propetamide, propetandrol, propicillin,
propikacin, propinetidine, propiolactone, propiomazine,
propipocaine, propiram, propisergide, propiverine, propizepine,
propofol, propoxate, propoxycaine, propoxyphene, propranolol,
propyl docetrizoate, propylene glycol, propylene glycol
monostearate, propyl gallate, propylhexedrine, propyliodone,
propylparaben, propylthiouracil, propyperone, propyphenazone,
propyromazine bromide, proquazone, proquinolate, prorenoate
potassium, proroxan, proscillaridin, prospidium chloride,
prostalene, prosulpride, prosultiamine, proterguride,
protheobromine, prothipendyl, prothixene, protiofate, protionamide,
protirelin, protizinic acid, protokylol, protoveratine,
protriptyline, proxazole, proxibarbal, proxibutene, proxicromil,
proxifezone, proxorphan, proxyphylline, prozapine, pseudoephedrine,
psilocybine, pumiteba, puromycin, pyrabrom, pyran copolymer,
pyrantel, pyrathiazine, pyrazinamide, pyrazofurin, pyricarbate,
pyridarone, pyridofylline, pyridostigmine bromide, pyridoxine,
pyrilamine, pyrimethamine, pyrimitate, pyrinoline, pyrithione zinc,
pyrithyldione, pyritidium bromide, pyritinol, pyronine,
pyrophenindane, pyrovalerone, pyroxamine, pyrrobutamine,
pyrrocaine, pyrroliphene, pyrrolnitrin, pyrvinium chloride,
pytamine, quadazocine, quadrosilan, quatacaine, quazepam,
quazinone, quazodine, quazolast, quifenadine, quillifoline,
quinacainol, quinacillin, quinacrine, quinaldine blue, quinapril,
quinaprilat, quinazosin, quinbolone, quincarbate, quindecamine,
quindonium bromide, quindoxin, quinestradol, quinestrol,
quinethazone, quinetolate, quinezamide, quinfamide, quingestanol
acetate, quingestrpne, quindine, quinine, quinocide, quinpirole,
quinterenol, quintiofos, quinuclium bromide, quinupramine,
quipazine, quisultazine, racefemine, racemethionine,
racemethorphan, racemetirosine, raclopride, ractopamine,
rafoxanide, ralitoline, raloxifene, ramciclane, ramefenazone,
ramipril, ramiprilat, ramixotidine, ramnodignin, ranimustine,
ranimycin, ranitidine, ranolazine, rathyronine, razinodil,
razobazam, razoxane, reboxetine, recainam, reclazepam, relomycin,
remoxipride, renanolone, rentiapril, repirinast, repromicin,
reproterol, recimetol, rescinnamine, reserpine, resorantel,
resorcinol, resorcinol monoacetate, retelliptine, retinol,
revenast, ribavirin, riboflavin, riboflavin 5'-phosphate,
riboprine, ribostamycin, ridazolol, ridiflone, rifabutin, rifamide,
rifampin, rifamycin, rifapentine, rifaximin, rilapine, rilmazafone,
rilmenidine, rilopirox, rilozarone, rimantadine, rimazolium
metilsulfate, rimcazole, rimexolone, rimiterol, rimoprogin,
riodipine, rioprostil, ripazepam, risocaine, risperidone,
ristianol, ristocetin, ritanserin, ritiometan, ritodrine,
ritropirronium bromide, ritrosulfan, robenidine, rocastine,
rociverine, rodocaine, rodorubicin, rofelodine, roflurante,
rokitamycin, roletamide, rolgamidine, rolicyclidine, rolicyprine,
rolipram, rolitetracycline, rolodine, rolziracetam, romifenone,
romifidine, ronactolol, ronidazole, ronifibrate, ronipamil, ronnel,
ropitoin, ropivacaine, ropizine, roquinimex, rosaprostol,
rosaramicin, rosaramicin butyrate, rosaramicin propionate,
rosoxacin, rosterolone, rotamicillin, rotoxamine, rotraxate,
roxarsone, roxatidine acetate, roxibolone, roxindole,
roxithromycin, roxolonium metilsulfate, roxoperonef rufloxacin,
rutamycin, rutin, ruvazone, sabeluzole, saccharin, salacetamide,
salafibrate, salantel, salazodine, salazossulfadimedine,
salazosulfamide, salazosulfathiazole, salethamide, salfluverine,
salicin, salicyl alcohol, salicylamide, salicylanilide, salicylic
acid, salinazid, salinomycin, salmefanol, salmeterol, salmisteine,
salprotoside, salsalate, salverine, sancycline, sangivamycin,
saperconazole, sarcolysin, sarmazenil, sarmoxicillin, sarpicillin,
saterinone, satranidazole, savoxepin, scarlet red, scopafungin,
scopolamine, seclazone, secnidazole, secobarbital, secoverine,
securinine, sedecamycin, seganserin, seglitide, selegiline,
selenium sulfide, selprazine, sematilide, semustine, sepazonium
chloride, seperidol, sequifenadine, serfibrate, sergolexole,
serine, sermetacin, serotonin, sertaconazole, sertraline,
setastine, setazindol, setiptiline, setoperone, sevitropium
mesilate, sevoflurane, sevopramide, siagoside, sibutramine,
siccanin, silandrone, silibinin, silicristin, silidianin, silver
sulfadiazine, simetride, simfibrate, simtrazene, simvastatin,
sinefungin, sintropium bromide, sisomicin, sitalidone, sitofibrate,
sitogluside, sodium benzoate, sodium dibunate, sodium ethasulfate,
sodium formaldehyde sulfoxylate, sodium gentisate, sodium
gualenate, sodium nitrite, sodium nitroprusside, sodium oxybate,
sodium phenylacetate, sodium picofosfate, sodium picosulfate,
sodium propionate, sodium stibocaptate, sodium stibogluconate,
sodium tetradecyl sulfate, sodium thiosulfate, sofalcone,
solasulfone, solpecainol, solypertine, somantadine, sopitazine,
sopromidine, soquinolol, sorbic acid, sorbinicate, sorbinil,
sorbitan monolaurate, sorbitan monooleate, sorbitan monopalmitate,
sorbitan monostearate, sorbitan trioleate, sorbitan tristearate,
sorbitol, sorndipine, sotalol, soterenol, spaglumic acid, sparfosic
acid, sparsomycin, sparteine, spectinomycin, spiclamine,
spiclomazine, spiperone, spiradoline, spiramide, spiramycin,
spirapril, spiraprilat, spirendolol, spirgetine, spirilene,
spirofylline, spirogermanium, spiromustine, spironolactone,
spiroplatin, spirorenone, spirotriazine, spiroxasone, spiroxatrine,
spiroxepin, spizofurone, stallimycin, stanolone, stanzolol, stearic
acid, stearyl alcohol, stearylsulfamide, steffimycin, stenbolone
acetate, stepronin, stercuronium iodide, stevaladil, stibamine
glucoside, stibophen, stilbamidine, stilbazium iodide, stilonium
iodide, stirimazole, stiripentol, stirocainide, stirifos,
streptomycin, streptonicozid, streptonigrin, streptovarycin,
streptozocin, strinoline, strychnine, styramate, subathizone,
subendazole, succimer, succinylcholine chloride,
succinylsulfathiazole, succisulfone, suclofenide, sucralfate,
sucrose octaacetate, sudexanox, sudoxicam, sufentanil, sufosfamide,
sufotidine, sulazepam, sulbactam, sulbactam pivoxil, sulbenicillin,
sulbenox, sulbentine, sulbutiamine, sulclamide, sulconazole,
sulfabenz, sulfabenzamide, sulfacarbamide, sulfacecole,
sulfacetamide, sulfachlorpyridazine, sulfachrysoidine,
sulfaclomide, sulfaclorazole, sulfaclozine, sulfacytine,
sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine,
sulfaethidole, sulfaguandide, sulfaguanole, sulfalene, sulfaloxic
acid, sulfamazone, sulfamerazine, sulfameter, sulfamethazine,
sulfamethizole, sulfamethoxazole, sulfamethoxypyridazine,
sulfamethoxypyridazine acetyl, sulfametomidine, sulfametrole,
sulfamonomethoxine, sulfamoxole, sulfanil amide, sulfanitran,
sulfaperin, sulfaphenazole, sulfaproxyline, sulfapyridine,
sulfaquinoxaline, sulfarsphenamine, sulfasalazine,
sulfasomizole,
sulfasuccinamide, sulfasymazine, sulfathiazole, sulfathiourea,
sulfatolamide, sulfatroxazole, sulfatrozole, sulfazamet,
sulfinalol, sulfinpyrazone, sulfiram, sulfisomidine, sulfisoxazole,
sulfisoxazole, sulfobromophthalein, sulfonethylmethane,
sulfonmethane, sulfonterol, sulforidazine, sulfoxone sodium,
sulicrinat, sulindac, sulisatin, sulisobenzone, sulmarin,
sulmazole, sulmepride, sulinidazole, sulocarbilate, suloctidil,
sulosemide, sulotroban, suloxifen, sulpiride, sulprosal,
sulprostone, sultamicillin, sulthiame, sultopride, sultosilic acid,
sultroponium, sulverapride, sumacetamol, sumatriptan, sumetizide,
sunagrel, suncillin, supidimide, suproclone, suprofen, suramin,
suricainide, suriclone, suxemerid, suxethonium chloride,
suxibuzone, symclosene, symetine, synephrine, syrisingopine,
taclamine, tacrine, taglutimide, talampicillin, talastine,
talbutal, taleranol, talinolol, talipexole, talisomycin,
talmetacin, talmetoprim, talniflumate, talopram, talosalate,
taloximine, talsupram, taltrimide, tameridone, tameticillin,
tametraline, tamitinol, tamoxipen, tampramine, tandamine,
taprostene, tartaric acid, tasuldine, taurocholic acid,
taurolidine, tauromustine, tauroselcholic acid, taurultam, taxol,
tazadolene, tazanolast, tazaburate, tazeprofen, tazifylline,
taziprinone, tazolol, tebatizole, tebuquine, teclothiazide,
teclozan, tedisamil, tefazoline, tefenperate, tefludazine,
teflurane, teflutixol, tegafur, telenzepine, temafloxacin,
temarotene, temazepam, temefos, temelastine, temocillin, temodox,
temozolomide, temurtide, tenamfetamine, tenilapine, teniloxazine,
tenilsetam, teniposide, tenocyclidine, tenonitrozole, tenoxicam,
tenylidone, teopranitol, teoprolol, tepirindole, tepoxalin,
terazosin, terbinafine, terbucromil, terbufibrol, terbuficin,
terbuprol, terbutaline, terciprazine, terconazole, terfenadine,
terfluranol, terguride, terizidone, ternidazole, terodiline,
terofenamate, teroxalene, teroxirone, terpin hydrate, tertatolol,
tesicam, tesimide, testolactone, testosterone, testosterone
cypionate, testosterone enanthate, testosterone ketolaurate,
testosterone phenylacetate, testosterone propionate, tetrabarbital,
tetrabenazine, tetracaine, tetrachloroethylene, tetracycline,
tetradonium bromide, tetraethylammonium chloride, tetrahydrozoline,
tetramethrin, tetramisole, tetrandrine, tetrantoin, tetrazepam,
tetriprofen, tetronasin 5930, tetroquinone, tetroxoprim,
tetrydamine, texacromil, thalicarpine, thalidomide, thebacon,
thebaine, thenalidine, thenium closylate, thenyldiamine,
theobromine, theodrenaline, theofibrate, theophylline,
thiabendazole, thiacetarsamide, thialbarbital, thiambutosine,
thiamine, thiamiprine, thiamphenicol, thiamylal, thiazesim,
thiazinamium chloride, thiazolsulforie, thiethyperazine, thihexinol
methylbromide, thimerfonate, thimerosal, thiocarbanidin,
thiocarzolamide, thiocolchioside, thiofuradene, thioguanine,
thioguanine alpha-deoxyriboside, thioguanine beta-deoxyriboside,
thioguanosine, thiohexamide, thioinosine, thiopental,
thiopropazate, thioproperazine, thioridazine, thiosalan, thiotepa,
thiotetrabarbital, thiothixene, thiouracil, thiphenamil,
thiphencillin, thiram, thonzonium bromide, thonzylamine,
thozalinone, threonine, thymidine, thymol, thymol iodide,
thymopentin, thyromedan, thyropropic acid, tiacrilast, tiadenol,
tiafibrate, tiamenidine, tiametonium iodide, tiamulin, tianafac,
tianeptine, tiapamil, tiapirinol, tiapride, tiaprofenic acid,
tiaprost, tiaramide, tiazofurin, tiazuril, tibalosin, tibenalast
sodium, tibenzate, tibezonium iodide, tibolone, tibric acid,
tibrofan, tic-mustard, ticabesone propionate, ticarbodine,
ticarcillin, ticarcillin cresyl, ticlatone, ticlopidine,
ticrynafen, tidiacic, tiemoium iodide, tienocarbine, tienopramine,
tienoxolol, tifemoxone, tiflamizole, tiflorex, tifluadom,
tiflucarbine, tiformin, tifurac, tigemonam, tigestol, tigloidine,
tilbroquinol, tiletamine, tilidine, tiliquinol, tilisolol,
tilmicosin, tilomisole, tilorone, tilozepine, tilsuprost,
timefurone, timegadine, timelotem, timepidium bromide, timiperone,
timobesone acetate, timofibrate, timolol, timonacic, timoprazole,
tinabinol, tinazoline, tinidazole, tinisulpride, tinofedrine,
tinoridine, tiocarlide, tioclomarol, tioconazole, tioctilate,
tiodazosin, tiodonium chloride, tiomergine, tiomesterone,
tioperidone, tiopinac, tiopronin, tiopropamine, tiospirone,
tiotidine, tioxacin, tioxamast, tioxaprofen, tioxidazole,
tioxolone, tipentosin, tipepidine, tipetropium bromide, tipindole,
tipredane, tiprenolol, tiprinast, tipropidil, tiprostanide,
tiprotimod, tiquinamide, tiquizium bromide, tiratricol,
tiropramide, tisocromide, tisopurine, tisoquone, tivandizole,
tixadil, tixanox, tixocortol pivalate, tizabrin, tianidine,
tizolemide, tizoprolic acid, tobramycin, tobuterol, tocainide,
tocamphyl, tocofenoxate, tocofibrate, tocophersolan, todralazine,
tofenacin, tofetridine, tofisoline, tofisopam, tolamolol,
tolazamide, tolazoline, tolboxane, tolbutamide, tolciclate,
toldimfos, tolfamide, tolfenamic acid, tolgabide, tolimidone,
tolindate, toliodium chloride, toliprolol, tolmesoxide, tolmetin,
tolnaftate, tolnapersine, tolnidamine, toloconium metilsulfate,
tolonidine, tolonium chloride, toloxatone, toloxychlorinol,
tolpadol, tolpentamide, tolperisone, toliprazole, tolpronine,
tolpropamine, tolpyrramide, tolquinzole, toirestat, toltrazuril,
tolufazepam, tolycaine, tomelukast, tomoglumide, tomoxetine,
tomoxiprole, tonazocine, topiramate, toprilidine, tonazocine,
topiramate, toprilidine, topterone, toquizine, torasemide,
toebafylline, toremifene, tosifen, tosufloxacin, tosulur,
toyocamycin, toyomycin, traboxepine, tracazolate, tralonide,
tramadol, tramazoline, trandolapril, tranexamic acid, tranilast,
transcainide, trantelinium bromide, tranylcypromine, trapencaine,
trapidil, traxanox, trazilitine, trazium esilate, trazodone,
trazolopride, trebenzomine, trecadrine, treloxinate, trenbolone
acetate, trengestone, trenizine, trosulfan, trepibutone, trepipam,
trepirium iodide, treptilamine, trequensin, trestolone acetate,
trethinium tosilate, trethocanoic acid, tretinoin, tretoquinol,
triacetin, triafungin, triamcinolone, triamcinolone acetonide,
triamcinolone acetonide-phosphate, triamcinolone benetonide,
triamcinolone diacetate, triamcinolone furetonide, triamcinolone
hexacetonide, triampyzine, triamterene, triazinate, triaziquone,
triazolam, tribendilol, tribenoside, tribromoethanol, tribromsalan,
tribuzone, triacetamide, trichlormethiazide, trichlormethine,
trichloroacetic acid, trichloroethylene, tricribine phosphate,
triclabendazole, triclacetamol, triclazate, triclobisonicum
chloride, triclocarban, triclodazol, triclofenol, piperazine,
triclofos, triclofylline, triclonide, triclosan, tricyclamol
chloride, tridihexethyl chloride, trientine, triethylenemelamine,
triethylenephosphoramide, trifenagrel, trifezolac, triflocin,
triflubazam, triflumidate, trifluomeprazine, trifluoperazine,
trifluperidol, triflupromazine, trifluridine, triflusal,
trigevolol, trihexyphenidyl, triletide, trilostane, trimazosin,
trimebutine, trimecaine, trimedoxime bromide, trimeperidine,
trimeprazine, trimetazidine, trimethadione, trimethamide,
trimethaphan camsylate, trimethidinium methosulfate,
trimethobenzamide, trimethoprim, trimetozine, trimetrexate,
trimexiline, trimipramine, trimoprostil, trimoxamine, trioxifene,
trioxsalen, tripamide, triparanol, tripelennamine, tripotassium
dicitratobismuthate, triprolidine, tritiozine, tritoqualine, trityl
cysteine, trixolane, trizoxime, trocimine, troclosene potassium,
trofosfamide, troleandomycin, trolnitrate, tromantadine,
tromethamine, tropabazate, tropanserin, tropapride, tropatepine,
tropenziline bromide, tropicamide, tropigline, tropiprine,
tropodifene, trospectomycin, trospium chloride, troxerutin,
troxipide, troxolamide, troxonium tosilate, troxypyrrolium
tosilate, troxypyrrolium tosilate, truxicurium iodide,
truxipicurium iodide, tryparsamide, tryptophan, tryptophane
mustard, tuaminoheptane, tubercidine, tubocurarine chloride,
tubulozole, tuclazepam, tulobutrol, tuvatidine, tybamate,
tylocrebin, tylosin, tyramine, tyropanic acid, tyrosine, ubenimex,
ubidecarenone, ubisindine, ufenamate, ufiprazole, uldazepam,
ulobetasol, undecoylium chloride, undecyclenic acid, uracil
mustard, urapidil, urea, uredepa, uredofos, urefibrate, urethane,
uridine, ursodeoxycholic acid, ursucholic acid, vadocaine,
valconazole, valdetamide, valdipromide, valine, valnoctamide,
valofane, valperinol, valproate pivoxil, valproic acid, valpromide,
valtrate, vancomycin hcl, vaneprim, vanillin, vanitolide,
vanyldisulfamide, vapiprost, vecuronium bromide, velnacrine
maleate, venlafaxine, veradoline, veralipride, verapamil, verazide,
verilopam, verofylline, vesnarinone, vetrabutine, vidarabine,
vidarabine phophate, vigabatrin, viloxazine, viminol, vinbarbital,
vinblastine, vinburnine, vincamine, vincanol, vincantril, vincofos,
vinconate, vincristine, vindrburnol, vindesine, vindepidine,
vinformide, vinglycinate, vinorelbine, vinpocetine, vinpoline,
vinrosidine, vintiamol, vintriptol, vinylbital, vinylether,
vinzolidine, viomycin, viprostol, viqualine, viquidil,
virginiamycin factors, viroxime, visnadine, visnafylline, vitamin
e, volazocine, warfarin, xamoterol, xanoxic acid, xanthinol
niacinate, xanthiol, xantifibrate, xantocillin, xenalipin, xenazoic
acid, xenbucin, xenipentone, xenthiorate, xenygloxal, xenyhexenic
acid, xenytropium bromide, xibenolol, xibornol, xilobam,
ximoprofen, xinidamine, xinomiline, xipamide, xipranolol,
xorphanol, xylamidine, xylazine, xylocoumarol, xylometazoline,
xyloxemine, yohimbic acid, zabicipril, zacopride, zafuleptine,
zaltidine, zapizolam, zaprinast, zardaverine, zenazocine mesylate,
zepastine, zeranol, zetidoline, zidapamide, zidometacin,
zidovudine, zilantel, zimeldine, zimidoben, zinc acetate, zinc
phenolsulfonate, zinc undecylenate, zindotrine, zindoxifene,
zinoconazole, zinterol, zinviroxime, zipeprol, zocainone,
zofenopril, zoficonazole, zolamine, zolazepam, zolenzepine,
zolertine, zolimidine, zoliprofen, zoloperone, zolpidem, zomebazam,
zomepirac, zometapine, zonisamide, zopiclone, zorubicin, zotepine,
zoxazolamine, zuclomiphene, zuclophenthixol, zylofuramine.
[0185] The following non-limitative examples serve to illustrate
the concept of multiple receptor specificity. Other combinations of
vectors, spacers and reporters and conjugation technologies leading
to multiple vector incorporation are also considered relevant to
this invention. Confirmation of the microparticulate nature of
products is performed using microscopy as described in
WO-A-9607434. Ultrasonic transmission measurements may be made
using a broadband transducer to indicate suspensions of products
giving an increased sound beam attenuation compared to a standard.
Flow dytometric analysis of products can be used to confirm
attachment of antibodies thereto. The ability of targeted agents to
bind specifically to cells expressing a target may be studied by
microscopy and/or using a flow chamber containing immobilised
cells, for example employing a population of cells expressing the
target structure and a further population of cells not expressing
the target. Radioactive, fluorescent or enzyme-labelled
streptavidin/avidin may be used to analyse biotin attachment.
EXAMPLE 1
Preparation and Biological Evaluation of Multiple-specific
Gas-containing Microbubbles of DSPS `Doped` with a Lipopeptide
Consisting of a Heparin Sulphate Binding Peptide (KRKR) and a
Fibronectin Peptide (WOPPRARI)
[0186] This example is directed at the preparation of targeted
microbubbles comprising multiple peptidic vectors arranged in a
linear sequence.
[0187] a) Synthesis of a Lipopeptide Consisting of a Heparin
Sulphate Binding Peptide (KRKR) and Fibronectin Peptide (WOPPRARI).
1
[0188] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Fmoc-Ile-Wang resin (Novabiochem)
on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids and palmitic acid were preactivated using HBTU before
coupling.
[0189] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT, 5% anisole and 5% H.sub.2O for 2 hours giving a
crude product yield of 150 mg.
[0190] Purification by preparative HPLC (Vydac 218TP1022 column) of
a 40 mg aliquot of crude material was carried out using a gradient
of 70 to 100% B over 40 min (A=0.1% TFA/water and B=MeOH) at a flow
rate of 9 mL/min. After lyophilization 16 mg of pure material was
obtained (Analytical HPLC; Gradient, 70-100% B where B=MeOH,
A=0.01% TFA/water: column--vydac 218TP54: Detection--UV 260 and
fluorescence, Ex.sub.280, Em.sub.350--product retention time=19.44
min). Further product characterization was carried out using MALDI
mass spectrometry; expected, M+H at 2198, found, at 2199.
[0191] b) Preparation of Gas-containing Microbubbles of DSPS
`Doped` with a Multiple-specific Lipopeptide Consisting of a
Heparin Sulphate Binding Peptide (KRKR) and Fibronectin Peptide
(WOPPRARI).
[0192] DSPS (Avanti, 4.5 mg) and lipopeptide from a) (0.5 mg) were
weighed into each of 2 vials and 0.8 mL of a solution of 1.4%
propylene glycol/2.4% glycerol was added to each vial. The mixture
was warmed to 80.degree. C. for 5 minutes (vials shaken during
warming). The samples were cooled to room temperature and the head
space flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 s and the microbubbles rolled overnight. Bubbles
were washed several times with deionised water and analysed by
Coulter counter (Size: 1-3 micron (87%), 3-5 micron (11.5%)) and
acoustic attenuation (frequency max att.: 3.5 MHz). The
microbubbles were stable at 120 mm Hg.
[0193] MALDI mass spectral analysis was used to confirm
incorporation into DSPS microbubbles as follows; ca. 0.05-0.1 mL of
microbubble suspension was transferred to a clean vial and 0.05-0.1
mL methanol added. The suspension was sonicated for 30 s and the
solution analysed by MALDI MS. Positive mode gave M+H at 2200,
expected for lipopeptide, 2198.
[0194] c) In Vitro Study of Gas-containing Microbubbles of DSPS
`Doped` with a Multiple-specific Lipopeptide Consisting of a
Heparin Sulphate Binding Peptide (KRKR) and Fibronectin Peptide
(WOPPRARI): Binding to Endothelial Cells Under Flow Conditions
[0195] The human endothelial cell line ECV 304, derived from a
normal umbilical cord (ATCC CRL-1998) was cultured in 260 mL Nunc
culture flasks (chutney 153732) in RPMI 1640 medium (Bio Whittaker)
to which L-Glutamine 200 mM, Penicillin/Streptomycin (10.000 U/mL
and 10.000 mcg/mL) and 10% Fetal Bovine Serum (Hyclone Lot no. AFE
5183) were added.
[0196] The cells were subcultured with a split ratio of 1:5 to 1:7
when reaching confluence.
[0197] Cover-glasses, 22mm in diameter (BDH, Cat no. 406/0189/40)
were sterilised and placed on the bottom of 12 well culture plates
(Costar) before cells in 0,5 mL complete medium with serum was
added on top.
[0198] When the cells reached confluence the coverslips were placed
in a custom made flow-chamber. The chamber consists of a groove
carved into a glass plate upon which the cover slip with cells was
placed with the cells facing the groove forming a flow channel.
[0199] Ultrasound microbubbles from section b) were passed from a
reservoir held at 37 degree Celsius through the flow chamber and
back to the reservoir using a peristaltic pump. The flow rate was
adjusted to simulate physiological relevant shear rates. The flow
chamber was placed under a microscope and the interaction between
the microspheres and cells viewed directly. A camera mounted on the
microscope was connected to a colour video printer and a
monitor.
[0200] A gradual accumulation of the microbubbles on the cells took
place which was dependent on the flow rate. By increasing the flow
rate the cells started to become detached from the coverslip, the
microbubbles were still bound to the cells. Control bubbles not
carrying the vector did not adhere to the endothelial cells and
disappeared from the cells under minimal flow conditions.
[0201] d) In Vivo Experiment in Dog
[0202] Case 1)
[0203] A 22 kg mongrel dog was anaesthetized with pentobarbital and
mechanically ventilated. The chest was opened by a midline
sternotomy, the anterior pericardium was removed, and a 30 mm
gelled silicone rubber spacer was inserted between the heart and a
P5-3 transducer of an ATL HDI-3000 ultrasound scanner. The scanner
was set for intermittent short axis imaging once in each
end-systole by delayed EGC triggering.
[0204] A net volume of 2 mL of microbubbles from b) were injected
as a rapid intravenous bolus. 3 seconds later, the imaged right
ventricle was seen to contain contrast material, another 3 seconds
later, the left ventricle was also filled, and a transient
attenuation shadow that obscured the view of the posterior parts of
the left ventricle was observed. A substantial increase in
brightness of the myocardium was seen, also in the portions of the
heart distal to the left ventricle when the attenuation shadow
subsided.
[0205] After passage of the inital bolus, the ultrasound scanner
was set to continuous, high frame rate high output power imaging, a
procedure known to cause destruction of utrasound contrast agent
bubbles in the imaged tissue regions. After a few seconds, the
scanner was adjusted back to its initial setting. The myocardium
was then darker, and closer to the baseline value. Moving the
imaged slice to a new position resulted in re-appearance of
contrast effects, moving the slice back to the initial position
again resulted in a tissue brightness again close to baseline.
[0206] Case 2) [Comparative]
[0207] A net volume of 2 mL microbubbles prepared in an identical
manner to b) above with the exception that no lipopeptide was
included in the preparation was injected, using the same imaging
procedure as above. The myocardial echo enhancement was far less
intense and of shorter duration than observed in case 1. At the
completion of the left ventricular attenuation phase, there was
also almost complete loss of myocardial contrast effects, and a
myocardial echo increases in the posterior part of the left
ventricle as in case 1 was not observed.
EXAMPLE 2
Multiple-specific Gas-containing Microbubbles of DSPS `Doped` with
RGDC-Mal-PEG.sub.2000-DSPE and a Lipopeptide Consisting of a
Heparin Sulphate Binding Peptide (KRKR) and Fibronectin Peptide
(WOPPRARI)
[0208] This example is directed at the preparation of targeted
microbubbles comprising multiple peptidic vectors.
[0209] a) Synthesis of 3-Maleimidopropionylamido-PEG.sub.200-acyl
Distearoyl Phosphatidylethanolamine (PE-PEG-MAL)
[0210] A mixture of distearoyl phosphatidyl ethanolamine (DSPE),
(37.40 mg, 0.005 mmol),
N-hydroxysuccinimido-PEG.sub.2000-maleimide, NHS-PEG-MAL, (100 mg,
0.25 mmol) and triethylamine (35 .mu.l, 0.25 mmol) in a solution of
chloroform/methanol (3:1) was stirred at room temperature for 24
hours. After evaporation of the solvents under reduced pressure,
the residue was purified by flash chromatography
(chloroform/methanol, 8:2). The product was obtained as a white
wax, 92 mg (66%) and structure was verified by NMR and
maldi-MS.
[0211] b) Synthesis of RGDC
[0212] The RGDC peptide was synthesised on a ABI 433A automated
peptide synthesiser (0.25 mmol scale, Fmoc-Cys(Trt)-Wang resin,
(Novabiochem). All amino acids were activated using HBTU. The crude
peptide was removed from the resin and simultaneously deprotected
in TFA containing 5% EDT, 5% phenol and 5% water. Following
evaporation of the excess cleavage solution the peptide was
precipitated and triturated several times with diethyl ether before
air drying. The crude peptide was purified by preparative hplc and
fractions containing pure product combined and freeze dried. Final
characterisation was performed using analytical hplc and MALDI
MS.
[0213] c) Preparation of Multiple-specific Gas-filled Microbubbles
Encapsulated by Phosphatidylserine and `Doped` with
RGDC-Mal-PEG.sub.3400-DSPE and a Lipopeptide Comprising a Heparin
Sulphate Binding Peptide (KRKR) and Fibronectin Peptide
(WOPPRARI).
[0214] DSPS (Avanti, 5.0 mg), lipopeptide (0.5 mg) from example 1
a) and PE-PEG-MAL (0.5 mg) from section a) was weighed into a clean
vial and 1.0 mL of a solution of 1.4% propylene glycol/2.4%
glycerol added. The mixture was sonicated for 3-5 mins, warmed to
80.degree. C. for 5 minutes then filtered through a 4.5 micron
filter. The mixture was cooled to room temperature and the head
space flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for
3 minutes. The infranatant was exchanged with 1 mL of PBS
containing 1 mg of the peptide RGDC and the pH adjusted to 8. The
conjugation reaction was allowed to proceed for 2 h. The bubbles
were washed in PBS then with water until all unreacted RGDC had
been removed from the infranatant as observed by MALDI-MS. The
microbubbles were further analysed by Coulter counter (98% between
1 and 7 micron).
[0215] d) In Vitro Binding Assay.
[0216] The binding of microbubbles to endothelial cells was carried
out under flow conditions using the in vitro assay described in
example 1. c). A gradual accumulation of the microbubbles on the
cells took place which was dependant on the flow rate. Control
bubbles not carrying the vectors did not adhere to the endothelial
cells detaching from the cells under minimal flow conditions.
EXAMPLE 3
Preparation of Multiple-specific Gas-containing Microbubbles
Encapsulated with DSPS and Thiolated Anti-CD62-Mal-PEG.sub.2000-PE
and Thiolated-anti-ICAM-1-Mal-PEG.sub.2000-PE
[0217] This example is directed at the preparation of microbubbles
comprising multiple antibody vectors for targeted ultrasound.
[0218] a) Preparation of Gas-containing Microbubbles Encapsulated
with DSPS and PE-PE.sub.22000-MAL
[0219] DSPS (Avanti, 4.5 mg) and PE-PEG.sub.2000-Maleimide from
example 2 a) (0.5 mg) were weighed into a clean vial and 1 mL of a
solution of 1.4% propylene glycol/2.4% glycerol added. The mixture
was warmed to 80.degree. C. for 5 minutes then filtered through a
4.5 micron filter. The sample was cooled to room temperature and
the head space flushed with perfluorobutane gas. The vials were
shaken in a cap mixer for 45 s and the microbubbles washed three
times with distilled water.
[0220] b) Thiolation of Anti-CD62 and Anti-ICAM-1 Antibodies
[0221] To 0.3 mg each of anti-CD62 and anti-ICAM-1 antibodies
dissolved in PBS buffer (pH 7, 0.5 mL) was added Traut's reagent
and the solutions stirred at room temperature for 1 h. Excess
reagent was separated from the modified protein on a NAP-5 column
(Pharmacia).
[0222] c) Conjuaation of Thiolated Anti-CD62 and Anti-ICAM-1
Antibodies to Gas-containing Microbubbles Encapsulated with DSPS
and DSPE-PEG.sub.2000-MAL
[0223] 0.5 mL of the mixed thiolated antibody preparation from b)
was added to an aliquot of microbubbles from a) and the conjugation
reaction allowed to proceed for 30 min on a roller table. Following
centrifugation at 2000 rpm for 5 min the infranatant was removed.
The microbubbles were washed a further three times with water.
[0224] The PEG spacer length may also be varied to include longer
e.g. PEG.sub.3400 and PEG.sub.5000 or shorter e.g. PEG.sub.600 or
PEG.sub.800 chains. Addition of a third antibody such as
thiolated-anti-CD34 is also envisaged. EXAMPLE 4
Targeted Multiple-specific Gas-containing Microbubbles of DSPS
Coated Non-covalently with Polylysine and a Fusion Peptide
Comprising a PS Binding Component and a Fibronectin Peptide
Sequence
NH.sub.2F.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.P.R.A.I.OH.
[0225] a) Synthesis of PS Binding/Fibronectin Fragment Fusion
Peptide
NH.sub.2F.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.P.R.A.I.OH.
[0226] The peptide was synthesised on an ABI 433A automatic peptide
synthesiser starting with Fmoc-Ile-Wang resin (Novabiochem) on a
0.1 mmol scale using 1 mmol amino acid cartridges. All amino acids
were preactivated using HBTU before coupling.
[0227] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT and 5% H.sub.2O for 2 hours giving a crude product
yield of 302 mg. Purification by preparative HPLC (Vydac 218TP1022
column) of a 25 mg aliquot of crude material was carried out using
a gradient of 20 to 40% B over 40 min (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) at a flow rate of 9 mL/min. After lyophilization
10 mg of pure material was obtained (Analytical HPLC; Gradient, 20
to 50% B where B=0.1% TFA/acetonitrile, A=0.01% TFA/water:
column--vydac 218TP54: Detection--UV 214 and 260 nm--product
retention time=12.4 min). Further product characterization was
carried out using MALDI mass spectrometry; expected, M+H at 2856,
found, at 2866.
[0228] b) Preparation of Microbubbles of DSPS Coated Non-covalently
with Polylysine and the PS Binding/Fibronectin Fragment Fusion
Peptide
NH.sub.2F.N.F.R.L.K.A.G.O.K.I.R.F.G.G.G.G.W.O.P.P.R.A.I.OH.
[0229] DSPS (5 mg, Avanti) was weighed into a clean vial along with
poly-L-lysine (Sigma, 0.2 mg) and peptide from a) above (0.2 mg).
To the vial was added 1.0 mL of a solution of 1.4% propylene
glycol/2.4% glycerol. The mixture was warmed to 80.degree. C. for 5
minutes. The sample was cooled to room temperature and the head
space flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 s and the microbubbles centrifuged at 1000 rpm for
3 minutes.
[0230] Following extensive washing with water, PBS and water the
final solution was examined for polylysine and peptide content
using MALDI MS. No polypeptide material was observed in the final
wash solution.
[0231] Acetonitrile (0.5 mL) was then added and the microbubbles
destroyed by sonication. Analysis of the resulting solution for
polylysine and PS-binding/fibronectin fusion peptide was then
carried out using MALDI MS. The results were as follows:
24 MALDI expected MALDI found Poly-L-lysine 786, 914, 1042, 1170
790, 919, 1048, 1177 DSPS-binding peptide 2856 2866
[0232] The spacer element contained within the PS
binding/Fibronectin fusion peptide (-GGG-) can also be replaced
with other spacers such as PEG.sub.2000 or poly alanine (-AAA-). It
is also envisaged that a form of pre-targeting may be employed,
whereby the DSPS binding/Fibronectin fragment fusion peptide is
firstly allowed to associate with cells via the fibronectin peptide
binding. This is followed by administration of PS microbubbles
which then bind to the PS binding peptide.
EXAMPLE 5
Multiple-specific Gas-containing Microbubbles Encapsulated with
Phosphatidylserine and Biotin-PEG.sub.3400-alanyl-cholesterol and
Functionalised with Streptavidin/biotinyl-endothelin-1 Peptide
(Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and
Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)
[0233] This example is directed at the preparation of targeted
ultrasound microbubbles whereby streptavidin is used as a linker
between biotinylated reporter(s) and vector(s).
[0234] a) Synthesis of Biotin-PEG.sub.3400-.beta.-Alanine
Cholesterol
[0235] To a solution of cholesteryl-.beta.-alanine hydrochloride
(15 mg, 0.03 mmol) in 3 mL chloroform/wet methanol (2.6:1), was
added triethylamine (42 mL, 0.30 mmol). The mixture was stired for
10 minutes at room temperature and a solution of
biotin-PEG.sub.3400-NHS (100 mg, 0.03 mmol) in 1,4-dioxan (1 mL)
was added dropwise. After stirring at room temperature for 3 h, the
mixture was evaporated to dryness and the residue purified by flash
chromatography to give white crystals, yield; 102 mg (89%). The
structure was verified by MALDI-MS and NMR.
[0236] b) Synthesis of Biotinylated Endothelin-1 Peptide
(Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH)
[0237] The peptide was synthesised on a ABI 433A automatic peptide
synthesiser starting with Fmoc-Trp(Boc)-Wang resin (Novabiochem) on
a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids were preactivated using HBTU before coupling.
[0238] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
anisole and 5% H.sub.2O for 2 hours giving a crude product yield of
75 mg. Purification by preparative HPLC (Vydac 218TP1022 column) of
a 20 mg aliquot of crude material was carried out using a gradient
of 30 to 80% B over 40 min (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) and a flow rate of 9 mL/min. After lyophilization
of the pure fractions 2 mg of pure material was obtained
(Analytical HPLC; Gradient, 30-80% B where B=0.1% TFA/acetonitrile,
A=0.01% TFA/water: column--vydac 218TP54: Detection--UV 214
nm--product retention time=12.6 min). Further product
characterization was carried out using MALDI mass spectrometry;
expected, M+H at 1077, found, 1077.
[0239] c) Synthesis of Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)
[0240] This peptide was synthesised and purified using similar
protocols to those described in section b) above. The pure product
was characterised by hplc and MALDI MS.
[0241] d) Preparation of Multiple-specific Gas-filled Microbubbles
Encapsulated with Phosphatidylserine and
Biotin-PEG.sub.3400-.beta.-Alani- ne Cholesterol
[0242] DSPS (Avanti, 4.5 mg) and biotin-PEG.sub.3400-.beta.-Alanine
cholesterol from section a) (0.5 mg) were weighed into a vial and
0.8 mL of a solution of 1.4% propylene glycol/2.4% glycerol added.
The mixture was warmed to 80.degree. C. for 5 minutes (vials shaken
during warming). The sample was cooled to room temperature and the
head space flushed with perfluorobutane gas. The vial was shaken in
a cap mixer for 45 s and the microbubbles rolled overnight.
[0243] The microbubble suspension was washed several times with
deionised water and analysed by Coulter counter and acoustic
attenuation.
[0244] e) Conjugation with Fluorescein Labelled Streptavidin and
Biotinylated Peptides from Section b) and c).
[0245] To the microbubble preparation from d) was added fluorescein
conjugated streptavidin (0.2 mg) dissolved in PBS (1 mL). The
bubbles were placed on a roller table for 3 h at room temperature.
Following extensive washing with water and analysis by fluorescence
microscopy the microbubbles were incubated in 1 mL of PBS
containing biotinyl-Endothelin-1 peptide (0.5 mg) and
biotinyl-Fibrin-anti-polymeran- t peptide (0.5 mg) from sections b)
and c) respectively for 2 h. Extensive washing of the microbubbles
was performed to remove unconjugated peptide.
EXAMPLE 6
Multiple-specific Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and a Biotinylated Lipopeptide Used to Prepare a
Streptavidin `Sandwich` with a Mixture of Biotinyl-endothelin-1
Peptide (Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and
Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)
[0246] a) Synthesis of Lipopeptide
Dipalmitoyl-lysinyl-tryptophanyl-lysiny-
l-lysinyl-lysinyl(biotinyl)-glycine
[0247] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Fmoc-Gly-Wang resin (Novabiochem)
on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids and palmitic acid were preactivated using HBTU before
coupling.
[0248] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT, 5% anisole and 5% H.sub.2O for 2 hours giving a
crude product yield of 150 mg. Purification by preparative HPLC
(Vydac 218TP1022 column) of a 40 mg aliquot of crude material was
carred out using a gradient of 70 to 100% B over 40 min (A=0.1%
TFA/water and B=MeOH) at a flow rate of 9 mL/min. After
lyophilization 14 mg of pure material (Analytical HPLC; Gradient,
70-100% B where B=MeOH, A=0.01% TFA/water: column--vydac 218TP54:
Detection 13 UV 260 and fluorescence, Ex280, Em350--product
retention time=22 min). Further product characterization was
carried out using MALDI mass spectrometry; expected, M+H at 1478,
found, 1471.
[0249] b) Preparation of Gas-containing Microbubbles of DSPS
`Doped` with the Biotinylated Lipopeptide Sequence from Section
a)
[0250] DSPS (Avanti, 4.5 mg) and lipopeptide from a) (0.5 mg) were
weighed into each of 2 vials and 0.8 mL of a solution of 1.4%
propylene glycol/2.4% glycerol was added to each vial. The mixture
was warmed to 80.degree. C. for 5 minutes (vials shaken during
warming). The samples were cooled to room temperature and the head
space flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 s and the microbubbles formed rolled overnight.
The microbubbles were washed several times with deionised water and
analysed by Coulter counter and acoustic attenuation.
[0251] MALDI mass spectral analysis was used to confirm
incorporation into DSPS microbubbles as described in example 1
b).
[0252] c) Preparation of Multiple-specific Gas-filled Microbubbles
Encapsulated with Phosphatidylserine and a Biotinylated Lipopeptide
and Functionalised with Streptavidin/biotinyl-endothelin-1 Peptide
(Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH)/biotinyl-fibrin-anti-polymerant
Peptide (Biotin-GPRPPERHOS.NH.sub.2)
[0253] The microbubble preparation from b) above was treated in an
analogous manner to that described in example 5 section e).
EXAMPLE 7
Multiple-specific Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and Biotin-DPPE Used to Prepare a Streptavidin
`Sandwich` with a Mixture of Biotinyl-endothelin-1 Peptide
(Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and
Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)
[0254] a) Preparation of Biotin Containing Microbubbles.
[0255] To a mixture of phosphatidylserine (5 mg, Avanti) and
biotin-DPPE (0.6 mg, Pierce) in a clean vial was added 5%
propyleneglycol-glycerol in water (1 mL). The dispersion was heated
to 80.degree. C. for 5 minutes and then cooled to ambient
temperature. The head space was then flushed with perfluorobutane
and the vial shaken in a cap-mixer for 45 seconds. After
centrifugation the infranatant was removed and the microbubbles
formed washed extensively with water.
[0256] b) Conjugation of Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and Biotin-DPPE with Streptavidin and a Mixture
of Biotinyl-endothelin-1 (Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) and
Biotinyl-Fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)
[0257] The procedure detailed in example 5 section e) was
followed.
EXAMPLE 8
Multiple-specific Gas-filled Microbubbles Encapsulated with
Phosphatidylserine, Streptavidin-Succ-PEG-DSPE and a Mixture of
Biotinylated Human Endothelium IgG Antibody and Biotinylated
Transferrin
[0258] a) Synthesis of Succ-PEG.sub.3400-DSPE
[0259] NH.sub.2-PEG.sub.3400-DSPE is carboxylated using succinic
anhydride, e.g. by a similar method to that described by Nayar, R.
and Schroit, A. J. in Biochemistry (1985) 24, 5967-71.
[0260] b) Preparation of Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0261] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Succ-PEG.sub.3400-DSPE (10-0.1 mol %) is added 5%
propyleneglycol-glycero- l in water (1 mL). The dispersion is
heated to not more than 80 .degree. C. for 5 minutes and then
cooled to ambient temperature. The dispersion (0.8 mL) is
transferred to a vial (1 mL) and the head space is flushed with
perfluorobutane. The vial is shaken in a cap-mixer for 45 seconds,
whereafter the sample is put on a roller table. After
centrifugation the infranatant is exchanged with water and the
washing is repeated.
[0262] c) Coupling of Streptavidin to Gas-filled Microbubbles
Encapsulated with Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0263] Streptavidin is covalently bound to Succ-PEG.sub.3400-DSPE
in the membrane by standard coupling methods using a water-soluble
carbodiimide. The sample is placed on a roller table during the
reaction. After centrifugation the infranatant is exchanged with
water and the washing is repeated. The functionality of the
attached streptavidin is analysed by binding, e.g. to fluorescently
labeled biotin, biotinylated antibodies (detected with a
fluorescently labeled secondary antibody) or biotinylated and
fluorescence- or radioactively-labeled oligonucleotides. Analysis
is performed by fluorescence microscopy or scintillation
counting.
[0264] d) Preparation of Multiiple-specific Gas-filled Microbubbles
Encapsulated with Phosphatidylserine and
Streptavidin-Succ-PEG.sub.3400-D- SPE Non-covalently Functionalised
with Biotinylated Human Transferrin and Human Endothelium IgG
Antibody
[0265] Microbubbles from section c) are incubated in a solution
containing human transferrin and human endothelium IgG antibody
biotinylated using the method described by Bayer et al., Meth.
Enzymol., 62, 308. The vector-coated microbubbles are washed as
described above.
EXAMPLE 9
Multiple-specific Gas-filled Microbubbles Encapsulated with
Phosphatidylserine/streptavidin-Succ-PEG-DSPE and the
Oligonucleotides Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and
biotin-GGCGCTGATGATGTTGTTGATTCTT
[0266] a) Synthesis of Succ-PEG.sub.3400-DSPE
[0267] Described in example 8 a)
[0268] b) Preparation of Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0269] Described in example 8b).
[0270] c) Coupling of Streptavidin to Gas-filled Microbubbles
Encapsulated with Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0271] Described in example 8 c).
[0272] d) Preparation of Gas-filled Microbubbles Encapsulated with
Phosphatidylserine/streptavidin-Succ-PEG-DSPE and the
Oligonucleotides Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and
Biotin-GGCGCTGATGATGTTGTTGATTCTT
[0273] Microbubbles from section c) are incubated in a solution
containing a mixture of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and
biotin-GGCGCTGATGATGTTG- TTGATTCTT. The oligonucleotide-coated
microbubbles are washed as described above. Binding of the
oligonucleotide to the bubbles is detected e.g. by using
fluorescent-labeled oligonucleotides for attachment to the bubbles,
or by hybridising the attached oligonucleotide to a labeled
(fluorescence or radioactivity) complementary oligonucleotide. The
functionality of the oligonucleotide-carrying microbubbles is
analysed, e.g. by hybridising the bubbles with immobilized
DNA-containing sequences complementary to the attached
oligonucleotide.
[0274] Other useful examples include an oligonucleotide
complementary to ribosomal DNA (of which there are many copies per
haploid genome) and an oligonucleotide complementary to an oncogene
(e.g. ras of which there is one copy per haploid genome) are
used.
EXAMPLE 10
Multiple-specific Gas-filled Microbubbles Encapsulated with
Phosphatidylserine and Phosphatidylethanolamine Covalently
Functionalised with the Fibronectin and Transferrin Proteins
[0275] a) Microbubbles Preparation.
[0276] DSPS (Avanti, 4.5 mg) and DSPE (Avanti, 1.0 mg) were weighed
into a clean vial and 1 mL of a solution of 1.4% propylene
glycol/2.4% glycerol added. The mixture was warmed to 80.degree. C.
for 5 minutes then filtered through a 4.5 micron filter. The sample
was cooled to room temperature and the head space flushed with
perfluorobutane gas. The vial was shaken in a cap mixer for 45 s
and the microbubbles washed two times with distilled water then
resuspended in 0.1 M sodium borate buffer pH 9.
[0277] b) Modification of Fibronectin/Transferrin.
[0278] Fibronectin (0.5 mg) and transferrin (1.3 mg) were mixed in
PBS and a solution containing NHS-fluorescin in DMSO added. The
mixture was stirred at room temperature for 1 hour then the protein
purified on a Superdex 200 column. The fluorescein-labelled protein
mixture in phosphate buffer pH 7.5 was freeze dried.
[0279] c) Microbubble Modification.
[0280] The freeze-dried product from b) was re-dissolved in 0.5 mL
water and to the fluorescein labelled fibronectin/transferrin
mixture was added 0.1 mmol of the crosslinker SDBP (Pierce). The
solution was incubated on ice for 2 hours, charged on a NAP-5
column and eluted with PBS. To this was added 1 mL of the
microbubble suspension from a) and incubation allowed to proceed
for 2 h at room temperature on a roller table. Unreacted material
was removed by allowing the microbubbles to float then replacing
the buffer with water, this process was repeated 3 times.
EXAMPLE 11
Preparation of Multiple-specific Hollow Polymer Particles
Incorporating Avidin in the Polymer Wall Conjugated with the
Oligonucleotide Biotin-GGCGCTGATGATGTTGTTGATTCTT and the
Endothelin-1 Peptide Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH
[0281] This example is directed at the preparation of polymeric
ultrasound contrast agents comprising multiple vectors attached to
non-surfactant for targeting/therapeutic applications.
[0282] a) Preparation of Polymer Particles Incorporating Avidin in
the Polymer Wall
[0283] Hollow polymer particles of P73 (as described in patent WO
96/07434) containing avidin were prepared by a process involving
the freeze-drying of an oil-in-water emulsion using the following
procedure: An oil solution was prepared by dissolving 0.25 g of the
biodegradable polymer P73 [poly(ethylidene
bis(16-hydroxyhexadecanoate)co(adipic acid)] in 5 mL of camphene at
60.degree. C. To 0.2 mL of the oil solution was added 2 mg avidin.
An aqueous solution was then prepared by dissolving 0.4 g of the
polymer, a-(16-hexadecanoyloxyhexadecanoyl)-w-methoxypolyoxy-
ethylene ester, in 20 mL of water at 60.degree. C. The oil solution
(0.2 mL) was then mixed with of the aqueous solution (0.8 mL) in a
vibromixer (Capmix) for 15 s to form the oil-in-water emulsion. The
emulsion was frozen in dry ice and methanol then dried at a
pressure of 200 mTorr for 24 h to remove excess solvent. The powder
was reconstituted as a suspension of hollow particles by addition
of 1.0 mL water. The resulting ultrasound contrast agent was
confirmed by microscopy observation, Coulter size distribution,
acoustic attenuation and resistance to external pressure.
[0284] b) Synthesis of Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH
[0285] Described in example 5 b).
[0286] c) Conjugation of Polymer Particles Incorporating
Avidin.
[0287] The particles from a) were centrifuged and the supernatant
replaced with 1 mL of PBS buffer pH 7.5 containing 0.2 mg of
biotin-GGCGCTGATGATGTTGTTGATTCTT and 0.2 mg of
biotin-D-Trp-Leu-Asp-Ile-I- le-Trp.OH from b) above. After
incubation for 24 h the particles were washed extensively with PBS
and water.
EXAMPLE 12
Functionalisation of Gas-filled Albumin Microspheres (GAM) with
Biotin for Multiple-specific Targeting
[0288] a) Preparation of Biotinylated Albumin Microspheres
[0289] A homogeneous suspension of GAM (6.times.10.sup.8
particles/mL) in 5 mg/mL albumin was used, with all manipulations
being carried out at room temperature. Two 10 mL aliquots were
centrifuged (170.times.g, 5 minutes) to promote floatation of the
microspheres and 8 mL of the underlying infranatant was removed by
careful suction and replaced by an equal volume of air-saturated
phosphate buffered saline, the preparations being rotated for 15-20
minutes to resuspend the microspheres. This procedure was repeated
twice, whereafter only negligible amounts of free
non-microsphere-associated albumin were assumed to remain. 50 .mu.l
of NHS-biotin (10 mM in dimethylsulphoxide) was added to one of the
aliquots (final concentration 50 .mu.M); the other (control)
aliquot received 50 .mu.l of dimethylsulphoxide. The tubes
containing the samples were rotated for 1 hour whereafter 20 .mu.l
portions of 50% aqueous glutaraldehyde were added to each tube to
crosslink the microspheres. After rotation for another hour the
tubes were positioned vertically overnight to allow floatation of
the microspheres. The next day, the suspensions were washed twice
with phosphate buffered saline containing 1 mg/mL human serum
albumin (PBS/HSA) and were resuspended in PBS/HSA after the last
centrifugation.
[0290] In order to determine the presence of microsphere-associated
biotin, streptavidin conjugated to horseradish peroxidase
(strep-HRP) was added to both suspensions and the tubes were
rotated for 1 hour to allow for reaction. The microspheres were
then washed three times, resuspended in 100 mM citrate-phosphate
buffer (pH 5) containing 0.1 mg/mL phenylenediamine dihydrochloride
and 0.01% hydrogen peroxide, and rotated for 10 minutes.
Development of a yellow-green colour was indicative of the presence
of enzyme. The following results were obtained:
25 Sample Colour development Biotinylated spheres + strp-HRP 2+
Control spheres + strp-HRP + This confirms that GAM were
biotinylated.
[0291] b) Multiple-specific Gas-containing Microparticles
[0292] The biotinylated microspheres are then used to prepare
multiple-specific targeting products in an analogous manner to
those exemplified in examples 5), 6) and 7).
EXAMPLE 13
Multiple-specific Gas-containing Microbubbles of DSPS
Functionalised with Heparin Sulphate Binding Peptide/Fibronectin
Peptide/RGD Peptide and Fluorescein.
[0293] a) Synthesis of a Lipopeptide Containing the RGD Sequence
and a Fluorescein Reporter Group: Dipalmitoyl-Lys-Lys-Lys-Lys
[Acetyl-Arg-Gly-Asp-Lys(Fluorescein)]Gly.OH 2
[0294] The lipopeptide was synthesised as described in example 1)
using commercially available amino acids and polymers. The
lipopeptide was cleaved from the resin in TFA containing 5% water,
5% phenol, 5% EDT for 2 h. Following evaporation in vacuo the crude
product was precipitated and triturated with diethyl ether.
Purification by preparative HPLC (Vydac 218TP1022 column) of a 40
mg aliquot of crude material was carried out using a gradient of 60
to 100% B over 40 min (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) at a flow rate of 9 mL/min. After lyophilization
10 mg of pure material (Analytical HPLC; Gradient, 60-100% B where
B=0.1% TFA/acetonitrile), A=0.01% TFA/water: column--vydac 218TP54:
Detection--UV 260--product retention time=20-22 min). Further
product characterization was carried out using MALDI mass
spectrometry; expected, M+H at 1922, found, at 1920.
[0295] b) Synthesis of a Lipopeptide Containing a Heparin Sulphate
Binding Sequence and a Fibronectin Peptide
[0296] Synthesis and purification described in example 1 a).
[0297] c) Preparation of Multiiple-specific Gas-containing
Microbubbles of DSPS Functionalised with a Heparin Sulphate Binding
Peptide a Fibronectin Peptide Acetyl-RGD Peptide and
Fluorescein.
[0298] DSPS (Avanti, 4 mg) and lipopeptide from a) (0.5 mg, 0.2
mmol) and lipopeptide from b) (0.5 mg) were weighed into each of 2
vials and 0.8 mL of a solution of 1.4% propylene glycol/2.4%
glycerol was added to each vial. The mixture was warmed to
80.degree. C. for 5 minutes (vials shaken during warming). The
samples were cooled to room temperature and the head space flushed
with perfluorobutane gas. The vials were shaken in a cap mixer for
45 s and the microbubbles formed rolled overnight. The microbubbles
were washed several times with deionised water and analysed by
MALDI mass spectrometry as described in example 1 b). The
microbubbles following analysis by microscopy were seen to consist
of a range of sizes between 1 and 5 micron. Furthermore the
microbubbles were fluorescent.
EXAMPLE 14
Multiple-specific Gas Containing Microbubbles of DSPS Covalently
Modified with CD71 FITC-labelled Anti-transferrin Receptor Antibody
and `Doped` with a Lipopeptide with Affinity for Endothelial
Cells
[0299] This example is directed at the preparation of multiple
vector targeted ultrasound agents.
[0300] a) Synthesis of an Endothelial Cell Binding Lipopeptide:
2-n-hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Lys-Ala-Leu-Lys-Ala--
Ala-Leu-Lys-Leu-Ala-NH.sub.2.
[0301] The lipopeptide shown below was synthesised on a ABI 433A
automatic peptide synthesiser starting with a Rink amide resin on a
0.1 mmol scale using 1 mmol amino acid cartridges. 3
[0302] All amino acids and 2-n-hexadecylstearic acid were
preactivated using HBTU before coupling. The simultaneous removal
of peptide from the resin and side-chain protecting groups was
carried out in TFA containing 5% EDT, and 5% H.sub.2O for 2 hours
giving a crude product yield of 150 mg. Purification by preparative
HPLC (Vydac 218TP1022 column) of a 40 mg aliquot of crude material
was carried out using a gradient of 90 to 100% B over 50 min
(A=0.1% TFA/water and B=MeOH) at a flow rate of 9 mL/min. After
lyophilization 10 mg of pure material was obtained (Analytical
HPLC; Gradient, 90-100% B where B=MeOH, A=0.01% TFA/water:
column--vydac 218TP54: Detection--UV 214 nm--product retention
time=23 min). Further product characterization was carried out
using MALDI mass spectrometry; expected, M+H at 2369, found, at
2373.
[0303] b) Preparation of Gas-containing Microbubbles of DSPS
`Doped` with a Endothelial Cell Binding Lipopeptide and
PE-PEG.sub.2000-MAL
[0304] DSPS (Avanti, 4.5 mg) and lipopeptide from a) (0.5 mg) along
with PE-PEG.sub.2000-Maleimide from example 2 (0.5 mg) were weighed
into a clean vial and 1 mL of a solution of 1.4% propylene
glycol/2.4% glycerol added. The mixture was warmed to 80.degree. C.
for 5 minutes then filtered through a 4.5 micron filter. The sample
was cooled to room temperature and the head space flushed with
perfluorobutane gas. The vials were shaken in a cap mixer for 45 s
and the microbubbles washed three times with distilled water.
[0305] c) Thiolation of FITC-labelled Anti-transferrin Receptor
Antibody.
[0306] FITC labelled CD71 anti-transferrin receptor Ab (100 mg/mL,
Becton Dickinson), 0.7 mL, in PBS was modified with Traut's reagent
(0.9 mg, Pierce) at room temperature for 1 h. Excess reagent was
separated from modified protein on a NAP-5 column (Pharmacia).
[0307] d) Conjugation of Thiolated FITC-labelled Anti-transferrin
Receptor Antibody to Gas-containing Microbubbles of DSPS `Doped`
with an Endothelial Cell Binding Lipopeptide and
DSPE-PEG.sub.2000-MAL
[0308] A 0.5 mL aliquot of the protein fraction (2 mL in total)
from c) above was added to the microbubbles from b) and the
conjugation reaction allowed to proceed for 10 min on a roller
table. Following centrifugation at 1000 rpm for 3 min the protein
solution was removed and the conjugation repeated a further two
times with 1 mL and 0.5 mL aliquots of protein solution
respectively. The bubbles were then washed four times in distilled
water and a sample analysed for the presence of antibody by flow
cytometry and microscopy. A fluorescent population of >92% was
observed.
[0309] Incorporation into the microbubbles of lipopeptide was
confirmed by MALDI mass spectrometry as described in example 1
b).
EXAMPLE 15
Preparation of Multiple-sepecific Transferrin/Avidin Coated
Gas-filled Microbubbles for Targeted Ultrasound Imaging
[0310] This example is directed to the preparation of microbubbles
containing multiple protein vectors for targeted
ultrasound/therapy.
[0311] a) Synthesis of a Thiol Functionalised Lipid Molecule:
Dipalmitoyl-Lys-Lys-Lys-Aca-Cys.OH 4
[0312] The lipid structure shown above was synthesised on a ABI
433A automatic peptide synthesiser starting with Fmoc-Cys(Trt)-Wang
resin (Novabiochem) on a 0.25 mmol scale using 1 mmol amino acid
cartridges. All amino acids and palmitic acid were preactivated
using HBTU coupling chemistry.
[0313] The simultaneous removal of peptide from the resin and
deprotection of side-chain protecting groups was carried out in TFA
containing 5% EDT, and 5% H.sub.2O for 2 hours giving a crude
product yield of 250 mg. Purification by preparative HPLC (Vydac
218TP1022 column) of a 40 mg aliquot of crude material was carried
out using a gradient of 90 to 100% B over 50 min (A=0.1% TFA/water
and B=MeOH) at a flow rate of 9 mL/min. After lyophilization 24 mg
of pure material was obtained (Analytical HPLC; Gradient, 70-100% B
where B=0.1% TFA/acetonitrile, A=0.01% TFA/water: column--vydac
218TP54: Detection--UV 214 nm-product retention time=23 min).
Further product characterization was carried out using MALDI mass
spectrometry; expected, M+H at 1096, found, at 1099.
[0314] b) Preparation of Gas-containing Microbubbles of DSPS
`Doped` with a Thiol Containing Lipid Structure:
[0315] DSPS (Avanti, 4.5 mg) and the lipid structure from a) above
(0.5 mg) were weighed into a clean vial and 0.8 mL of a solution
containing 1.4% propylene glycol/2.4% glycerol in water added. The
mixture was warmed to 80.degree. C. for 5 minutes (vials shaken
during warming) and filtered while still hot through a 40 micron
filter. The samples were cooled to room temperature and the head
space flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 s and the microbubbles placed on roller table
overnight. Bubbles were washed several times with deionised water
and analysed for thiol group incorporation using Ellmans
Reagent.
[0316] c) Modification of Transferrin and Avidin with
Fluorescein-NHS and Sulpho-SMPB.
[0317] To a mixture of 2 mg of transferrin (Holo, human, Alpha
Therapeutic Corp) and 2 mg of avidin (Sigma) in PBS (1 mL) was
added 0.5 mL DMSO solution containing 1 mg Sulpho-SMPB (Pierce) and
0.5 mg Fluorescein-NHS (Pierce). The mixture was stirred for 45
minutes at room temperature then passed through a Sephadex 200
column using PBS as eluent. The protein fraction was collected and
stored at 4.degree. C. prior to use.
[0318] d) Microbubble Conjugation with Modified
Transferrin/Avidin.
[0319] To the thiol containing microbubbles from b) was added 1 mL
of the modified transferrin/avidin protein solution c). After
adjusting the pH of the solution to 9 the conjugation reaction was
allowed to proceed for 2 h at room temperature. Following extensive
washing with deionised water the microbubbles were analysed by
Coulter counter (81% between 1 and 7 micron) and fluorescence
microscopy (highly fluorescent microbubbles were observed).
EXAMPLE 16
Preparation of Functionalised Gas-filled Microbubbles for Targeted
Ultrasound Imaging
[0320] This example is directed to the preparation of microbubbles
having a reactive group on the surface for non-specific targeting,
principally utilising disulphide exchange reactions to effect
binding to a multiplicity of cellular targets.
[0321] DSPS (Avanti, 5.0 mg) and the thiol containing lipid
structure from example 15 a)(1.0 mg) were weighed into a clean vial
and 0.8 mL of a solution containing 1.4% propylene glycol/2.4%
glycerol in water added. The mixture was warmed to 80.degree. C.
for 5 minutes (vials shaken during warming) and filtered while
still hot through a 40 micron filter. The samples were cooled to
room temperature and the head space flushed with perfluorobutane
gas. The vials were shaken in a cap mixer for 45 s and the
microbubbles placed on roller table overnight. Bubbles were washed
several times with deionised water and analysed for thiol group
incorporation using Ellmans Reagent.
EXAMPLE 17
Multiple-specific Gas-containing Microbubbles of DSPS Comprising a
Lipopeptide for Endothelial Cell Targeting and a Captopril
Containing Molecule
[0322] This example is directed to the preparation of ultrasound
agents for combined targeting and therapeutic applications.
[0323] a) Synthesis of a Lipopeptide Functionalised with Captopril:
5
[0324] The structure shown above was synthesised using a manual
nitrogen bubbler apparatus starting with Fmoc protected Rink Amide
MBHA resin (Novabiochem) on a 0.125 mmol scale. All amino acids
were purchased from Novabiochem and palmitic acid from Fluka.
Coupling was carried out using standard TBTU/HOBt/DIEA protocols.
Bromoacetic acid was coupled through the side-chain of Lys as a
symmetrical anhydride using DIC preactivation. Captopril (Sigma)
dissolved in DMF was introduced on the solid-phase using DBU as
base.
[0325] Simultaneous removal of the peptide from the resin and
deprotection of side-chain protecting groups was carried out in TFA
containing 5% EDT, 5% water and 5% ethyl methyl sulphide for 2 h.
An aliquot of 10 mg of the crude material was purified by
preparative liquid chromatography (Vydac 218TP1022 column) using a
gradient of 70 to 100% B over 60 min (A=0.1% TFA/water and
B=0.1%
[0326] TFA/acetonitrile) at a flow rate of 10 mL/min. After
lyophilization a yield of 2 mg of pure material was obtained
(analytical HPLC: gradient 70-100% B over 20 min, A=0.1% TFA/water
and B=0.1% TFA/acetonitrile; flow rate 1 mL/min; column Vydac
218TP54; detection UV 214 nm; retention time 26 min). Further
characterisation was carried out using MALDI mass spectrometry,
giving M+H at 1265 as expected.
[0327] b) Synthesis of a Lipopeptide with Affinity for Endothelial
Cells:
Dipalmitoyl-Lys-Lys-Lys-Aca-Ile-Arg-Arg-Val-Ala-Arg-Pro-Pro-Leu-NH.sub.2
6
[0328] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Rink amide resin (Novabiochem) on
a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids and palmitic acid were preactivated using HBTU before
coupling.
[0329] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT and 5% H.sub.2O for 2 hours giving a crude product
yield of 160 mg. Purification by preparative HPLC (Vydac 218TP1022
column) of a 35 mg aliquot of crude material was carried out using
a gradient of 70 to 100% B over 40 min (A=0.1%
[0330] TFA/water and B=MeOH) at a flow rate of 9 mL/min. After
lyophilization 20 mg of pure material was obtained (Analytical
HPLC; Gradient, 70-100% B where B=MeOH, A=0.01% TFA/water:
column--vydac 218TP54: Detection--UV 214 and 260 nm--product
retention time=16 min). Further product characterization was
carried out using MALDI mass spectrometry; expected, M+H at 2050,
found, at 2055.
[0331] c) Preparation of Gas-containing Microbubbles of DSPS
Comprising a Lipopeptide for Endothelial Cell Targeting and a
Captopril Containing Molecule for Drug Delivery
[0332] DSPS (Avanti, 4.5 mg), product from a) (0.5 mg) and product
from b) (0.5 mg) were weighed into a vial and 1.0 mL of a solution
of 1.4% propylene glycol/2.4% glycerol was added to each vial. The
mixture was warmed to 80.degree. C. for 5 minutes (vials shaken
during warming). The samples were cooled to room temperature and
the head space flushed with perfluorobutane gas. The vials were
firstly shaken in a cap mixer for 45 s then rolled for 1 h followed
by extensive washing with deionised water. No detectable levels of
starting material were found in the final wash solution as
evidenced by MALDI MS.
[0333] MALDI mass spectral analysis was used to confirm
incorporation of the products from section a) and b) into the
microbubbles as described in example 1 b).
[0334] d) In Vitro Study of Gas-containing Microbubbles of DSPS
Comprising a Lipopepitde for Endothelial Cell Targeting and a
Captopril Containing Molecule for Therapeutic Applications.
[0335] The in vitro assay described in example 1 c) was used to
examine cell binding under flow conditions. A gradual accumulation
of the microbubbles on the cells took place which was dependant on
the flow rate. By increasing the flow rate the cells started to
become detached from the coverslip, the microbubbles were still
bound to the cells. Control bubbles not carrying the vector did not
adhere to the endothelial cells and disappeared from the cells
under minimal flow conditions.
EXAMPLE 18
Preparation of Multiple-specific Gas-containing Microbubbles of
DSPS Loaded with a Lipopeptide Comprising a Helical Peptide with
Affinity for Cell Membranes and the Peptide Antibiotic Polymixin B
Sulphate
[0336] This example is directed at the preparation of targeted
microbubbles comprising multiple peptidic vectors having a combined
targeting and a therapeutic application.
[0337] a) Synthesis of a Lipopeptide Comprising a Helical Peptide
with Affinity for Cell
Membranes:hexadecylstearyl-Lys-Leu-Ala-Leu-Lys-Leu-Ala--
Leu-Lys-Ala-Leu-Lys-Ala-Ala-Leu-Lys-Leu-Ala-NH.sub.2.
[0338] Described in example 14 a).
[0339] b)--Preparation of Multiiple-specific Gas-containing
Microbubbles.
[0340] DSPS (Avanti, 5.0 mg), lipopeptide from a)(0.3 mg)and
polymixin B sulphate (Sigma, 0.5 mg) were weighed into a clean vial
and 1.0 mL of a solution of 1.4% propylene glycol/2.4% glycerol
added. The mixture was sonicated for 3-5 mins, warmed to 80.degree.
C. for 5 minutes then filtered through a 4.5 micron filter. The
mixture was cooled to room temperature and the head space flushed
with perfluorobutane gas. The vial was shaken in a cap mixer for 45
s and the microbubbles centrifuged at 1000 rpm for 3 minutes. The
microbubbles were washed in water until no polymixin B sulphate or
lipopeptide could be detected in the infranatant by MALDI-MS.
Microscopy showed that the size distribution of the bubble
population was between 1-8 micron as desired.
[0341] To the washed bubbles (ca. 0.2 mL) was added methanol (0.5
mL) and the mixture placed in a sonic bath for 2 min. The resulting
clear solution, following analysis by MALDI-MS, was found to
contain both lipopeptide and polymixin B sulphate (expected 1203,
found 1207).
EXAMPLE 19
Preparation of Multiple-specific Gas-containing Microbubbles of
DSPS `Doped` with a Lipopeptide Comprising a IL-1 Receptor Binding
Sequence and Modified with a Branched Structure Containing the Drug
Methotrexate
[0342] This example is directed at the preparation of targeted
microbubbles comprising multiple vectors for
targeted/therapeutic/drug release applications.
[0343] a) Synthesis of a Lipopeptide Comprising an Interleukin-1
Receptor Binding Peptide:
Dipalmitoyl-Lys-Gly-Asp-Trp-Asp-Gln-Phe-Gly-Leu-Trp-Ara--
Gly-Ala-Ala.OH 7
[0344] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Fmoc-Ala-Wang resin (Novabiochem)
on a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids and palmitic acid were preactivated using HBTU before
coupling.
[0345] The simultaneous removal of lipopeptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
H.sub.20, 5% anisole, 5% phenol and 5% EDT for 2 hours giving a
crude product yield of 150 mg. Purification by preparative HPLC
(Vydac 218TP1022 column) of a 30 mg aliquot of crude material was
carried out using a gradient of 90 to 100% B over 40 min (A=0.1%
TFA/water and B=MeOH) at a flow rate of 9 mL/min. After
lyophilization 4 mg of pure material was obtained (Analytical HPLC;
Gradient, 90-100% B over 20 min where B=MeOH, A=0.01% TFA/water:
column--vydac 218TP54: Detection--UV 214 nm; product retention
time=23 min). Further product characterization was carried out
using MALDI mass spectrometry; expected, M+H at 2083, found, at
2088.
[0346] b) Synthesis of a Branched Methotrexate Core Structure
Containing a Thiol Moiety. 8
[0347] The methotrexate structure was synthesised on a ABI 433A
automatic peptide synthesiser starting with Fmoc-Cys(Trt) Tentagel
resin on a 0.1 mmol scale. The simultaneous removal of product from
the resin and deprotection of protecting groups was carried out in
TFA containing 5% EDT and 5% H.sub.2O for 2 hours giving a crude
product yield of 160 mg. Purification by preparative HPLC (Vydac
218TP1022 column) of a 30 mg aliquot of crude material was carried
out using a gradient of 10 to 30% B over 40 min (A=0.1% TFA/water
and B=0.1% TFA/acetonitrile) and a flow rate of 9 mL/min. After
lyophilization of the pure fractions 9 mg of pure material was
obtained (Analytical HPLC; Gradient, 5-50% B where B=0.1%
TFA/acetonitrile, A=0.01% TFA/water: column--vydac 218TP54:
Detection--UV 214 nm--product retention time=9.5 min). Further
product characterization was carried out using MALDI mass
spectrometry; expected, M+H at 1523, found, 1523.
[0348] c)--Preparation of Multiiple-specific Gas-containing
Microbubbles.
[0349] DSPS (Avanti, 4.5 mg.) and thiol containing lipopeptide from
example 15 a) (0.5 mg) and lipopeptide from a) (0.2 mg) above were
weighed into a clean vial and 1.0 mL of a solution of 1.4%
propylene glycol/2.4% glycerol added. The mixture was sonicated for
3-5 mins, warmed to 80.degree. C. for 5 minutes then filtered
through a 4.5 micron filter. The mixture was cooled to room
temperature and the head space flushed with perfluorobutane gas.
The vials were shaken in a cap mixer for 45 s and the microbubbles
centrifuged at 1000 rpm for 3 minutes following which the
infranatant was discarded.
[0350] d) Conjugation of Methotrexate Branched Structure to
Thiolated Microbubbles.
[0351] The methotrexate structure from b) above (0.5 mg) was
dissolved in PBS pH 8.0. The solution was then added to the thiol
containing bubbles from c) and disulphide bond formation allowed to
proceed for 16 h. Following extensive washing with PBS and water
the bubbles were analysed by microscopy and MALDI MS.
[0352] It is also considered relevant that the disulphide bond
linking the methotrexate structure to the microbubble may be
reduced in vivo liberating the free drug molecule. This in
combination with a tumour specific vector is a drug delivery
system. A physiologically relevant reducing agent such as
glutathione may be used to bring about drug release.
EXAMPLE 20
Preparation of Microbubbles Coated with Poly-L-lysine Complexed to
Fluorescein Labeled DNA Fragments from Plasmid pBR322
[0353] This example is directed to the preparation of microbubbles
for gene therapy/anti-sense applications. It is envisaged that
specific targeting may be achieved by further doping of microbubble
membranes with vector modified lipid structures as described in
example 1.
[0354] a) Preparation of DSPS Gas-containing Microbubbles
[0355] DSPS (Avanti, 4.5 mg) was weighed into a clean vial. 1.0 mL
of a solution of 1.4% propylene glycol/2.4% glycerol was added and
the mixture sonicated for 2 min then warmed to 80.degree. C. for 5
minutes. Immediately following warming the solution was filtered
through a 4 micron filter. The sample was cooled to room
temperature and the head space flushed with perfluorobutane gas.
The vial was shaken in a cap mixer for 45 s. Bubbles were then
washed once with deionised water and the infranatant discarded. The
microbubbles were then resuspended in 0.5 mL water.
[0356] b) Preparation of Poly-L-lysine/DNA Complex and Loading of
DSPS Microbubbles
[0357] To 1 mg of poly-L-lysine (70-150 kD) in a clean vial was
added 0.1 mL of a fluorescein labeled digest of plasmid pBR322
(Biorad) dissolved in TE buffer (10 mM tris-HCl, pH 8). The
solution was made up to a total of 0.6 mL by addition of water and
the pH adjusted to 8.
[0358] Complexation was allowed to proceed for 1 h then 0.05 mL of
the polylysine-DNA solution was added to the microbubble suspension
from a) above. After. 1 h microscopy was used to show that the
bubbles were fluorescent confirming the presence of DNA.
EXAMPLE 21
Preparation of Multiple-specific Gas-filled Microbubbles Containing
a Branched Core Peptide Comprising a Dabsylated-atherosclerotic
Plaque Binding Sequence and RGDS
[0359] This example is directed to the preparation of microbubbles
having a thiol group on the surface for modification with thiol
containing vectors for targeting/drug delivery and drug
release.
[0360] a) Synthesis of the Branched Peptide
Dabsyl-Tyr-Arg-Ala-Leu-Val-Asp- -Thr-leu-Lys-Lys
(NH2-Arg-Gly-Asp-Ser)-Gly-Cys.OH 9
[0361] The peptide was synthesised on a ABI 433A automatic peptide
synthesiser starting with Fmoc-Cys(Trt)-Tentagel resin on a 0.1
mmol scale using 1 mmol amino acid cartridges. All amino acids were
preactivated using HBTU before coupling.
[0362] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT and 5% H.sub.2O for 2 hours giving a crude product
yield of 160 mg. Purification by preparative HPLC (Vydac 218TP1022
column) of a 30 mg aliquot of crude material was carried out using
a gradient of 10 to 60% B over 40 min (where A=0.1% TFA/water and
B=acetonitrile) at a flow rate of 9 mL/min. After lyophilization
2.5 mg of pure material was obtained (Analytical HPLC; Gradient,
10-50% B over 20 min where B=0.1% TFA/acetonitrile and A=0.01%
TFA/water: column--vydac 218TP54: Detection--UV 214 and 435
nm--product retention time=21 min). Further product
characterization was carried out using MALDI mass spectrometry;
expected, M+H at 2070, found, at 2073.
[0363] b) Preparation of Thiol Containing Gas-filled
Microbubbles.
[0364] As described in example 15 a) and b).
[0365] c) Oxidative Coupling of Thiolated Microbubbles with
Multiple-specific Peptide Via Disulphide Bond Formation.
[0366] The infranatant from the microbubbles from b) above was
discarded and replaced with a solution of dabsyl-peptide from a) (1
mg) in 0.7 mL dil. ammonia solution (pH 8). To this was added 0.2
mL of a stock solution containing 6 mg of potassiumferricyanate
dissolved in 2 mL of water. The vial was placed on a roller table
and-thiol oxidation allowed to proceed for 2 h. The bubbles were
then washed extensively with water until the infranatant was free
of the dabsyl-peptide as evidenced by hplc and MALDI MS.
[0367] Detection of microbubble bound peptide was carried out by
reduction of the disulphide bond using the water souble reducing
agent tris-(2-carboxyethyl)-phosphine. Following reduction the
infranatant was found to contain free dabsyl-peptide as evidenced
by hplc and MALDI MS.
[0368] Other physiological relevant reducing agents such as reduced
glutathione are also considered useful for initiating release.
EXAMPLE 22
Gas-containing Microparticles Comprising Polymer from Ethylidene
Bis(16-hydroxyhexadecanoate) and Adipoyl Chloride and
Biotin-amidocaproate-Ala Covalently Attached to the Polymer
[0369] a) Synthesis of Z-Ala-polymer
(3-O-(carbobenzyloxy-L-alanyl)-polyme- r)
[0370] The polymer is prepared from ethylidene
bis(16-hydroxyhexadecanoate- ) and adipoyl chloride as described in
WO-A-9607434, and a polymer fraction with molecular weight 10000 is
purified using gel permeation chromatography (GPC). 10 g of the
material (corresponding to 1 mmol OH groups), Z-alanine (5 mmol)
and dimethylaminopyridine (4 mmol) are dissolved in dry
dimethylformamide/tetrahydrofuran and dicyclohexylcarbodiimide is
then added. The reaction mixture is stirred at ambient temperature
overnight. Dicyclohexylurea is filtered off and the solvent is
removed using rotary evaporation. The product is purified by
chromatography, fractions containing the title compound are
combined and the solvent is removed using rotary evaporation. The
structure of the product is confirmed by NMR.
[0371] b) Synthesis of Ala-polymer (3-O-(L-alanyl)-polymer)
[0372] Z-Ala-polymer (0.1 mmol) is stirred in
toluene/tetrahydrofuran and glacial acetic acid (15% of the total
volume) and hydrogenated in the presence of 5% palladium on
charcoal for 2 hours. The reaction mixture is filtered and
concentrated in vacuo.
[0373] c) Synthesis of Biotinamidocaproate-Ala-polymer
[0374] A solution of Biotinamidocaproate N-hydroxysuccinimide ester
in tetrahydrofuran is added to H.sub.2N-Ala-polymer dissolved in a
mixture of tetrahydrofuran and dimethylformamide and 0.1 M sodium
phosphate buffer having a pH of 7.5. The reaction mixture is heated
to 30.degree. C. and stirred vigorously; the reaction is followed
by TLC to completion. The solvent is evaporated and the crude
product is used without further purification.
[0375] d) Gas-containing Particles Comprising
Biotin-amidocaproate-Ala-pol- ymer and PEG 10000 Methyl Ether
16-hexadecanoyloxyhexadecanoate
[0376] 10 mL of a 5% w/w solution of
biotin-amidocaproate-Ala-polymer in (-)-camphene maintained at
60.degree. C. is added to 30 mL of an 1% w/w aqueous solution of
PEG 10000 methyl ether 16-hexadecanoyloxyhexadecanoat- e (prepared
as described in WO-A-9607434) at the same temperature. The mixture
is emulsified using a rotor stator mixer (Ultra Turax.RTM. T25) at
a slow speed for several minutes, and thereafter is frozen in a dry
ice/methanol bath and lyophilized for 48 hours, giving the title
product as a white powder.
[0377] e) Acoustic Characterisation and Microscopy of the
Product
[0378] Confirmation of the microparticulate nature of the product
is performed using light microscopy as described in WO-A-9607434.
Ultrasonic transmission measurements using a 3.5 MHz broadband
transducer indicate that a particle suspension of <2 mg/mL gives
a sound beam attenuation of at least 5 dB/cm.
[0379] f) Multiple-specific Microparticles
[0380] The biotinylated microspheres are then used to prepare
multiple-specific targeting products similar to those exemplified
in examples 5), 6) and 7).
EXAMPLE 23
Preparation of Multiple-specific Gas-containing Microbubbles
Encapsulated with DSPS and
Biotin-PEG.sub.3400-acyl-phosphatidylethanolamine and
Functionalised with Streptavidin, Oligonucleotide
Biotin-GAAAGGTAGTGGGGTC- GTGTGCCGG and Biotinylated
Fibrin-anti-polymerant Peptide (Biotin-GPRPPERHOS.NH.sub.2)
[0381] a) Synthesis of Biotin-PEG.sub.3400-acvl-phosphatidyl
Ethanolamine
[0382] A mixture of dipalmitoyl phosphatidyl ethanolamine, (21.00
mg, 0.03 mmol), biotin-PEG-CO.sub.2--NHS, (100 mg, 0.03 mmol) and
triethylamine (42 .mu.l, 0.30 mmol) in a solution of
chloroform/methanol (3:1) was stirred at room temperature for 2
hours. After evaporation of the solvents under reduced pressure,
the residue was flash chromatographed (methylene
chloride/methanol/water, 40:8:1). The product was obtained as a
yellow gum, 112 mg (94%) and structure verified by NMR and
MALDI-MS.
[0383] b) Binding of Fluorescein-conjugated Streptavidin to Gas
Filled Microbubbles
[0384] Gas-containing microbubbles were prepared by mixing DSPS and
biotin-PEG.sub.3400-acyl-phosphatidyl ethanolamine as described in
example 5 a).
[0385] The microbubble suspension was divided into 0.2 mL aliquots
and fluorescein conjugated streptavidin added as shown in the table
below. The samples were incubated on a roller table for 15 or 30
minutes at ambient temperature before removal of excess protein by
washing in PBS.
[0386] Results:
26 Added Particle Streptavidin Incubation % Fluo- median Aliquot
(.mu.g/200: 1 time (amb. rescent diameter no. sample) temp.)
particles (microns) 1 0 2.0 -- 2 0 -- 12 (foam) 3 0.2 30 min 7.8
3.9 (3 .times. 10.sup.-9 mmol) 4 2 30 min 26.2 4.2 (3 .times.
10.sup.-8 mmol) 5 10 15 min 30.5 na .sup. (1.5 .times. 10.sup.-7
mmol).sup. 6 20 30 min 97.9 5.2 (3 .times. 10.sup.-7 mmol) 7 40 15
min 96.7 5.1 (6 .times. 10.sup.-7 mmol) 8 DSPS 20 15 min 0.6 3.7
control (3 .times. 10.sup.-7 mmol)
[0387] The samples were analysed by flow cytometry and Coulter
Counter. The results are summarized in the table above.
[0388] c) Conjugation of Streptavin Coated Microbubbles with the
Oligonucleotide Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and Biotinylated
Fibrin-anti-polymerant Peptide Biotin-GPRPPERHOS
[0389] The particles from aliquot no. 6 above were centrifuged and
the supernatant replaced with 1 mL of PBS buffer pH 7.5 containing
0.2 mg of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG and 0.2 mg of
biotin-GPRPPERHQS (example 5 c). After incubation for 24 h the
particles were washed extensively with PBS and water.
[0390] It is envisaged that other biotinylated vectors or
therapeutic agents may be conjugated to streptavidin or avidin
coated microbubbles using this procedure.
EXAMPLE 24
Preparation of Microbubbles Encapsulated with DSPS and
Functionalised with a Thrombi-targeting Lipopeptide and the
Thrombolytic Enzyme Tissue Plasminogen Activator
[0391] This example is directed at the preparation of thrombus
targeted US agents comprising a therapeutic thromolytic agent.
[0392] a) Synthesis of a Lipopeptide with Affinity for Thrombi
(Diplamitoyl-Lys-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.NH.s-
ub.2). 10
[0393] The lipopeptide was synthesised on a ABI 433 A automatic
peptide synthesiser starting with Rink amide resin (Novabiochem) on
a 0.1 mmol scale using 1 mmol amino acid cartridges. All amino
acids and palmitic acid were preactivated using HBTU before
coupling.
[0394] The simultaneous removal of peptide from the resin and
side-chain protecting groups was carried out in TFA containing 5%
phenol, 5% EDT, 5% anisole and 5% H.sub.2O for 2 h giving a crude
product yield of 80 mg. Purification by preparative HPLC (Vydac
218TP1022 column) of a 20 mg aliquot of the crude material was
carried out. After lyophilization 6 mg of pure material was
obtained. The product was characterized by MALDI mass spectrometry
and analytical HPLC.
[0395] b) Modification of Tissue Plasminogen Activator with
Sulpho-SMPB.
[0396] A solution of 0.1 mL of ammonium carbonate buffer containing
0.1 mg of t-PA (Sigma) was made up to 0.2 mL by the addition of
water. To this solution was added 0.4 mg of Sulpho-SMPB (Pierce)
dissolved in 0.05 mL DMSO. The protein solution was left standing
at room temperature for 45 min then purification carried out on a
Superdex 200 column. The product was eluted in PBS and the modified
protein fraction collected.
[0397] c) Preparation of Microbubbles Encapsulated with
DSPS/thrombi-binding Lipopeptide and Thiol Containing Lipoeptide
and Conjugation to Modified Tissue Plasminogen Activator.
[0398] DSPS (Avanti, 5.0 mg) was weighed into a clean vial along
with 0.5 mg of the lipopeptide from a) and 0.5 mg of the thiol
containing lipopeptide from example 15 a). To this was added 1.0 mL
of a solution of 1.4% propylene glycol/2.4% glycerol and the
mixture sonicated for 2 min then warmed to 80.degree. C. for 5
minutes. Immediately following warming the solution was filtered
through a 4 micron filter. The sample was cooled to room
temperature and the head space flushed with perfluorobutane gas.
The vials were shaken in a cap mixer for 45 s and the microbubbles
washed 2 times with deionised water. The infranatant was discarded
and replaced with a 1 mL aliquot of the protein solution from b)
above. The conjugation reaction was allowed to proceed for 1 h. The
bubbles were centrifuged and infranatant exchanged with a further 1
mL of protein solution. The incubation step was repeated until all
protein solution was used up. The microbubbles were then washed
extensively with water and analysed by Coulter counter. The
microbubbles were tested in the flow chamber assay described in
example 1 c). Microbubbles modified with protein were found to bind
in higher numbers than those comprising either lipopeptide/DSPS or
DSPS alone.
[0399] It is envisaged that the targeting/therapeutic/ultrasound
activities of these microbubbles be evaluated in models of in vitro
and in vivo thrombogenisis.
EXAMPLE 25
Multiple-specific PFB Gas-filled Microbubbles Encapsulated with
DSPS and a Lipopeptide Comprising a Heparin Sulphate Binding
Peptide (KRKR) and a Fibronectin Peptide (WOPPRARI) for Targeting
and a Lipopeptide Containing Atenolol for Therapeutic
Applications
[0400] a) Synthesis of a Lipopeptide Consisting of a Heparin
Sulphate Binding Peptide (KRKR) and a Fibronectin Peptide
(WOPPRARI)
[0401] Synthesis and purification described in example 1 a).
[0402] b) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0403] i) Synthesis of methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0404] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.03
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 h. The reaction
mixture was cooled, and excess epichlorohydrin was distilled off
(rotavapor). The residue was taken up in ethyl acetate, washed with
brine and dried (Na.sub.2SO.sub.4). The solution was filtered and
concentrated. The dark residue was chromatographed (silica,
hexane/ethyl acetate 7:3) to give 2.25 g (34%) of a colourless oil.
.sup.1H (300 MHz) and .sup.13C NMR (75 MHz) spectra were in
accordance with the structure.
[0405] ii) Synthesis of methyl 4-
[2-hydroxy-3-[(1-methylethyl)amino]-prop- oxy]phenylacetate
[0406] A mixture of methyl 4-[(2,3-epoxy)propoxy]phenylacetate
(2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 mol) and water
(1.35 ml, 74.7 mmol) was stirred at room temperature overnight. The
reaction mixture was concentrated (rotavapor) and the oily residue
was dissolved in chloroform and dried (Na.sub.2SO.sub.4).
Filtration and concentration gave quantitative yield of a yellow
oil that was used in the next step without further purification.
The structure was verified by .sup.1H and .sup.13C NMR
analysis.
[0407] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phe- nylacetic acid
hydrochloride
[0408] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]- phenylacetate (563
mg, 2.00 mmol) in 6 M hydrochloric acid (15 ml) was heated at 100
.degree. C. for 4 h. The reaction mixture was concentrated
(rotavapor) and the residue was taken up in water and lyophilised.
.sup.1H and .sup.13C NMR spectra were in accordance with the
strucure and MALDI mass spectrometry gave a M+H at 268 as
expected.
[0409] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]-propox- y]phenylacetic
acid
[0410] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]-propoxy]phe- nylacetic acid
hydrochloride (2.0 mmol) in water (2 ml) was added to a solution of
sodium bicarbonate (0.60 g, 7.2 mmol) in water/dioxane (2:1, 15
ml). A solution of di-tert-butyl dicarbonate (0.48 g, 2.2 mmol) in
dioxane (5 ml) was added. Progress of the reaction was monitored by
TLC analysis (silica, CHCl.sub.3/MeOH/AcOH 85:10:5), and portions
of di-tert-butyl dicarbonate were added until conversion was
complete.
[0411] The reaction mixture was poured onto water saturated with
potassium hydrogensulphate and organic material was extracted into
ethyl acetate. The organic phase was washed with water and brine,
dried (Na.sub.2SO.sub.4) and filtered to give 0.6 g of crude
material. The product was purified by chromatography (silica,
CHCl.sub.3/MeOH/AcOH 85:10:5). The solution was concentrated and
the residue was taken up in glacial acetic acid and lyophilised.
Yield 415 mg (56%), white solid. The structure was confirmed by
.sup.1H and .sup.13C NMR analysis.
[0412] c) Synthesis of a Lipopeptide Functionalised with Atenolol
11
[0413] The structure shown above was synthesised on a manual
nitrogen bubbler starting with Fmoc protected Rink Amide MBHA resin
(Novabiochem) on a 0.125 mmol scale, using amino acids from
Novabiochem, palmitic acid from Fluka and the compound from a).
Coupling was carried out using standard TBTU/HOBt/DIEA
protocols.
[0414] Simultaneous removal of the peptide from the resin and
deprotection of side-chain protecting groups was carried out in TFA
containing 5% EDT and 5% water for 2 h. Crude material was
precipitated from ether and purified by preparative liquid
chromatography (Vydac 218TP1022 column) using a gradient of 70 to
100% B over 60 min (A=0.1% TFA/water and B=0.1% TFA/acetonitrile)
at a flow rate of 10 ml/min. After lyophilisation a yield of 38 mg
of pure material was obtained (analytical HPLC: gradient 70-100% B
over 20 min, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow
rate 1 ml/min, column Vydac 218TP54, detection UV 214 nm, retention
time 25 min). Further characterisation was carried out using MALDI
mass spectrometry (ACH matrix), giving M+H at 1258, expected
1257.
[0415] d) Preparation of Gas-filled Microbubbles of DSPS Comprising
a Lipopeptide Cosisting of a Heparin Sulphate Binding Peptide
(KRKR) and a Fibronectin Peptide (WOPPRARI) and a Lipopeptide
Containing Atenolol
[0416] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 5.0 mg), product from a)
(0.5 mg) and product from c) (0.5 mg) in a vial. The mixture was
sonicated for 5 min and then heated at 80 .degree. C. for 5 min
(vial was shaken during warming). The solution was filtered and
cooled. Head space was flushed with perfluorobutane gas and the
vial was shaken in a cap mixer for 45 s followed by extensive
washing with deionised water.
[0417] Incorporation of atenolol containing lipopeptide into the
bubbles was confirmed by MALDI-MS as described in example 1 b).
[0418] e) In Vitro Study of Multiiple-specific Gas-filled
Microbubbles.
[0419] In vitro analysis of the microbubble suspension was carried
out as described in example 1 c). A gradual accumulation of the
microbubbles on the cells took place which was dependent on the
flow rate. By increasing the flow rate the cells started to become
detached from the coverslip, the microbubbles were still bound to
the cells. Control bubbles not carrying the vector did not adhere
to the endothelial cells and disappeared from the cells under
minimal flow conditions.
EXAMPLE 26
PFB Gas-filled Microbubbles of DSPS Containing a Cholesterol Ester
of Chlorambucil for Diagnostic and Therapeutic Applications
[0420] This example is directed at non-specific modification of a
multiplicity of cell receptors on endothelial cells.
[0421] a) Synthesis of Cholesterol
4-[4-[bis(2-chloroethyl)amino]-phenyl]b- utanoate
[0422] DIC (170 .mu.l, 1.10 mmol) was added to a solution of
chlorambucil (Sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15
ml). The mixture was stirred at room temperature for 0.5 h and
added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and
DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction
mixture was stirred overnight and then poured onto 5% sodium
bicarbonate. The phases were separated and the organic phase was
washed with brine and dried (MgSO.sub.4). The solution was filtered
and concentrated and the product was purified by column
chromatography (silica, chloroform) to give 560 mg (83%) yield of
colouless oil. The product was characterised by MALDI mass
spectrometry, giving M+H at 674 as expected. Further
characterisation was carried out using .sup.1H (500 MHz) and
.sup.13C (125 MHz) NMR analysis, giving spectra in accordance with
the structure.
[0423] b) Preparation of Gas-containing Microbubbles of DSPS
Comprising a Cholesteryl Ester of Chlorambucil for Diagnostic
and/or Therapeutic Applications
[0424] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 4.5 mg) and product from a)
(0.5 mg) in a vial. The mixture was sonicated for 5 min and then
heated at 80 .degree. C. for 5 min (vial was shaken during warming)
and cooled. Head space was flushed with perfluorobutane gas and the
vial was shaken in a cap mixer for 45 s followed by extensive
washing with deionised water. MALDI mass spectrometry showed no
detectable level of compound from a) in the final wash solution.
Incorporation of chlorambucil cholesteryl ester into the bubbles
was confirmed by MALDI-MS as follows: ca 50 .mu.l of microbubbles
were transferred to a clean vial containing ca 100 .mu.l of 90%
methanol. The mixture was sonicated for 30 s and analysed by
MALDI-MS, giving a M+H peak at 668 corresponding to structure from
a).
[0425] In combination with a tumour specific vector these
microbubbles are considered useful as targeted drug delivery
agents.
EXAMPLE 27
Multiple-specific Gas-filled Microbubbles of DSPS Comprising a
Lipopeptide Containing Atenolol and a Cholesterol Derivative of
Chlorambucil for Diagnostic and Therapeutic Applications
[0426] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0427] As described in example 25 section b).
[0428] b) Synthesis of a Lipopeptide Functionalised with
Atenolol
[0429] As described in example 25 section c).
[0430] c) Synthesis of Cholesteryl
4-[4-[bis(2-chloroethyl)amino]phenyl]bu- tanoate
[0431] As described in example 25 section d).
[0432] d) Preparation of Microbubbles of DSPS Comprising a
Lipopeptide Containing Atenolol and a Cholesteryl Ester of
Chloambucil
[0433] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 5.0 mg), product from b)
(0.5 mg) and c) (0.5 mg) and in a vial. The mixture was sonicated
for 5 min and then warmed to 80.degree. C. for 5 min (vial was
shaken during warming). The solution was filtered and cooled. Head
space was flushed with perfluorobutane gas and the vial was shaken
in a cap mixer for 45 s followed by extensive washing with
deionised water. Incorporation of atenolol containing lipopeptide
and chlorabucil analogue into the bubble membrane was confirmed by
MALDI-MS as described in example 1 c).
[0434] e) In Vitro Study of Multiiple-specific PFB Gas-containing
Microbubbles of DSPS Comprising a Lipopeptide Containing Atenolol
and a Cholesterol Derivative of Chlorambucil for Diagnostic and
Therapeutic Applications
[0435] The in vitro assay described in example 1 c) was used to
assess cellular binding under flow conditions. A gradual
accumulation of the microbubbles on the cells took place which was
dependant on the flow rate. By increasing the flow rate the cells
started to become detached from the coverslip, the microbubbles
were still bound to the cells. Control bubbles not carrying the
vector did not adhere to the endothelial cells and disappeared from
the cells under minimal flow conditions.
EXAMPLE 28
Multiple-specific Gas-filled Microbubbles of DSPS Comprising a
Lipopeptide Containing Atenolol for Cell Targeting and a Lipophilic
Thiol Ester of Captopril for Therapeutic Use
[0436] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0437] As described in example 25 section b).
[0438] b) Synthesis of a Lipopeptide Functionalised with
Atenolol
[0439] As described in example 25 section c).
[0440] c) Synthesis of Cholanic Acid Thiol Ester of Captopril
[0441] A mixture of 5-.beta.-cholanic acid (Sigma, 361 mg, 1.00
mmol) and DIC (77 .mu.l, 0.50 mmol) in dichloromethane (5 ml) was
stirred for 10 min and then added to a solution of captopril
(Sigma, 130 mg, 0.600 mmol) and DBU (180 .mu.l, 1.20 mmol) in
dichloromethane (10 ml). The reaction mixture was stirred overnight
and then poured onto dilute hydrochloric acid. Chloroform (30 ml)
was added. The phases were separated and the organic phase was
washed with water and brine and dried (MgSO.sub.4). After
filtration and concentration the crude material was chromatographed
(silica, chloroform/methanol/acetic acid 95:4:1). The product was
lyophilised from a acetonitrile/water/ethanol mixture. Yield 137 mg
(49%) of off-white solid. The structure was verified by .sup.1H
(500 MHz) and .sup.13C (125 MHz) NMR spectroscopy. Further
characterisation was carried out using MALDI mass spectrometry,
giving a M+Na peak in positive mode at m/z 584.
[0442] d) Preparation of Gas-filled Microbubbles of DSPS Comprising
a Lipopeptide Containing Atenolol for Cell Targeting and a
Lipophilic Thiol Ester of Captopril for Therapeutic Use.
[0443] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 5.0 mg) and product from b)
(0.5 mg) and c) (0.5 mg) in a vial. The mixture was sonicated for 5
min and then heated at 80 .degree. C. for 5 min (vial was shaken
during warming) and cooled. Head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45 s
followed by extensive washing with deionised water. MALDI mass
spectrometry showed no detectable level of compound from b) and c)
in the final wash solution. Incorporation of compounds from b) and
from c) into the bubbles was confirmed by MALDI-MS as follows. Ca.
50 .mu.l of microbubbles were transferred to a clean vial
containing ca 100 .mu.l of 90% methanol. The mixture was sonicated
for 30 s and analysed by MALDI-MS (ACH-matrix),giving peaks
according to structures from b) and c), respectively.
[0444] e) In Vitro Study of Gas-containing Microbubbles from d)
[0445] The in vitro assay described in example 1 c) was used to
assess cellular binding under flow conditions. A gradual
accumulation of the microbubbles on the cells took place which was
dependent on the flow rate. By increasing the flow rate the cells
started to become detached from the coverslip, the microbubbles
were still bound to the cells. Control bubbles not carrying the
vector did not adhere to the endothelial cells and disappeared from
the cells under minimal flow conditions.
EXAMPLE 29
Gas-filled Microbubbles of Phosphatidylserine Comprising
Biotinamide-PEG-B-Ala-Cholesterol and a Cholesteryl Ester of
Chlorambucil for Diagnostic and Therapeutic Applications
[0446] a) Synthesis of Cholesterol N-Boc-.beta.-alaninate
[0447] DIC (510 .mu.l) was added to a solution of Boc-.beta.-Ala-OH
(1.25 g, 6.60 mmol) in dichloromethane (15 ml) under an inert
atmosphere. The reaction mixture was stirred for 30 min and then
transferred to a flask containing a solution of cholesterol (1.16
g, 3.00 mmol) and DMAP (367 mg, 3.00 mmol) in dichloromethane (15
ml). The reaction mixture was stirred for 2 h and then mixed with
an aqueous solution of potassium hydrogensulphate. The phases were
separated and the aqueous phase extracted with chloroform. The
combined organic phases were washed with aqueous potassium
hydrogensulphate and water and dried over MgSO.sub.4. After
filtration and evaporation the crude product was chromatographed
(silica, chloroform/methanol 99:1) to give 1.63 g (97%) of white
solid. The structure was confirmed by .sup.1H NMR (500 MHz).
[0448] b) Synthesis of Cholesterol .beta.-alaninate
Hydrochloride
[0449] A solution of compound from a) (279 mg, 0.500 mmol) in 1 M
hydrochloric acid in 1,4-dioxan (5 ml) was stirred at room
temperature for 4 h. The reaction mixture was concentrated to give
a quantitative yield of cholesteryl .beta.-alaninate hydrochloride.
The structure was confirmed by 1H NMR (500 MHz) analysis and by
MALDI mass spectrometry, giving a M+Na peak at 482, expected
481.
[0450] c) Biotin-PEG.sub.3400-.beta.-Ala-Cholesterol
[0451] To a solution of cholesteryl .beta.-alaninate hydrochloride
(15 mg, 0.03 mmol) in chloroform/wet methanol (2.6:1, 3 ml) was
added triethylamine (42 .mu.l, 0.30 mmol). The mixture was stirred
for 10 minutes at room temperature and a solution of
biotin-PEG3400-NHS (100 mg, 0.03 mmol) in 1,4-dioxane (1 ml) was
added dropwise. After stirring at room temperature for 3 hours, the
mixture was evapourated to dryness and the residue purified by
flash chromatography to give white crystals, yield; 102 mg (89%).
The structure was verified by MALDI-MS and by NMR analysis.
[0452] d) Synthesis of Cholesterol
4-[4-[bis(2-chloroethyl)amino]phenyl]bu- tanoate
[0453] DIC (170 .mu.l, 1.10 mmol) was added to a solution of
chlorambucil (Sigma, 669 mg, 2.20 mmol) in dry dichloromethane (15
ml). The mixture was stirred at room temperature for 0.5 h and
added to a solution of cholesterol (Aldrich, 387 mg, 1.00 mmol) and
DMAP (122 mg, 1.00 mmol) in dichloromethane (10 ml). The reaction
mixture was stirred overnight then poured into a solution of 5%
sodium bicarbonate. The organic phase was washed with brine and
dried over MgSO.sub.4. The solution was filtered and concentrated
and the product was purified by column chromatography (silica,
chloroform) to give 560 mg (83%) yield of colouless oil. The
product was characterised by MALDI mass spectrometry, giving M+H at
674 as expected. Further characterisation was carried out using
.sup.1H (500 MHz) and .sup.13C (125 MHz) NMR analysis, giving
spectra in accordance with the structure.
[0454] e) Preparation of Gas-filled Microbubbles
[0455] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 5 mg) and product from c)
(0.5 mg) and d) (0.5 mg) in a vial. The mixture was sonicated for 5
min and then heated at 80.degree. C. for 5 min (vial was shaken
during warming) and cooled. Head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45 s
followed by extensive washing with deionised water. MALDI mass
spectrometry showed no detectable level of compound from c and d)
in the final wash solution.
[0456] Incorporation of compounds from c) and d) into the bubbles
was confirmed by MALDI-MS as described in example 1 b).
EXAMPLE 30
Gas-filled Microbubbles of DSPS Comprising a Lipopeptide Containing
Chlorambucil for Diagnostic and Therapeutic Applications
[0457] This example is directed at the preparation of
functionalised microbubbles with non-specific affinity for a
multiplicity of cell surface molecules.
[0458] a) Synthesis of a Lipopeptide Containing Chlorambucil 12
[0459] The structure shown above was synthesised on a manual
nitrogen bubbler starting with Fmoc protected Rink Amide MBHA resin
(Novabiochem) on a 0.125 mmol scale. Standard amino acids were
purchased from Novabiochem and palmitic acid from Fluka. Coupling
was carried out using standard TBTU/HOBt/DIEA protocol.
Chlorambucil (Sigma) was coupled through the side-chain of Lys as a
symmetrical anhydride using DIC preactivation.
[0460] Simultaneous removal of the peptide from the resin and
deprotection of side-chain protecting groups was carried out in TFA
containing 5% EDT, 5% water and 5% ethyl methyl sulphide for 2 h.
An aliqout of 10 mg of the crude material was purified by
preparative liquid chromatography (Vydac 218TP1022 column) using a
gradient of 70 to 100% B over 60 min (A=0.1% TFA/water and B 0.1%
TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophilisation
a yield of 30 mg of pure material was obtained (analytical HPLC:
gradient 70-100% B over 20 min, A=0.1% TFA/water and B=0.1%
[0461] TFA/acetonitrile; flow rate 1 ml/min; column Vydac 218TP54;
detection UV 214 nm; retention time 26.5 min). Further
characterisation was carried out using MALDI mass spectrometry,
giving M+H at 1295, expected 1294.
[0462] b) Preparation of Gas-filled Microbubbles Comprising a
Lipopeptide Containing Chlorambucil for Diagnostic and Therapeutic
Applications
[0463] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 4.5 mg) and product from a)
(0.5 mg) in a vial. The mixture was sonicated for 5 min and then
heated at 80 .degree. C. for 5 min (vial was shaken during warming)
and cooled. Head space was flushed with perfluorobutane gas and the
vial was shaken in a cap mixer for 45 s followed by extensive
washing with deionised water. MALDI mass spectrometry showed no
detectable level of compound from a) in the final wash
solution.
[0464] Incorporation of chlorambucil containing lipopeptide into
the bubbles was confirmed by MALDI-MS as follows. Ca 50 .mu.l of
microbubbles were transferred to a clean vial containing ca 100
.mu.l of 90% methanol. The mixture was sonicated for 30 s and
analysed by MALDI-MS (ACH-matrix), giving a M+H peak at 1300,
expected at 1294-and a M+Na peak at 1324, expected 1317.
[0465] c) In Vitro Study of Gas-containing Microbubbles of DSPS
`Doped` with a Lipopeptide Containing Chlorambucil for Diagnostic
and Therapeutic Applications
[0466] The microbubbles were evaluated using the in vitro flow
assay described in example 1 c). A gradual accumulation of the
microbubbles on the cells took place which was dependant on the
flow rate. By increasing the flow rate the cells started to become
detached from the coverslip, the microbubbles were still bound to
the cells. Control bubbles not carrying the vector did not adhere
to the endothelial cells and disappeared from the cells under
minimal flow conditions.
EXAMPLE 31
Gas-filled Microbubbles of DSPS Comprising a Lipopeptide Containing
Atenolol and a Lipophilic Derivative of Captopril for Diagnostic
and Therapeutic Applications
[0467] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0468] As described in example 25) b)
[0469] b) Synthesis of
N-[(S)-3-hexadecylthio-2-methylpropionyl]proline
[0470] DIEA (188 .mu.l, 1.10 mmol) was added to a solution of
1-iodohexadecane (176 mg, 0.500 mmol), captopril (120 mg, 0.550
mmol) and DBU (165 .mu.l, 1.10 mmol) in tetrahydrofuran (5 ml). The
mixture was heated at 70.degree. C. for 2 h and then concentrated.
The residue was poured onto water saturated with potassium
hydrogensulphate and organic material was extracted into
chloroform. The organic phase was washed with water and dried
(MgSO.sub.4). The product purified by chromatography (silica,
CHCl.sub.3/MeOH/AcOH 85:10:5) and lyophilised to give 105 mg (48%)
of white solid material. The structure was verified by 1H (500 MHz)
and 13C (125 MHz) analysis and further characterised by MALDI mass
spectrometry, giving M-H in negative mode at m/z 440 as
expected.
[0471] c) Preparation of Gas-filled Microbubbles of DSPS Comprising
a Lipopeptide Containing Atenolol and a Lipophilic Derivative of
Captopril for Diagnostic and Therapeutic Applications
[0472] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (Avanti, 4.5 mg), product from b)
(0.5 mg) and c) in a vial. The mixture was sonicated for 5 min and
then heated at 80.degree.0 C. for 5 min (vial was shaken during
warming) and cooled. Head space was flushed with perfluorobutane
gas and the vial was shaken in a cap mixer for 45 s followed by
extensive washing with deionised water. MALDI mass spectrometry
showed no detectable level of compound from b)or c) in the final
wash solution. Incorporation of compound b) and c) containing
lipopeptide into the bubbles was confirmed by MALDI-MS as described
in example 1 b).
[0473] d) In Vitro Study of Gas-containing Microbubbles of DSPS
Comprising a Lipopeptide Containing Atenolol and a Lipophilic
Derivative of Captopril for Diagnostic and Therapeutic
Applications
[0474] The microbubbles were evaluated using the in vitro flow
assay described in example 1 c). A gradual accumulation of the
microbubbles on the cells took place which was dependant on the
flow rate. By increasing the flow rate the cells started to become
detached from the coverslip, the microbubbles were still bound to
the cells. Control bubbles not carrying the vector did not adhere
to the endothelial cells and disappeared from the cells under
minimal flow conditions.
EXAMPLE 32
Floatation of Endothelial Cells by DSPS Microbubbles Comprising a
Multiple-specific Lipopeptide that Binds to the Endothelial
Cells
[0475] This example was carried out to show that the invention
could also be used for cell separation.
[0476] The human endothelial cell line ECV 304, derived from a
normal umbilical cord (ATCC CRL-1998) was cultures in Nunc culture
flasks (chutney 153732) in RPMI 1640 medium (Bio Whitaker) to which
L-Glutamine 200 mM, Penicillin/Streptomycin (10.000 U/ml and 10.00
mcg/ml) and 10% Fetal Calf Serum (Hyclone Lot no AFE 5183) were
added. The cells were subcultured following trypsination with a
split ratio of 1:5 to 1:7 when reaching confluence. 2 mill. cells
from trypsinated confluent cultures were added to a set of 5
centrifuge tubes followed by either control microbubbles of DSPS,
microbubbles from example 1 or microbubbles of DSPS doped with the
endothelial cell binding lipopeptide from example 14 a) at a
concentration of 2, 4, 6, 8 or 10 mill bubbles per tube. The cells
at the bottom of the tubes after centrifugation at 400 g for 5
minutes were counted by Coulter counter. It was found that binding
of four or more microbubbles to a cell brought about floatation.
Furthermore all cells were floated by the endothelial cell binding
lipopeptide bubbles while around 50% were floated with microbubbles
from example 1).
EXAMPLE 33
Gene Transfer by PFB Gas-filled Microbubbles
[0477] This example is directed at the preparation of targeted
microbubbles for gene transfer.
[0478] a) Preparation of DSPS Lipopeptide Bubbles/PFB Gas Coated
with Polyl-L-lysine
[0479] DSPS (4,5 mg) and lipopeptide from 17 b) (0.5 mg) were
weighed in two 2-ml vials. To each vial, 0.8 ml
propyleneglycol/glycerol (4%) in water was added. The solution was
heated at 80.degree. C. for 5 minutes and shaken. The solution was
then cooled to ambient temperature and the headspace flushed with
perfluorobutane. The vials were shaken on a Capmix (Espe Capmix,
4450 oscillations/min) for 45 seconds and put on a roller table for
5 minutes. The content of the vials were mixed and the sample
washed by centrifugation at 2000 rpm for 5 minutes. The infranatant
was removed and the same volume of distilled water added. The
washing procedure was repeated once.
[0480] poly-L-lysine HBr (Sigma, 20.6 mg) was dissolved in 2 mL
water then an aliquot (0.4 mL) made up to 2 mL water. To 1.2 mL of
the diluted poly-L-lysine solution was added 0.12 mL of the
DSPS-lipopeptide bubble suspension. Following incubation excess
polylysine was removed by extensive washing with water.
[0481] b) Transfection of Cells
[0482] Endothelial cells (ECV 304) were cultured in 6 well plates
to a uniform subconfluent layer. A transfection mixture consisting
of 5 .mu.g DNA (an Enhanced Green Fluorescent Protein vector from
CLONTECH) and 50 .mu.l of microbubble suspension from a) in RPMI
medium at a final volume of 250 .mu.l was prepared. The mixture was
left standing for 15 min at room temperature then 1 mL of complete
RPMI medium added. The medium was removed from the cell culture
dish, and the DNA-microbubble mixture added to the cells.
[0483] The cells were incubated in a cell culture incubator
(37.degree. C.).
[0484] c) Ultrasonic Treatment
[0485] After 15 minutes incubation, selected wells were exposed to
continious wave ultrasound of 1 MHz, 0.5 W/cm.sup.2, for 30
seconds.
[0486] d) Incubation and Examination
[0487] The cells were further incubated in the cell culture
incubator (37 .degree. C.) for approximately 41/2 hours. The medium
containing DNA-microbubbles was then removed by aspiration, and 2
ml complete RPMI medium was added. The cells were incubated for
40-70 hours before examination. Most of the medium was then
removed, and the cells were examined by fluorescence microscopy.
The results were compared to the results from control experiments
were DNA or DNA-polylysine were added to the cells.
Sequence CWU 1
1
20 1 4 PRT Artificial Sequence Description of Artificial Sequence
Heparin sulphate binding peptide 1 Lys Arg Lys Arg 1 2 8 PRT
Artificial Sequence Description of Artificial Sequence Fibronectin
peptide 2 Trp Gln Pro Pro Arg Ala Arg Ile 1 5 3 13 PRT Artificial
Sequence Description of Artificial Sequence Lipopeptide consisting
of heparin sulphate binding peptide and fibronectin peptide 3 Lys
Lys Arg Lys Arg Trp Gln Pro Pro Arg Ala Arg Ile 1 5 10 4 4 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
RGDC sequence 4 Arg Gly Asp Cys 1 5 24 PRT Artificial Sequence
Description of Artificial Sequence Synthetic fusion peptide
comprising a PS binding component and a fibronectin peptide
sequence 5 Phe Asn Phe Arg Leu Lys Ala Gly Gln Lys Ile Arg Phe Gly
Gly Gly 1 5 10 15 Gly Trp Gln Pro Pro Arg Ala Ile 20 6 6 PRT
Artificial Sequence Description of Artificial Sequence Biotinylated
endothelin-1 peptide 6 Trp Leu Asp Ile Ile Trp 1 5 7 10 PRT
Artificial Sequence Description of Artificial Sequence Biotinylated
fibrin-antipolymerant peptide 7 Gly Pro Arg Pro Pro Glu Arg His Gln
Ser 1 5 10 8 6 PRT Artificial Sequence Description of Artificial
Sequence Lipopeptide 8 Lys Trp Lys Lys Lys Gly 1 5 9 25 DNA
Artificial Sequence Description of Artificial Sequence Biotinylated
synthetic oligonucleotide 9 gaaaggtagt ggggtcgtgt gccgg 25 10 25
DNA Artificial Sequence Description of Artificial Sequence
Biotinylated synthetic oligonucleotide 10 ggcgctgatg atgttgttga
ttctt 25 11 5 PRT Artificial Sequence Descriptionof Artificial
Sequence Lipopeptide containing the RGD sequence and a fluorescein
reporter group 11 Lys Lys Lys Lys Gly 1 5 12 18 PRT Artificial
Sequence Description of Artificial Sequence Synthetic endothelial
cell binding lipopeptide 12 Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala
Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala 13 5 PRT Artificial
Sequence Description of Artificial Sequence Thiol functionalised
lipid molecule 13 Lys Lys Lys Xaa Cys 1 5 14 4 PRT Artificial
Sequence Description of Artificial Sequence Synthetic lipopeptide
functionalised with captopril 14 Lys Lys Lys Lys 1 15 13 PRT
Artificial Sequence Description of Artificial Sequence Synthetic
lipopeptide functionalised with captopril 15 Lys Lys Lys Xaa Ile
Arg Arg Val Ala Arg Pro Pro Leu 1 5 10 16 14 PRT Artificial
Sequence Description of Artificial Sequence Lipopeptide comprising
an interleukin 1 receptor binding peptide 16 Lys Gly Asp Trp Asp
Gln Phe Gly Leu Trp Arg Gly Ala Ala 1 5 10 17 12 PRT Artificial
Sequence Description of Artificial Sequence Core peptide comprising
dabsylated-atherosclerotic plaque binding sequence and RGDS 17 Tyr
Arg Ala Leu Val Asp Thr Leu Lys Lys Gly Cys 1 5 10 18 15 PRT
Artificial Sequence Description of Artificial Sequence Lipopeptide
with an affinity for thrombi 18 Lys Asn Asp Gly Asp Phe Glu Glu Ile
Pro Glu Glu Tyr Leu Gln 1 5 10 15 19 4 PRT Artificial Sequence
Description of Artificial Sequence Lipopeptide functionalised with
atenolol 19 Lys Lys Lys Lys 1 20 4 PRT Artificial Sequence
Description of Artificial Sequence Lipopeptide containing
chlorambucil 20 Lys Lys Lys Lys 1
* * * * *