U.S. patent application number 09/765614 was filed with the patent office on 2002-08-01 for diagnostic/therapeutic agents.
This patent application is currently assigned to NYCOMED IMAGING AS. Invention is credited to Cuthbertson, Alan, Hellebust, Halldis, Hoff, Lars, Hogset, Anders, Klaveness, Jo, Lovhaug, Dagfinn, Naevestad, Anne, Rongved, Pal, Solbakken, Magne, Tolleshaug, Helge.
Application Number | 20020102215 09/765614 |
Document ID | / |
Family ID | 27581685 |
Filed Date | 2002-08-01 |
United States Patent
Application |
20020102215 |
Kind Code |
A1 |
Klaveness, Jo ; et
al. |
August 1, 2002 |
Diagnostic/therapeutic agents
Abstract
Targetable diagnostic and/or therapeutically active agents, e.g.
ultrasound contrast agents, having reporters comprising gas-filled
microbubbles stabilized by monolayers of film-forming surfactants,
the reporter being coupled or linked to at least one vector.
Inventors: |
Klaveness, Jo; (Oslo,
NO) ; Rongved, Pal; (Oslo, NO) ; Hogset,
Anders; (Oslo, NO) ; Tolleshaug, Helge; (Oslo,
NO) ; Naevestad, Anne; (Oslo, NO) ; Hellebust,
Halldis; (Oslo, NO) ; Hoff, Lars; (Oslo,
NO) ; Cuthbertson, Alan; (Oslo, NO) ; Lovhaug,
Dagfinn; (Oslo, NO) ; Solbakken, Magne; (Oslo,
NO) |
Correspondence
Address: |
BACON & THOMAS, PLLC
4th Floor
625 Slaters Lane
Alexandria
VA
22314-1176
US
|
Assignee: |
NYCOMED IMAGING AS
|
Family ID: |
27581685 |
Appl. No.: |
09/765614 |
Filed: |
January 22, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09765614 |
Jan 22, 2001 |
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08960054 |
Oct 29, 1997 |
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08960054 |
Oct 29, 1997 |
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08958993 |
Oct 28, 1997 |
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60049264 |
Jun 6, 1997 |
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60049265 |
Jun 6, 1997 |
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60049268 |
Jun 7, 1997 |
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Current U.S.
Class: |
424/9.52 ;
514/44R |
Current CPC
Class: |
A61K 47/62 20170801;
A61K 49/223 20130101; A61K 51/1255 20130101; A61K 49/0002 20130101;
A61K 49/0058 20130101; A61K 47/6925 20170801; Y10S 977/928
20130101; A61K 51/1227 20130101; A61K 49/0047 20130101; A61K 51/088
20130101; A61K 49/0054 20130101; A61K 47/542 20170801; A61K 49/0043
20130101; A61K 49/0091 20130101; A61K 49/0056 20130101 |
Class at
Publication: |
424/9.52 ;
514/44 |
International
Class: |
A61K 049/00; A61K
048/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 28, 1996 |
GB |
9622366.4 |
Oct 28, 1996 |
GB |
9622367.2 |
Oct 28, 1996 |
GB |
9622368.0 |
Jan 15, 1997 |
GB |
9700699.3 |
Apr 24, 1997 |
GB |
9708265.5 |
Jun 6, 1997 |
GB |
9711842.6 |
Jun 6, 1997 |
GB |
9711846.7 |
Claims
1. A targetable diagnostic and/or therapeutically active agent
comprising a suspension in an aqueous carrier liquid of a reporter
comprising gas-filled microbubbles stabilised by monolayers of
film-forming surfactant, said agent further comprising at least one
vector.
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 any of the preceding claims wherein the
film-forming surfactant material comprises a non-polymeric and
non-polymerisable wall-forming surfactant material, a polymer
surfactant material or a phospholipid.
6. An agent as claimed in claim 5 wherein at least 75% of the
film-forming surfactant material comprises phospholipid molecules
individually bearing net overall charge.
7. An agent as claimed in claim 6 wherein at least 75% of the
film-forming surfactant material comprises one or more
phospholipids selected from phosphatidylserines,
phosphatidylglycerols, phosphatidylinositols, phosphatidic acids
and cardiolipins.
8. An agent as claimed in claim 7 wherein at least 80% of said
phospholipids comprise phosphatidylserines.
9. An agent as claimed in any of the preceding claims wherein the
film-forming surfactant material comprises a lipopeptide.
10. An agent as claimed in any of the preceding claims wherein the
vector is selected from antibodies; cell adhesion molecules; cell a
adhesion molecule receptors; cytokines; growth factors; peptide
hormones and pieces thereof; non-peptide agonists/antagonists and
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; and small bioactive molecules.
11. An agent as claimed in any of the preceding claims 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.
12. An agent as claimed in claim 11 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.
13. An agent as claimed in any of the preceding claims wherein the
vector or vectors are sited such that they are not readily exposed
to the target.
14. An agent as claimed in any of the preceding claims wherein the
vector is covalently coupled or linked to the reporter.
15. An agent as claimed in any of claims 1 to 13 wherein the vector
is coupled or linked to the reporter through electrostatic charge
interactions.
16. An agent as claimed in any of claims 1 to 13 wherein the vector
is coupled or linked to the reporter by means of avidin-biotin
and/or streptavidin-biotin interactions.
17. An agent as claimed in any of the preceding claims which
further contains moieties which are radioactive or are effective as
X-ray contrast agents, light imaging probes or spin labels.
18. An agent as claimed in any one of the preceding claims further
comprising a therapeutic compound.
19. An agent as claimed in claim 18 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.
20. An agent as claimed in claim 18 or claim 19 wherein said
therapeutic compound is covalently coupled or linked to the
reporter through disulphide groups.
21. An agent as claimed in claim 18 or claim 19 wherein a
lipophilic or lipophilically-derivatised therapeutic compound is
linked to the surfactant monolayers stabilising the gas-filled
microbubbles of the reporter through hydrophobic interactions.
22. 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.
23. A combined formulation as claimed in claim 22 wherein said
pre-targeting vector is a monoclonal antibody.
24. A combined formulation comprising: i) a first administrable
composition comprising an agent as claimed in any of claims 1 to
21; and ii) a second administrable composition comprising a
substance capable of displacing or releasing said agent from its
target.
25. A combined formulation comprising: i) a first administrable
composition comprising an agent as claimed in claim 20; 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.
26. A process for the preparation of a targetable diagnostic and/or
therapeutically active agent as defined in claim 1 which comprises
either coupling or linking at least one vector to a reporter
comprising gas-filled microbubbles stabilised by monolayers of
film-forming surfactant or generating gas-filled reporter
microbubbles using film-forming surfactant having at least one
vector attached thereto.
27. A process as claimed in claim 26 wherein a therapeutic compound
is also combined with the reporter.
28. A process as claimed in claim 27 wherein a therapeutic compound
containing thiol groups is linked to thiol group-containing
surfactant monolayers stabilising the gas-filled microbubbles of
the reporter by reaction under oxidative conditions so as to
generate disulphide groups.
29. Use of an agent as claimed in any of claims 1 to 21 as a
targetable ultrasound contrast agent.
30. 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 any of claims 1 to 21 and generating an ultrasound,
magnetic resonance, X-ray, radiographic or light image of at least
a part of said body.
31. A method as claimed in claim 30 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 as
claimed in any of claims 1 to 21, said agent comprising a vector
having affinity for said pre-targeting vector.
32. A method as claimed in claim 31 wherein said pre-targeting
vector is a monoclonal antibody.
33. A method as claimed in claim 30 which comprises the steps: i)
administering to said body an agent as claimed in any of claims 1
to 21; and thereafter ii) administering a substance capable of
displacing or releasing said agent from its target.
34. A method as claimed in any of claims 30 to 33 wherein said
agent further comprises a therapeutic compound.
35. A method as claimed in claim 34 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.
36. A method for in vitro investigation of targeting by an agent as
defined in any of claims 1 to 21 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.
37. A method as claimed in claim 36 wherein the flow rate of
carrier liquid is controlled to simulate shear rates encountered in
vivo.
Description
[0001] This application claims benefit under 35 U.S.C. 119(e) of
provisional applications serial No. 60/049,264 and 60/049,265 both
filed Jun. 6, 1997; and No. 60/049,268 filed Jun. 7, 1997.
[0002] This invention relates to diagnostic and/or therapeutically
active agents, more particularly to diagnostic and/or
therapeutically active agents incorporating moieties which interact
with or have 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.
[0003] It is well known that ultrasound 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] The molecules and/or structure to which the agent is
intended to bind will hereinafter be referred to as the target. In
order to obtain specific imaging of or a therapeutic effect at 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/or treated, and is able to bind specifically
or selectively to a given vector molecule.
[0009] The vector is attached or linked to the reporter moiety in
order to bind these moieties to the region/structure to be imaged
and/or treated. 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.
[0010] 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 is 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, Oct. 3-5, 1984, Buri, P. and Gumma,
A. (Ed), Elsevier, Amsterdam 1984).
[0011] 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
reporter 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).
[0012] 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 Nov. 13-16, 1995.
[0013] 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.
[0014] The present invention is based on the finding that
gas-filled microbubbles stabilised by monolayers of film-forming
surfactant material are particularly useful reporters in targeted
diagnostic and/or therapeutic agents. Thus, for example, the
flexibility and deformability of such thin monolayer membranes
substantially enhances the echogenicity of such reporters relative
to liposome systems containing lipid bilayers or multiples of such
bilayers. This may permit the use of very low doses of the reporter
material to achieve high ultrasound contrast efficacy, with
consequent safety benefits.
[0015] 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-filled microbubbles stabilised by
monolayers of film-forming surfactant material, said agent further
comprising at least one vector.
[0016] The term "monolayer" is used herein to denote that the
amphiphilic surfactant moieties form monolayer films or membranes
similar to so-called Langmuir-Blodgett films at the gas-liquid
interfaces, with the lipophilic parts of the amphiphiles aligning
towards the gas phase and the hydrophilic parts interacting with
the water phase.
[0017] As indicated in WO-A-9729783, it is believed that
electrostatic repulsion between charged phospholipid membranes
encourages the formation of stable and stabilising monolayers at
microbubble-carrier liquid interfaces. The flexibility and
deformability of such thin membranes are believed to enhance the
echogenicity of products according to the invention disclosed
therein relative to gas-filled liposomes comprising one or more
lipid bilayers. The amount of phospholipid used to stabilise such
microbubble-containing aqueous suspensions may be as low as that
necessary for formation of single monolayers of surfactant around
each gas microbubble, the resulting film-like structure stabilising
the microbubbles against collapse or coalescence. Microbubbles with
a liposome-like surfactant bilayer are believed not to be obtained
when such low phospholipid concentrations are used.
[0018] 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-vascularised and
inflamed tissue areas. The present invention is particularly suited
to imaging changes which occur in normal blood vessels situated in
areas of tissue necrosis.
[0019] In a further embodiment of the present invention, one or
more vectors may be attached to or included within the reporter in
a manner such that the vectors are not readily exposed to the
target or target receptors. Increased tissue specificity may
therefore be achieved by applying an additional process to expose
the vectors, for example by exposing the agent after administration
to external ultrasound so as to modify the diffusibility of the
moieties containing the vectors.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1: Flow cytometric comparison of negative control
microbubbles of DSPS (left curve) with bubbles conjugated with CD71
FITC-labelled anti-transferrin antibody (filled curve, right)
showing that 92% of the population fluoresce.
[0021] FIG. 2: Flow cytometry data--comparison with negative
control bubbles (left curve). 98% of the bubbles were calculated to
be fluorescent.
[0022] Any biocompatible gas may be present in the reporter, 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,
dichlorotetrafluoroethan- e, 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,
perfluorotrimethylcyclobutanes, perfluorocyclopentane,
perfluoromethylcyclopentane, 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.
[0023] The gas may comprise a substance such as butane,
cyclobutane, n-pentane, isopentane, neopentane, cyclopentane,
perfluoropentane, perfluorocyclopentane, perfluorohexane or a
mixture containing one or more such gases which is liquid at
handling or processing temperatures but gaseous at body
temperature, e.g. as described in the aforementioned WO-A-9416739,
since the film-forming surfactant monolayers in reporter units
according to the invention may stabilise the resulting microbubbles
against uncontrollable growth.
[0024] In principle, any appropriate film-forming surfactant may be
employed to form the gas-encapsulating monolayers, including
non-polymeric and non-polymerisable wall-forming surfactant
materials, e.g. as described in WO-A-9521631; polymer surfactant
material, e.g. as described in WO-A-9506518; and phospholipids,
e.g. as described in WO-A-9211873, WO-A-9217212, WO-A-9222247,
WO-A-9428780, WO-A-9503835 or WO-A-9729783. Advantageously 75%,
preferably substantially all, of the film-forming surfactant
present in agents according to the invention is incorporated into
monolayers at the gas-liquid interfaces.
[0025] 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
dimyristoylphosphatidylcholine, dipalmitoylphosphatidyl- choline 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.
[0026] It has been found that the use of phospholipids
predominantly (e.g. at least 75%) comprising molecules individually
bearing net overall charge may be particularly advantageous,
especially when used as essentially the sole amphiphilic component
of the reporter, and may convey valuable benefits in terms of
parameters such as product stability and acoustic properties.
Without wishing to be bound by theoretical considerations, it is
believed that electrostatic repulsion between charged phospholipid
membranes may encourage the formation of stable monolayers at the
gas-liquid interfaces; as noted above, the flexibility and
deformability of such thin membranes will enhance the echogenicity
of reporters used in accordance with the invention relative to
gas-filled liposomes comprising one or more lipid bilayers.
[0027] The use of charged phospholipids may also provide reporters
with advantageous properties regarding, for example, stability,
dispersibility and resistance to coalescence without recourse to
additives such as further surfactants and/or viscosity enhancers,
thereby ensuring that the number of components administered to the
body of a subject upon injection of the contrast agents is kept to
a minimum. Thus, for example, the charged surfaces of the
microbubbles may minimise or prevent their aggregation as a result
of electrostatic repulsion.
[0028] Desirably at least 75%, preferably substantially all of
phospholipid material used in reporters in agents of the invention
consists of molecules bearing a net overall charge under conditions
of preparation and/or use, which charge may be positive or, more
preferably, negative. Representative positively charged
phospholipids include esters of phosphatidic acids such as
dipalmitoylphosphatidic acid or distearoylphosphatidic acid with
aminoalcohols such as hydroxyethylethylenediamine. Examples of
negatively charged phospholipids include 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 cardiolipins. The fatty acyl groups of such phospholipids will
typically each contain about 14-22 carbon atoms, for example as in
palmitoyl and stearoyl groups. Lyso forms of such charged
phospholipids are also useful in accordance with the invention, the
term "lyso" denoting phospholipids containing only one fatty acyl
group, this preferably being ester-linked to the 1-position carbon
atom of the glyceryl moiety. Such lyso forms of charged
phospholipids may advantageously be used in admixture with charged
phospholipids containing two fatty acyl groups.
[0029] Phosphatidylserines represent particularly preferred
phospholipids of use in agents according to the invention and
preferably constitute a substantial part, e.g. at least 80% of the
phospholipid content thereof, for example 85-92%. While we do not
wish to be bound by theoretical considerations, it may be that
ionic bridging between the carboxyl and amino groups of adjacent
serine moieties contributes to the stability of such reporter
systems. Preferred phosphatidylserines include saturated (e.g.
hydrogenated or synthetic) natural phosphatidylserine and synthetic
distearoylphosphatidylserine, dipalmitoylphosphatidylserine and
diarachidoylphosphatidylserine.
[0030] Other potentially useful lipids include
phosphatidylethanolamines optionally admixed with one or more
lipids such as stearic acid, palmitic acid, stearylamine,
palmitylamine, cholesterol, bisalkyl glycerols, sphingoglycolipids,
synthetic lipids such as N,N-dimethyl-N-octadecyl-1-o-
ctadecanammonium chloride or bromide (DODAC, DODAB), and/or maleic
acid bisalkylesters.
[0031] Additional 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. Such lipid structures may be
coupled by amide bond formation to amino acids containing one or
more amino groups; the resulting lipid-modified amino acids (e.g.
dipalmitoyllysine or distearoyl-2,3-diaminopropionic acid) may be
useful precursors for the attachment of functionalised spacer
elements having coupling sites for conjugation of one or more
vector molecules.
[0032] Further useful stabilisers include lipopeptides comprising a
lipid attached to a peptide linker portion which is suitably
functionalised for coupling to one or more vector molecules. A
particular preference is the inclusion of a positively charged
peptide linker element (e.g. comprising two or more lysine
residues) capable of anchoring through electrostatic interaction
with reporter microbubbles stabilised by negatively charged
phospholipid or other surfactant membranes.
[0033] Another embodiment of the invention comprises 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, may bind to
cell surface receptors via disulphide exchange reactions. The
reversible nature of such reactions means that microbubble flow may
be controlled by altering the redox environment. Similarly,
functionalised microbubbles with membranes comprising activated
esters such as N-hydroxysuccinimide esters may be used to react
with amino groups found on a multiplicity of cell surface
molecules.
[0034] Previously proposed microbubble-containing contrast agents
based on phospholipids, for example as described in WO-A-9409829,
are typically prepared by contacting powdered surfactant, e.g.
freeze-dried preformed liposomes or freeze-dried or spray-dried
phospholipid solutions, with air or other gas and then with aqueous
carrier, agitating to generate a microbubble suspension which must
then be administered shortly after its preparation. Such processes,
however, suffer the disadvantages that substantial agitational
energy must be imparted to generate the required dispersion and
that the size and size distribution of the microbubbles are
dependent on the amount of energy applied and so cannot in practice
be controlled.
[0035] The reporters or agents according to the present invention,
on the other hand, may advantageously be prepared by generating a
gas microbubble dispersion in an appropriate surfactant (e.g.
phospholipid)-containing aqueous medium, which may if desired
previously have been autoclaved or otherwise sterilised, and then,
preferably after washing and/or size fractionation of the
thus-formed microbubbles, subjecting the dispersion to
lyophilisation, e.g. in the presence of one or more
cryoprotectants/lyoprotectants, to yield a dried product which is
readily reconstitutable in water/aqueous solutions to generate
consistently reproducible microbubble dispersions. This process is
described in greater detail in WO-A-9729783, the contents of which
are incorporated herein by reference; the ability to remove bubbles
of unwanted size and excess surfactant material render this process
of substantial advantage over processes such as those described in
the aforementioned WO-A-9409829 and in prior art such as
WO-A-9608234 (where bubbles are generated on site prior to
injection by shaking a suspension of different phospholipids and
viscosity enhancers such as propylene glycol and glycerol).
[0036] The above-described process may be used to generate reporter
microbubbles with a very narrow size distribution, e.g. such that
over 90% (e.g. at least 95%, preferably at least 98%) of the
microbubbles have volume mean diameter in the range 1-7 .mu.m and
less than 5% (e.g. not more than 3%, preferably not more than 2%)
of the microbubbles have volume mean diameter above 7 .mu.m. The
washing step may be used to ensure that the reporter is
substantially free of unwanted components such as excess lipids or
viscosity enhancers. Agents containing reporters prepared in this
way may exhibit the following advantages over prior art contrast
agent materials:
[0037] Echogenicity per dose may be greatly enhanced since
substantially all of the surfactant material participate in
stabilisation of the microbubbles as monolayers. In vivo ultrasound
tests in dogs have shown that ultrasound contrast agents prepared
as above may produce an increase in backscattered signal intensity
from the myocardium of 15 dB following intravenous injection of
doses as low as 0.1 .mu.l microbubbles/kg body weight.
[0038] Safety in vivo is improved for the same reasons, since such
agents may, for example, be administered in doses such that the
amount of phospholipid injected is as low as 0.1-10 .mu.g/kg body
weight, e.g. 1-5 .mu.g/kg. The use of such low levels of surfactant
may clearly be of substantial advantage in minimising possible
toxic side effects.
[0039] The high efficacy/dose ratio is also particularly
advantageous in targeting applications, since it is generally
understood that rather low amounts of reporter will accumulate at
sites of interest when using products comprising vectors having
affinity for such sites. These preferred reporters according to the
invention may therefore considerably improve contrast at sites of
interest compared to known targetable ultrasound contrast agents.
Their high efficacy may effectively make it possible to "see"
single microbubbles using ultrasound, giving a sensitivity close to
or potentially even higher than that of scintigraphy, which
currently is probably the most useful technique in targeting,
although the resolution in scintigraphic pictures is not
impressive.
[0040] A particular advantage of phosphatidylserine-based agents is
their biocompatibility; thus no acute toxic effects such as changes
in blood pressure or heart rate have been observed in animal tests
on dogs injected with intravenous boluses of
phosphatidylserine-based contrast agents prepared as described
above at doses of up to ten times a normal imaging dose.
[0041] The use of charged phospholipids may also be of advantage in
that they will contain functional groups such as carboxyl or amino
which permit ready linking of vectors, if desired by way of linking
units. It should be noted that other functional groups may also be
incorporated into such systems by mixing a lipid containing a
desired functional group with the film-forming surfactant prior to
microbubble generation.
[0042] It is generally unnecessary to incorporate additives such as
emulsifying agents and/or viscosity enhancers such as are commonly
employed in many existing contrast agent formulations into agents
of the invention. As noted above, this is of advantage in keeping
to a minimum the number of components administered to the body of a
subject and ensuring that the viscosity of the agents is as low as
possible. Since preparation of the agents typically involves a
freeze drying step as discussed above, it may however be
advantageous to include a cryoprotectant/lyoprotectant or bulking
agent, for example an alcohol, e.g. an aliphatic alcohol such as
t-butanol; a polyol such as glycerol; a carbohydrate, e.g. a sugar
such as sucrose, mannitol, trehalose or a cyclodextrin, or a
polysaccharide such as dextran; or a polyglycol such as
polyethylene glycol. The use of physiologically well-tolerated
sugars such as sucrose is preferred.
[0043] Lyophilised dried products prepared as described above are
especially readily reconstitutable in water, requiring only minimal
agitation such as may, for example, be provided by gentle
hand-shaking for a few seconds. The size of the microbubbles so
generated is consistently reproducible and is independent of the
amount of agitational energy applied, in practice being determined
by the size of the microbubbles formed in the initial microbubble
dispersion; surprisingly this size parameter is substantially
maintained in the lyophilised and reconstituted product. Thus,
since the size of the microbubbles in the initial dispersion may
readily be controlled by process parameters such as the method,
speed and duration of agitation, the final microbubble size may
readily be controlled.
[0044] The lyophilised dried products have also proved to be
storage stable for at least several months under ambient
conditions. The microbubble dispersions generated upon
reconstitution in water are stable for at least 8 hours, permitting
considerable flexibility as to when the dried product is
reconstituted prior to injection.
[0045] The high efficacy of these preferred reporters may make it
possible to use smaller bubbles than usual while still generating
ultrasound contrast effects significantly above the minimum
detection levels of current ultrasound imaging equipment. Such
smaller bubbles have potential advantages such as reduced clogging
of vessels, longer circulation times, greater ability to reach
targets, and lower accumulation in lungs or other non-target
organs, and their use and agents containing them constitute further
features of the invention.
[0046] It may also be possible to use such smaller bubbles to
exploit the enhanced ultrasound contrast effects of bubble
clusters. It is known from theory that the ultrasound contrast
effect of a specific number of bubbles with total volume V in a
dilute dispersion increases when the bubbles aggregate to form a
larger gas phase with the same total volume V. It may therefore be
possible to use small bubbles which give substantially no
ultrasound contrast until they are clustered (as may occur in
target areas in preference to non-target sites having low densities
of target molecules). Small bubbles may also be designed to fuse,
e.g. through interbubble binding promoted by interaction with the
target, so as to enhance contrast in target areas. Interbubble
crosslinking and consequent clustering may also be effected if the
reporter, in addition to carrying a vector leading to retention at
specific sites, has unreacted linker moieties capable of reaction
with functional groups on other bubbles.
[0047] Within the context of the present invention, the reporter
unit will usually remain attached to the vectors. However, in one
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 pre-targeting vector
molecule (when the pre-targeting vector is an antibody, the
reporter may be coupled to an immunoglobulin-binding molecule, such
as protein A or an anti-immunoglobulin antibody). The 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.
[0048] Again in the context of the present invention, for example
in assessment of blood perfusion rates in targeted areas such as
the myocardium, it is of interest to measure the rate at which
contrast agents bound to the target are displaced or released
therefrom. This may be achieved in a controlled manner by
administration of an additional vector and/or other substance able
to displace or release the contrast agent from its target.
[0049] 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 above modalities. Surprisingly excellent second harmonic
signals have been obtained from targeted monolayer-stabilised
microspheres 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.
[0050] 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.
[0051] 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 stabilising membranes. Thus,
the therapeutic compound may be linked to a part of the membrane,
for example through covalent or ionic bonds, or may be physically
mixed into the stabilising 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 drug release may be varied depending on the type of
therapeutic application, using a specific amount of ultrasound
energy from the transducer.
[0052] The therapeutic 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 bond or linker, and then incorporate this
derivative into the material used to prepare the reporter, as
described above.
[0053] Representative therapeutics suitable for use in the present
drug delivery compositions include any known therapeutic drugs or
active analogues thereof containing thiol groups which may be
coupled to thiol-containing microbubbles under oxidative conditions
yielding disulphide groups. In combination with a vector or vectors
such drug/vector-modified microbubbles may be allowed to accumulate
in target tissue; administration of a reducing agent such as
reduced glutathione may then liberate the drug molecule from the
targeted microbubble in the vicinity of the target cell, increasing
the local concentration of the drug and enhancing its therapeutic
effect. Alternatively the composition may initially be prepared
without the therapeutic, which may then be coupled to or coated on
the microbubbles immediately prior to use; thus, for example, a
therapeutic may be added to a suspension of microbubbles in aqueous
media and shaken in order to attach or adhere the therapeutic to
the microbubbles.
[0054] 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 cationic polylysine. This method has the
advantage that the vector or vectors used for targeted delivery are
not directly attached to the polylysine 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.
[0055] Alternatively free polylysine chains are firstly modified
with drug or vector molecules then condensed onto the negative
surface of targeted microbubbles.
[0056] 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, FXa, tissue factor, heparins, hirudin, hirulog,
argatroban, DEGR-rFVIIa and annexin V: inhibitors of fibrin
formation and promoters of fibrinolysis such as t-PA, urokinase,
Plasmin, 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.
[0057] Lipophilic derivatives of drugs linked to the microbubble
membrane 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, such as cholesterol or a related compound. 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, such as 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, for example atenolol or
chlorambucil, include amide and ester formation by coupling
respectively with amines and alcohols possessing appropriate
physical properties. A preferred embodiment comprises attachment of
cholesterol to a therapeutic compound by forming a degradable ester
bond.
[0058] 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 synthesise a basement membrane
around the new vessels.
[0059] 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.
[0060] 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.
[0061] 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.
[0062] By targeting gas-filled microbubbles containing a
prodrug-activating enzyme to areas of pathology, one may image
targeting the enzyme, making it possible to visualise when the
microbubbles 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.
[0063] Another alternative is to incorporate the prodrug,
prodrug-activating enzyme and vector in the same microbubbles 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 microbubbles by
external ultrasound after the desired targeting has been
achieved.
[0064] Therapeutics may easily be delivered in accordance with the
invention to diseased or necrotic areas, for example in the heart,
general vasculature, and to the liver, spleen, kidneys and other
regions such as the lymph system, body cavities or gastrointestinal
system.
[0065] 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 flotation and repeated washing.
[0066] Coupling of a reporter unit to a desired vector (and/or
therapeutic drug) may be achieved by covalent or non-covalent
means, usually involving interaction with one or more functional
groups located on the reporter and/or vector and/or any intervening
linker group/spacer element. 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.
[0067] Covalent coupling of reporter and vector may therefore be
effected using linking agents containing reactive moities 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
vector 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).
[0068] Examples of reactive moieties capable of reaction with amino
groups include alkylating and acylating agents. Representative
alkylating agents include:
[0069] 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;
[0070] 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;
[0071] iii) aryl halides such as reactive nitrohaloaromatic
compounds;
[0072] iv) alkyl halides as described by McKenzie, J. A. et al. in
J. Protein Chem. (1988) 7, 581;
[0073] 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;
[0074] vi) epoxide derivatives such as epichlorohydrin and
bisoxiranes,which may react with amino, sulfhydryl or phenolic
hydroxyl groups;
[0075] vii) chlorine-containing derivatives of s-triazines, which
are very reactive towards nucleophiles such as amino, sufhydryl and
hydroxy groups;
[0076] 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;
[0077] ix) squaric acid diethyl esters as described by Tietze, L.
F. in Chem. Ber. (1991) 124, 1215; and
[0078] 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.
[0079] Representative amino-reactive acylating agents include:
[0080] 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;
[0081] 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;
[0082] iii) Acid halides;
[0083] iv) Active esters such as nitrophenylesters or
N-hydroxysuccinimidyl esters;
[0084] v) acid anhydrides such as mixed, symmetrical or
N-carboxyanhydrides;
[0085] vi) other useful reagents for amide bond formation as
described by Bodansky, M. et al. in `Principles of Peptide
Synthesis` (1984) Springer-Verlag;
[0086] 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;
[0087] viii) azlactones attached to polymers such as
bis-acrylamide, e.g. as described by Rasmussen, J. K. in Reactive
Polymers (1991) 16, 199; and
[0088] 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.
[0089] 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.
[0090] 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.
[0091] 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
cabodiimides, 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 Woodwards 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.
[0092] 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 .alpha.-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.
[0093] Vector-reporter coupling may also be effected using enzymes
as zero-length linking agents; thus, for example, transglutaminase,
peroxidase and xanthine oxidase may be used to produce linked
products. Reverse proteolysis may also be used for linking through
amide bond formation.
[0094] Non-covalent vector-reporter 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 a negatively charged membrane surface may also
increase non-specifically the affinity of a microbubble for a cell
through charge interactions.
[0095] Alternatively, a vector may be coupled to a protein 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; a conjugate of a vector with such a
phosphatidylserine-bindi- ng 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.
[0096] 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
may 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 may 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 destabilise 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 destabilising
binding moieties are not selected. Furthermore the selection may be
done under shear conditions, for example 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 (by chemical
conjugation or via peptide synthesis, or at the DNA-level for
recombinant vectors) to a vector molecule, constituting a general
tool for attaching any vector molecule to the microspheres.
[0097] A vector which comprises or is coupled to a peptide,
lipo-oligosaccharide 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 Hi 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.
[0098] 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. Alternatively,
avidin or streptavidin may be attached directly to the surface of
reporter microparticles.
[0099] 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 be crosslinked by antibody molecules and lead to formation of
multi-bubble cross-linked assemblies of potentially increased
echogenicity.
[0100] 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.
[0101] 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.
[0102] 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 in accordance with the invention, as may agents
such as biotin/avidin systems which induce non-covalent
reporter-vector linking and agents which induce hydrophobic or
electrostatic interactions.
[0103] 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.
[0104] 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.
[0105] Contrast agents according to the present invention are
therefore useful in all imaging modalities since contrast elements
such as X-ray contrast agents, light imaging probes, spin labels or
radioactive units may readily be incorporated in or attached to the
reporter units.
[0106] 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 PEGs, 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.
[0107] Appropriate molecular weights for PEG spacers used in
accordance with the invention may, for example, be between 120
Daltons and 20 kdaltons.
[0108] 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). 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).
[0109] 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.
[0110] 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, oligosaccharides and oligonucleotides, which may or
may not contain enzyme cleavage sites.
[0111] 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--
[0112] [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.2 are 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.
[0113] 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. Volfova, I., Rihova,
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.).
[0114] Other potentially useful polymeric spacer materials
include:
[0115] 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;
[0116] ii) block copolymers of polymethacrylates with biodegradable
polyesters (see e.g. San Roman, J. and Guillen-Garcia, P. in
Biomaterials (1991) 12, 236-241);
[0117] 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);
[0118] 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);
[0119] 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);
[0120] 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);
[0121] 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);
[0122] viii) polyesters consisting of alternating units of ethylene
glycol and terephthalic acid, e.g. Dacron.sup.R, which are
non-degradable but highly biocompatible;
[0123] 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);
[0124] 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);
[0125] 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);
[0126] 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;
[0127] 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
[0128] 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).
[0129] The following tables list linking agents and agents for
protein modification which may be useful in preparing targetable
agents in accordance with the invention.
1 Heterobifunctional linking agents 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 Linking agent Reactivity Comments Homobifunctional
linking agents BS --NH.sub.2 BMH --SH BASED (1) photoreactive
iodinable disuiphide 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 Biotinylation
agents biotin-BMCC --SH biotin-DPPE* preparation of biotinylated
liposomes biotin-LC-DPPE* preparation of biotinylated liposomes
biotin-HPDP --SH disuiphide 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 disuiphide linker photoactivatable biotin
nucleic acids sulfo-NHS-biotin --NH.sub.2 water-soluble
sulfo-NHS-LC-biotin --NH.sub.2 Notes: DPPE =
dipalmitoylphosphatidylethanolami- ne; LC = long chain Agents for
protein modification 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
[0130] Other potentially useful protein modifications 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.
[0131] Vectors which may be usefully employed in targetable agents
according to the invention include the following:
[0132] 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
bi- and multi-specific antibodies, i.e. antibodies having
specificity for two or more different antigens 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.
[0133] 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.
[0134] 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.
[0135] iv) Oligonucleotides and modified oligonucleotides which
bind DNA or RNA through Watson-Crick or other type's 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 any possible target
molecules and which therefore may be employed as vectors for
targeting.
[0136] v) DNA-binding drugs may behave similarly to
oligonuclotides, but may exhibit biological acitvity and/or toxic
effects if taken up by cells.
[0137] vi) Protease substrates/inhibitors. Proteases are involved
in many pathological conditions. Many substrates/inhibitors are
non-peptidic but, at least in the case of inhibitors, are often
bioactive.
[0138] vii) Vector molecules may be generated 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.
[0139] 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.
[0140] ix) Proteins or peptides which bind to glucosamioglycan side
chains e.g. heparan sulphate, including glucosoaminoglycan-binding
portions of larger molecules, as binding to glucosoaminoglycans
does not result in a biological response. Proteoglycans are not
found on red blood cells, which eliminates undesirable adsorption
to these cells.
[0141] Other peptide vectors and lipopeptides thereof of particular
interest for targeted ultrasound imaging are listed below:
Atherosclerotic plaque binding peptides such as YRALVDTLK (SEQ ID
NO:26), YAKFRETLEDTRDRMY (SEQ ID NO:27) and RALVDTEFKVKQEAGAK (SEQ
ID NO:28); Thrombus binding peptides such as NDGDFEEIPEEYLQ (SEQ ID
NO:29) and GPRG (SEQ ID NO:30), Platelet binding peptides such as
PLYKKIIKKLLES (SEQ ID NO:31); and cholecystokinin,
.alpha.-melanocyte-stimulating hormone, heat stable enterotoxin 1,
vasoactive intestinal peptide, synthetic alpha-M2 peptide from the
third heavy chain complementarity-determininig region and analogues
thereof for tumour targeting.
[0142] The following tables identify various vectors which may be
targeted to particular types of targets and indicated areas of use
for targetable diagnostic and/or therapeutic agents according to
the invention which contain such vectors.
2 Protein and peptide vectors - antibodies Vector type Target
Comments/areas of use Ref antibodies CD34 vascular diseases in
general, 1 (general) normal vessel wall (e.g " myocardium),
activated " endothelium, immune cells " ICAM-1 " 1 " ICAM-2 " 1 "
ICAM-3 " 1 " E-selectin " 1 " P-selectin " 1 " PECAM " 1 "
Integrins, " 2 e.g. VLA-1, VLA-2, VLA- 3, VLA-4, VLA-5, VLA- 6,
.beta..sub.1.alpha..sub.7, .beta..sub.1.alpha..sub.8,
.beta..sub.1.alpha..sub.v, LFA-l, Mac- 1, CD41a, etc. " GlyCAM
Vessel wall in lymph nodes 3 (quite specific for lymph nodes) "
MadCam 1 " 3 " fibrin Thrombi 4 " Tissue Activated endothelium, 5
Factor tumours " Myosin Necrosis, myocardial infaction 6 " CEA
Tumours 7 (carcino- embryonal antigen) " Mucins Tumours 8 "
Multiple Tumours 9 drug resistance protein " Prostate Prostate
cancer specific antigen " Cathepsin B Tumours (proteases of various
10 kinds are often more or less specifically overexpressed in a
variety of tumours - Cathepsin B is such a protease) " Transferrin
Tumors, 11 receptor vessel wall MoAb 9.2.27 Tumours 12 Antigen
upregulated on cell growth VAP-1 Adhesion molecule 13 Band 3
Upregulated during protein phagocytic activity antibodies CD34
(sialomu endothelial cells cin antibodies CD31 (PECAM- endothelial
cells 1) antibodies intermediate filaments necrotic cells/tissue
CD44 tumour cells a antibodies .beta.-micro- general b antibodies
globulin MHC class 1 general b antibodies integrin tumours;
angiogenesis c antibodies .alpha.v.beta.3
REFERENCES
[0143] 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.
[0144] b) I. Roitt, J. Brostoff, and D. Male. 1985. Immunology,
London: Gower Medical Publishing, p. 4.7
[0145] c) Stromblad, S., and D. A. Cheresh. 1996. "Integrins,
angiogenesis and vascular cell survival". Chemistry & Biology
3: 881-885.
3 Vector Type Target Comments/areas of use Ref Protein and peptide
vectors - cell adhesion molecules etc. L-selectin CD34 vascular
diseases in 3 MadCAM1 general, normal vessel wall GlyCam 1 (e.g
myocardium), activated endothelium, Lymph nodes Other selectins
carbohydrate vascular diseases in 14 ligands general, normal vessel
wall (sialyl Lewis x) (e.g myocardium), activated heparan sulfate
endothelium RGD-peptides integrins " 2 PECAN PECAN, Endothelium, 15
and other Cells in immune system Integrins, Laminin, Endothelium,
16 e.g. VLA-1, VLA- collagen, Vessel wall 2, VLA-3, VLA-4,
fibronectin, etc. VLA-5, VLA-6, VCAM-1, thrombo-
.beta..sub.1.alpha..sub.7, .beta..sub.1 .alpha..sub.8, spondin,
.beta..sub.1.alpha..sub.v, LFA-1, vitronectin etc. Mac-1, CD41a,
etc. Integrin Integrins, Cells in immune system 17 receptors, e.g.
VLA-1, VLA- vessel wall 18 e.g. Laminin, 2, VLA-3, VLA-4, etc.
collagen, VLA-5, VLA-6, fibronectin, .beta..sub.1.alpha..sub.7,
.beta..sub.1.alpha..sub.8, VCAM-1, .beta..sub.1.alpha..sub.v,
LFA-1, thrombospondin, Mac-1, CD41a, vitronectin etc. etc. Nerve
cell proteoglycans 19 adhesion N-CAM molecule (N-CAM) (homophilic)
integrin .alpha.v.beta.3 CD31 (PECAM-1) endothelial cells
RGD-peptides integrins angiogenesis c Vectors comprising
cytokines/growth factors/peptide hormones and fragments thereof
Epidermal growth EGF-receptor or Tumours 20 factor related
receptors Nerve growth NGF-receptor Tumours 21 factor Somatostatin
ST-receptor Tumours 22 Endothelin Endothelin- Vessel wall receptor
Interleukin-1 IL-1-receptor Inflammation, activated 23 cells of
different kinds Interleukin-2 IL-2-receptor " 24 Chemokines (ca.
Chemokine Inflammation 25 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 26 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 27
growth factors Proteoglycans Angiogenesis Schwann cell
proteoglycans 28 growth factor specific receptors Miscellaneous
protein and peptide vectors Streptavidin Kidney Kidney diseases 29
Bacterial Fibronectin Vessel wall 30 fibronectin binding proteins
Fc-part of Fc-receptors Monocytes 31 antibodies macrophages liver
Transferrin transferrin- Tumours 11 receptor vessel walls
Streptokinase/ thrombi thrombi tissue plasminogen activator
Plasminogen, Fibrin Thrombi, 32 plasmin tumours Mast cell
proteoglycans 33 proteinases Elastase proteoglycans 34 Lipoprotein
proteoglycans 35 lipase Coagulation proteoglycans 36 enzymes
Extracellular proteoglycans 37 superoxide dismutase Heparin
cofactor proteoglycans 38 II Retinal survival proteoglycans 39
factor specific receptors Heparin-binding proteoglycans 40 brain
mitogen specific receptors Apolipoprotein, proteoglycans 41 e.g.
specific apolipoprotein B receptors (e.g., LDL receptor)
Apolipoprotein E LDL receptor 42 proteoglycans Adhesion-
proteoglycans 43 promoting proteins, e.g. Purpurin Viral coat
proteoglycans 44 proteins, e.g. HIV, Herpes Microbial fibronectin,
45 adhesins, e.g. collagen, "Antigen 85" fibrinogen, complex of
vitronectin, mycobacteria heparan sulfate .beta.-amyloid
proteoglycans .beta.-amyloid accumulates in 46 precursor
Alzheimer's disease Tenascin, heparan sulfate, 47 e.g. tenascin C
integrins Vectors comprising non-peptide agonists/antagonists or
non-bioactive binders of receptors for cytokines/growth
factors/peptide hormones/cell adhesion molecules Several
agonists/antagonists 48 are known for such factors 49 acting
through G-protein coupled receptors 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 50 analogues vessel wall etc.
Vectors comprising anti-angiogenic factors 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
Vectors comprising angiogenic factors acidic fibroblast growth EC
of tumors K 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 growth EC of
tumors K factor Bradykinin EC of tumors K Calcitonin gene-related
EC of tumors K peptide epidermal growth factor EC of tumors K
Fibrin tumors K Fibrinogen tumors K Heparin EC of tumors K
histamine EC of tumors K hyaluronic acid or fragments EC of tumors
K thereof Interleukin-1.alpha. EC of tumors K laminin, laminin
fragments EC of tumors K nicotinamide EC of tumors K platelet
activating factor EC of tumors K Platelet-derived endothelial EC of
tumors K growth factor prostaglandins E1, E2 EC of tumors K
spermine EC of tumors K spermine EC of tumors K Substance P EC of
tumors K transforming growth factor-.alpha. EC of tumors K
transforming growth factor-.beta. EC of tumors K Tumor necrosis
factor-.alpha. EC of tumors K vascular endothelial growth EC of
tumors K factor/vascular permeability factor vitronectin A Vector
molecules other than recognized angiogenetic factors with known
affinity for receptors associated with angiogenesis angiopoietin
tumors, B inflammation .alpha..sub.2-antiplasmin tumors,
inflammation combinatorial libraries, tumors, for instance:
compounds from inflammation compounds that bind to basement
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 factor, tumors, E basic inflammation hepatocyte growth
factor tumors, I 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 A factor
antagonists inflammation angiogenesis 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, e g,,
TIMP-2 U metalloproteinases inflammation thrombin tumors, H
inflammation thrombin-receptor-activat- tumors, H ing
tetradecapeptide inflammation thymidine phosphorylase tumors, D
inflammation tumor growth factor tumors, ZA inflammation
Receptors/targets associated with angiogenesis 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 tumors, G
receptors inflammation fibronectin tumors P Flk-1/KDR, Flt-4
tumors, VEGF receptor D inflammation FLT-1 (fins-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-G- 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.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, inflammation heat 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 Oligonucleotide vectors Oligonucleotides DNA
made Tumours 51 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 51 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 51 complementary to agent infections DNA of infecting
agent. Triple or As in above As in above examples 51
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
Modified oligonucleotide vectors Phosphorothioate As for As for
unmodified oligos 51 oligos unmodified oligos 2'-O-methyl " " 51
substituted oligos circular oligos " " 51 oligos " " 51 containing
hairpin structure to decrease degradation oligos with " " 51
terminal phosphorothioate 2'-fluoro oligos " " 51 2'-amino oligos "
" 51 DNA-binding " Increased binding affinity 52 drugs conjugated
as compared to pure oligos to oligos (for examples, see below)
Peptide Nucleic " Increased binding affinity 53 Acids (PNAs, and
stability compared to oligonucleotidss standard oligos. with a
peptide backbone) Nucleoside and nucleotide vectors Adenosine or
Adenosine Vessel wall 54 analogues receptors Heart ADP, UDP, UTP
Various Many tissues, e.g. brain, 55 and others nucleotide spinal
cord, kidney, spleen receptors Receptors comprising DNA-binding
drugs 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. Receptors
comprising protease substrates Peptidic or non- Cathepsin B
Tumours, a variety of 10 peptidic which may more or less substrates
specifically overexpress proteases of various kinds, e.g. Cathepsin
B Receptors comprising protease inhibitors Peptidic or non-
Cathepsin B Tumours, a variety of 10 peptidic which may more or
less inhibitors specifically overexpress e.g. N-acetyl- proteases
of various kinds, Leu-Leu- e.g. 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 56 (extracellular
protease inhibitors) Antithrombin proteoglycans, 57 Coagulation
factors Vectors from combinatorial libraries Antibodies with Any of
above Any diseased or normal 58, structure targets - or may
structure of interest, e.g. 59, determined be unknown when thrombi,
tumours or walls 60 during make functional of myocardial
vessels
generation selection of process vector binding to chosen diseased
structure Peptides with " " 58, sequence 59, determined 60 during
generation process Oligonucleotides " " 58, with sequence 59,
determined 60 during generation process Modifications of " " 58,
oligos obtained 59, as above 60 Other chemicals " " 58, with
structure 59, determined 60 during generation process Carbohydrate
vectors neo- macrophages general activation/ glycoproteins
inflammation oligosaccharides Asialo- liver 61 with terminal
glycoprotein galactose receptor Hyaluronan aggrecan (a 62
proteoglycan) "link proteins" cell - surface receptors: CD44
Mannose Blood brain barrier, 63 Brain tumours and other diseases
causing changes in BBB Bacterial " 64 glycopeptides (Glyco) Lipid
vectors GM1 gangliosides cholera bacteria diagnosis/treatment of in
the cholera gastrointestinal tract platelet PAF receptors diagnosis
of inflammation activating factor (PAF) antagonists Prostoglandin
Prostoglandin diagnosis of inflammation antagonists of receptors
inflammation Thromboxane Leukotriene diagnosis of inflammation
antagonists of receptors inflammation Small molecule vectors
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
[0146] Representative examples of drugs useful in accordance with
the invention include: abamectin, abundiazole, acaprazine,
acabrose, acebrochol, aceburic acid, acebutolol, acecainide,
acecarbromal, aceclidine, aceclofenac, acedapsone, acediasulfone,
acedoben, acefluranol, acefurtiamine, acefylline clofibrol,
acefylline piperazine, aceglatone, aceglutamide, aceglutamide
aluminium, acemetacin, acenocoumarol, aceperone, acepromazine,
aceprometazine, acequinoline, acesulfame, acetaminophen,
acetaminosalol, acetanilide, acetarsone, acetazolamide,
acetergamine, acetiamine, acetiromate, acetohexamide,
acetohydroxamic acid, acetomeroctol, acetophenazine, acetorphine,
acetosulfone, acetriozate, acetryptine, acetylcholine chloride,
acetylcolchinol, acetylcysteine, acetyldigitoxin, acetylleucine,
acetylsalicyclic acid, acevaltrate, acexamic acid, acifran,
acipimox, acitemate, acitretin, acivicin, aclantate, aclarubicin,
aclatonium napadisilate, acodazole, aconiazide, aconitine,
acoxatrine, acridorex, acrihellin, acrisorcin, acrivastine,
acrocinide, acronine, actinoquinol, actodigin, acyclovir,
adafenoxate, adamexine, ademetionine, adenosine phosphate,
adibendan, adicillin, adimolol, adinazolam, adiphenine, aditeren,
aditoprim, adrafinil, adrenalone, afloqualone, afurolol, aganodine,
ajmaline, aklomide, alacepril, alafosfalin, alanine mustard,
alanosine, alaproclate, alazanine triclofenate, albendazole,
albendazole oxide, albuterol, albutoin, alclofenac, alcometasone
dipropionate, alcloxa, alcuronium chloride, aldioxa, aldosterone,
alepride, aletamine, alexidine, alfacalcidol, alfadex, alfadolone,
alfaprostol, alfaxalone, alfentanil, alfuzosin, algestone
acetonide, algestone acetophenide, alibendol, aliconazole,
alifedrine, aliflurane, alimadol, alinidine, alipamide, alitame,
alizapride, allantoin, alletorphine, allobarbital, alloclamide,
allocupreide, allomethadione, allopurinol, allylestrenol, allyl
isothicyanate, allylprodine, allylthiourea, almadrate sulfate,
almasilate, almecillin, almestrone, alminoprofen, almitrine,
almoxatone, alonacic, alonimid, aloxistatin, alozafone, alpertine,
alphacetylmethadol, alphameprodine, alphamethadol, alphaprodine,
alphavinylaziridinoethyl acetate, alpidem, alpiropride, alprazolam,
alprenolol, alprostadil, alrestatin, altanserin, altapizone,
alteconazole, althiazide, altrenogest, altretamine, aluminium
acetate, aluminium clofibrate, aluminium subacetate, alverine,
amadinone acetate, amafolone, amanozine, amantadine, amantanium
bromide, amantocillin, ambasilide, ambazone, ambenonium chloride,
ambenoxan, ambroxol, ambruticin, ambucaine, ambucetamide,
ambuphylline, ambuside, ambutonium bromide, amcinafal, amcinafide,
amcinonide, amdinocillin, amdinocillin pivoxil, amebucort,
amedalin, ametantrone, amezepine, amezinium metilsulfate, amfenac,
amfepentorex, amfetaminil, amflutizole, amfonelic acid,
amicarbalide, amicibone, amicloral, amicycline, amidantel,
amidapsone, amidephrine, amiflamine, amifloverine, amifloxacin,
amifostine, amikacin, amikhelline, amiloride, aminacrine,
amindocate, amineptine, aminobenzoic acid, aminocaproic acid,
aminoethyl nitrate, aminoglutethimide, aminohippuric acid,
aminometradine, aminopentamide, aminophylline, aminopromazine,
aminopterin, aminopyrine, aminoquinol, aminoquinuride, aminorex,
aminosalicyclic acid, aminothiadiazole, aminothiazole, amiodarone,
amiperone, amipheazole, amipizone, amiprilose, amiquinsin,
amisometradine, amisulpride, amiterol, amithiozone, amitraz,
amitriptyline, amitriptylinoxide, amixetrine, amlexanox,
amlodipine, amobarbital, amodiaquine, amogastrin, amolanone,
amonofide, amoproxan, amopyroquin, amorolfine, amocanate,
amosulalol, amotriphene, amoxapine, amoxecaine, amoxicillin,
amoxydramine camsilate, amperozide, amphecloral, amphenidone,
amphetamine, amphotalide, amphotericin B, ampicillin, ampiroxicam,
amprolium, ampyrimine, ampyzine, amquinate, amrinone, amsacrine,
amygdalin, amylene, amylmetacresol, amyl nitrite, anagestone
acetate, anagrelide, anaxirone, anazocine, anazolene, ancarolol,
ancitabine, androstanediol, androstanol propionate,
androstenetrione, androstenonol propionate, anethole, anguidine,
anidoxime, anilamate, anileridine, aniline, anilopam, anipamil,
aniracetam, anirolac, anisacril, anisindione, anisopirol,
anisoylbromacrylic acid, anitrazafen, anpirtoline, ansoxetine,
antafenite, antazoline, antazonite, anthelmycin, anthiolimine,
anthralin, anthramycin, antienite, antimony potassium tartrate,
antimony thioglycollate, antipyrine, antrafenine, apalcillin,
apazone, apicycline, apomorphine, apovincamine, apraclonidine,
apramycin, aprindine, aprobarbital, aprofene, aptazapine,
aptocaine, arabinosylmercaptopurine, aranotin, arbaprostil,
arbekacin, arclofenin, arfendazam, arginine, arginine glutamat,
arildone, arnolol, aronixil, arotinolol, arpinocid, arpromidine,
arsanilic acid, arsthinol, artemisinin, articaine, asaley, ascorbic
acid, ascorbyl palmitate, asocainol, aspartame, aspartic acid,
asperlin, aspoxicillin, astemizole, atamestane, atenolol,
atipamezole, atiprosin, atolide, atracurium besilate, atromepine,
atropine, atropine oxide, auranof in, aurothoiglucose,
aurothioglycanide, avilamycin-A, avridine, axamozide, azabon,
azabuperone, azacitodine, azaclorzine, azaconazole, azacosterol,
azacyclonol, azaftozine, azaguanidine, azaloxan, azamethonium
bromide, azamulin, azanator, azanidazole, azaperone, azapicyl,
azaprocin, azaquinzole, azaribine, azarole, azaserine, azaspirium
chloride, azastene, azastrptonigrin, azatodine, azathioprine,
azauridine, azelastine, azepexole, azepindole, azetepa,
azidamfenicol, azidocillin, azimexon, azintamide, azipramine,
azithromycin, azlocillin, azolimine, azosemide, azotomycin,
aztreonam, azumolene, bacampicillin, baclofen, bacmecillinam,
balsalazide, bamaluzole, bambuterol, bamethan, bamifylline,
bamipine, bamnidazole, baquiloprim, barbexaclone, barbital,
barucainide, batilol, bazinaprine, becanthone, beclamide,
beclobrate, beclomethasone dipropionate, beclotiamine, befiperide,
befunolol, befuraline, bekanamycin, belarizine, beloxamide,
bemarinone, bemegride, bemetizide, bemitradine, benactyzine,
benafentrine, benanserin, benapryzine, benaxibine, benazepril,
bencianol, bencisteine, benclonidine, bencyclane, bendamustine,
bendazac, bendazol, benderizine, bendroflumethiazide, benethamide
penicillin, benexate, benflorex, benfosformin, benfotiamine,
benfurodil hemisuccinate, benhepazone, benidipine, benmoxin,
benolizime, benorilate, benorterone, benoxafos, benoxaprofen,
benoxinate, benperidol, benproperine, benrixate, bensalan,
benserazide, bensuldazic acid, bentazepam, bentemazole, bentiamine,
bentipimine, bentiromide, benurestat, benzaldehyde, benzalkonium
chloride, benzaprinoxide, benzarone, benzbromarone, benzestrol,
benzethidine, benzethonium chloride, benzetimide, benzilonium
bromide, benzindopyrine, benziodarone, benzmalecene, benznidazole,
benzobarbital, benzocaine, benzoclidine, benzoctamide, benzodepa,
benzododecinium chloride, benzoic acid, benzoin, benzonatate,
benzopyrronium bromide, benzoquinium chloride, benzotript,
benzoxiquine, benzoxonium chloride, benzoyl peroxide, benzoylpas,
benzphetamine, benzpiperylon, benzpyrinium bromide, benzquercin,
benzquinamide, benzthiazide, benztropine, benzydamine,
benzylpenicillin, benzylsulfamide, beperidium iodide, bephenium
naphtoate, bepiastine, bepridil, beraprost, berberine sulfate,
bermastine, bermoprofen, berythromycin, besulpamide, beslunide,
beta carotene, betacetylmethadol, betahistine, betaine,
betameprodine, betamethadol, betamethasone, betamethasone acetate,
betamethasone acibutate, betamethasone benzoate, betamethasone
dipropionate, betamethasone phosphate, betamethasone valerate,
betamicin, betaprodine, betaxolol, betazole, bethanechol chloride,
bethanidine, betiatide, betoxycaine, bevantolol, bevonium
metilsulfate, bezafibrate, bezitramide, bialamicol, bibenzonium
bromide, bibrocathol, bicifadine, biclodil, biclofibrate,
biclotymol, bicozamycin, bidimazium iodine, bietamiverine,
bietaserpine, bifemelane, bifepramide, bifluranol, bifonazole,
binedaline, binfloxacin, binfibrate, bioallethrin, bioresmethrin,
biotin, bipenamol, biperiden, biphenamine, biriperone, bisacodyl,
bisantrene, bis(aziridinyl) butanediol, bisbendazole,
bisbentiamine, bisfenazone, bisfentidine, bismuth betanaphthol,
bismuth-triglycollamate, bismuth subgallate, bismuth subsalicylate,
bisorbin, bisoprolol, bisorcic, bioxatin acetate, bispyrithione
magsulfex, bithionol, bithionoloxide, bitipazone, bitoterol,
bitoscantate, bleomycin, bluensomycin, bofumustine, bolandiol
dipropionate, bolasterone, bolazine, boldenone undecylenate,
bolenol, bolmantalate, bometolol, bopindolol, bornaprine,
bornaprolol, bornelone, botiacrine, boxidine, brallobarbital,
brazergoline, brefonalol, bremazocine, brequinar, bretylium
tosylate, brindoxime, brivundine, brobactam, broclepride,
brocresine, brocrinat, brodimoprim, brofaromine, brofezil,
brofoxine, brolaconazole, brolamfetamine, bromacrylide,
bromadoline, bromamid, bromazepam, bromchlorenone, bromebric acid,
bromerguride, brometenamine, bromfenac, bromhexine, bromindione,
bromisovalum, bromociclen, bromocriptine, bromodiphenhydramine,
bromofenofos, bromopride, bromoxandide, bromperidol, bromperidol
decanoate, brompheniramine, bronopol, broparestrol, broperamole,
bropirimine, broquinaldol, brosotamide, brosuximide, brotianide,
brotizolam, brovanexine, brovincamine, broxaldine, broxaterol,
broxitalamic acid, broxuridine, broxyquinoline, bruceantin,
brucine, bucainide, bucetin, buciclovir, bucillamine, bucindolol,
bucladesine, buclizine, buclosamide, bucloxic acid, bucolome,
bucricaine, bucromarone, bucrylate, bucumolol, budesonide,
budipine, budotitane, budralazine, bufenadrine, bufeniode,
bufetolol, bufexamac, bufezolac, buflomedil, bufogenin, buformin,
bufrolin, bufuralol, bumadizone, bumecaine, bumepidil, bumetanide,
bumetrizole, bunaftine, bunamidine, bunamiodyl, bunaprolast,
bunazosin, bunitrolol, bunolol, buparvaquone, bupicomide,
bupivacaine, bupranolol, buprenorphine, bupropion, buquineran,
buquinolate, buquiterine, buramate, burodiline, buspirone,
busulfan, butabarbital, butacaine, butacetin, butaclamol,
butadiazamide, butafosfan, butalamine, butalbital, butamben,
butamirate, butamisole, butamoxane, butanediol cyclic sulfite,
butanilicaine, butanixin, butanserin, butantrone, butaperazine,
butaprost, butaverine, butedronate, buterizine, butetamate,
butethamine, buthiazide, butibufen, butidrine, butikacin,
butilfenin, butinazocine, butinoline, butirosin, butixirate,
butobendine, butoconazole, butocrolol, butoctamide, butofilolol,
butonate, butopamine, butopiprine, butoprozine, butopyrammonium
iodide, butorphanol, butoxamine, butoxylate, butriptyline,
butropium bromide, butylated hydroxyanisole, butylated
hydroxytoluene, butylparaben, butynamine, buzepide metiodide,
cabastine, cabergoline, cadralazine, cafaminol, cafedrine,
caffeine, calcifediol, calcitrol, calcium citrate, calcium
dobesilate, calcium glubionate, calcium gluceptate, calcium
gluconate, calcium glycerophosphate, calcium hypophosphite, calcium
lactate, calcium lactobionate, calcium levulinate, calcium
mandelate, calcium pantothenate, calcium phosphate dibasic, calcium
phophate tribasic, calcium saccharate, calcium stearate,
calusterone, camazepam, cambendazole, camiverine, camostast,
camphotamide, camptothecin, camylofin, canbisol, cannabinol,
canrenoic acid, canrenone, cantharidine, capobenic acid,
capreomycin, caproxamine, capsaicine, captamine, captodiame,
captopril, capuride, caracemide, caramiphen, carazolol, carbachol,
carbadox, carbaldrate, carbamazepine, carbamide peroxide, carbantel
lauryl sulfate, carbaril, carbarsone, carbaspirin calcium,
carbazeran, carbazochrome, carbazachrome salicylate, carbazachrome
sulfonate, carbazocine, carbeniciltin, carbenicillin indanyl,
carbencillin phenyl, carbenoxolone, carbenzide, carbestrol,
carbetapentane, carbidopa, carbimazole, carbinoxamine, carbiphene,
carbocloral, carbocysteine, carbofenotion, carbol-fuschin,
carbomycin, carboplatin, carboprost, carboprost methyl, carboquone,
carbromal, carbubarb, carburazepam, carbutamide, carbuterol,
carcainium chloride, carebastine, carfentanil, carfimate,
carisoprodol, carmantadine, carmetizide, carmofur, carmustine,
carnidazole, carnitine, carocainide, caroverine, caroxazone,
carperidine, caperone, carphenazine, carpindolol, carpiramine,
carprofen, carpronium chloride, carsalam, cartazolate, carteolol,
carubicin, carumonam, carvedilol, carzenide, carzolamide, cathine,
cathinone, cefaclor, cefadroxil, cefalonium, cefaloram,
cefamandole, cefamandole naftate, cefaparole, cefatrizine,
cefazaflur, cefazedone, cefazolin, cefbuperazone, cefcanel,
cefcanel daloxate, cefedrolor, cefempidone, cefepime, cefetamet,
cefetrizole, cefvitril, cefixime, cefmenoxime, cefmepidium
chloride, cefmetazole, cefminox, cefodizime, cefonizid,
cefoperazone, ceforanide, cefotaxime, cefotetan, cefotiam,
cefoxazole, cefoxitin, cefpimizole, cefpiramide, cefpirome,
cefpodoxime, cefpodoxime proxetil, cefquinome, cefrotil,
cefroxadine, cefsulodin, cefsumide, ceftazidime, cefteram,
ceftezole, ceftiofur, ceftiolene, ceftioxide, ceftizoxime,
ceftriaxone, cefuracetime, cefuroxime, cefuraxime axetil,
cefurzonam, celiprolol, cephacetrile, cephalexin, cephaloglycin,
cephaloridine, cephalothin, cephapirin, cephradine, cetaben,
cetamolol, cethexonium chloride, cetiedil, cetirizine, cetocycline,
cetohexazine, cetophenicol, cetotiamine, cetoxime, cetraxate,
chaulmosulfone, chendiol, chiniofon, chlophedianol, chloracyzine,
chloral betaine, chloral hydrate, chloralose, chlorambucil,
chloramine, chloramphenicol, chloramphenicol palmitate,
chloramphenicol succinate, chlorazanil, chlorbenzoxamine,
chlorbetamide, chlorcyclizine, chlordantoin, chlordiazepoxide,
chlordimorine, chlorhexadol, chlorhexidine, chlorhexidine
phosphanilate, chlorindanol, chlorisondamine chloride,
chlormadinone acetate, chlormerodrin, chlormezanone, chlormidazole,
chloronaphazine, chloroazodin, chlorobutanol, chlorocresol,
chlorodihydroxyandrostenone, chloroethyl mesylate,
5-chloro-3'-fluoro-2'3-dideoxyuridine, chloroguanide,
chlorophenothane, chloroprednisone acetate, chloroprocaine,
chloropyramine, chloroquine, chloroserpidine, chlorothen,
chlorothiazide, chlorotriansene, chloroxine, chloroxylenol,
chlorozotocin, chlorphenesin, chlorphenesin carbamate,
chlorpheniramine, chlorphenoctium amsonate, chlorphenoxamine,
chlorphentermine, chlorproethazine, chlorproguanil, chlorpromazine,
chlorpropamide, chlorprothixene, chlorquinaldol, chlortetracycline,
chlorthalidone, chlorthenoxazine, chlorzoaxazone, chloecalciferol,
cholic acid, choline chloride, choline glycerophosphate,
chromocarb, chromonar, ciadox, ciamexon, cianergoline, cianidol,
cianopramine, ciapilome, cicaprost, cicarperone, ciclactate,
ciclafrine, ciclazindol, cicletanine, ciclomenol, ciclonicate,
ciclonium bromide, ciclopirox, ciclopramine, cicloprofen,
cicloprolol, ciclosidomine, ciclotizolam, ciclotropium bromide,
cicloxilic acid, cicloxolone, cicortonide, cicrotic acid,
cidoxepin, cifenline, cifostodine, ciglitazone, ciheptolane,
ciladopa, cilastatine, cilazapril, cilazaprilat, cilobamine,
cilofungin, cilostamide, cilostazol, ciltoprazine, cimaterol,
cimemoxin, cimepanol, cimetidine, cimetropium bromide, cimoxatone,
cinchonine, cinchophen, cinecromen, cinepaxadil, cinepazet,
cinepazic acid, cinepazide, cinfenine, cinfenoac, cinflumide,
cingestol, cinitapride, cinmetacin, cinnamaverine, cinnamedrine,
cinnarizine, cinnarizine clofibrate, cinnofuradione, cincotramide,
cinodine, cinolazepam, cinoquidox, cinoaxin, cinoxate, cinoxolone,
cinooxopazide, cinperene, cinprazole, cinpropazide, cinromide,
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, colforsin, 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, cyheptamide, 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,
dexetozoline, 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, dinazafone, 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,
etilefrine 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, fenclofenac, 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, fursultiamine, 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, isometheptene, 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, lasalocid,
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, lydinycin,
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, medroxyprogestrone
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, methaqualone, 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, methylprednisolone 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, murocainide,
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,
nifurethazone, 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 bromide, 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,
prednylidene, 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, prodolic acid,
profadol, profexalone, proflavine, proflazepam, progabide,
progesterone, proglumetacin, proglumide, proheptazine,
proligestone, proline, prolintane, prolonium iodide, promazine,
promegestone, promestriene, 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, quingestrone, 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, roxoperone, 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,
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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,
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succinylsulfathiazole, succisulfone, suclofenide, sucralfate,
sucrose octaacetate, sudexanox, sudoxicam, sufentanil, sufosfamide,
sufotidine, sulazepam, sulbactam, sulbactam pivoxil, sulbenicillin,
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sulfabenz, sulfabenzamide, sulfacarbamide, sulfacecole,
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sulfaclomide, sulfaclorazole, sulfaclozine, sulfacytine,
sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine,
sulfaethidole, sulfaguandide, sulfaguanole, sulfalene,sulfaloxic
acid, sulfamazone, sulfamerazine, sulfameter, sulfamethazine,
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sulfaperin,sulfaphenazole, sulfaproxyline, sulfapyridine,
sulfaquinoxaline, sulfarsphenamine, sulfasalazine, sulfasomizole,
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sulfatolamide, sulfatroxazole, sulfatrozole, sulfazamet,
sulfinalol, sulfinpyrazone, sulfiram, sulfisomidine, sulfisoxazole,
sulfisoxazole, sulfobromophthalein, sulfonethylmethane,
sulfonmethane, sulfonterol, sulforidazine, sulfoxone sodium,
sulicrinat, sulindac, sulisatin, sulisobenzone, sulmarin,
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sulosemide, sulotroban, suloxifen, sulpiride, sulprosal,
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sunagrel, suncillin, supidimide, suproclone, suprofen, suramin,
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taloximine, talsupram, taltrimide, tameridone, tameticillin,
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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,
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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, thiazolsulfone, 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, tolrestat, toltrazuril,
tolufazepam, tolycaine, tomelukast, tomoglumide, tomoxetine,
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topiramate, toprilidine, topterone, toquizine, torasemide,
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toyocamycin, toyomycin, traboxepine, tracazolate, tralonide,
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cysteine, trixolane, trizoxime, trocimine, troclosene potassium,
trofosfamide, troleandomycin, trolnitrate, tromantadine,
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tropenziline bromide, tropicamide, tropigline, tropiprine,
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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.
[0147] The following non-limitative examples serve to illustrate
the 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 microbubble suspensions
giving an increased sound beam attenuation compared to a standard.
Flow cytometric analysis of products can be used to confirm
attachment of macromolecules thereto. The ability of targeted
microbubbles to bind specifically to cells expressing a target may
be studied in vitro 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
Adhesion of Poly-L-lysine-coated Phosphatidylserine-encapsulated
Microbubbles to Endothelial Cells
[0148] Poly-L-lysine (8 mg) having a molecular weight of 115 kDa
was dissolved in water (400 .mu.l). Freshly redispersed
microbubbles of phosphatidylserine-encapsulated perfluorobutane (40
.mu.l) were incubated in either water (400 Al) or the poly-L-lysine
solution for 15 minutes at room temperature. Zeta potential
measurements confirmed that the poly-L-lysine-coated microbubbles
were positively charged while the uncoated bubbles were negatively
charged. A cell adhesion study using human endothelial cells grown
in culture dishes was performed with the above-described
microbubbles, the uncoated microbubbles being used as a control.
Microscopy of the endothelial cells after incubation showed a much
increased number of poly-L-lysine-coated microbubbles adhering to
endothelial cells in comparison to the uncoated microbubbles.
EXAMPLE 2
Gas-filled Microbubbles Comprising Phosiphatidylserine and
RGDC-Mal-PEG.sub.3400-DSPE (SEQ ID NO:1)
[0149] a) Synthesis of Boc-NH-PEG3400-DSPE (t-butyl Carbamate
Poly(Ethylene Glycol) Distearoylphosphatidylethanolamine)
[0150] DSPE (distearoylphosphatidylethanolamine) (31 mg, Sygena
Inc.) was added to a solution of Boc-NH-PEG3400-SC (t-butyl
carbamate poly(ethylene glycol)-succinimidyl carbonate) (150 mg) in
chloroform (2 ml), followed by triethylamine (33 .mu.l). The
mixture formed a clear solution after stirring at 41.degree. C. for
10 minutes. The solvent was rotary evaporated and the residue taken
up in acetonitrile (5 ml). The thus-obtained dispersion was cooled
to 4.degree. C. and centrifuged, whereafter the solution was
separated from the undissolved material and evaporated to dryness.
The structure of the resulting product was confirmed by NMR.
[0151] b) Synthesis of H.sub.2N-PEG.sub.3400-DSPE
(Amino-poly(Ethylene
Glycol)-distearoylphosphatidylethanolamine)
[0152] Boc-NH-PEG.sub.3400-DSPE (167 mg) was stirred in 4 M
hydrochloric acid in dioxane (5 ml) for 2.5 hours at ambient
temperature. The solvent was removed by rotary evaporation and the
residue was taken up in chloroform (1.5 ml) and washed with water
(2.times.1.5 ml). The organic phase was removed by rotary
evaporation. TLC (chloroform/methanol/water 13:5:0.8) gave the
title product with Rf=0.6; the structure of the product, which was
ninhydrin positive, was confirmed by NMR.
[0153] c) Synthesis of Mal-PEG.sub.3400-DSPE (3-maleimidopropionate
Poly(Ethylene Glycol)distearoyldhosphatidylethanolamine)
[0154] A solution of N-succinimidyl-3-maleimidopropionate (5.6 mg,
0.018 mmol) in tetrahydrofuran (0.2 ml) is added to
H.sub.2N-PEG.sub.3400-DSPE (65 mg, 0.012 mmol) dissolved in
tetrahydrofuran (1 ml) and 0.1 M sodium phosphate buffer pH 7.5 (2
ml). The reaction mixture is heated to 30.degree. C. and the
reaction is followed to completion by TLC, whereafter the solvent
is evaporated.
[0155] d) Synthesis of RGDC-Mal-PEG3400-DSPE (SEQ ID NO:1)
[0156] Mal-PEG3.sub.400-DSPE (0.010 mmol) in 0.1 M sodium phosphate
buffer having a pH of 7.5 is added to the peptide RGDC (SEQ ID
NO:1) (0.010 mmol). The reaction mixture is heated to 37.degree. C.
if necessary and the reaction is followed by TLC to completion,
whereafter the solvent is removed.
[0157] e) Preparation of Gas-Filled Microbubbles Encapsulated by
Phosphatidylserine and RGDC-Mal-PEG.sub.3400-DSPE (SEQ ID NO:1)
[0158] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Mal-PEG.sub.3400-DSPE (10-0.1 mol %) is added 5% propylene
glycol-glycerol 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 then 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 0.1 M sodium phosphate buffer having a pH of 7.5.
The peptide RGDC (SEQ ID NO:1), dissolved in 0.1 M sodium phosphate
buffer having a pH of 7.5, is added to the washed microbubbles,
which are placed on the roller table. The washing procedure is then
repeated.
[0159] f) Alternative Preparation of Gas-Filled Microbubbles
Encapsulated by Phosphatidylserine and RGDC-Mal-PEG.sub.3400-DSPE
(SEQ ID NO:1)
[0160] To phosphatidylserine (5 mg) is added 5% propylene
glycol-glycerol 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 0.1 M sodium phosphate buffer having a pH of 7.5.
RGDC-Mal-PEG.sub.3400-DSPE (SEQ ID NO:1) dissolved in 0.1 M sodium
phosphate buffer having a pH of 7.5 is added to the washed
microbubbles, which are then placed on the roller table. The
washing procedure is repeated following incorporation of the
RGDC-Mal-PEG.sub.3400-DSPE (SEQ ID NO:1) into the microbubble
membranes.
EXAMPLE 3
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine,
Phosphatidylcholine and
Biotin-Amidocaproate-PEG.sub.3400-Ala-Cholesterol
[0161] a) Synthesis of Z-Ala-Cholesterol
(3-O-(Carbobenzyloxy-L-Alanyl)Cho- lesterol)
[0162] Cholesterol (4 mmol), Z-alanine (5 mmol) and
dimethylaminopyridine (4 mmol) were dissolved in
dimethylformamide/tetrahydrofuran (20 ml+5 ml) and
dicyclohexylcarbodiimide was added. The reaction mixture was
stirred at ambient temperature overnight. Dicyclohexylurea was
filtered off and the solvent was rotary evaporated. The residue was
taken up in chloroform, undissolved dicyclohexylurea was filtered
off and the solvent was removed by rotary evaporation. The residue
was placed on a column of silica gel, and Z-Ala-cholesterol was
eluted with toluene/petroleum ether (20:2) followed by
toluene/diethyl ether (20:2). The fractions containing the title
compound were combined and the solvent was removed by rotary
evaporation. The structure of the product was confirmed by NMR.
[0163] b) Synthesis of Ala-Cholesterol
(3-O-(L-Alanyl)Cholesterol)
[0164] Z-Ala-cholesterol (0.48 mmol) is placed in tetrahydrofuran
(20 ml) and glacial acetic acid (3 ml) and hydrogenated in the
presence of 5% palladium on charcoal for 2 hours. The reaction
mixture is filtered and concentrated in vacuo.
[0165] c) Synthesis of Boc-NH-PEG3400-Ala-Cholesterol
[0166] Ala-cholesterol is added to a solution of
Boc-NH-PEG.sub.3400-SC (t-butyl carbamate poly(ethylene
glycol)succinimidyl carbonate) in chloroform, followed by
triethylamine. The suspension is stirred at 41 oC for 10 minutes.
The crude product is purified by chromatography.
[0167] d) Synthesis of H.sub.2N-PEG3400-Ala-Cholesterol
[0168] Boc-NH-PEG.sub.3400-Ala-cholesterol is stirred in 4 M
hydrochloric acid in dioxane for 2.5 hours at ambient temperature.
The solvent is removed by rotary evaporation and the residue is
taken up in chloroform and washed with water. The organic phase is
rotary evaporated to dryness. The crude product may be purified by
chromatography.
[0169] e) Synthesis of
Biotinamidocaproate-PEG3400-Ala-Cholesterol
[0170] A solution of biotinamidocaproate N-hydroxysuccinimide ester
in tetrahydrofuran is added to
H.sub.2N-PEG.sub.3400-Ala-cholesterol dissolved in tetrahydrofuran
and 0.1 M sodium phosphate buffer having a pH of 7.5 (2 ml). The
reaction mixture is heated to 30.degree. C. and the reaction is
followed to completion by TLC, whereafter the solvent is
evaporated.
[0171] f) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine, Phosphatidylcholine and
Biotinamidocaproate-PEG.sub.3- 400-Ala-Cholesterol
[0172] To a mixture (5 mg) of phosphatidylserine and
phosphatidylcholine (in total 90-99.9 mole) and
biotinamidocaproate-PEG.sub.3400-Ala-choleste- rol (10-0.1 mole) is
added 5% propylene glycol-glycerol 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 then
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.
[0173] g) Alternative Preparation of Gas-Filled Microbubbles
Encapsulated With Phosphatidylserine, Phosphatidylcholine and
Biotinamidocaproate-PEG.- sub.3400-Ala-Cholesterol
[0174] To a mixture (5 mg) of phosphatidylserine and
phosphatidylcholine is added 5% propylene glycol-glycerol 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 then 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.
Biotinamidocaproate-PEG.sub.3400-Ala-cholesterol dissolved in water
is added to the washed microbubbles, which are placed on a roller
table for several hours. The washing procedure is repeated
following incorporation of the
biotinamidocaproate-PEG.sub.3400-Ala-cholesterol into the
microbubble membranes.
EXAMPLE 4
Gas-Filled Microbubbles Comprising Phosphatidylserine,
Phosphatidylcholine,
Biotin-Amidocaproate-PEG.sub.3400-Ala-Cholesterol and
Drug-Cholesterol
[0175] a) Synthesis of Drug-Cholesterol
[0176] Cholesterol (4 mmol), a drug having an acid group and
dimethylaminopyridine (4 mmol) are dissolved in
dimethylformamide/tetrahy- drofuran (20 ml+5 ml) and
dicyclohexylcarbodiimide is added. The reaction mixture is stirred
at ambient temperature overnight. Dicyclohexylurea is filtered off
and the solvent is rotary evaporated. The title compound is
purified by chromatography.
[0177] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine, Phosphatidylcholine,
Biotinamidocaproate-PEG.sub.3400- -Ala-Cholesterol and
Drug-Cholesterol
[0178] To a mixture (5 mg) of phosphatidylserine and
phosphatidylcholine (in total 90-99.9mol %) and
biotinamidocaproate-PEG3400-Ala-cholesterol (prepared as in Example
3) and drug-cholesterol (in total 10-0.31 mol %) is added 5%
propylene glycol-glycerol 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.
EXAMPLE 5
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Thiolated-anti-CD34-Mal-PEG.sub.3400-DSPE
[0179] a) Preparation of Thiolated Anti-CD34 Antibodies
[0180] Thiolation of anti-CD34 antibodies may be effected as
described by Hansen, C. B. et al. (1995) Biochim. Biophys. Acta
1239, 133-144.
[0181] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and
Thiolated-anti-CD34-Mal-PEG.sub.3400-DSPE
[0182] To a mixture (5 mg) of phosphatidylserine (90-99.9mol %) and
Mal-PEG.sub.3400-DSPE (10-0.1 mol %, prepared as in Example 2) is
added 5% propylene glycol-glycerol 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 an appropriate
buffer and coupling of the thiolated antibody to the microbubbles
is performed, e.g. as described by Goundalkar, A., Ghose, T. and
Mezei, M. in J. Pharm. Pharmacol. (1984) 36 465-66 or Hansen, C. B.
et al.(1995) Biochim. Biophys. Acta 1239 133-144. The microbubbles
are then placed on a roller table for several hours and are washed.
Flow cytometric analysis of the resulting microbubbles (employing a
fluorescently labeled secondary antibody) is used to confirm
attachment of the anti-CD34 antibody to the bubbles. The ability of
the bubbles to bind specifically to CD34-expressing cells is
studied by microscopy employing one population of cells expressing
CD34 and one population that do not express CD34.
EXAMPLE 6
Biotin Attached to Gas-Filled Microbubbles
[0183] Biotin may be attached to microbubbles in many different
ways, e.g. in a similar way to that described by Corley, P. and
Loughrey, H. C. in (1994) Biochim. Biophys. Acta 1195, 149-156. The
resulting bubbles are analysed by flow cytometry, e.g. by employing
fluorescent streptavidin to detect attachment of biotin to the
bubbles. Alternatively radioactive or enzyme-labelled
streptavidin/avidin is used to analyse biotin attachment.
EXAMPLE 7
Gas-Filled Microbubbles Encapsulated With
Distearoylphosphatidylserine and Biotin-DPPE
[0184] To distearoylphosphatidylserine (DSPS) (22.6 mg) was added
4% propylene glycol-glycerol in water (4 ml). The dispersion was
heated to not more than 80.degree. C. for five minutes and then
cooled to ambient temperature. An aqueous dispersion of biotin-DPPE
(1.5 mg) in 4% propylene glycol-glycerol (1 ml) was added and the
sample was put on a roller table for 1-2 hours. The suspension was
filled into vials and the head spaces were flushed with
perfluorobutane. The vials were shaken for 45 seconds, whereafter
they were put on a roller table. After centrifugation for 7 minutes
the infranatant was exchanged with water and the washing was
repeated twice. Normal phase HPLC with an Evaporative Light
Scattering Detector confirmed that the membranes of the
microbubbles contained 4 mol % biotin-DPPE. The mean particle
diameter of the microbubbles was 4 .mu.m measured by Coulter
Counter. Ultrasound transmission measurements using a 3.5 MHz
broadband transducer showed that a particle dispersion of <2
mg/ml gave a sound beam attenuation higher than 5 dB/cm.
EXAMPLE 8
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Biotinylated Antibody Non-covalently Bound to
Streptavidin-Succ-PEG-DSPE
[0185] a) Synthesis of Succ-PEG.sub.3400-DSPE
[0186] NH.sub.2-PEG.sub.3400-DSPE (prepared as in Example 2) 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, 59G7-71.
[0187] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0188] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Succ-PEG.sub.3400-DSPE (10-0.1 mol %) is added 5% propylene
glycol-glycerol in water (1 ml). The dispersion is heated to not
more than 80.degree. C. for 5 minutes and then coooled 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. Alternatively the
microbubbles may be prepared as described in Example 2(f).
[0189] c) Coupling of Streptavidin to Gas-Filled Microbubbles
Encapsulated With Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0190] Streptavidin is covalently bound to Succ-PEG.sub.3400-DSPE
in the microbubble membranes 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.
[0191] d) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Biotin Non-Covalently Bound to
Streptavidin-Succ-PEG3400-DSPE
[0192] Microbubbles from Example 8(c) are incubated in a solution
containing biotinylated vectors, e.g. biotinylated antibodies. The
vector-coated microbubbles are washed as described above.
EXAMPLE 9
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Biotinylated Oligonucleotide Non-Covalently Bound to
Streptavidin-Succ-PEG-DSPE
[0193] a) Synthesis of Succ-PEG.sub.3400-DSPE
[0194] NH.sub.2-PEG.sub.3400-DSPE (prepared as in Example 2) 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.
[0195] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0196] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Succ-PEG.sub.3400-DSPE (10-0.1 mol %) is added 5% propylene
glycol-glycerol 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. Alternatively the
microbubbles may be prepared as described in Example 2(f).
[0197] c) Coupling of Streptavidin to Gas-Filled Microbubbles
Encapsulated With Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0198] Streptavidin is covalently bound to Succ-PEG.sub.3400-DSPE
in the microbubble membraness 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 analyzed 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.
[0199] d) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and a Biotinylated Oligonucleotide
Non-Covalently Bound to Streptavidin-Succ-PEG.sub.3400-DSPE
[0200] Microbubbles from Example 9(c) are incubated in a solution
containing a biotinylated oligonucleotide. The
oligonucleotide-coated bubbles 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. As examples, 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) may be used.
EXAMPLE 10
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Folate-PEG-Succ-DSPE
[0201] a) Preparation of Folate-PEG-Succ-DSPE
[0202] Folate-PEG-Succ-DSPE is synthesised as described by Lee, R.
J. and Low, P. S. in (1995) Biochimica. Biophysica. Acta 1233,
134-144.
[0203] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Folate-PEG-Succ-DSPE
[0204] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and folate-PEG-DSPE (10-0.1 mol %) is added 5% propylene
glycol-glycerol in water (1 ml). The dispersion is heated to not
more than 80.degree. C. for 5 minutes and is 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. Alternatively the
microbubbles are prepared as described in Example 2(e) or 2(f).
Analysis of folate attachment may for example be done by
microscopic study of the binding of the folate-containing
microbubbles to cells expressing different levels of folate
receptors.
EXAMPLE 11
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Thiolated-anti-CD34-Mal-PEG.sub.3400-DSPE,
Thiolated-anti-ICAM-1-Mal-PEG.- sub.3400-DSPE and
Thiolated-anti-E-Selectin-Mal-PEG.sub.3400-DSPE
[0205] a) Preparation of Thiolated-anti-CD34 Antibodies
[0206] Thiolation of anti-CD34 antibodies may be effected as
described by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta
1239, 133-144.
[0207] b) Preparation of Thiolated-anti-ICAM-1 Antibodies
[0208] Thiolation of anti-ICAM-1 antibodies may be effected as
described by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta
1239, 133-144.
[0209] c) Preparation of Thiolated-anti-E-selectin Antibodies
[0210] Thiolation of anti-E-selectin antibodies may be effected as
described by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta
1239, 133-144.
[0211] d) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Thiolated-anti-CD34-Mal-PEG.sub.3400-DSPE,
Thiolated-anti-ICAM-1-Mal-PEG.sub.3400-DSPE,
Thiolated-anti-E-Selectin-Ma- l-PEG.sub.3400-DSPE
[0212] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Mal-PEG.sub.3400-DSPE (10-0.1 mol %, prepared as in Example 2)
is added 5% propylene glycol-glycerol in water (1 ml). The
dispersion is heated to not more than 80.degree. C. for 5 minutes
and is 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 an appropriate
buffer, and coupling of the antibodies from Example 11(a), 11(b)
and 11(c) to the microbubbles is performed, e.g. as described by
Goundalkar, A., Ghose, T. and Mezei, M. in J. Pharm. Pharmacol.
(1984) 36, 465-466 or by Hansen, C. B. et al. in (1995) Biochim.
Biophys. Acta 1239, 133-144. The microbubbles are placed on a
roller table for several hours and are then washed.
EXAMPLE 12
The Peptide FNFRLKAGOKIRFGAAAWEPPRARI (SEQ ID NO:2) Attached to
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine
[0213] The peptide FNFRLKAGQKIRFGAAAWEPPRARI (SEQ ID NO:2),
comprising phosphatidylserine-binding and heparin-binding sections,
is synthesised. The peptide is added to preformed
phosphatidylserine-encapsulated perfluorobutane microbubbles and
thoroughly mixed.
EXAMPLE 13
Fibronectin Covalently Bound to Gas-Filled Microbubbles
Encapsulated With Phosphatidylserine and
Phosphatidylethanolamine
[0214] a) Microbubbles Preparation
[0215] DSPS (25 mg) and DSPE (5.0 mg) were weighed into a clean
vial and 5 ml of a solution of 1.4% propylene glycol/2.4% glycerol
was added. The mixture was warmed to 80.degree. C. for 5 minutes.
The sample was cooled to room temperature and the head space was
flushed with perfluorobutane gas. The vials were shaken in a cap
mixer for 45 seconds and the microbubbles were twice washed with
distilled water then resuspended in 0.1 M sodium borate buffer, pH
9.
[0216] b) Modification of Fibronectin
[0217] Fibronectin (1.0 mg) in 5 ml 0.01 M Hepes buffer, pH 8, was
added to 0.1 mmol of the crosslinker SDBP. The mixture was
incubated on ice for 2 hours.
[0218] c) Microbubble Modification.
[0219] To the protein solution from (b) was added the microbubble
suspension from (a) and incubation was allowed to proceed for 2
hours at room temperature on a roller table. Unreacted material was
removed by allowing the microbubbles to float and then replacing
the buffer with 0.1 M sodium borate buffer, pH 9. This process was
repeated three times.
[0220] d) In vitro Aanalysis.
[0221] The microbubbles were tested in the in vitro assay detailed
in Example 21. A gradual accumulation of microbubbles binding to
the cells was observed.
EXAMPLE 14
Gas-filled Microbubbles Encapsulated With Phosphatidylserine, and
3.beta.-FN-(N',N'-Dimethylaminoethane)Carbamoyllcholesterol
[0222] a) Synthesis of
3.beta.-[N-(N',N'-Dimethylaminoethane)Carbamoyl]cho- lesterol
(DC-chol) (Farhood, H., Gao. X, Barsoum, J. and Huang. L. Anal.
Biochem. 225. 89-93 (1995))
[0223] To a stirred solution of 2-dimethylaminoethylamine (19.40
mg, 24:1, 0.22 mmol) and triethylamine (310 l, 2.23 mmol) in
dichloromethane (3 ml) at room temperature was slowly added a
solution of cholesteryl chloroformate (100 mg, 0.22 mmol) in
1,4-dioxane. When the reaction was completed, the mixture was
evaporated to dryness and the residue was purified by flash
chromatography (CHCl.sub.3/MeOH, 4:1). A white solid was obtained,
yield 105 mg (95%). The structure was verified by NMR and
MALDI.
[0224] b) Preparation of Microbubble Dispersion
[0225] Monolayer-encapsulated microbubbles containing
perfluorobutane are made from a mixture of 90% phosphatidylserine
and 10% (DC-chol) by weighing DSPS (4.5 mg) and (DC-chol) (0.5 mg)
into a 2 ml vial. 0.8 ml propylene glycol/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 was flushed with perfluorobutane. The vial was shaken
on a cap-mixer at 4450 oscillations/minute for 45 seconds and put
on a roller table. The sample was washed by centrifuging at 2000
rpm for 5 minutes. The infranatant was removed by a syringe and
distilled water was added to the same volume. The headspace was
again flushed with perfluorobutane and the sample was kept on a
roller table until a homogeneous appearance was obtained. The
washing procedure was repeated again.
EXAMPLE 15
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
WEPPRARI-PE (SEQ ID NO:3)
[0226] Phosphatidylethanolamine (PE) is reacted with an equimolar
amount of the crosslinker
N-hydroxysuccinimidyl-2,3-dibromopropionate in a 1:1 mixture of
dioxane and 0.02 M HEPES buffer, pH 8.0. Following incubation for 2
hours on ice, an equimolar amount of the heparin-binding peptide
WEPPRARI (SEQ ID NO:3) is added, the pH is brought to 9 by the
addition of 0.2 M disodium tetraborate, and the incubation is
continued for 2 hours at room temperature. The reaction product is
purified by chromatography. Monolayer-encapsulated microbubbles
containing perfluorobutane are made from a mixture of 80-95%
phosphatidylserine (PS) and 5-20% of peptide-substituted PE.
EXAMPLE 16
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Inactivated Human Thrombin-Succ-PEG.sub.3400-DSPE
[0227] a) Inactivation of Human Thrombin
[0228] Human thrombin was inactivated by incubation with a 20%
molar excess of D-Phe-L-Pro-L-Arg-chloromethyl ketone in 0.05 N
HEPES buffer, pH 8.0, at 37.degree. C. for 30 minutes.
[0229] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Succ-PEG.sub.3400-DSPE
[0230] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %)
and Succ-PEG.sub.3400-DSPE (10-0.1 mol %, prepared as in Example
9(a)) was added 5% propylene glycol-glycerol in water (1 ml). The
dispersion was heated to not more than 80.degree. C. for 5 minutes
and was then cooled to ambient temperature. The dispersion (0.8 ml)
was transferred to a vial (1 ml) and the head space was flushed
with perfluorobutane. The vial was shaken in a cap-mixer for 45
seconds, whereafter the sample was put on a roller table. After
centrifugation the infranatant was exchanged with water and the
washing was repeated. Alternatively the microbubbles may be
prepared as described in Example 2(f).
[0231] c) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine and Inactivated Human
Thrombin-Succ-PEG.sub.3400-DSPE
[0232] Inactivated human thrombin was covalently bound to
Succ-PEG.sub.3400-DSPE in the microbubbles from Example 16(b) by
standard coupling methods using a water-soluble carbodiimide. The
sample was placed on a roller table during the reaction. After
centrifugation the infranatant was exchanged with water and the
washing was repeated.
EXAMPLE 17
Gas-Filled Microbubbles Having Methotrexate and Prodrug-Activating
Enzyme Attached
[0233] a) Methotrexate Attached via a Peptide Linker to Gas-Filled
Micrububbles
[0234] Methods for attaching aminoacids to the anticancer drug
methotrexate (MTX) are well described in the literature (see e.g.
Huennekens, F. M. (1994), TIBTECH 12, 234-239 and references
therein). Instead of a single amino acid a peptide may be attached
to MTX using the same technology. Such a peptide may constitute a
linker for the attachment of MTX to the surface of microbubbles.
One class of such linkers comprises peptides of the general
structure (MTX)-F-K/R-X-R-Z-C where X is any amino acid and Z is a
hydrophobic amino acid. A specific example of such a linker is
(MTX)-F-K-L-R-L-C (SEQ ID NO:4). The SH-- group in the Cys-residue
is employed for attachment of the MTX-peptide to the microbubbles
(e.g. composed of phosphatidylserine and Mal-PEG-DSPE) using
standard technology, e.g. as in Example 2. A linker of this kind is
expected to be cleaved by the enzyme cathepsin B which often is
selectively overexpressed outside and on the surface of tumour
cells (Panchal, R. G. et al. (1996), Nat. Biotechnol. 14, 852-856).
Thus, the potential prodrug (MTX)-F-K/R-X-R would be liberated
selectively in tumours. This prodrug can further be activated to
the active drug MTX by the action of carboxypeptidases, either
present endogeneously in the tumour or targeted to the tumour e.g.
by tumour-associated antibodies (see below).
[0235] b) Prodrug-Activating Enzyme Covalently Attached to the
Surface of Gas-Filled Microbubbles
[0236] An example of a prodrug-activating enzyme is
carboxypeptidase A (CPA), which may be conjugated to the surface of
microbubbles encapsulated by, for example, a mixture of
phosphatidylserine and phosphatidylethanolamine, e.g. by using a
3400 Da poly(ethylene glycol) chain bearing an N-hydroxysuccinimide
group at both ends (Perron, M. J. and Page, M., Br. J. Cancer 73,
281-287); the microbubbles may be prepared by standard methods.
Microbubbles containing CPA may be targeted to areas of pathology
by incorporating a suitable targeting vector in the CPA-containing
bubbles. Alternatively CPA may be attached directly to a vector
(e.g. an antibody), for example by the method as described above.
In this latter case the CPA-vector conjugate will be attached to
the surface of the microbubbles as described in Hansen, C. B. et
al. (1995) Biochim. Biophys. Acta 1239 133-144. Examples of the
many possible prodrug-enzyme pairs are described in e.g.
Huennekens, F. M. (1994) TIBTECH 12, 234-239.
EXAMPLE 18
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine,
Thiolated-anti-CEA-Mal-PEG.sub.3400-DSPE and the Anticancer Prodrug
3',5'-O-Dipamitoyl-5-Fluoro-2'-Deoxyuridine
[0237] a) Preparation of Thiolated Anti-CEA Antibodies
[0238] Thiolation of anti-CEA antibodies may be effected as
described by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta
1239, 133-144.
[0239] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine, Thiolated-anti-CEA-Mal-PEG.sub.3400-DSPE and
the Anticancer Prodrug
3',5'-O-Dipamitoyl-5-Fluoro-2'-Deoxyuridine
[0240] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %),
Mal-PEG.sub.3400-DSPE (10-0.1 mol %, prepared as in Example 2) and
the anticancer prodrug 3',5'-O-dipamitoyl-5-fluoro-2'-deoxyuridine
(Mori, A. et al. (1995) Cancer Chemother. Pharmacol. 35, 447-456)
is added 5% propylene glycol-glycerol in water (1 ml). The
dispersion is heated to not more than 80.degree. C. for 5 minutes
and is 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 an approperiate
buffer, and coupling of the antibody to the microbubble is
performed, e.g. as described by Goundalkar, A., Ghose, T. and
Mezei, M. in J. Pharm. Pharmacol. (1984) 36 465-466 or by Hansen,
C. B. et al. in (1995) Biochim. Biophys. Acta 1239 133-144. The
microbubbles are placed on a roller table for several hours and are
then washed.
EXAMPLE 19
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine,
Thiolated-anti-CEA-Mal-PEG.sub.3400-DSPE and the Anticancer Prodrug
N-trifluoroacetyl-adriamycin-14-valerate
[0241] a) Preparation of thiolated anti-CEA antibodies Thiolation
of anti-CEA antibodies may be effected as described by Hansen, C.
B. et al. in (1995) Biochim. Biophys. Acta 1239 133-144.
[0242] b) Preparation of Gas-Filled Microbubbles Encapsulated With
Phosphatidylserine, Thiolated-anti-CEA-Mal-PEG.sub.3400-DSPE and
the Anticancer Prodrug N-trifluoroacetyl-adriamycin-14-valerate
[0243] To a mixture (5 mg) of phosphatidylserine (90-99.9 mol %),
Mal-PEG.sub.3400-DSPE (10-0.1 mol %, prepared as in Example 2) and
the anticancer prodrug N-trifluoroacetyl-adriamycin-14-valerate
(Mori, A. et al. (1993) Pharm. Res. 10, 507-514), is added 5%
propylene glycol-glycerol in water (1 ml). The dispersion is heated
to not more than 80.degree. C. for 5 minutes and is 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 an appropriate buffer, and coupling of the antibody
to the microbubble is performed, e.g. as described by Goundalkar,
A., Ghose, T. and Mezei, M. in J. Pharm. Pharmacol. (1984) 36
465-66 or by Hansen, C. B. et al. in (1995) Biochim. Biophys. Acta
1239 133-144. The microbubbles are placed on a roller table for
several hours and are then washed.
EXAMPLE 20
Method of Use
[0244] An agent comprising phosphatidylserine-encapsulated
microbubbles having inactivated human
thrombin-Succ-PEG.sub.3400-DSPE incorporated into the encapsulating
membrane is lyophilised from 0.01 M phosphate buffer, pH 7.4. The
product is redispersed in sterile water and injected intravenously
into a patient with suspected venous thrombosis in a leg vein. The
leg is examined by standard ultrasound techniques. The thrombus is
located by increased contrast as compared with surrounding
tissue.
EXAMPLE 21
Preparation and Biological Evaluation of Gas-Containing
Microbubbles of DSPS `Doped` With a Lipopeptide Comprising a
Heparin Sulphate Binding Peptide (KRKR) (SEQ ID NO:5) and a
Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6)
[0245] This example is directed at the preparation of targeted
microbubbles comprising multiple peptidic vectors arranged in a
linear sequence.
[0246] a) Synthesis of a Lipopeptide Consisting of a Heparin
Sulphate Binding Peptide (KRKR) (SEQ ID NO:5) and Fibronectin
Peptide (WOPPRARI) (SEQ ID NO:6) 1
[0247] The lipopeptide was synthesised on an ABI 433A automatic
peptide synthesiser starting with Fmoc-Ile-Wang resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic 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% 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 of a 40 mg
aliquot of crude material was carried out using a gradient of 70 to
100% B over 40 minutes (A=0.1% TFA/water and B=MeOH) at a flow rate
of 9 ml/min. After lyophilisation, 16 mg of pure material were
obtained (analytical HPLC, gradient 70-100% B where B=MeOH, A=0.01%
TFA/water: detection--UV 260 and fluorescence, Ex280,
Em350--product retention time=19.44 minutes). Further product
characterisation was carried out using MALDI mass spectrometry:
expected M+H at 2198, found at 2199.
[0248] b) Preparation of Gas-Filled Microbubbles of DSPS `Doped`
With a Multiple-Specific Lipopeptide Consisting of a Heparin
Sulphate Binding Peptide (KRKR) (SEQ ID NO:5) and Fibronectin
Peptide (WOPPRARI) (SEQ ID NO:6)
[0249] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) were weighed
into each of two vials and 0.8 ml of a solution of 1.4% propylene
glycol/2.4% glycerol was added to each vial. The mixtures were
warmed to 80.degree. C. for 5 minutes (vials shaken during
warming). The samples were cooled to room temperature and the head
spaces flushed with perfluorobutane gas. The vials were shaken in a
cap mixer for 45 seconds and rolled overnight. The resulting
microbubbles 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 at maximum
attenuation: 3.5 MHz). The microbubbles were stable at 120 mm Hg.
MALDI mass spectral analysis was used to confirm incorporation of
lipopeptide 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 was added. The suspension was sonicated for 30 seconds
and the solution was analysed by MALDI MS. Positive mode gave M+H
at 2200 (expected for lipopeptide, 2198).
[0250] c) In vitro Study of Gas-Filled Microbubbles of DSPS `doped`
With a Multiple-Specific Lipopeptide Consisting of a Heparin
Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) and Fibronectin
Peptide (WOPPRARI) (SEQ ID NO:6): Binding to Endothelial Cells
Under Flow Conditions
[0251] 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 to which
L-glutamine (200 mM), penicillin/streptomycin (10,000 U/ml and
10,000 .mu.g/ml) and 10% fetal bovine serum were added. The cells
were subcultured with a split ratio of 1:5 to 1:7 when reaching
confluence. Cover-glasses, 22 mm in diameter, were sterilised and
placed on the bottom of 12 well culture plates, whereafter cells in
0.5 ml complete medium with serum were added above the plates. When
the cells reached confluence the coverslips were placed in a
custom-made flow chamber consisting of a groove carved into a glass
plate upon which the cover slip with cells was placed, with the
cells facing the groove, so as to form a flow channel. Microbubbles
prepared as in (b) were passed from a reservoir held at 37.degree.
C. through the flow chamber and back to the reservoir using a
peristaltic pump. The flow rate was adjusted to simulate
physiologically relevant shear rates. The flow chamber was placed
under a microscope and the interaction between the microbubbles and
cells was viewed directly. A camera mounted on the microscope was
connected to a colour video printer and a monitor. A gradual
accumulation of microbubbles on the cells took place at a rate
dependent on the flow rate. On further increasing the flow rate,
cells started to become detached from the coverslip, but the
microbubbles remained bound to the cells. Control bubbles not
carrying the vector did not adhere to the endothelial cells and
disappeared from the chamber under minimal flow conditions.
[0252] d) In vivo Experiment in Dog
[0253] Case 1)
[0254] A 22 kg mongrel dog was anaesthetised 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. A net volume of 2 ml of
microbubbles from (b) was injected as a rapid intravenous bolus; 3
seconds later, the imaged right ventricle was seen to contain
contrast material, and another 3 seconds later the left ventricle
was also filled and a transient attenuation shadow which obscured
the view of the posterior parts of the left ventricle was observed.
Substantial increases in brightness were seen in the myocardium
and, when the attenuation shadow subsided, in the portions of the
heart distal to the left ventricle. 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 ultrasound contrast agent microbubbles 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
close to baseline.
[0255] Case 2) [Comparative]
[0256] 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 that observed in Case 1. At
the completion of the left ventricular attenuation phase, there was
also almost complete loss of myocardial contrast effects, and the
myocardial echo increases in the posterior part of the left
ventricle noted in Case 1 were not observed.
EXAMPLE 22
Preparation of Gas-Filled Microbubbles Encapsuled With DSPS
Comprising Thiolated Anti-CD34-MAL-PEG.sub.2000-PE
[0257] a) Preparation of Gas-Filled Microbubbles Encapsuled With
DSPS and PE-PEG2000-Mal
[0258] DSPS (4.5 mg, 3.9 mmol) and PE-PEG.sub.2000-Mal from Example
50 (0.5 mg) were weighed into a clean vial and 1 ml of a solution
of 1.4% propylene glycol/2.4% glycerol was 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 was flushed with perfluorbutane gas. The vials were
shaken in a cap mixer for 45 seconds and the resulting microbubbles
were washed three times with distilled water.
[0259] b) Thiolation of Anti-CD34 Antibodies
[0260] To 0.3 mg of anti-CD34 antibody dissolved in 0.5 ml
phosphate buffered saline (PBS), pH7, was added 0.3 mg Traut's
reagent and the solution was stirred at room temperature for 1
hour. Excess reagent was separated from the modified protein on a
NAP-5 column.
[0261] c) Conjugation of Thiolated Anti-CD34 Antibody to Gas-Filled
Microbubbles Encapsuled With DSPS and Comprising
DSPE-PEG.sub.2000-MAL
[0262] 0.5 ml of the thiolated antibody praparation from (b) was
added to an aliquot of microbubbles from (a) and the conjugation
reaction was allowed to proceed for 30 minutes on a roller table.
Following centifugation at 2000 rpm for 5 minutes the infranatant
was removed. The microbubbles were washed a further three times
with water.
[0263] d) Detection of the Antibody Encapsulated in the
Microbubbles Using a FITC-Conjugated Secondary Antibody
[0264] To the microbubble suspension from (c) was added 0.025 mL
FITC-conjugated goat-anti-mouse antibody. The mixture was incubated
in the dark at room temperature for 30 minutes on a roller table
and was then centrifuged at 2000 rpm for 5 minutes. The infranatant
was then removed and the microbubbles were washed a further three
times with water. Flow cytometric analysis of the microbubble
suspension showed that 98% of the population was fluorescent.
EXAMPLE 23
Preparation of Gas-Filled Microbubbles Encapsuled With DSPS
Comprising Thiolated Anti-CD62-MAL-PEG.sub.2000-PE
[0265] An identical procedure to that described in Example 22 was
used to prepare microbubbles comprising anti-CD62 antibodies.
EXAMPLE 24
Preparation of Gas-Filled Microbubbles Encapsuled With DSPS
Comprising Thiolated Anti-ICAM-1-MAL-PEG.sub.2000-PE
[0266] An identical procedure to that described in Example 22 was
used to prepare microbubbles comprising anti-ICAM-1 antibodies.
EXAMPLE 25
Preparation of Gas-Filled Microbubbles Encapsulated With DSPS and
Thiolated Anti-CD62-Mal-PEG.sub.2000-PE and
Thiolated-anti-ICAM-1-Mal-PEG- .sub.2000-PE
[0267] This example is directed to the preparation of microbubbles
comprising multiple antibody vectors for targeted ultrasound
imaging.
[0268] a) Preparation of Gas-Filled Microbubbles Encapsulated With
DSPS and PE-PEG.sub.2000-Mal
[0269] DSPS (4.5 mg) and PE-PEG.sub.2000 -Mal 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 was added. The mixture was
warmed to 80.degree. C. for 5 minutes and then filtered through a
4.5 micron filter. The sample was cooled to room temperature and
the head space was flushed with perfluorobutane gas. The vials were
shaken in a cap mixer for 45 seconds and the microbubbles were
washed three times with distilled water.
[0270] b) Thiolation of Anti-CD62 and Anti-ICAM-1 Antibodies
[0271] 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 were stirred at room temperature for 1 hour.
Excess reagent was separated from the modified protein on a NAP-5
column.
[0272] c) Conjugation of Thiolated Anti-CD62 and Anti-ICAM-1
Antibodies to Gas-Filled Microbubbles Encapsulated With DSPS and
DSPE-PEG.sub.2000-Mal
[0273] 0.5 ml of the mixed thiolated antibody preparation from (b)
was added to an aliquot of microbubbles from (a) and the
conjugation reaction was allowed to proceed for 30 minutes on a
roller table. Following centrifugation at 2000 rpm for 5 minutes,
the infranatant was removed. The microbubbles were washed a further
three times with water.
[0274] The PEG spacer length may 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 possible.
EXAMPLE 26
Targeted Gas-Filled Microbubbles Comprising DSPS Coated
Non-Covalently With Polylysine and a Fusion Peptide Comprising a
PS-Binding Component and a Fibronectin Peptide Sequence
FNFRLKAGOKIRFGGGGWOPPRAI (SEQ ID NO:8)
[0275] a) Synthesis of PS-Binding/Fibronectin Fragment Fusion
Peptide FNFRLKAGOKIRFGGGGWOPPRAI (SEQ ID NO:8)
[0276] The peptide was synthesised on an ABI 433A automatic peptide
synthesiser starting with Fmoc-Ile-Wang resin on a 0.1 mmol scale
using 1 mmol amino acid cartridges. All amino acids 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% phenol, 5% EDT and 5% H.sub.2O for
2 hours, giving a crude product yield of 302 mg. Purification by
preparative HPLC of a 25 mg aliquot of crude material was carried
out using a gradient of 20 to 40% B over 40 minutes (A=0.1%
TFA/water and B=0.1% TFA/acetonitrile) at a flow rate of 9 ml/min.
After lyophilisation 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: detection--UV 214 and 260
nm--product retention time=12.4 minutes). Further product
characterization was carried out using MALDI mass spectrometry:
expected M+H at 2856, found at 2866.
[0277] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
Coated Non-Covalently With Polylysine and the
PS-Binding/Fibronectin Fragment Fusion Peptide
FNFRLKAGOKIRFGGGGWOPPRAI (SEQ ID NO:8)
[0278] DSPS (5 mg) was weighed into a clean vial along with
poly-L-lysine (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 was
flushed with perfluorobutane gas. The vials were shaken in a cap
mixer for 45 seconds and the resulting microbubbles were
centrifuged at 1000 rpm for 3 minutes. 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. Acetonitrile (0.5
ml) was then added and the microbubbles were 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:
4 MALDI expected MALDI found Poly-L-lysine 786, 914, 790, 919,
1042, 1170 1048, 1177 DSPS-binding peptide 2856 2866
[0279] The spacer element contained within the
PS-binding/fibronectin fusion peptide (-GGG-) may also be replaced
with other spacers such as PEG.sub.2000 or poly alanine (-AAA-). A
form of pre-targeting may also be employed, whereby the
DSPS-binding/fibronectin fragment fusion peptide is firstly allowed
to associate with cells via fibronectin peptide binding, followed
by administration of PS microbubbles which then bind to the
PS-binding peptide.
EXAMPLE 27
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and
Biotin-PEG-Alanyl-Cholesterol and Functionalised With
Streptavidin/Biotinyl-endothelin-1 Peptide
(biotin-D-Trp-Leu-Asp-Ile-Ile-- Trp.OH) (SEQ ID NO:9) and
Biotinyl-fibrin-anti-polymerant Peptide (Biotin-GPRPPERHOS.NH2)
(SEQ ID NO:10)
[0280] 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).
[0281] a) Synthesis of Biotin-PEG.sub.3400-b-Alanine
Cholesterol
[0282] To a solution of cholesteryl-b-alanine hydrochloride (as
described in Example 59) (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-dioxane (1 ml) was added dropwise. After stirring at room
temperature for 3 hours, 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.
[0283] b) Synthesis of Biotinylated Endothelin-1 Peptide
(Biotin-D-Trp-Leu-Asp-Ile-Ile-Trp.OH) (SEQ ID NO:9)
[0284] The peptide was synthesised on a ABI 433A automatic peptide
synthesiser starting with Fmoc-Trp(Boc)-Wang resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids 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% anisole and 5% H.sub.2O for 2
hours giving a crude product yield of 75 mg. Purification by
preparative HPLC of a 20 mg aliquot of crude material was carried
out using a gradient of 30 to 80% B over 40 minutes (A=0.1%
TFA/water and B=0.1% TFA/acetonitrile) and a flow rate of 9 ml/min.
After lyophilisation 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: detection--UV 214 nm--product
retention time=12.6 minutes). Further product characterization was
carried out using MALDI mass spectrometry: expected M+H at 1077,
found at 1077.
[0285] c) Synthesis of Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2)(SEQ ID NO:10)
[0286] This peptide was synthesised and purified using similar
protocols to those described in (b) above. The pure product was
characterised by HPLC and MALDI MS.
[0287] d) Preparation of Multiple-Specific Gas-Filled Microbubbles
Encapsulated With Phosphatidylserine and
Biotin-PEG.sub.3400-b-Alanine Cholesterol
[0288] DSPS (4.5 mg) and biotin-PEG.sub.3400-b-alanine cholesterol
from (a) (0.5 mg) were weighed into a vial and 0.8 ml of a solution
of 1.4% propylene glycol/2.4% glycerol was 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 was flushed with perfluorobutane gas. The vial was shaken in
a cap-mixer for 45 seconds and the vial was rolled overnight. The
microbubble suspension was washed several times with deionised
water and analysed by Coulter counter and acoustic attenuation.
[0289] e) Conjugation With Fluorescein-Labelled Streptavidin and
Biotinylated Peptides from (b) and (c)
[0290] 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 hours 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 (b) and (c)
respectively for 2 hours. Extensive washing of the microbubbles was
performed to remove unconjugated peptide.
EXAMPLE 28
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) (SEQ ID NO:9) and
Biotinyl-fibrin-anti-polymerant Peptide
(biotin-GPRPPERHOS.NH.sub.2) (SEQ ID NO:10)
[0291] a) Preparation of Biotin-Containing Microbubbles
[0292] To a mixture of phosphatidylserine (5 mg) and biotin-DPPE
(0.6 mg) in a clean vial was added 5% propylene glycol-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 was shaken in a
cap-mixer for 45 seconds. After centrifugation the infranatant was
removed and the microbubbles were washed extensively with
water.
[0293] 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) (SEQ
ID NO:9) and Biotinyl-fibrin-anti-polymerant Peptide
(Biotin-GPRPPERHOS.NH.sub.2) (SEQ ID NO:10)
[0294] The procedure detailed in Example 27 was followed.
EXAMPLE 29
PFB Gas-Containing Microbubbles of DSPS Functionalised With Heparin
Sulphate Binding Peptide/Fibronectin Peptide/RGD Peptide and
Fluorescein
[0295] a) Synthesis of a Lipopeptide Containing the RGD Sequence
and a Fluorescein Reporter Group: Dipalmitoyl-Lys-Lys-Lys-Lys
[Acetyl-Arg-Gly-Asp- 2
[0296] The lipopeptide was synthesised as described in Example
21(a) using commercially available amino acids and polymers. The
lipopeptide was cleaved from the resin in TFA containing 5% water,
5% phenol and 5% EDT for 2 hours. Following evaporation in vacuo
the crude product was precipitated and triturated with diethyl
ether. Purification by preparative HPLC of a 40 mg aliquot of crude
material was carried out using a gradient of 60 to 100% B over 40
minutes (A=0.1% TFA/water and B=0.1% TFA/acetonitrile) at a flow
rate of 9 ml/min. After lyophilisation 10 mg of pure material
(analytical HPLC, gradient 60-100% B where B=0.1% TFA/acetonitrile,
A=0.01% TFA/water: detection--UV 260--product retention time=20-22
minutes). Further product characterisation was carried out using
MALDI mass spectrometry: expected M+H at 1922, found at 1920.
[0297] b) Synthesis of a Lipopeptide Containing a Heparin
Sulphate-Binding Sequence and a Fibronectin Peptide
[0298] Synthesis and purification were carried out as described in
Example 21 (a).
[0299] c) Preparation of Multiple-Specific Gas-Filled Microbubbles
of DSPS Functionalised With a Heparin Sulphate-Binding Peptide, a
fibronectin Peptide, Acetyl-RGD Peptide and Fluorescein
[0300] DSPS (4 mg, 3.9 mmol), lipopeptide from (a) (0.5 mg, 0.2
mmol) and lipopeptide from (b) (0.5 mg) were weighed into each of
two vials and 0.8 ml of a solution of 1.4% propylene glycol/2.4%
glycerol was added to each vial. The mixtures were warmed to
80.degree. C. for 5 minutes (vials shaken during warming). The
samples were cooled to room temperature and the head spaces were
flushed with perfluorobutane gas. The vials were shaken in a cap
mixer for 45 seconds and then rolled overnight. The microbubbles so
obtained were washed several times with deionised water and
analysed by MALDI mass spectrometry as described in Example 21(b).
The microbubbles were investigated by microscopy and were seen to
have a range of sizes between 1 and 5 microns. Furthermore the
microbubbles were fluorescent.
EXAMPLE 30
Gas-Filled Microbubbles Comprising DSPS Covalently Modified With
CD71 FITC-Labelled Anti-Transferrin Receptor Antibody and `Doped`
With a Lipopeptide With Affinity for Endothelial Cells
[0301] This example is directed at the preparation of multiple
vector targeted ultrasound agents.
[0302] 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 (SEQ ID NO:12)
[0303] 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
[0304] 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 of a 40 mg aliquot of crude material was carried out using a
gradient of 90 to 100% B over 50 minutes (A=0.1% TFA/water and
B=MeOH) at a flow rate of 9 ml/min. After lyophilisation, 10 mg of
pure material was obtained (analytical HPLC, gradient 90-100% B
where B=MeOH, A=0.01% TFA/water: detection--UV 214 nm--product
retention time=23 minutes). Further product characterisation was
carried out using MALDI mass spectrometry: expected M+H at 2369,
found at 2373.
[0305] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
`Doped` With a Endothelial Cell-Binding Lipopeptide and
PE-PEG.sub.2000-Mal
[0306] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) along with
PE-PEG.sub.2000-Mal from Example 50 (0.5 mg) were weighed into a
clean vial and 1 ml of a solution of 1.4% propylene glycol/2.4%
glycerol was added. The mixture was warmed to 80.degree. C. for 5
minutes and then filtered through a 4.5 micron filter. The sample
was cooled to room temperature and the head space was flushed with
perfluorobutane gas. The vial was shaken in a cap mixer for 4%
seconds and the resulting microbubbles were washed three times with
distilled water.
[0307] c) Thiolation of FITC-Labelled Anti-Transferrin Receptor
Antibody
[0308] FITC-labelled CD71 anti-transferrin receptor Ab (100 mg/ml
in PBS, 0.7 ml) was reacted with Traut's reagent (0.9 mg) at room
temperature for 1 hour. Excess reagent was separated from modified
protein on a NAP-5 column.
[0309] d) Conjugation of Thiolated FITC-Labelled Anti-Transferrin
Receptor Antibody to Gas-Filled Microbubbles Comprising DSPS
`Doped` With an Endothelial Cell-Binding Lipopeptide and
DSPE-PEG.sub.2000-Mal
[0310] 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 was allowed to proceed for 10 minutes on a
roller table. Following centrifugation at 1000 rpm for 3 minutes
the protein solution was removed and the conjugation repeated twice
more 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 (see FIG. 1).
[0311] Incorporation of lipopeptide into the microbubbles was
confirmed by MALDI mass spectrometry as described in Example 21
(b).
EXAMPLE 31
Gas-Filled Microbubbles Comprising DSPS, a Lipopeptide for
Endothelial Cell Targeting and a Captopril-Containing Molecule
[0312] This example is directed to the preparation of ultrasound
agents for combined targeting and therapeutic applications.
[0313] a) Synthesis of a Lipopeptide Functionalised With Captopril
4
[0314] The structure shown above was synthesised using a manual
nitrogen bubbler apparatus starting with Fmoc-protected Rink Amide
MBHA resin on a 0.125 mmol scale. 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 dissolved in DMF was introduced on the
solid-phase using DBU as base. 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 hours. An aliquot of 10 mg of the crude material was
purified by preparative liquid chromatography using a gradient of
70 to 100% B over 60 minutes (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) at a flow rate of 10 ml/min. After lyophilisation
a yield of 2 mg of pure material was obtained (analytical HPLC,
gradient 70-100% B over 20 minutes, A=0.1% TFA/water and B=0.1%
TFA/acetonitrile, flow rate 1 ml/min., detection UV 214 nm,
retention time 26 minutes). Further characterisation was carried
out using MALDI mass spectrometry, giving M+H at 1265 as
expected.
[0315] 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
(SEQ ID NO:14) 5
[0316] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Rink amide resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic 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% phenol, 5%
EDT and 5% H20 for 2 hours, giving a crude product yield of 160 mg.
Purification by preparative HPLC of a 35 mg aliquot of crude
material was carried out using a gradient of 70 to 100% B over 40
minutes (A=0.1% TFA/water and B=MeOH) at a flow rate of 9 ml/min.
After lyophilisation, 20 mg of pure material was obtained
(analytical HPLC, gradient 70-100% B where B=MeOH, A=0.01%
TFA/water: detection--UV 214 and 260 nm--product retention time=16
minutes). Further product characterisation was carried out using
MALDI mass spectrometry: expected M+H at 2050, found at 2055.
[0317] c) Preparation of Gas-Filled Microbubbles Comprising DSPS, a
Lipopeptide for Endothelial Cell Targeting and a
Captopril-Containing Molecule for Drug Delivery
[0318] DSPS (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. The mixture was
warmed to 80.degree. C. for 5 minutes (vial shaken during warming).
The sample was cooled to room temperature and the head space was
flushed with perfluorobutane gas. The vial was firstly shaken in a
cap-mixer for 45 seconds then rolled for 1 hour, whereafter the
contents were extensively washed with deionised water. No
detectable level of starting material was found in the final wash
solution as evidenced by MALDI MS. MALDI mass spectral analysis was
used to confirm incorporation of the products from (a) and (b) into
the microbubbles as described in Example 21(b).
[0319] d) In vitro Study of Gas-Filled Microbubbles Comprising
DSPS, a Lipopepitde for Endothelial Cell Targeting and a
Captopril-Containing Molecule for Therapeutic Applications
[0320] The in vitro assay decribed in Example 21(c) was used to
examine cell binding under flow conditions. A gradual accumulation
of microbubbles on the cells took place, depending on the flow
rate. On further increasing the flow rate cells started to become
detached from the coverslip, but the microbubbles remained bound to
the cells. Control microbubbles not carrying the vector did not
adhere to the endothelial cells and disappeared from the chamber
under minimal flow conditions.
EXAMPLE 32
Preparation of Gas-Filled Microbubbles Comprising DSPS Loaded with
a Lipopeptide Comprising a Helical Peptide With Affinity for Cell
Membranes and the Peptide Antibiotic Polymixin B Sulphate
[0321] This example is directed to the preparation of targeted
microbubbles comprising multiple peptidic vectors having a combined
targeting and therapeutic application.
[0322] 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 (SEQ ID
NO:12)
[0323] This is prepared as described in Example 30(a).
[0324] b) Preparation of Multiple-Specific Gas-Filled
Microbubbles
[0325] DSPS (5.0 mg), lipopeptide from (a)(0.3 mg) and polymixin B
sulphate (0.5 mg) were weighed into a clean vial and 1.0 ml of a
solution of 1.4% propylene glycol/2.4% glycerol was added. The
mixture was sonicated for 3-5 minutes, warmed to 80.degree. C. for
5 minutes and then filtered through a 4.5 micron filter. The
mixture was cooled to room temperature and the head space was
flushed with perfluorobutane gas. The vial was shaken in a
cap-mixer for 45 seconds and the resulting microbubbles were
centrifuged at 1000 rpm for 3 minutes. The microbubbles were washed
with 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 in the desired range
of 1-8 micron. To the washed bubbles (ca. 0.2 ml) was added
methanol (0.5 ml), and the mixture was placed in a sonicator bath
for 2 minutes. The resulting clear solution, on analysis by
MALDI-MS, was found to contain both lipopeptide and polymixin B
sulphate (expected 1203, found 1207).
EXAMPLE 33
Preparation of Gas-Filled Microbubbles Comprising DSPS `Doped` With
a Lipopeptide Comprising a IL-1 Receptor-Binding Sequence and
Modified With a Branched Structure Containing the Drug
Methotrexate
[0326] This example is directed to the preparation of targeted
microbubbles comprising multiple vectors for targeted/therapeutic
applications.
[0327] a) Synthesis of a Lipopeptide Comprising an Interleukin-1
Receptor-Binding Peptide:
Dipalmitoyl-Lys-Gly-Asp-Trp-Asp-Gln-Phe-Gly-Leu-
-Trp-Arg-Gly-Ala-Ala.OH (SEQ ID NO:15) 6
[0328] The lipopeptide was synthesised on a ABI 433A automatic
peptide synthesiser starting with Fmoc-Ala-Wang resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges.
[0329] All amino acids and palmitic acid were preactivated using
HBTU before coupling. The simultaneous removal of lipopeptide from
the resin and side-chain protecting groups was carried out in TFA
containing 5% H.sub.2O, 5% anisole, 5% phenol and 5% EDT for 2
hours, giving a crude product yield of 150 mg. Purification by
preparative HPLC of a 30 mg aliquot of crude material was carried
out using a gradient of 90 to 100% B over 40 minutes (A=0.1%
TFA/water and B=MeOH) at a flow rate of 9 mlmin. After
lyophilisation, 4 mg of pure material was obtained (analytical
HPLC, gradient 90-100% B over 20 minutes where B=MeOH, A=0.01%
TFA/water: Detection--UV 214 nm--product retention time=23
minutes). Further product characterisation was carried out using
MALDI mass spectrometry: expected M+H at 2083, found at 2088.
[0330] b) Synthesis of a Branched Methotrexate Core Structure
Containing a Thiol Moiety 7
[0331] The methotrexate structure was synthesised on an 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 of a 30
mg aliquot of crude material was carried out using a gradient of 10
to 30% B over 40 minutes (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) and a flow rate of 9 ml/min. After lyophilisation
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: detection--UV 214 nm--product retention time=9.5
minutes). Further product characterisation was carried out using
MALDI mass spectrometry: expected M+H at 1523, found at 1523.
[0332] c) Preparation of Multiple-Specific Gas-filled
Microbubbles
[0333] DSPS (4.5 mg), thiol-containing lipopeptide from Example
64(a) (0.5 mg) and lipopeptide from (a) (0.2 mg) were weighed into
a clean vial and 1.0 ml of a solution of 1.4% propylene glycol/2.4%
glycerol was added. The mixture was sonicated for 3-5 minsutes,
warmed to 80.degree. C. for 5 minutes and then filtered through a
4.5 micron filter. The mixture was cooled to room temperature and
the head space was flushed with perfluorobutane gas. The vial was
shaken in a cap mixer for 45 seconds and the resulting microbubbles
were centrifuged at 1000 rpm for 3 minutes, whereafter the
infranatant was discarded.
[0334] d) Conjugation of Methotrexate Branched Structure to
Thiolated Microbubbles
[0335] 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 microbubbles from (c) and disulphide bond
formation was allowed to proceed for 16 hours. Following extensive
washing with PBS and water the bubbles were analysed by microscopy
and MALDI MS.
[0336] The disulphide bond linking the methotrexate structure to
the microbubbles may be reduced in vivo to liberate the free drug
molecule, so that such microbubbles in combination with a tumour
specific vector comprise a drug delivery system. A physiologically
acceptable reducing agent such as glutathione may be used to bring
about drug release.
EXAMPLE 34
Preparation of Gas-Filled Microbubbles Coated With Poly-L-lysine
Complexed to Fluorescein-Labeled DNA Fragments from Plasmid
pBR322
[0337] This example is directed to the preparation of microbubbles
for gene therapy/anti-sense applications. Specific targeting may be
achieved by further doping of microbubble membranes with
vector-modified lipid structures as described in Example 21.
[0338] a) Preparation of DSPS-Encapsulated Gas-Filled
Microbubbles
[0339] DSPS (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 was sonicated for 2 minutes and 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 was flushed with perfluorobutane
gas. The vial was shaken in a cap mixer for 45 seconds. The
resulting microbubbles were then washed once with deionised water
and the infranatant was discarded. The microbubbles were then
resuspended in 0.5 ml water.
[0340] b) Preparation of Poly-L-lysine/DNA Complex and Loading of
DSPS-Encapsulated Microbubbles
[0341] 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
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 was
adjusted to 8. Complexing was allowed to proceed for 1 hour, after
which 0.05 mL of the polylysine-DNA solution was added to the
microbubble suspension from (a) above. After 1 hour microscopy was
used to show that the bubbles were fluorescent, confirming the
presence of DNA.
EXAMPLE 35
Preparation of Gas-Filled Microbubbles Containing a Branched Core
Peptide Comprising a Dabsylated-Atherosclerotic Plague-Binding
Sequence and RGDS
[0342] 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.
[0343] a) Synthesis of the Branched Peptide
Dabsyl-Tyr-Ara-Ala-Leu-Val-Asp- -Thr-leu-Lys-Lys
(NH.sub.2-Arg-Gly-Asp-Ser) -Gly-Cys.OH (SEQ ID NO:16) 8
[0344] The peptide was synthesised on an 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. 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. P urification by
preparative HPLC of a 30 mg aliquot of crude material was carried
out using a gradient of 10 to 60% B over 40 minutes (where A=0.1%
TFA/water and B=acetonitrile) at a flow rate of 9 ml/min. After
lyophilisation, 2.5 mg of pure material was obtained (analytical
HPLC, gradient 10-50% B over 20 minutes where B=0.1%
TFA/acetonitrile and A=0.01% TFA/water: detection--UV 214 and 435
nm--product retention time=21 minutes). Further product
characterisation was carried out using MALDI mass spectrometry:
expected M+H at 2070, found at 2073.
[0345] b) Preparation of Thiol-Containing Gas-Filled
Microbubbles
[0346] These were prepared as described in Example 64(a).
[0347] c) Oxidative Coupling of Thiolated Microbubbles With
Multiple-Specific Peptide via Disulphide Bond Formation
[0348] 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 dilute ammonia solution (pH 8). To this was added
0.2 ml of a stock solution containing 6 mg of potassium
ferricyanate dissolved in 2 ml of water. The vial was placed on a
roller table and thiol oxidation allowed to proceed for 2 hours.
The bubbles were then washed extensively with water until the
infranatant was free of the dabsyl-peptide as evidenced by HPLC and
MALDI MS. 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.
[0349] Other physiologically acceptable reducing agents such as
reduced glutathione may also be useful for initiating release.
EXAMPLE 36
Preparation of Gas-Filled Microbubbles Encapsulated With DSPS and
Biotin-PEG.sub.3400-acyl-phosphatidylethanolamine and
Functionalised With Streptavidin, Oligonucleotide
Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and Biotinylated
Fibrin-anti-polymerant Peptide (Biotin-GPRPPERHOS.NH.sub.2)(SEQ ID
NO:10)
[0350] a) Synthesis of Biotin-PEG.sub.3400-acyl-phosphatidyl
Ethanolamine
[0351] 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 was verified by NMR and
MALDI-MS.
[0352] b) Binding of Fluorescein-Conjugated Streptavidin to
Gas-Filled Microbubbles
[0353] Gas-filled microbubbles were prepared by mixing DSPS and
biotin-PEG.sub.3400-acyl-phosphatidylethanolamine as described in
previous examples. The microbubble suspension was divided into 0.2
ml aliquots and fluorescein-conjugated streptavidin was 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. The samples were analysed by flow
cytometry and Coulter Counter. The results are summarized in the
table below.
[0354] Results:
5 Added Particle Streptavidin Incubation % median Aliquot (mg/200:1
time (amb. Fluorescent 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 (1.5 .times. 10.sup.-7 ) mmol 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
[0355] c) Conjugation of Streptavin-Coated Microbubbles With the
Oligonucleotide Biotin-GAAAGGTAGTGGGGTCGTGTGCCGG (SEQ ID NO:17) and
Biotinylated Fibrin-anti-polymerant Peptide Biotin-GPRPPERHOS (SEQ
ID NO:10)
[0356] The particles from aliquot no. 6 above were centrifuged and
the supernatant was replaced with 1 ml PBS buffer, pH 7.5,
containing 0.2 mg of biotin-GAAAGGTAGTGGGGTCGTGTGCCGG (SEQ ID
NO:17) and 0.2 mg of biotin-GPRPPERHQS (SEQ ID NO:10) (prepared as
in Example 27(b) and (c)). After incubation for 24 hours the
particles were washed extensively with PBS and water.
[0357] Other biotinylated vectors or therapeutic agents may be
conjugated to streptavidin- or avidin-coated microbubbles using
this procedure.
EXAMPLE 37
Preparation of Gas-Filled Microbubbles Encapsulated With DSPS and
Functionalised With a Thrombi-Targeting Lipopeptide and the
Thrombolytic Enzyme Tissue Plasminogen Activator
[0358] This example is directed at the preparation of thrombus
targeted ultrasound contrast agents comprising a therapeutic
thromolytic agent.
[0359] a) Synthesis of a Lipopeptide With Affinity for Thrombi
(Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.-
NH.sub.2) (SEQ ID NO:18) 9
[0360] The lipopeptide was synthesised on an ABI 433 A automatic
peptide synthesiser starting with Rink amide resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic 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% phenol, 5%
EDT, 5% anisole and 5% H.sub.2O for 2 hours, giving a crude product
yield of 80 mg. Purification by preparative HPLC of a 20 mg aliquot
of the crude material was carried out. After lyophilisation, 6 mg
of pure material was obtained. The product was characterised by
MALDI mass spectrometry and analytical HPLC.
[0361] b) Modification of Tissue Plasminogen Activator With
Sulpho-SMPB
[0362] A solution of 0.1 ml of ammonim carbonate buffer containing
0.1 mg of t-PA was made up to 0.2 ml by the addition of water. To
this solution was added 0.4 mg of Sulpho-SMPB (dissolved in 0.05 ml
DMSO. The protein solution was left standing at room temperature
for 45 minutes, whereafter purification was carried out on a
Superdex 200 column. The product was eluted in PBS and the modified
protein fraction was collected.
[0363] c) Preparation of Gas-Filled Microbubbles Encapsulated With
DSPS/Thrombi-Binding Lipopeptide and Thiol-Containing Lipoeptide
and Conjugation of Modified Tissue Plasminogen Activator
[0364] DSPS (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 64(a). To this was added 1.0 ml of a
solution of 1.4% propylene glycol/2.4% glycerol and the mixture was
sonicated for 2 minutes and 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 seconds and the resulting
microbubbles were washed twice 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 hour. The microbubbles were centrifuged
and the infranatant was 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 21(c).
Microbubbles modified with protein were found to bind in higher
numbers than those comprising either lipopeptide/DSPS or DSPS
alone.
[0365] The targeting/therapeutic/ultrasound activities of these
microbubbles be evaluated in models of both in vitro and in vivo
thrombogenisis.
EXAMPLE 38
Preparation of Gas-Filled Microbubbles Comprising DSPS Loaded With
a Lipopeptide Comprising a Helical Peptide With Affinity for Cell
Membranes
[0366] This example is directed to the preparation of targeted
microbubbles comprising a peptidic vector for targeting of cell
membrane structures.
[0367] a) Synthesis of a Lipopeptide Comprising a Helical Peptide
With Affinity for Cell Membranes (SEQ ID NO:12) 10
[0368] The lipopeptide was synthesised on an ABI 433A automatic
peptide synthesiser starting with Rink amide resin on a 0.2 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
2-n-hexadecylstearic acid were preactivated using HBTU before
coupling. The simultaneous removal of lipopeptide from the resin
and side-chain protecting groups was carried out in TFA containing
5% H.sub.2Ofor 2 hours, giving a crude product yield of 520 mg.
Purification by preparative HPLC of a 30 mg aliqout of crude
material was carried out using a gradient of 90 to 100% B over 40
minutes (A=0.1% TFA/water and B=MeOH) at a flow rate of 9 ml/min.
After lyophilisation, 10 mg of pure material was obtained
(analytical HPLC, gradient 90-100% B over 20 minutes where B=MeOH,
A=0.01% TFA/water: detection--UV 214 nm--product retention time=23
minutes). Further product characterisation was carried out using
MALDI mass spectrometry: expected M+H at 2369, found at 2375.
[0369] b) Preparation of Gas-Filled Microbubbles
[0370] DSPS (4.5 mg) and lipopeptide from (a)(0.5 mg) were weighed
into a clean vial and 1.0 ml of a solution of 1.4% propylene
glycol/2.4% glycerol was added. The mixture was sonicated for 3-5
minutes, warmed to 80.degree. C. for 5 minutes and then filtered
through a 4.5 mm filter. The mixture was cooled to room temperature
and the head space was flushed with perfluorobutane gas. The vial
was shaken in a cap mixer for 45 seconds and the resulting
microbubbles were centrifuged at 1000 rpm for 3 minutes. The
microbubbles were then washed with water until no lipopeptide could
be detected by MALDI-MS. Coulter counter, acoustic attenuation and
pressure stability studies were performed. To an aliquot of the
washed bubbles (ca. 0.2 ml) was added methanol (0.5 ml), and the
mixture was placed in a sonicator bath for 2 minutes. The resulting
clear solution, on analysis by MALDI-MS, was found to contain the
lipopeptide.
[0371] c) In vitro and in vivo Tests
[0372] The microbubbles had similar characteristics in vitro and in
vivo as was found for the microbubbles made in Example 21.
EXAMPLE 39
Gas-Filled Microbubbles Encapsulated With Phosphatidylserine and a
Biotinylated Lipopeptide
[0373] a) Synthesis of Lipopeptide
Dipalmitoyl-lysinyl-tryptophanyl-lysiny-
l-lysinyl-lysinyl(biotinyl)-glycine (SEQ ID NO:19)
[0374] The lipopeptide was synthesised on an ABI 433A automatic
peptide synthesiser starting with Fmoc-Gly-Wang resin on a 0.1 mmol
scale using immol amino acid cartridges. All amino acids and
palmitic 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% 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 of a 40 mg
aliqout of crude material was carred out using a gradient of 70 to
100% B over 40 minutes (A=0.1% TFA/water and B=MeOH) at a flow rate
of 9 ml/min. After lyophilisation. 14 mg of pure material
(analytical HPLC, gradient 70-100% B where B=MeOH, A=0.01%
TFA/water: detection--UV 260 and fluorescence, Ex280,
Em350--product retention time=22 minutes). Further product
characterisation was carried out using MALDI mass spectrometry:
expected M+H at 1478, found at 1471.
[0375] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
`Doped` With the Biotinylated Lipopeptide Sequence from (a)
[0376] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg, 0.2 mmol)
were weighed into each of two vials, and 0.8 ml of a solution of
1.4% propylene glycol/2.4% glycerol was added to each vial. The
mixtures were warmed to 80.degree. C. for 5 minutes (vials shaken
during warming). The samples were cooled to room temperature and
the head spaces were flushed with perfluorobutane gas. The vials
were shaken in a cap mixer for 45 seconds and then rolled
overnight. The resulting microbubbles were washed several times
with deionised water and analysed by Coulter counter and acoustic
attenuation. MALDI mass spectral analysis was used to confirm
incorporation of lipopeptide into DSPS microbubbles as follows: ca.
50-100 ml of microbubbles were transferred to a clean vial and
50-100 ml water were added. The mixture was sonicated for 30
seconds and spotted onto a clean target disc (1 ml+0.5 ml ACH
matrix). Positive mode gave M+H at 1474, expected for lipopeptide
at 1478.
EXAMPLE 40
Preparation of Multiple-Specific Gas-Filled Microbubbles Comprising
DSPS Loaded With a Lipopeptide Comprising a Non-Bioactive
Interleukin-1 Receptor-Binding Peptide
[0377] This example is directed to the preparation of targeted
microbubbles comprising a non-bioactive peptidic vector for
targeting at the IL-1 recptor which does not induce signal
tranduction or prevent IL-1 binding.
[0378] a) Synthesis of a Lipopeptide Comprising a non-Bioactive
Interleukin-1 Receptor-Binding Peptide (SEQ ID NO:15) 11
[0379] The lipopeptide was synthesised on an ABI 433A automatic
peptide synthesiser starting with Fmoc-Ala-Wang resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic acid were preactivated using HBTU before coupling. The
simultaneous removal of lipopeptide from the resin and side-chain
protecting groups was carried out in TFA containing 5% H.sub.2O, 5%
anisole, 5% phenol and 5% EDT for 2 hours, giving a crude product
yield of 150 mg. Purification by preparative HPLC of a 30 mg
aliquot of crude material was carried out using a gradient of 90 to
100% B over 40 minutes (A=0.1% TFA/water and B=MeOH) at a flow rate
of 9 ml/min. After lyophilisation, 4 mg of pure material was
obtained (analytical HPLC, gradient 90-100% B over 20 minutes where
B=MeOH, A=0.01% TFA/water: Detection--UV 214 nm--product retention
time=23 minutes). Further product characterisation was carried out
using MALDI mass spectrometry: expected M+H at 2083, found at
2088.
[0380] b) Preparation of Gas-Filled Microbubbles
[0381] DSPS (4.5 mg) and lipopeptide from (a) (0.5 mg) were weighed
into a clean vial and 1.0 ml of a solution of 1.4% propylene
glycol/2.4% glycerol was added. The mixture was sonicated for 3-5
mins, warmed to 80.degree. C. for 5 minutes and then filtered
through a 4.5 micron filter. The mixture was cooled to room
temperature and the head space was flushed with perfluorobutane
gas. The vials were shaken in a cap mixer for 45 seconds and the
resulting microbubbles were centrifuged at 1000 rpm for 3 minutes.
The microbubbles were then washed with water until no lipopeptide
could be detected by MALDI-MS. To the washed microbubbles (ca. 0.2
ml) was added methanol (0.5 ml), and the mixture was placed in a
sonicator bath for 2 minutes. The resulting clear solution, on
analysis by MALDI-MS, was found to contain lipopeptide (expected
2083, found 2088).
EXAMPLE 41
Preparation of Perfluoropropane-Filled Microbubbles Comprising
DSPC, DSPS and Endothelial Cell-Binding Lipopeptide for Targeted
Ultrasound Imaging
[0382] To 0.8 ml of a solution containing DSPC:DSPS (3:1) (5mg/ml)
in propylene glycol/glycerol (4% in water) was added 0.5 mg of the
lipopeptide from Example 31(b). The mixture was heated to
80.degree. C. for 5 minutes and shaken. The solution was then
cooled to ambient temperature and the headspace was flushed with
perfluoropropane. The vial was shaken on a cap-mixer for 45 seconds
and placed on a roller table for 5 minutes. The sample was
centrifuged at 2000 rpm for 5 minutes and the infranatant was
removed and replaced with distilled water. The headspace was again
flushed with perfluoropropane and the sample was kept on a roller
table until a homogeneous appearance was obtained. The washing
procedure was repeated. The resulting ultrasound contrast agent was
characterised by Coulter counter analysis, acoustic attenuation
measurements and resistance to external pressure. The microbubbles
were tested in the in vitro assay as detailed in Example 21. A
gradual accumulation of microbubbles binding to the cells was
observed.
EXAMPLE 42
Preparation of Sulphur Hexafluoride-Containing Microbubbles
Comprising DSPC, DSPS and Endothelial Cell-Binding Lipopeptide for
Targeted Ultrasound Imaging
[0383] To 0.8 ml of a solution containing DSPC:DSPS (3:1) (5mg/ml)
in propylene glycol/glycerol (4% in water) was added 0.5 mg of the
lipopeptide from Example 31(b). The mixture was heated to
80.degree. C. for 5 minutes and shaken. The solution was then
cooled to ambient temperature and the headspace was flushed with
sulphur hexafluoride gas. The vial was shaken on a cap-mixer for 45
seconds and placed on a roller table for 5 minutes. The sample was
centrifuged at 2000 rpm for 5 minutes and the infranatant was
removed and replaced with distilled water. The headspace was again
flushed with sulphur hexafluoride and the sample was kept on a
roller table until a homogenous appearance was obtained. The
washing procedure was repeated.
[0384] The resulting ultrasound contrast agent was confirmed by
Coulter counter, acoustic attenuation measurements and resistance
to external pressure.
EXAMPLE 43
Preparation of Gas-Filled Microbubbles Comprising DSPG and
Endothelial Cell-Binding Lipopeptide for Targeted Ultrasound
Imaging
[0385] To 0.8 ml of a solution containing DSPG (Smg/ml) in
propylene glycol/glycerol (4% in water) was added 0.5 mg of the
lipopeptide from Example 31(b). The mixture was heated to
80.degree. C. for 5 minutes and shaken. The solution was then
cooled to ambient temperature and the headspace was flushed with
perfluorobutane. The vial was shaken on a cap-mixer for 45 seconds
and placed on a roller table for 5 minutes. The sample was
centrifuged at 2000 rpm for 5 minutes and the infranatant was
removed and replaced with distilled water. The headspace was again
flushed with perfluorobutane and the sample was kept on a roller
table until a homogenous appearance was obtained. The washing
procedure was repeated. The resulting ultrasound contrast agent was
characterised by Coulter counter analysis, acoustic attenuation
measurements and resistance to external pressure. The microbubbles
were tested in the in vitro assay as detailed in Example 21: a
gradual accumulation of microbubbles binding to the cells was
observed.
EXAMPLE 44
Preparation of Perfluoropropane-Filled Microbubbles Comprising DSPG
and Endothelial Cell Binding Lipopeptide for Targeted Ultrasound
Imaging
[0386] To 0.8 ml of a solution containing DSPG (5 mg/ml) in
propylene glycol/glycerol (4% in water) was added 0.5 mg of the
lipopeptide from Example 31(b). The mixture was heated to
80.degree. C. for 5 minutes and then shaken. The solution was then
cooled to ambient temperature and the headspace was flushed with
perfluoropropane. The vial was shaken on a cap-mixer for 45 seconds
and placed on a roller table for 5 minutes. The sample was
centrifuged at 2000 rpm for 5 minutes and the infranatant was
removed and replaced with distilled water. The headspace was again
flushed with perfluorobutane and the sample was kept on a roller
table until a homogeneous appearance was obtained. The washing
procedure was repeated. The resulting ultrasound contrast agent was
characterised by Coulter counter analysis, acoustic attenuation
measurements and resistance to external pressure. The microbubbles
were tested in the in vitro assay as detailed in Example 21: a
gradual accumulation of microbubbles binding to the cells was
observed.
EXAMPLE 45
Preparation of Sulphur Hexafluoride-Containing Microbubbles
Comprising DSPG and Endothelial Cell-Binding Lipopeptide for
Targeted Ultrasound Imaging
[0387] To 0.8 ml of a solution containing DSPG (5mg/ml) in
propylene glycol/glycerol (4% in water) was added 0.5 mg of the
lipopeptide from Example 31(b). The mixture was heated to
80.degree. C. for 5 minutes and shaken. The solution was then
cooled to ambient temperature and the headspace was flushed with
sulphur hexafluoride gas. The vial was shaken on a cap-mixer for 45
seconds and placed on a roller table for 5 minutes. The sample was
centrifuged at 2000 rpm for 5 minutes and the infranatant was
removed and replaced with distilled water. The headspace was again
flushed with sulphur hexafluoride and the sample was kept on a
roller table until a homogeneous appearance was obtained. The
washing procedure was repeated. The resulting ultrasound contrast
agent was characterised by Coulter counter analysis, acoustic
attenuation measurements and resistance to external pressure.
EXAMPLE 46
Targeted Gas-Filled Microbubbles Comprising DSPS Coated
Non-Covalently With Polylysine
[0388] DSPS (5 mg) was weighed into a clean vial along with
poly-L-lysine (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 vial was shaken in a cap mixer for 45 seconds and the resulting
microbubbles were centrifuged at 1000 rpm for 3 minutes. Following
extensive washing with water, PBS and water, the final solution was
examined for polylysine content using MALDI MS. No polypeptide
material was observed in the final wash solution. Acetonitrile (0.5
ml) was then added and the microbubbles were sonicated until all
bubbles had burst. Analysis of the resulting solution for
polylysine was again carried out using MALDI MS. The results were
as follows:
6 MALDI expected MALDI found Poly-L-lysine 786, 914, 1042, 1170
790, 919, 1048, 1177
EXAMPLE 47
Preparation of Functionalised Gas-Filled Microbubbles for Targeted
Ultrasound Imaging (SEQ ID NO:20)
[0389] 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.
[0390] a) Synthesis of a Thiol-Functionalised Lipid Molecule (SEQ
ID NO:20) 12
[0391] The lipid structure shown above was synthesised on an ABI
433A automatic peptide synthesiser starting with Fmoc-Cys(Trt)-Wang
resin on a 0.25 mmol scale using 1 mmol amino acid cartridges. All
amino acids and palmitic acid were preactivated using HBTU coupling
chemistry. 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 of a 40
mg aliquot of crude material was carried out using a gradient of 90
to 100% B over 50 minutes (A=0.1% TFA/water and B=MeOH) at a flow
rate of 9 ml/min. After lyophilisation, 24 mg of pure material was
obtained (analytical HPLC, gradient 70-100% B where B=0.1%
TFA/acetonitrile, A=0.01% TFA/water: detection--UV 214 nm--product
retention time=23 minutes). Further product characterisation was
carried out using MALDI mass spectrometry: expected M+H at 1096,
found at 1099.
[0392] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
`Doped` With a Thiol-Containing Lipid Structure
[0393] DSPS (4.5 mg) and the lipid structure from (a) above (0.5
mg, 0.4 mmol) were weighed into a clean vial and 0.8 ml of a
solution containing 1.4% propylene glycol/2.4% glycerol in water
was added. The mixture was warmed to 80.degree. C. for 5 minutes
(vial shaken during warming) and filtered while still hot through a
40 mm filter. The sample was cooled to room temperature and the
head space was flushed with perfluorobutane gas. The vial was
shaken in a cap mixer for 45 seconds and then placed on roller
table overnight. The resulting microbubbles were washed several
times with deionised water and analysed for thiol group
incorporation using Ellmans Reagent.
EXAMPLE 48
Preparation of Gas-Filled Microbubbles Comprising DSPS Doped With a
Thrombus-Binding Lipopeptide
[0394] a) Synthesis of a Lipopeptide With Affinity for Thrombi
(Dipalmitoyl-Lys-Asn-Asp-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln.-
NH.sub.2) (SEQ ID NO:18) 13
[0395] The lipopeptide was synthesised on an ABI 433 A automatic
peptide synthesiser starting with Rink amide resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic 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% phenol, 5%
EDT, 5% anisole and 5% H.sub.2O for 2 hours, giving a crude product
yield of 80 mg. Purification by preparative HPLC of a 20 mg aliquot
of the crude material was carried out. After lyophilisation, 6 mg
of pure material were obtained. The product was characterised by
MALDI mass spectrometry and analytical HPLC.
[0396] b) Preparation of Thromi-Targeting Ultrasound
Microbubbles
[0397] DSPS (4.5 mg) and lipopeptide from (a) (1.0 mg) were weighed
into a vial and 0.8 ml of a solution of 1.4% propylene glycol/2.4%
glycerol was added. The mixture was warmed to 80.degree. C. for 5
minutes and then filtered through a 4 micron filter. After cooling
to room temperature the head space was flushed with perfluorobutane
gas. The vial was shaken in a cap mixer for 45 seconds and the
resulting microbubbles were washed extensively with deionised
water. The microbubbles were characterised by microscopy and
Coulter counter analysis. MALDI-MS was used to confirm the presence
of lipopeptide as described in previous examples.
EXAMPLE 49
Preparation of Transferrin-Coated Gas-Filled Microbubbles for
Targeted Ultrasound Imaging
[0398] a) Synthesis of a Thiol-Functionalised Lipid Molecule (SEQ
ID NO:20) 14
[0399] The lipid structure shown above was synthesised on an ABI
433A automatic peptide synthesiser starting with Fmoc-Cys(Trt)-Wang
resin on a 0.25 mmol scale using 1 mmol amino acid cartridges. All
amino acids and palmitic acid were preactivated using HBTU before
coupling. 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 of a 40
mg aliquot of crude material was carried out using a gradient of 90
to 100% B over 50 minutes (A=0.1% TFA/water and B=MeOH) at a flow
rate of 9 ml/min. After lyophilisation, 24 mg of pure material was
obtained (analytical HPLC, gradient 70-100% B where B=0.1%
TFA/acetonitrile, A=0.01% TFA/water: detection--UV 214 nm--product
retention time=23 minutes). Further product characterisation was
carried out using MALDI mass spectrometry: expected M+H at 1096,
found at 1099.
[0400] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
`Doped` With a Thiol-Containing Lipid Structure
[0401] DSPS (4.5 mg) and lipid structure from (a) above (0.5 mg,
0.4 mmol) were weighed into a clean vial and 0.8 ml of a solution
of 1.4% propylene glycol/2.4% glycerol was added. The mixture was
warmed to 80.degree. C. for 5 minutes (vial shaken during warming)
and filtered while still hot through a 40 mm filter. The sample was
cooled to room temperature and the head space was flushed with
perfluorobutane gas. The vial was shaken in a cap mixer for 45
seconds and then placed on roller table overnight. The resulting
microbubbles were washed several times with deionised water and
analysed for thiol group incorporation using Ellmans Reagent.
[0402] c) Modification of Transferrin With Fluorescein-NHS and
Sulpho-SMPB
[0403] To 4 mg of transferrin (Holo, human) in PBS (1 ml) was added
0.5 ml DMSO solution containing 1 mg Sulpho-SMPB and 0.5 mg
fluorescein-NHS. The mixture was stirred for 45 minutes at room
temperature and 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.
[0404] d) Microbubble Conjuration With Transferrin
[0405] To the thiol-containing microbubbles from (b) was added 1 ml
of the modified transferrin protein solution from (c). After
adjusting the pH of the solution to 9 the conjugation reaction was
allowed to proceed for 2 hours at room temperature. Following
extensive washing with deionised water the microbubbles were
analysed by Coulter counter (97% between 1 and 5 mm) and
fluorescence microscopy (highly fluorescent microbubbles).
EXAMPLE 50
Gas-Filled Microbubbles Comprising DSPS Incorporating
PE-PEG.sub.2000-Mal Conjugated to Thiolated Trypsin Fluorescein
[0406] a) Synthesis of Boc-NH-PEG.sub.2000-DSPE (t-butyl Carbamate
Poly(Ethylene Glycol)Distearoylphosphatidylethanolamine)
[0407] DSPE (31 mg) was added to a solution of
Boc-NH-PEG.sub.2000-SC (150 mg) in chloroform (2 ml), followed by
triethylamine (33 .mu.l). The mixture was stirred at 41.degree. C.
for 10 minutes until the starting material had dissolved. The
solvent was rotary evaporated and the residue was taken up in
acetonitrile (5 ml). The resulting dispersion was cooled to
4.degree. C. and centrifuged, whereafter the solution was filtered
and evaporated to dryness. The structure of the resulting product
was confirmed by NMR.
[0408] b) Synthesis of H.sub.2N-PEG.sub.2000-DSPE
(Amino-poly(Ethylene
Glycol)-distearoylphosphatidylethanolamine)
[0409] Boc-NH-PEG.sub.2000-DSPE (167 mg) was stirred in 4 M
hydrochloric acid in dioxane (5 ml) for 2.5 hours at ambient
temperature. The solvent was removed by rotary evaporation and the
residue was taken up in chloroform (1.5 ml) and washed with water
(2.times.1.5 ml). The organic phase was evaporated in vacuo. TLC
analysis (chloroform/methanol/water 13:5:0.8) gave a single
ninhydrin positive spot with Rf=0.6; confirmation of the structure
was obtained by NMR.
[0410] c) Synthesis of Mal-PEG.sub.2000-DSPE (3-maleimidopropionate
Poly(Ethylene Glycol)Distearoylphosphatidylethanolamine)
[0411] A solution of N-succinimidyl-3-maleimidopropionate (5.6 mg,
0.018 mmol) in tetrahydrofuran (0.2 ml) was added to
H.sub.2N-PEG-.sub.2000-DSP- E (65 mg, 0.012 mmol) dissolved in
tetrahydrofuran (1 ml) and 0.1 M sodium phosphate buffer pH 7.5 (2
ml). The mixture was warmed to 30.degree. C. and the reaction was
followed to completion by TLC, whereafter the solvent was removed
in vacuo. The title material was purified on a flash silica column
using 80:20 chloroform:methanol as eluent. The structure of the
pure product was confirmed by NMR and mass spectrometry.
[0412] d) Preparation of Gas-Filled Microbubbles of DSPS `Doped`
With PE-PEG.sub.2000 -Mal
[0413] DSPS (4.5 mg) and PE-PEG.sub.2000-Mal from (c) above (0.5
mg) were weighed into a clean vial and 1 ml of a solution of 1.4%
propylene glycol/2.4% glycerol was added. The mixture was warmed to
80.degree. C. for 5 minutes and then filtered through a 4.5 mm
filter. The sample was cooled to room temperature and the head
space was flushed with perfluorobutane gas. The vial was shaken in
a cap mixer for 45 seconds and the resulting microbubbles were
washed three times with distilled water.
[0414] e) Preparation of Fluorescein-Labelled Trypsin
[0415] To 5 mg of trypsin in PBS (1 ml) was added 0.2 ml DMSO
solution containing 1 mg of fluorescein-NHS. The mixture was
stirred for 45 minutes at room temperature. A Sephadex 200 column
was then charged with the modified protein mixture and product was
eluted at a flow rate of 1 ml/min using PBS. The protein fraction
(5 ml) was collected and stored at 4.degree. C.
[0416] f) Preparation of Thiolated, Fluorescein-Labelled
Trypsin
[0417] To the protein fraction from (e) was added 1 mg of Traut's
reagent and the mixture stirred at room temperature for a further 1
hour. 4 ml of the Traut's-modified product was then charged on a
Sephadex 200 column and the product was eluted with PBS. The
protein fraction containing maximum fluorescent intensity was
collected in a total volume of 6 ml.
[0418] g) Conjugation of Microbubbles With Thiolated,
Fluorescein-Labelled Trypsin
[0419] Microbubbles from (d) were incubated on a roller table in 1
ml of protein solution from (f) above. The conjugation was allowed
to proceed at pH 7.3-7.8 for 10 minutes before centrifugation and
removal of the infranatant. The process was repeated a further
three times, after which the bubbles were washed four times with
water to remove unconjugated protein.
[0420] D. Bubbles contained active enzyme as evidenced by the
cleavage of an Arg-pNA derivative in PBS.
[0421] E. Analysis of the bubbles by Coulter and measurement of
echogenicity was carried out.
[0422] Bubbles were Pressure Stable (see FIG. 2)
7 FEK-022-015 Total 0.83 concentration Diameter 1-3 mm 40 Diameter
3-5 mm 28 Diameter 5-7 mm 13 Freq of max Atten. 3.3 Atten at 2. Mhz
4.9 Atten at 3.5 Mhz 7.8 Atten at 5.0 MHz 7.2
EXAMPLE 51
Gas-Filled Microbubbles Comprising DSPS and a Captopril-Containing
Molecule for Diagnostic and Therapeutic Applications
[0423] a) Synthesis of a Lipopeptide Functionalised With Captopril
(SEQ ID NO:13) 15
[0424] The structure shown above was synthesised by the manual
`bubbler` method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale. Coupling was carried out using standard
TBTU/HOBt/DIEA protocol. Bromoacetic acid was coupled through the
side-chain of Lys as a symmetrical anhydride using DIC
preactivation. Captopril dissolved in DMF was introduced on the
solid phase using DBU as base. 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 hours. An aliquot of 10 mg of the crude material was
purified by preparative liquid chromatography using a gradient of
70 to 100% B over 60 minutes (A=0.1% TFA/water and B=0.1%
TFA/acetonitrile) at a flow rate of 10 ml/min. After
lyophilisation, a yield of 2 mg of pure material was obtained
(analytical HPLC, gradient 70-100% B over 20 minutes, A=0.1%
TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/min,
detection UV 214 nm, retention time 26 minutes). Further
characterisation was carried out using MALDI mass spectrometry,
giving M+H at 1265 as expected.
[0425] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Compound Containing Captopril
[0426] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
warmed to 80.degree. C. for 5 minutes (vial was shaken during
warming). The vial was then cooled and the head space was flushed
with perfluorobutane gas. The vial was shaken in a cap mixer for 45
seconds and the resulting microbubbles were extensively washed with
deionised water. MALDI mass spectrometry showed no detectable level
of compound from (a) in the final wash solution. Incorporation of
captopril-containing lipopeptide into the microbubbles 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 seconds and analysed by
MALDI mass spectrometry, giving a M+H peak corresponding to
lipopeptide from (a).
EXAMPLE 52
Gas-Filled Microbubbles Comprising DSPS and a Vector With Affinity
for Adrenergic Receptors for Diagnostic and Therapeutic
Applications
[0427] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0428] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0429] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0430] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0431] 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.
[0432] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0433] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino[propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4hours. 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.
[0434] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0435] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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.
[0436] b) Synthesis of a Lipopeptide Functionalised With Atenolol
(SEQ ID NO:21) 16
[0437] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using the compound from (a). Coupling was
carried out using standard TBTU/HOBt/DIEA protocols. 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 hours. Crude material was precipitated from
ether and purified by preparative liquid chromatography using a
gradient of 70 to 100% B over 60 minutes (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 minutes, A=0.1%
TFA/water and B=0.1% TFA/acetonitrile, flow rate 1 ml/minute,
detection UV 214 nm, retention time 25 minutes). Further
characterisation was carried out using MALDI mass spectrometry (ACH
matrix), giving M+H at 1258, expected 1257.
[0438] c) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing Atenolol
[0439] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (b) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes, heated at
80.degree. C. for 5 minutes (vial was shaken during warming) and
then cooled. The head space was flushed with perfluorobutane gas
and the vial was shaken in a cap mixer for 45 seconds, whereafter
the contents were extensively washed with deionised water. MALDI
mass spectrometry showed no detectable level of compound from (b)
in the final wash solution. Incorporation of atenolol-containing
lipopeptide into the microbubbles 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 seconds and analysed by MALDI-MS (ACH-matrix),
giving a M+H peak at 1259 corresponding to lipopeptide (b).
[0440] d) In vitro Analysis
[0441] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 53
Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide
Consisting of a Heparin Sulphate-Binding Peptide (KRKR) (SEQ ID
NO:5) and a Fibronectin Peptide (WOPPRARI) (SEQ ID NO:6) for
Targeting and a Lipopeptide Containing Atenolol for Therapeutic
Application
[0442] a) Synthesis of a Lipopeptide Consisting of a Heparin
Sulphate-Binding Peptide (KRKR) (SEQ ID NO:5) and a Fibronectin
Peptide (WOPPRARI) (SEQ ID NO:6) 17
[0443] The lipopeptide was synthesised on an ABI 433A automatic
peptide synthesiser starting with Fmoc-Ile-Wang resin on a 0.1 mmol
scale using 1 mmol amino acid cartridges. All amino acids and
palmitic 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% 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 of a 40 mg
aliquot of crude material was carried out using a gradient of 70 to
100% B over 40 minutes (A=0.1% TFA/water and B=MeOH) at a flow rate
of 9 ml/min. After lyophilisation, 16 mg of pure material was
obtained (analytical HPLC, gradient 70-100% B where B=MeOH, A=0.01%
TFA/water: detection--UV 260 and fluorescence, Ex280, Em350
--product retention time=19.44 minutes). Further product
characterisation was carried out using MALDI mass spectrometry:
expected M+H at 2198, found at 2199.
[0444] b) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0445] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0446] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0447] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0448] 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.
[0449] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0450] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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.
[0451] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0452] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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 13C NMR analysis.
[0453] c) Synthesis of a Lipopeptide Functionalised With Atenolol
(SEQ ID NO:21) 18
[0454] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids, palmitic acid
and the compound from (a). Coupling was carried out using standard
TBTU/HOBt/DIEA protocols. 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 hours.
Crude material was precipitated from ether and purified by
preparative liquid chromatography using a gradient of 70 to 100% B
over 60 minutes (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 minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow
rate 1 ml/minute, detection UV 214 nm, retention time 25 minutes).
Further characterisation was carried out using MALDI mass
spectrometry (ACH matrix), giving M+H at 1258, expected 1257.
[0455] d) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Consisting of a Heparin Sulphate-Binding Peptide
(KRKR) (SEQ ID NO:5), a Fibronectin Peptide (WOPPRARI) (SEQ ID
NO:6) and a Lipopeptide Containing Atenolol
[0456] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (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 minutes and then heated at 80.degree. C. for 5 minutes (vial
was shaken during warming). The solution was filtered and cooled.
The head space was flushed with perfluorobutane gas and the vial
was shaken in a cap mixer for 45 seconds, whereafter the contents
were extensively washed with deionised water. Incorporation of
atenolol-containing lipopeptide into the microbubbles 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 seconds and analysed by
MALDI-MS (ACH matrix), giving two M+H peaks at 2202 and 1259,
corresponding to lipopeptide (a) and to lipopeptide (c)
respectively.
[0457] e) In vitro Analysis
[0458] The microbubbles were tested in the in vitro assay as
detailed in example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 54
Gas-Filled Microbubbles Comprising DSPS and a Lipophilic Derivative
of Atenolol With Affinity for Adrenergic Receptors for Diagnostic
and Therapeutic Applications
[0459] a) Synthesis of
N-hexadecyl-4-[2-hydroxy-3-F(1-methyethyl)amino]pro-
poxy]phenylacetamide
[0460] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0461] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0462] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0463] 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.
[0464] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0465] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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.
[0466] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0467] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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.
[0468] v) Synthesis of N'-Boc,
N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)-
amino]propoxy]phenylacetamide
[0469] A solution of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]ph- enylacetic
acid (92 mg, 0.25 mmol) and hexadecylamine (60 mg, 0.25 mmol) in
DMF (5 ml) was cooled to 0.degree. C. HOBt (39 mg, 0.25 mmol) and
N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (water
soluble carbodiimide) (48 mg, 0.25 mmol) were added. The reaction
mixture was stirred at 0.degree. C. for 1 hour and then at room
temperature overnight. The reaction mixture was poured onto water
(25 ml) containing sodium carbonate (2.5 g) and sodium chloride
(4.0 g). Precipitated material was filtered off, washed with water
and taken up in chloroform. The chloroform phase was washed with 5%
sodium carbonate and water and dried (Na.sub.2SO.sub.4). The
solution was filtered and concentrated to give 150 mg of
yellow-white crude material. The product was purified by column
chromatography (silica, chloroform/methanol 95:5) to give 118 mg
(80%) of white material. The structure was verified by .sup.1H (500
MHz) and .sup.13C (125 MHz) NMR. The product was further
characterised by MALDI mass spectrometry, giving a M+Na peak at 614
as expected.
[0470] vi) Synthesis of
N-hexadecyl-4-[2-hydroxy-3-[(1-methyl-ethyl)amino]-
propoxy]phenylacetamide
[0471] To a solution of
N'-Boc-N-hexadecyl-4-[2-hydroxy-3-[(1-methyl-ethyl-
)amino]propoxy]phenylacetamide (10 mg) in dichloromethane (9 ml)
was added trifluoroacetic acid (1 ml). The reaction mixture was
stirred for 2 hours at room temperature. TLC (silica,
chloroform/methanol 95:5) showed complete conversion of starting
material. Solvents were evaporated off and the residue was taken up
in water/acetonitrile and lyophilised to give a quantitative yield
of white solid material. The structure was verified by .sup.1H (500
MHz) and .sup.13C (125 MHz) NMR analysis and further characterised
by MALDI mass spectrometry, giving M+H at 492 and M+Na at 514 as
expected.
[0472] b) Preparation of Gas-filled Microbubbles Comprising DSPS
and
N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide
for Diagnostic and Therapeutic Applications
[0473] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and
N-hexadecyl-4-[2-hydroxy-3-[(1-me-
thylethyl)amino]propoxy]phenylacetamide (0.5 mg) in a vial. The
mixture was sonicated for 5 minutes and then heated at 80.degree.
C. for 5 minutes (vial was shaken during warming). The solution was
filtered and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed with
deionised water. Incorporation of compound from (a) into the
microbubbles 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 seconds and
analysed by MALDI-MS, giving a M+H peak at 492 corresponding to
N-hexadecyl-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenylacetamide.
EXAMPLE 55
Gas-Filled Microbubbles Encapsulated With DSPS and a Compound
Containing Folic Acid for Diagnostic Applications
[0474] a) Synthesis of a Lipopeptide Containing Folic Acid 19
[0475] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids, palmitic acid
and folic acid. Coupling was carried out using standard
TBTU/HOBt/DIEA protocols. 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 hours.
Crude material was precipitated from ether and analysed by MALDI
mass spectrometry, giving a M+H peak corresponding to the structure
at 1435, expected 1430. The material was further characterised by
analytical HPLC, gradient 70-100% B over 20 minutes, A=0.1%
TFA/water and B=0.1 TFA/acetonitrile, flow rate 1.0 ml/minute,
giving a product peak with retention time 27 minutes detected at UV
368 nm.
[0476] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing Folic Acid
[0477] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5
mg) in a vial. Dilute ammonia (to pH 8) and DMSO (40 .mu.l) were
added and the mixture was sonicated for 5 minutes and then heated
at 80.degree. C. for 5 minutes (vial was shaken during warming).
The solution was filtered and cooled. The head space was flushed
with perfluorobutane gas and the vial was shaken in a cap mixer for
45 seconds, whereafter the contents were extensively washed with
deionised water. Incorporation of structure from (a) 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 seconds and
analysed by MALDI-MS (ACH matrix), giving a M+H peak at 1238
corresponding to structure from (a).
[0478] c) In vitro Analysis
[0479] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 56
Gas-Filled Microbubbles Comprising DSPS and a Cholesterol Ester of
Chlorambucil for Diagnostic and Therapeutic Applications
[0480] a) Synthesis of Cholesterol
4-F4-Fbis(2-chloroethyl)aminolphenyllbu- tanoate DIC (170 .mu.l,
1.10 mmol) was added to a solution of chlorambucil (669 mg, 2.20
mmol) in dry dichloromethane (15 ml). The mixture was stirred at
room temperature for 0.5 hour and added to a solution of
cholesterol (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%) 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.
[0481] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Cholesterol Ester of Chlorambucil for Diagnostic and/or
Therapeutic Applications
[0482] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed 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 seconds and analysed by MALDI-MS,
giving a M+H peak at 668 corresponding to structure from
EXAMPLE 57
Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide
Containing Atenolol and a Cholesterol Derivative of Chlorambucil
for Diagnostic and Therapeutic Applications
[0483] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0484] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0485] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0486] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0487] 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.
[0488] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0489] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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
structure and MALDI mass spectrometry gave a M+H at 268 as
expected.
[0490] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0491] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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.
[0492] The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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 13C NMR analysis.
[0493] b) Synthesis of a Lipopeptide Functionalised With Atenolol
(SEQ ID NO:21) 20
[0494] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids, palmitic acid
and the compound from (a). Coupling was carried out using standard
TBTU/HOBt/DIEA protocols. 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 hours.
Crude material was precipitated from ether and purified by
preparative liquid chromatography using a gradient of 70 to 100% B
over 60 minutes (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 minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow
rate 1 ml/minute, detection UV 214 nm, retention time 25 minutes).
Further characterisation was carried out using MALDI mass
spectrometry (ACH matrix), giving M+H at 1258, expected 1257.
[0495] c) Synthesis of Cholesterol
4-[4-[bis(2-chloroethyl)amino]phenyl]bu- tanoate
[0496] DIC (170 .mu.l, 1.10 mmol) was added to a solution of
chlorambucil (669 mg, 2.20 mmol) in dry dichloromethane (15 ml).
The mixture was stirred at room temperature for 0.5 hour and added
to a solution of cholesterol (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%) 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.
[0497] d) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing Atenolol and a Cholesteryl Ester of
Chloambucil
[0498] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (5.0 mg), product from (b) (0.5 mg)
and product from (c) (0.5 mg) in a vial. The mixture was sonicated
for 5 minutes and then warmed to 80.degree. C. for 5 minutes (vial
was shaken during warming). The solution was filtered and cooled.
The head space was flushed with perfluorobutane gas and the vial
was shaken in a cap mixer for 45 seconds, whereafter the contents
were extensively washed with deionised water. Incorporation of
compounds (b) and (c) into the microbubbles 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 seconds and analysed by MALDI-MS
(ACH-matrix), giving a M+H peak corresponding to lipopeptide (b)
and cholesteryl ester (c). e) In vitro analysis The microbubbles
were tested in the in vitro assay as detailed in Example 21. A
gradual accumulation of microbubbles binding to the cells was
observed.
EXAMPLE 58
Gas-Fiiled Microbubbles Comprising DSPS and a Lipopeptide
Containing Atenolol for Cell Targeting and a Lipophilic Thiol Ester
of Captopril for Therapeutic Use
[0499] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0500] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0501] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0502] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0503] 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.
[0504] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0505] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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.
[0506] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0507] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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.
[0508] b) Synthesis of a Lipopeptide Functionalised With Atenolol
(SEQ ID NO:21) 21
[0509] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids, palmitic acid
and the compound from (a). Coupling was carried out using standard
TBTU/HOBt/DIEA protocols. 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 hours.
Crude material was precipitated from ether and purified by
preparative liquid chromatography using a gradient of 70 to 100% B
over 60 minutes (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 minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow
rate 1 ml/minute, detection UV 214 nm, retention time 25 minutes).
Further characterisation was carried out using MALDI mass
spectrometry (ACH matrix), giving M+H at 1258, expected 1257.
[0510] c) Synthesis of Cholanic Acid Thiol Ester of Captopril
[0511] A mixture of 5-.beta.-cholanic acid (361 mg, 1.00 mmol) and
DIC (77 .mu.l, 0.50 mmol) in dichloromethane (5 ml) was stirred for
10 minutes and then added to a solution of captopril (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.
[0512] d) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing Atenolol for Cell Targeting and a
Lipophilic Thiol Ester of Captopril for Therapeutic Use
[0513] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (5.0 mg) and products from (b) (0.5
mg) and (c) (0.5 mg) in a vial. The mixture was sonicated for 5
minutes and then heated at 80.degree. C. for 5 minutes (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
seconds 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 compounds from
(b) and (c) into the microbubbles 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 seconds and analysed by MALDI-MS (ACH-matrix),
giving peaks according to structures from (b) and (c)
respectively.
[0514] e) In vitro Analysis
[0515] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 59
Gas-Filled Microbubbles Comprising Phosphatidylserine and
Biotinamide-PEG-.beta.-Ala-Cholesterol and a Cholesterol Ester of
Chlorambucil for Diagnostic and Therapeutic Applications
[0516] a) Synthesis of Cholesterol N-Boc-.beta.-alaninate
[0517] 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 minutes 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 hours and then
poured onto an aqeous solution of potassium hydrogen sulphate.
After phase separation the aqueous phase was extracted with
chloroform. The combined organic phases were washed with aqueous
potassium hydrogen sulphate and water and dried (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).
[0518] b) Synthesis of Cholesteryl .beta.-Alaninate
Hydrochloride
[0519] A solution of compound from (a) (279 mg, 0.500 mmol) in 1M
hydrochloric acid in 1,4-dioxane (5 ml) was stirred at room
temperature for 4 hours. The reaction mixture was concentrated to
give a quantitative yield of cholesteryl .beta.-alaninate
hydrochloride. The structure was confirmed by .sup.1H NMR (500 MHz)
analysis and by MALDI mass spectrometry, giving a M+Na peak at 482,
expected 481.
[0520] c) Biotin-PEG.sub.3400-.beta.B-Ala-Cholesterol
[0521] 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-PEG.sub.3400-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 evaporated to dryness and the residue was purified
by flash chromatography to give white crystals, yield 102 mg (89%).
The structure was verified by MALDI-MS and by NMR analysis.
[0522] d) Synthesis of cholesterol
4-[4-[bis(2-chloroethyl)amino]phenyl]bu- tanoate
[0523] DIC (170 .mu.l, 1.10 mmol) was added to a solution of
chlorambucil (669 mg, 2.20 mmol) in dry dichloromethane (15 ml).
The mixture was stirred at room temperature for 0.5 hour and added
to a solution of cholesterol (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.
[0524] e) Preparation of Gas-Filled Microbubbles
[0525] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (5 mg) and products from (c) (0.5
mg) and (d) (0.5 mg) in a vial. The mixture was sonicated for 5
minutes and then heated at 80.degree. C. for 5 minutes (vial was
shaken during warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed with
deionised water. MALDI mass spectrometry showed no detectable level
of compound from (c) or (d) in the final wash solution.
Incorporation of compounds from (c) and (d) into the microbubbles
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 seconds and analysed by
MALDI-MS (ACH-matrix), giving M+H peaks corresponding to compounds
from (c) and (d).
EXAMPLE 60
Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide
Containing a Derivative of Bestatin for Diagnostic and Therapeutic
Applications
[0526] a) Synthesis of a Lipopeptide Containing a Derivative of
Bestatin (SEQ ID NO:23) 22
[0527] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids and palmitic
acid. Coupling was carried out using standard TBTU/HOBt/DIEA
protocols. 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 hours. Crude material was
precipitated from ether and purified by preparative liquid
chromatography using a gradient of 70 to 100% B over 60 minutes
(A=0.1% TFA/water and B=0.1% TFA/acetonitrile) at a flow rate of 10
ml/min. After lyophilisation, a yield of 12 mg of pure material was
obtained (analytical HPLC, gradient 70-100% B over 20 minutes,
A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1
ml/minute, detection UV 214 nm, retention time 25 minutes). Further
characterisation was carried out using MALDI mass spectrometry (ACH
matrix), giving M+H at 1315, expected 1314.
[0528] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing a Derivative of Bestatin for
Diagnostic and Therapeutic Applications
[0529] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds and the contents were extensively washed with deionised
water. MALDI mass spectrometry showed no detectable level of
compound from (b) in the final wash solution. Incorporation of
atenolol-containing lipopeptide into the microbubbles 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 seconds and analysed by
MALDI-MS (ACH-matrix), giving a M+H peak at 1320, expected at 1314,
corresponding to lipopeptide from (a).
[0530] c) In vitro Analysis
[0531] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 61
Gas-Filled Microbubbles Comprising DSPS and a Lipopeptide
Containing Chlorambucil for Diagnostic and Therapeutic
Applications
[0532] a) Synthesis of a Lipopeptide Containing Chlorambucil (SEQ
ID NO:24) 23
[0533] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids and palmitic
acid. Coupling was carried out using standard TBTU/HOBt/DIEA
protocol. Chlorambucil was coupled through the side-chain of Lys as
a symmetrical anhydride using DIC preactivation. 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 hours. An aliqout
of 10 mg of the crude material was purified by preparative liquid
chromatography using a gradient of 70 to 100% B over 60 minutes
(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 minutes,
A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1
ml/minute, detection UV 214 nm retention time 26.5 minutes).
Further characterisation was carried out using MALDI mass
spectrometry, giving M+H at 1295, expected 1294.
[0534] b) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Lipopeptide Containing Chlorambucil for Diagnostic and
Therapeutic Applications
[0535] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (a) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed with
deionised water. MALDI mass spectrometry showed no detectable level
of compound from (a) in the final wash solution. 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 seconds 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.
[0536] c) In vitro Analysis
[0537] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 62
Gas-Filled Microbubbles Comprising DSPS, a Lipopeptide Containing
Atenolol and a Lipophilic Derivative of Captopril for diagnostic
and Therapeutic Applications
[0538] a) Synthesis of a Protected Atenolol Derivative Suitable for
Solid Phase Coupling
[0539] i) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0540] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0541] ii) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propox- y]phenylacetate
[0542] 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.
[0543] iii) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic Acid
Hydrochloride
[0544] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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.
[0545] iv) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
Acid
[0546] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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.
[0547] b) Synthesis of a Lipopeptide Functionalised With Atenolol
(SEQ ID NO:21) 24
[0548] The structure shown above was synthesised by the manual
bubbler method starting with Fmoc-protected Rink Amide MBHA resin
on a 0.125 mmol scale, using appropriate amino acids, palmitic acid
and the compound from (a). Coupling was carried out using standard
TBTU/HOBt/DIEA protocols. 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 hours.
Crude material was precipitated from ether and purified by
preparative liquid chromatography using a gradient of 70 to 100% B
over 60 minutes (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 minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow
rate 1 ml/minute, detection UV 214 nm, retention time 25 minutes).
Further characterisation was carried out using MALDI mass
spectrometry (ACH matrix), giving M+H at 1258, expected 1257.
[0549] c) Synthesis of
N-[(S)-3-hexadecylthio-2-methylpropionyl]proline
[0550] 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 hours and then
concentrated. The residue was poured onto water saturated with
potassium hydrogen sulphate and organic material was extracted into
chloroform. The organic phase was washed with water and dried
(MgSO.sub.4). The product was 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 .sup.1H (500
Mhz) and .sup.13C (125 Mhz) NMR analysis and further characterised
by MALDI mass spectrometry, giving M-H in negative mode at m/z 440
as expected.
[0551] d) Preparation of Gas-Filled Microbubbles Comprising DSPS, a
Lipopeptide Containing Atenolol and a Lipophilic Derivative of
Captopril for Diagnostic and Therapeutic Applications
[0552] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and products from (b) (0.5
mg) and (c) in a vial. The mixture was sonicated for 5 minutes and
then heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed with
deionised water. MALDI mass spectrometry showed no detectable level
of compound from (b) or (c) in the final wash solution.
Incorporation of compounds (b) and (c) into the microbubbles 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 seconds and analysed by
MALDI-MS (ACH-matrix), giving M+H peaks corresponding to structures
(b) and (c) respectively.
[0553] e) In vitro Analysis
[0554] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of microbubbles
binding to the cells was observed.
EXAMPLE 63
Gas-Filled Microbubbles Comprising DSPS and a Cholesterol
Derivative of Atenolol for Diagnostic and Therapeutic
Applications
[0555] a) Synthesis of Methyl
4-[(2,3-epoxy)propoxy]phenylacetate
[0556] A mixture of methyl 4-hydroxyphenylacetate (4.98 g, 0.030
mol), epichlorohydrin (23.5 ml, 0.30 mol) and pyridine (121 .mu.l,
1.5 mmol) was stirred at 85.degree. C. for 2 hours. 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.
[0557] b) Synthesis of Methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetate
[0558] A mixture of methyl 4-[(2,3-epoxy)propoxy]phenylacetate
(2.00 g, 9.00 mmol), isopropylamine (23 ml, 0.27 mo) 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.
[0559] c) Synthesis of
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phenyl- acetic Acid
Hydrochloride
[0560] A solution of methyl
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]p- henylacetate (563
mg, 2.00 mmol) in 6M hydrochloric acid (15 ml) was heated at
100.degree. C. for 4 hours. 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.
[0561] d) Synthesis of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]- phenylacetic
Acid
[0562] A solution of the
4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy]phen- ylacetic 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. The reaction mixture was poured onto water saturated with
potassium hydrogen sulphate 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.
[0563] e) Synthesis of Cholesteryl N-Boc-.beta.-alaninate
[0564] 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 minutes 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 hours and then
poured onto an aqeous solution of potassium hydrogen sulphate.
After phase separation the aqueous phase was extracted with
chloroform. The combined organic phases were washed with aqueous
potassium hydrogen sulphate and water and dried (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).
[0565] f) Synthesis of Cholesterol .beta.-Alaninate
Hydrochloride
[0566] A solution of compound from (a) (279 mg, 0.500 mmol) in 1M
hydrochloric acid in 1,4-dioxane (5 ml) was stirred at room
temperature for 4 hours. 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.
[0567] g) Synthesis of Cholesterol
N-Boc-4-[2-hydroxy-3-(1-methylethyl)ami-
no]propoxy]phenylacetyl-.beta.-alaninate.
[0568] To a solution of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy- ]phenylacetic
acid (55 mg, 0.15 mmol) and cholesteryl .beta.-alaninate
hydrochloride (74 mg, 0.15 mmol) in DMF (5 ml) was added DIEA (26
ml, 0.15 mmol). HOBt (23 mg, 0.15 mmol) and water-soluble
carbodiimide (WSC) (29 mg, 0.15 mmol) were added. The reaction
mixture was stirred at room temperature overnight and then poured
onto water (25 ml) containing sodium carbonate (2.5 g) and sodium
chloride (4.0 g). Precipitated material was extracted into
chloroform. The organic phase was washed with water and dried
(MgSO.sub.4). After filtration and concentration, crude material
(132 mg) was purified by column chromatography (silica,
chloroform/methanol/acetic acid, 95:4:1). Pooled fractions were
concentrated, taken up in glacial acetic acid and lyophilised.
Yield 83 mg (69%), yellow-white solid. Structure was confirmed by
.sup.1H NMR analysis.
[0569] h) Synthesis of Cholesterol
4-[2-hydroxy-3-[(1-methylethyl)amino]pr-
opoxy]phenylacetyl-.beta.-alaninate Trifluoroacetate
[0570] To a solution of
N-Boc-4-[2-hydroxy-3-[(1-methylethyl)amino]propoxy-
]phenylacetyl-.beta.-alaninate (40 mg, 0.05 mmol) in dry
dichloromethane (4 ml) was added trifluoroacetic acid (2 ml). The
reaction mixture was stirred for 2 hours and then concentrated. The
product was lyophilised from a acetonitrile/water mixture to give a
quantitative yield of white-yellow material. The product was
characterised by MALDI mass spectrometry giving M+H at 708 as
expected.
[0571] i) Preparation of Gas-Filled Microbubbles Comprising DSPS
and a Cholesterol Derivative of Atenolol for Diagnostic and
Therapeutic Applications
[0572] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (h) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds, whereafter the contents were extensively washed with
deionised water. MALDI mass spectrometry showed no detectable level
of compound from (b) in the final wash solution. Incorporation of
compound from (h) into the microbubbles was confirmed by MALDI mass
spectrometry.
[0573] j) In vitro Analysis
[0574] The microbubbles were tested in the in vitro assay as
detailed in Example 21. A gradual accumulation of bubbles binding
to the cells was observed.
EXAMPLE 64
Preparation of Multiple-Sepecific Transferrin/Avidin-Coated
Gas-Filled Microbubbles for Targeted Ultrasound Imaging
[0575] This example is directed to the preparation of microbubbles
containing vectors for targeted ultrasound/therapy.
[0576] a) Synthesis of a Thiol-Functionalised Lipid Molecule:
Dipalmitoyi-Lys-Lys-Lys-Aca-Cys.OH (SEQ ID NO:20) 25
[0577] The lipid structure shown above was synthesised on an ABI
433A automatic peptide synthesiser starting with Fmoc-Cys(Trt)-Wang
resin on a 0.25 mmol scale using 1 mmol amino acid cartridges. All
amino acids and palmitic acid were preactivated using HBTU coupling
chemistry. 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 of a 40
mg aliquot of crude material was carried out using a gradient of 90
to 100% B over 50 minutes (A=0.1% TFA/water and B MeOH) at a flow
rate of 9 ml/min. After lyophilisation, 24 mg of pure material was
obtained (analytical HPLC, gradient 70-100% B where B=0.1%
TFA/acetonitrile, A=0.01% TFA/water: detection--UV 214 nm--product
retention time=23 minutes). Further product characterisation was
carried out using MALDI mass spectrometry: expected M+H at 1096,
found at 1099.
[0578] b) Preparation of Gas-Containing Microbubbles Comprising
DSPS `Doped` With a Thiol-Containing Lipid Structure
[0579] DSPS (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 was added.
The mixture was warmed to 80.degree. C. for 5 minutes (vial shaken
during warming) and filtered while still hot through a 40 micron
filter. The sample was cooled to room temperature and the head
space was flushed with perfluorobutane gas. The vial was shaken in
a cap mixer for 45 seconds and then placed on aroller table
overnight. The resulting microbubbles were washed several times
with deionised water and analysed for thiol group incorporation
using Ellmans Reagent.
[0580] c) Modification of Transferrin and Avidin With
Fluorescein-NHS and Sulpho-SMPB
[0581] To a mixture of 2 mg of transferrin (Holo, human) and 2 mg
of avidin in PBS (1 ml) was added 0.5 ml of a DMSO solution
containing 1 mg Sulpho-SMPB and 0.5 mg fluorescein-NHS. 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.
[0582] d) Microbubble Conjugation With Modified
Transferrin/Aidin
[0583] To the thiol-containing microbubbles from (b) was added 1 ml
of the modified transferrin/avidin protein solution from (c). After
adjusting the pH of the solution to 9, the conjugation reaction was
allowed to proceed for 2 hours 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 65
Gene Transfer by Gas-Filled Microbubbles
[0584] This example is directed at the preparation of targeted
microbubbles for gene transfer.
[0585] a) Preparation of Gas-Filled Microbubbles Comprising DSPS
and Lipopeptide Coated With Poly-L-lysine
[0586] DSPS (4.5 mg) and lipopeptide from Example 41 (0.5 mg) were
weighed in two 2 ml vials. To each vial, 0.8 ml propylene
glycol/glycerol (4%) in water was added. Each solution was heated
at 80.degree. C. for 5 minutes, shaken and then cooled to ambient
temperature, whereafter the headspaces were flushed with
perfluorobutane. The vials were shaken on a cap-mixer at 4450
oscillations/minute for 45 seconds and put on a roller table for 5
minutes. The content of the vials were mixed and the resulting
sample was washed by centrifugation at 2000 rpm for 5 minutes. The
infranatant was removed and the same volume of distilled water was
added. The washing procedure was repeated once. Poly-L-lysine HBr
(20.6 mg) was dissolved in 2 ml water, then an aliquot (0.4 ml) was
made up to 2 ml with water. To 1.2 ml of the diluted poly-L-lysine
solution was added 0.12 ml of the DSPS-lipopeptide microbubble
suspension. Following incubation, excess polylysine was removed by
extensive washing with water.
[0587] b) Transfection of Cells
[0588] 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 minutes at room temperature then 1 ml of
complete RPMI medium was added. The medium was removed from the
cell culture dish and the DNA-microbubble mixture was added to the
cells. The cells were incubated in a cell culture incubator
(37.degree. C.).
[0589] c) Ultrasonic Treatment
[0590] After 15 minutes incubation, selected wells were exposed to
continious wave ultrasound of 1 MHz, 0.5 W/cm.sup.2, for 30
seconds.
[0591] d) Incubation and Examination
[0592] The cells were further incubated in the cell culture
incubator (37.degree. C.) for approximately 4.5 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 where DNA or
DNA-polylysine were added to the cells.
EXAMPLE 66
Flotation of Endothelial Cells by Microbubbles With Vectors That
Specifically Bind to the Endothelial Cells
[0593] This experiment was carried out to show that the present
invention can be used for separation of cells to which the
microbubbles are targeted. The human endothelial cell line ECV 304,
derived from a normal umbilical cord (ATCC CRL-1998) was cultured
in Nunc culture flasks (chutney 153732) in RPMI 1640 medium to
which L-glutamine (200 mM), penicillin/streptomycin (10,000 U/ml
and 10,00 .mu.g/ml) and 10% fetal calf serum were added. The cells
were subcultured following trypsination with a split ratio of 1:5
to 1:7 when reaching confluence. 2 million cells from trypsinated
confluent cultures were added to each set of five centrifuge tubes.
Then control microbubbles or microbubbles binding to endothelial
cells, made as described in Example 21 and in Example 38, were
added at 2, 4, 6 ,8 or 10 million bubbles per tube. The cells at
the bottom of the tubes after centrifugation at 400 g for 5 minutes
were counted with a Coulter counter. It was found the 4 or more
microbubbles binding to a cell brought the cells to the top of the
fluid in the centrifugation tube. All cells were floated by the
microbbbles from Example 38 whereas about 50% were floated with the
microbubbles from Example 21.
EXAMPLE 67
Gas-Filled Microbubbles of Distearoylphosphatidylserine Comprising
a Lipopeptide Containing a Vector With Affinity for Endothelin
Receptors for Targeted Ultrasound Imaging
[0594] a) Synthesis of
4'-[(3,4-dimethyl-5-isoxazolyl)sulfamoyl]succinanil- ic Acid
[0595] To a solution of sulfisoxazole (267 mg, 1.00 mmol) in DMF
(10 ml) was added succinic anhydride (1.00 g, 10.0 mmol) and
4-dimethylaminopyridine (122 mg, 1.00 mmol). The reaction mixture
was stirred at 80.degree. C. for 2 hours and then concentrated. The
residue was taken up in 5% aqueous sodium bicarbonate solution and
extracted with ethyl acetate. The aqueous solution was acidified
with dilute hydrochloric acid and organic material was extracted
into ethyl acetate. The organic phase was washed with dilute
hydrochloric acid, water and brine, treated with active charcoal
and dried (MgSO.sub.4). The solution was filtered and concentrated
to give 280 mg (76%) of white solid. The structure was verified by
.sup.1H (300 MHz) and .sup.13C (75 MHz) NMR spectroscopy. Further
characterisation was carried out using MALDI mass spectrometry (ACH
matrix), giving a M+Na peak at m/z 390 and a M+K peak at m/z 406 as
expected.
[0596] b) Synthesis of a Lipopeptide Functionalised With
Sulfisoxazole (SEQ ID NO:25) 26
[0597] The structure shown above was synthesised on a manual
nitrogen bubbler apparatus starting with Fmoc-protected Rink Amide
BMHA resin on a 0.125 mmol scale, using appropriate amino acids,
palmitic acid and the compound from (a). Coupling was carried out
using standard TBTU/HOBt/DIEA protocols. 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 hours. Crude material was precipitated from ether. The
product was analysed by analytical HPLC, gradient 70-100% B over 20
minutes, A=0.1% TFA/water and B=0.1% TFA/acetonitrile, flow rate 1
ml/minute, detection UV 214 nm, retention time 27 minutes). Further
characterisation was carried out using MALDI mass spectrometry,
giving a M+H at m/z 1359, expected 1356.
[0598] c) Preparation of Gas-Filled Microbubbles Comprising the
Compound From (b)
[0599] A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml)
was added to a mixture of DSPS (4.5 mg) and product from (b) (0.5
mg) in a vial. The mixture was sonicated for 5 minutes and then
heated at 80.degree. C. for 5 minutes (vial was shaken during
warming) and cooled. The head space was flushed with
perfluorobutane gas and the vial was shaken in a cap mixer for 45
seconds followed by extensive washing with deionised water. MALDI
mass spectrometry showed no detectable level of compound from (b)
in the final wash solution. Incorporation of isoxazole-containing
lipopeptide into the microbubbles 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 seconds and analysed by MALDI-MS (ACH-matrix),
giving a m+H peak at m/z 1359 corresponding to lipopeptide (b).
Sequence CWU 1
1
31 1 4 PRT Artificial Sequence Description of Artificial
SequenceRGDC-Mal-PEG3400-DSPE 1 Arg Gly Asp Cys 1 2 25 PRT
Artificial Sequence Description of Artificial SequencePeptide
comprising phosphatidylserine-binding and heparin-binding sections
2 Phe Asn Phe Arg Leu Lys Ala Gly Gln Lys Ile Arg Phe Gly Ala Ala 1
5 10 15 Ala Trp Glu Pro Pro Arg Ala Arg Ile 20 25 3 8 PRT
Artificial Sequence Description of Artificial
SequenceHeparin-binding peptide 3 Trp Glu Pro Pro Arg Ala Arg Ile 1
5 4 6 PRT Artificial Sequence Description of Artificial
SequenceLinker sequence 4 Phe Lys Leu Arg Leu Cys 1 5 5 4 PRT
Artificial Sequence Description of Artificial SequenceHeparin
sulphate binding peptide 5 Lys Arg Lys Arg 1 6 8 PRT Artificial
Sequence Description of Artificial SequenceFibronectin peptide 6
Trp Gln Pro Pro Arg Ala Arg Ile 1 5 7 13 PRT Artificial Sequence
Description of Artificial SequenceLipopeptide consisting of a
heparin sulphate binding peptide and a fibronectin peptide 7 Lys
Lys Arg Lys Arg Trp Gln Pro Pro Arg Ala Arg Ile 1 5 10 8 24 PRT
Artificial Sequence Description of Artificial SequenceFibronectin
peptide sequence 8 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 9 6
PRT Artificial Sequence Description of Artificial
SequenceBiotinylated endothelin-1 peptide 9 Trp Leu Asp Ile Ile Trp
1 5 10 10 PRT Artificial Sequence Description of Artificial
SequenceBiotinylated fibrin-anti-polymerant peptide 10 Gly Pro Arg
Pro Pro Glu Arg His Gln Ser 1 5 10 11 5 PRT Artificial Sequence
Description of Artificial SequenceLipopeptide containing RGD
sequence and fluorescein reporter group 11 Lys Lys Lys Lys Gly 1 5
12 18 PRT Artificial Sequence Description of Artificial
SequenceEndothelial 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 4
PRT Artificial Sequence Description of Artificial
SequenceLipopeptide functionalised with captopril 13 Lys Lys Lys
Lys 1 14 13 PRT Artificial Sequence Description of Artificial
SequenceLipopeptide with an affinity for endothelial cells 14 Lys
Lys Lys Xaa Ile Arg Arg Val Ala Arg Pro Pro Leu 1 5 10 15 14 PRT
Artificial Sequence Description of Artificial SequenceLipopeptide
comprising an interleukin-1 receptor binding peptide 15 Lys Gly Asp
Trp Asp Gln Phe Gly Leu Trp Arg Gly Ala Ala 1 5 10 16 12 PRT
Artificial Sequence MOD_RES (1) Dabsyl-Tyr 16 Tyr Arg Ala Leu Val
Asp Thr Leu Lys Lys Gly Cys 1 5 10 17 25 DNA Artificial Sequence
Description of Artificial SequenceSynthetic oligonucleotide 17
gaaaggtagt ggggtcgtgt gccgg 25 18 15 PRT Artificial Sequence
Description of Artificial SequenceLipopeptide with affinity for
thrombi 18 Lys Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr Leu
Gln 1 5 10 15 19 6 PRT Artificial Sequence Description of
Artificial SequenceLipopeptide 19 Lys Trp Lys Lys Lys Gly 1 5 20 5
PRT Artificial Sequence Description of Artificial
SequenceThiol-functionalised lipid molecule 20 Lys Lys Lys Xaa Cys
1 5 21 4 PRT Artificial Sequence Description of Artificial
SequenceLipopeptide functionalised with atenolol 21 Lys Lys Lys Lys
1 22 4 PRT Artificial Sequence Description of Artificial
SequenceLipopeptide containing folic acid 22 Lys Lys Lys Lys 1 23 4
PRT Artificial Sequence Description of Artificial
SequenceLipopeptide containing a derivative of bestatin 23 Lys Lys
Lys Lys 1 24 4 PRT Artificial Sequence Description of Artificial
SequenceLipopeptide containing chlorambucil 24 Lys Lys Lys Lys 1 25
4 PRT Artificial Sequence Description of Artificial
SequenceLipopeptide functionalised with sulfisoxazole 25 Lys Lys
Lys Lys 1 26 9 PRT Artificial Sequence Description of Artificial
SequenceAtherosclerotic plaque-binding peptide 26 Tyr Arg Ala Leu
Val Asp Thr Leu Lys 1 5 27 16 PRT Artificial Sequence Description
of Artificial SequenceAtherosclerotic plaque-binding peptide 27 Tyr
Ala Lys Phe Arg Glu Thr Leu Glu Asp Thr Arg Asp Arg Met Tyr 1 5 10
15 28 17 PRT Artificial Sequence Description of Artificial
SequenceAtherosclerotic plaque-binding peptide 28 Arg Ala Leu Val
Asp Thr Glu Phe Lys Val Lys Gln Glu Ala Gly Ala Lys 1 5 10 15 29 14
PRT Artificial Sequence Description of Artificial SequenceThrombus
binding peptide 29 Asn Asp Gly Asp Phe Glu Glu Ile Pro Glu Glu Tyr
Leu Gln 1 5 10 30 4 PRT Artificial Sequence Description of
Artificial SequenceThrombus binding peptide 30 Gly Pro Arg Gly 1 31
13 PRT Artificial Sequence Description of Artificial
SequencePlatelet binding peptide 31 Pro Leu Tyr Lys Lys Ile Ile Lys
Lys Leu Leu Glu Ser 1 5 10
* * * * *