Note: Descriptions are shown in the official language in which they were submitted.
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POLYMERS
The present invention relates to polymers useful as
therapeutic and diagnostic agents and to processes for
their preparation. In particular, the invention relates
to amino acid based biodegradable polymers for use in
targeting of diagnostic imaging and therapeutic agents.
The polymers in accordance with the invention are
suitable for use in a variety of applications where
specific delivery is desirable, and are particularly
suited for the delivery of biologically active agents.
However, a preferred use of the polymers of the
invention is in the enhancement of images of selected
mammalian organs, tissues and cells in vivo using MR,
X-ray, ultrasound, light and nuclear imaging techniques
by virtue of their enhanced imaging properties and site
specificity. The polymers are especially suited for use
as intravascular contrast agents and blood pool agents
in such imaging techniques. As such they may be used in
imaging blood vessels, e.g. in magnetic resonance
angiography, in the measurement of blood flow and
volume, in the identification and characterization of
lesions by virtue of differences in vascularity from
normal tissue, in the imaging of the lungs for the
evaluation of pulmonary disease and in blood perfusion
studies.
Medical imaging techniques, such as MRI and X-ray,
have become extremely important tools in the diagnosis
and treatment of disease. Some imaging of internal
parts relies on inherent attributes of those parts, such
~ as bones, to be differentiated from surrounding tissue
in a particular type of imaging, such as X-ray. Other
organs and anatomical components are only visible when
specifically highlighted by particular imaging
techniques.
One such technique with the potential to provide
SUBSTITUTE SHEET (RULE 26)
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images of a wide variety of anatomical components
involves biotargeting image-enhancing metals. Such a
procedure has the possibility of creating or enhancing
images of specific organs and/or tumors or other such
localized sites within the body, while reducing the
background and potential interference created by
simultaneous highlighting of non-desired sites.
It has been recognized for many years that
chelating various metals increases the physiologically
tolerable dosage of such metals and so permits their use
in vivo to enhance images of body parts. One chelate
complex which has been the subject of much study is Gd-
DTPA. However, despite its satisfactory relaxivity and
safety, this has several disadvantages. Due to its low
molecular weight, Gd-DTPA is rapidly cleared from the
blood stream. This severely limits the imaging window,
the number of optimal images that can be taken after
each injection, and increases the agent's required dose
and relative toxicity. Moreover, such simple metal
chelate image enhancers, without further modification,
do not generally provide any significant site
specificity.
The attachment of metal chelates to tissue or organ
targeting molecules, e.g. biomolecules such as proteins,
in order to produce site specific therapeutic or
diagnostic agents has been widely suggested. Many such
bifunctional chelating agents, i.e. agents which by
virtue of the chelant moiety are capable of strongly
binding a therapeutically or diagnostically useful metal
ion and by virtue of the site-specific molecular
component are capable of selective delivery of the
chelated metal ion to the body site of interest, are
known or have been proposed. However, drawbacks of
conjugating metal chelates to protein carriers for use
in MR imaging include inappropriate biodistribution,
toxicity and short blood half-life. Their use in MR
imaging is therefore limited. In addition, proteins
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provide a defined structure not subject to wide
synthetic variation.
Site-specific uses of various imaging techniques
are enhanced by the use of a multiplicity of the
appropriate metal ion conjugated to a site-directed
macromolecule and numerous attempts have been made to
produce bifunctional polychelants with increased numbers
of chelant moieties per site-specific macromolecule.
For site-specific image enhancement however it is
important that the site specificity of the tissue or
organ targeting moiety of such chelates of bifunctional
chelants should not be destroyed by the conjugation to
the targeting moiety of the chelant moiety. Where the
bifunctional chelant contains only one chelant moiety
this is not generally a severe problem. However, when
attempts have been made to produce bifunctional
polychelants by conjugating several chelant moieties
onto a single site-specific macromolecule, it has been
found not only may the maximum achievable chelant:site-
specific macromolecule ratio be relatively limited but
as the ratio achieved increases, so the site-specificity
of the resulting bifunctional polychelant decreases.
In order to overcome the problems of attaching
larger numbers of chelant moieties to a site-specific
macromolecule without destroying its site-specificity,
i.e. without disturbing its binding site(s), there have
been many proposals for the use of a backbone molecule
to which large numbers of chelant moieties can be
attached to produce a polychelant, one or more of which
can then be conjugated to the site-specific
macromolecule to produce the bifunctional polychelant.
Bifunctional polychelants in which the chelant
moieties are residues of open chain PAPCAs, such as EDTA
and DTPA, and in which the backbone molecule is a
polyamine such as polylysine or polyethyleneimine have
been produced.
WO-A-90/12050 describes techniques for producing
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polychelants comprising macrocyclic chelating moieties,
such as polylysine-polyDOTA, and for the preparation of
corresponding bifunctional polychelants. This document
also suggests the use of starburst dendrimers, such as a
sixth generation PAMAM starburst dendrimer as the
skeleton for such polychelants. WO-A-93/06868 similarly
describes polychelants comprising dendrimeric backbone
molecules linked to a plurality of macrocyclic chelant
moieties, e.g. DOTA residues. These in turn may be
conjugated to a site-directed molecule, e.g. a protein.
However, to date starburst dendrimers have found little
use in imaging.
Thus, there still exists a need for other polymeric
contrast agents, e.g. MR, X-ray, ultrasound, light-based
and nuclear, which contain relatively large amounts of
metal per molecule, are of a molecular weight which
enables them to be circulated within the blood for
extended periods of time and which exhibit improved
biodistribution.
The present invention lies in the recognition that
co-polymers of amino acids carrying or attached to one
or more reporter groups, e.g. chelating moieties,
fluors, or absorbers, are particularly suitable for
diagnostic and therapeutic use by virtue both of their
structures and of their substantial uniformity in terms
of molecular weight distribution. Moreover, by virtue
of their relatively high molecular weights such
compounds can function as effective blood pool agents
without requiring attachment to site-directed
biomolecules.
Thus viewed from one aspect the invention provides
a compound comprising a linear, branched or dendrimeric
polymer backbone with linked thereto at least one
reporter moiety, said polymer backbone comprising a
plurality of amine-containing acids, e.g. amino acid
residues or similar non-native amine-containing acids;
with the proviso that when the polymer backbone is
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linear, the reporter moiety comprises an iodinated
contrast agent, an ultrasound contrast agent, a light-
. based reporter or a metal chelator other than DOTA, DTPA
or similar polyaminopolycarboxylic acids. When the
polymer backbone is linear, the reporter moiety
preferably comprises an iodinated contrast agent or TMT.
As used herein, the term "reporter moiety" is
intended to define any atom, ion or molecule which may
be linked to the polymer backbone to produce an effect
which is detectable by any chemical, physical or
biological examination. A reporter moiety may thus be
either a therapeutic or diagnostic agent, e.g. a
contrast agent or pharmacologic agent. Where two or
more reporter moieties are attached to a given polymer
backbone, these may be identical or different. Thus,
these may comprise any combination of diagnostic and/or
therapeutic agents. The number of attached reporter
moieties depends on the structure of the polymer
backbone, in particular the degree of any branching, but
generally will be in the range of from 3 to 200,
preferably up to 100, e.g. up to 50.
Dendrimeric (or cascade) polymers are preferred as
the backbone moiety. These are formed from monomers
which act as branching sites and with each successive
branching a new "generation" is formed. The dendrimeric
backbone molecule preferably comprises a multiplicity of
native or non-native, preferably native amino acid
residues arranged to extend radially outwards from a
central core moiety. These amino acid residues may be
terminally bonded directly, or optionally via a linking
group, to one or more reporter groups. Alternatively,
these may be terminally branched by the addition of
further amino acid residues. A backbone molecule
wherein a central branched core has itself been
terminally branched once is termed a first-generation
backbone molecule. Further terminal branching of the
amino acid residues of first-generation backbone
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molecules provides second, third, fourth etc. generation
backbones. With each successive round of branching, the
number of attachment points available for bonding to
reporter groups increases. Depending on the nature of
the central core moiety, branching from this may extend
radially in one or more directions, resulting in either
radially asymmetrical or symmetrical dendrimers.
Preferably, the dendrimer backbone molecules are
radially asymmetrical.
Dendrimeric polymers comprising a plurality of
native or non-native, preferably native amino acid
residues form a further aspect of the invention.
Conveniently, these comprise from 3 to 200 amino acid
residues, e.g. from 3 to 100 amino acid residues
extending radially from a central core moiety.
Whilst the core moiety may itself comprise one or
more amino acid residues, other core moieties are
contemplated. Typically, the core moiety may be any
molecule to which a multiplicity of successive amino
acid residues may be attached and may itself comprise a
reporter moiety. Suitable core moieties include
HZNCOCHzCH2CONH2 , and
Y (CH2) m-X
H-N- (CHz) n,-C- (CHZ) a,-X
Y
in which m=0-4 ;
Y represents hydrogen or an alkyl or aryl group,
a . g . a C1_6 alkyl group; and
X represents a -COZH, -SOZC1 or -CHzBr group,
as well as modifications thereto and derivatives
thereof.
In one embodiment of the invention, the dendrimer
core may itself comprise a reporter moiety. Thus, in
another aspect, the invention provides a compound
comprising a dendrimeric polymer backbone extending
~.___ .~.,..~..._.~.,_.r.._ ,
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WO 98!32469 PCT/GB98100270
radially from a reporter moiety, said polymer backbone
comprising a plurality of amino acid residues.
Preferably, biodegradable linking groups serve to
link the reporter moieties to the polymer backbone. In
' 'this way, biodegradation of the compound at the targeted
site results in release of the reporter moieties, e.g.
an ionic or non-ionic contrast agent at the site of
interest. Examples of suitable linking groups include
amide, ether, thioether, guanidyl, acetal, ketal and
phosphoester groups. Linkage between the backbone and
the reporter groups is preferably via an amide bond, the
amide nitrogen deriving from the backbone molecule and
the amide carbonyl deriving from a carboxyl or carboxyl
derivative on the reporter group.
The advantage of a biodegradable polymer is that it
will not accumulate at the injection site, e.g. during
lymphographic procedures, or in tissues, e.g. the liver
during angiographic procedures provided its degradation
rate is tuned to the required imaging time.
Biodegradability of the compounds of the invention can
be adjusted by selection of particular linker and
peptide cluster compounds. Moreover, if desired, the
biodegradability of the linkers and polymer backbones
can be optimised in vitro using purified enzymes and/or
biological fluids/tissues. The use of amino acid
monomers which themselves are rapidly cleared may
further aid clearance after imaging.
Preferred polymer backbones are those comprising
from 3 to 200 amino acid residues, preferably from 3 to
100 amino acid residues and having a molecular weight of
from 300 to 20,000 daltons. These are preferably bonded
via peptide bonds, thereby ensuring the biodegradability
of the polymer and subsequent elimination from the body.
The polyamino acid may be a polymer of a single species
or at least two different species of amino acids, or may
be a block copolymer. Preferably the polyamino acid is
poly-1-aspartic acid.
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_ g _
Particularly preferred compounds in accordance with
the invention are those of formula I:
o
R
\ 2
wherein n is an integer of from 1 to 100; and
R represents a reporter group or a biodegradable linker-
reporter adduct.
In a preferred embodiment of the invention, the
reporter moieties are chelating agents. These are
capable of chelating metal ions with a high level of
stability, and may be metallated with the appropriate
metal ion(s), e.g. to enhance images in MRI, gamma
scintigraphy or X-ray or to deliver cytotoxic doses of
radioactivity to kill undesirable cells such as tumors.
Conveniently, the chelating agents are contrast agents
comprising at least one paramagnetic metal ion.
Alternatively, the chelating agents may be used in their
unmetallated or undermetallated state for absorption of
available metal ions in vivo, e.g. in metal
detoxification.
The reporter moieties may also comprise therapeutic
agents, e.g. antibiotic, analgesic, anti-inflammatory or
other bioactive agents. Prolonged circulation in the
blood of polymers carrying such agents substantially
prolongs their therapeutic effect. Proteolysis of the
... . ...~..._ ~...,.. _~ _... ... ._. r
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WO 98132469
_ g _
linking groups provides a release of therapeutic agent.
Selection of a particular linking group thus provides
the potential for a timed release of therapeutic agent
at the desired site of interest.
If desired, the compounds in accordance with the
invention can be attached by well-known methods to one
or more site-directed molecules or targeting agents,
e.g. a protein, to form bifunctional polymers which can
enhance images and/or deliver cytotoxic doses of
radioactivity to the targeted cells, tissues, organs,
and/or body ducts. Targeting of contrast agents to the
site of interest in this way increases the effectiveness
of the imaging method. Such agents accumulate at the
site of interest which is dependent upon the specificity
of the targeting agent. Alternatively) the polymers may
be used as blood pool agents without being coupled to
site directed molecules.
For those compounds of the invention comprising a
dendrimeric backbone moiety, any terminal amino acid
residues may thus be bonded either directly or via a
biodegradable linking group to either a reporter or a
targeting agent. Preferably, where the core moiety is
itself a reporter group, each terminal amino acid
residue is bound via a biodegradable linking group to a
targeting agent. In this way, a compound comprising
more than one targeting agent can be provided.
Conveniently the number of targeting agents will be from
1 to 128, preferably from 1 to 16, e.g. from 1 to 4.
In an alternative embodiment of the invention those
compounds comprising a dendrimeric polymer backbone may
comprise a targeting agent or site-directed
macromolecule as the core moiety. The resulting peptide
cluster may in turn be linked to one or more reporter
moieties. Viewed from a yet further aspect, the
invention thus provides a compound comprising a
dendrimeric polymer backbone extending radially from a
targeting agent, said polymer backbone comprising a
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plurality of amino acid residues with linked thereto at
least one reporter moiety.
The heat stable STa enterotoxin from E.coli as
described in WO-A-95/11694 is particularly suitable as a
core targeting agent. Attached Figure l illustrates a
compound of the invention in which the STa peptide is
linked to a poly-1-aspartic acid cluster (Asp3) which in
turn is linked to a plurality of TMT reporter molecules.
The polymers in accordance with the invention are
in and of themselves useful entities in medical
diagnosis and therapy, due in part to their unique
localization in the body. The size of the polymer,
typically 200 to 100,000 daltons, particularly 200 to
50,000 daltons, especially 10,000 to 40,000 daltons,
radically alters its biodistribution. Selection of
particular linking groups and/or variations in the
polyamino acid sequence also affects the biodistribution
of the polymers and the attached reporter or targeting
agents.
The compounds of the invention generally have
extended intravascular residence times, e.g. of the
order of hours, although this can be specifically
tailored according to the desired use of the compounds
by selection of appropriate linking agents and/or
modification of the polyamino acid sequence of the
backbone polymer. Usually the compounds will eventually
clear into the extracellular fluid (ECF) space and
undergo renal excretion. Since the compounds remain
primarily in the intravascular system for a
diagnostically useful residence time, they are suitable
for a range of uses from blood pool and cardiac
perfusion imaging, CNS tumour detection and volume
determination to thrombus detection and angiography. As
blood pool agents they are particularly suited to use in
studies of blood flow or volume, especially in relation
to lesion detection and myocardial perfusion studies.
The conventional monomeric MRI contrast agents which
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rapidly disperse into the extracellular/extravascular
space cannot readily be used for these purposes.
' Moreover in view of their enhanced relaxivity, the
polymers according to the invention can be administered
at significantly reduced dosages relative to current
monomeric MRI contrast agents such as GdDTPA and GdDOTA,
thus providing a significantly improved safety margin in
their use.
The invention thus provides compounds which are
able to provide MR contrast enhancement of the blood
pool for long periods of time, which have a specificity
towards accumulation in various body tissues, which
provide relatively large amounts of metal and whose
molecular weight can be synthetically tailored to
produce an agent of desired composition, molecular
weight and size.
Furthermore, by suitable selection of chelated
species, chelates according to the invention may be
produced which are capable of functioning as X-ray
agents, e.g. by choosing tungsten, and also as MR
contrast agents by choosing an appropriate metal ion
e.g. a lanthanide ion.
Attachment of the compounds to a site-directed
molecule results in even greater in vivo target
specificity. The site-directed molecule is preferably
an antibody, antibody fragment, other protein or other
macromolecule which will travel in viva to that site to
deliver the chelated metals. In the present invention
the capacity of this site-directed macromolecule to
travel and/or bind to its target is not compromised by
the addition of the chelated metals. The number of
chelates per molecule is sufficient to enhance the image
of that particular target.
Suitable chelating agents for attachment to the
polymer backbone include both linear and macrocyclic
PAPCAs. Examples of suitable PAPCAs include
ethylenediamine tetraacetic acid (EDTA),
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diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-
tetraazacyclododecanetetraacetic acid (DOTA), 1,4,7,10-
tetraazacyclododecane-1,4,7-triacetic acid (D03A), 1-
oxa-4,7,10-triazacyclododecanetriacetic acid (DOXA),
1,4,7-triazacyclononanetriacetic acid (NOTA) and
1,4,8,11-tetraazacyclotetradecanetetraacetic acid
(TETA) .
Other chelating agents suitable for attachment to
the polymer backbone include terpyridines such as
described in US-A-5367080, e.g. 4'-(3-amino-4-methoxy-
phenyl)-6,6"-bis(N',N'-dicarboxymethyl-N-
methylhydrazino)-2,2':6',2"-terpyridine (THT) and
4'-(3-amino-4-methoxy-phenyl)-6,6'"-bis(N,N-
di(carboxymethyl)aminomethyl]-2,2':6',2"-terpyridine
( TMT ) .
Metals that can be incorporated, through chelation,
include lanthanides and other metal ions, including
isotopes and radioisotopes thereof, such as, for
example, Mg, Ca, Sc, Ti, B, V, Cr, Mn, Fe, Co, Ni, Cu,
Zn, Ga, Sr, Y, Zr, Tc, Ru, In, Hf, W, Re, Os, Pb and Bi.
The choice of metal ion for chelation will depend upon
the desired therapeutic or diagnostic application.
For use in X-ray contrast imaging, the reporter
moiety may comprise an ionic or non-ionic iodinated
monocyclic or bis-cyclic X-ray contrast agent. By mono
and bis-cyclic is meant that the contrast agents contain
either one or two iodinated rings. Generally, the
iodinated rings will be di- or tri-iodinated, e.g. tri-
iodinated aryl rings, in particular phenyl rings.
Examples of iodinated contrast agents for use in
accordance with the invention include iohexol, iopentol,
iopamidol and iodixanol. Conveniently, one or more
iodinated contrast agents may be conjugated to form an
alternating co-polymer which in turn can be attached to
the polymer backbone. An example of the synthesis of
such a co-polymer from iodixanol is shown below:
u-_.., .~._.~.....,r.~.,.e-....-..~_..-. . ~.
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» ~J~-I'i,I o
~ I
_ 1 H
~ ~N ~ / I~-Ia
V
H
a ~.COta)s'_N~
HO ~
O
I
HO H H ti p
HO~TI / N~~~)~~.N
~t
HO
O
I ' I
HO H
HO~H
o~
Of;
H
OH
HO ti
HO ~ N O
I
~ H
~ ~N 1 / IV N ~ N ~~
O
I O
~N
H
TFA
H
fp Ii ~ Y
O
O 1
I ''
H
~HO~
~ ~N ~ / N ~~ / N ~~
O
I
~Nli~
The bifunctional agents in accordance with the
invention involve coupling the compounds to a site-
directed molecule. The site-directed molecules may be
any of the molecules that naturally concentrate in a
selected target organ, tissue, cell or group of cells,
or other location in a mammalian body, in vivo. These
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can include amino acids, oligopeptides (e. g.
hexapeptides), molecular recognition units (MRU's),
single chain antibodies (SCR's), proteins, non-peptide
organic molecules, Fab fragments, and antibodies.
Examples of site-directed molecules include
polysaccharides (e. g. CCK and hexapeptides), proteins
(such as lectins, asialofetuin, polyclonal IgG, blood
clotting proteins (e.g. hirudin), lipoproteins and
glycoproteins), hormones, growth factors, and clotting
factors (such as PF4). Exemplary site-directed proteins
include E.coli heat stable enterotoxin STa and its
analogues, polymerized fibrin fragments (e. g., E1), serum
amyloid precursor (SAP) proteins, low density
lipoprotein (LDL) precursors, serum albumin, surface
proteins of intact red blood cells, receptor binding
molecules such as estrogens, liver-specific
proteins/polymers such as galactosyl-neoglycoalbumin
(NGA) (see Vera et al. in Radiology 151: 191 (1984)) N-
(2-hydroxy-propyl)methacrylamide (HMPA) copolymers with
varying numbers of bound galactosamines (see Duncan et
al., Biochim. Biophys. Acta 880:62 (1986)), and allyl
and 6-aminohexyl glycosides (see Wong et al., Carbo.
Res. 170:27 (1987)), and fibrinogen.
The site-directed protein can also be an antibody.
The choice of antibody, particularly the antigen
specificity of the antibody, will depend on the desired
use of the conjugate. Monoclonal antibodies are
preferred over polyclonal antibodies.
Human serum albumin (HSA) is a preferred protein
for the study of the vascular system. HSA is available
commercially from a number of sources including Sigma
Chemical Co. Preparation of antibodies that react with
a desired antigen is well known. Antibody preparations
are available commercially from a variety of sources.
Fibrin fragment E1 can be prepared as described by Olexa
et al. in J. Biol. Chem. 254:4925 (1979). Preparation
of LDL precursors and SAP proteins is described by de
_..__._..~.,-, .t"r."...._....._-_rv ..."~.
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Beer et al. in J. Immunol. Methods 50:17 (1982). The
above described articles are incorporated herein by
reference in their entirety.
The compounds in accordance with the invention are
conveniently prepared by conjugation of a linear,
branched or dendrimeric backbone comprising a plurality
of amino acid residues to one or more reporter groups in
a non-reactive solvent. Linkage of the reporter groups
to the backbone molecule may be effected through any
reactive group and standard coupling techniques are
known in the art. Preferred reaction conditions, e.g.
temperature, solvents etc. depend primarily on the
particular reactants and can be readily determined by
those skilled in the art.
Methods for metallating any chelating agents
present are within the level of skill in the art.
Metals can be incorporated into a chelant moiety by any
one of three general methods: direct incorporation,
template synthesis and/or transmetallation. Direct
incorporation is preferred.
Methods for attaching the polymer backbones to
antibodies and other proteins are within the level of
skill in the art. Such methods are described in Pierce
1989 Handbook and General Catalog and the references
cited therein, Blatter et al, Biochem., 24:1517 (1985)
and Jue et al, Biochem., 17:5399 (1978).
The polymer backbone itself may be synthesised in
accordance with conventional peptide synthesis
techniques. Suitable methods for forming the amino acid
units are described in, for example, "Synthesis of
Optically Active a-Amino Acids" by Robert M. Williams
(Pergamon Press, 1989). In general, the reactive side
chain groups present, e.g. amino, thiol and/or carboxy,
will be protected during the coupling of the individual
amino acids, although it is possible to leave some side
chain groups unprotected, e.g. hydroxy, primary amide
groups, during the entire synthetic procedure.
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The final step in the synthesis of a compound in
accordance with the invention will be the deprotection
of a fully protected or partly protected derivative of
such a compound and such a process forms part of the
invention. Thus, the present invention provides a
process for producing a compound as hereinbefore
described, said process comprising deprotecting a
partially or fully protected derivative thereof.
In building up the peptide chain, it is in
principle possible to start either at the C-terminal or
the N-terminal. However, only the C-terminal starting
procedure is in common use. This is due to difficulties
encountered when synthesising in the N to C direction
which include an unacceptably high degree of
racemisation (see Konig & Geiger, Chemische Berichte
103:2024-2033, 1970).
Contrary to expectation, it has been found that the
peptide compounds for use in accordance with the
invention may be produced in good yield and high purity
(c 0.1% racemisation per step) by synthesising in the
amino to carboxy direction. This method of synthesis
has been found to be particularly effective in preparing
the dendrimeric polymer backbones. In particular, these
have been found to be more stable than those dendrimers
derived from the more conventional Michael addition
chemistry. Moreover, synthesising the polymer backbones
in the amino to carboxy direction has been found to
produce discrete polymers which are substantially non
cross-linked and which have particularly low levels of
racemic impurities.
Thus, in another aspect the invention further
provides a process for the preparation of a compound
comprising a linear, branched or dendrimeric polymer
backbone with linked thereto at least one reporter
moiety, said polymer backbone comprising a plurality of
amino acid residues, said process comprising:
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(a) stepwise linking of successive protected amino acid
residues in the amino to carboxy direction to form a
polymer backbone;
(b) linking the polymer backbone to one or more reporter
moieties, optionally via a linking group; and
(c) deprotecting any protected group.
Thus, one can start at the N-terminal by reaction
of a suitably protected derivative of, for example,
aspartic acid with a suitably protected derivative of a
second aspartic acid molecule. The first aspartic acid
derivative will have a protected amino group and a free
carboxyl group while the other reactant will have either
a free or activated a-amino group and a protected
carboxyl group. After coupling, the intermediate may be
purified, e.g. by chromatography, and then selectively
deprotected to permit addition of further amino acid
residues. This procedure is continued until the
required amino acid sequence is completed.
A wide range of protecting groups for amino acids
are known. Suitable amine protecting groups include
carbobenzoxy (Z- or Cbz), t-butoxycarbonyl (Boc-) and 9-
fluorenylmethoxycarbonyl (Fmoc-). Carboxyl protecting
groups which may be used include benzyl (-Bzl) and t-
butyl (-tBu) .
A wide range of procedures exist for removing
amine- and carboxyl-protecting groups. Amine protecting
groups such as Boc and carboxyl protecting groups such
as -tBu may be removed simultaneously by acid treatment,
e.g. with trifluoroacetic acid.
The coupling of free amino and carboxyl groups may,
for example, be effected using N,N'-dicyclohexyl
carbodiimide (DCC). Other coupling agents which may be
used include 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) and 2-(11-H-benzotriazolyl-1-yl)-
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1,1,3-tetramethyluranium tetrafluoroborate (TBTU).
The coupling reactions may be effected at ambient
temperatures, conveniently in a suitable solvent system,
e.g. tetrahydrofuran, dimethylformamide,
dimethylsulphoxide or a mixture of these solvents.
It may be convenient to carry out the peptide
synthesis on a solid phase resin support. Amino acids
are added stepwise to a growing peptide chain linked to
an insoluble matrix, such as polystyrene beads. One
advantage of this solid-phase method is that the desired
product at each stage is bound to beads which can be
rapidly filtered and washed so the need to purify
intermediates is obviated. A number of suitable solid
phase supports are known in the art, e.g. 4-hydroxy
benzyl alcohol resin which has been modified to form an
ester with succinic anhydride.
The compounds of the invention, especially the
bifunctional polymers, may be administered to patients
for imaging in amounts sufficient to yield the desired
contrast with the particular imaging technique.
Generally dosages of from 0.001 to 5.0 mmoles of
chelated imaging metal ion per kilogram of patient
bodyweight are effective to achieve adequate contrast
enhancements. For most MRI applications preferred
dosages of imaging metal ion will be in the range of
from 0.02 to 1.2 mmoles/kg bodyweight while for X-ray
applications dosages of from 0.5 to 1.5 mmoles/kg are
generally effective to achieve X-ray attenuation.
Preferred dosages for most X-ray applications are from
0.8 to 1.2 mmoles of the lanthanide or heavy metal/kg
bodyweight.
The dosage of the compounds of the invention for
therapeutic use will depend upon the condition being
treated, but in general will be of the order of from 1
pmol/kg to 1 mmol/kg bodyweight.
The compounds of the present invention may be
formulated with conventional pharmaceutical or
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veterinary aids, for example emulsifiers, fatty acid
esters, gelling agents, stabilizers, antioxidants,
osmolality adjusting agents, buffers, pH adjusting
agents, etc., and may be in a form suitable for
parenteral or enteral administration, for example
injection or infusion or administration directly into a
body cavity having an external escape duct, for example
the gastrointestinal tract, the bladder or the uterus.
Thus the compounds of the present invention may be in
conventional pharmaceutical administration forms such as
tablets, capsules, powders, solutions, suspensions,
dispersions, syrups, suppositories etc. However,
solutions, suspensions and dispersions in
physiologically acceptable carrier media, for example
water for injections, will generally be preferred.
The compounds according to the invention may
therefore be formulated for administration using
physiologically acceptable carriers or excipients in a
manner fully within the skill of the art. For example,
the compounds, optionally with the addition of
pharmaceutically acceptable excipients, may be suspended
or dissolved in an aqueous medium, with the resulting
solution or suspension then being sterilized.
For MRI and for X-ray imaging of some portions of
the body the most preferred mode for administering metal
chelates as contrast agents is parenteral, e.g.,
intravenous administration. Parenterally administrable
forms, e.g. intravenous solutions, should be sterile and
free from physiologically unacceptable agents, and
should have low osmolality to minimize irritation or
other adverse effects upon administration, and thus the
contrast medium should preferably be isotonic or
slightly hypertonic. Suitable vehicles include aqueous
vehicles customarily used for administering parenteral
solutions such as Sodium Chloride Injection, Ringer's
Injection, Dextrose Injection, Dextrose and Sodium
Chloride Injection, Lactated Ringer's Injection and
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other solutions such as are described in Remington's
Pharmaceutical Sciences, 15th ed., Easton: Mack
Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and
The National Formulary XIV, 14th ed. Washington:
American Pharmaceutical Association (1975). The
solutions can contain preservatives, antimicrobial
agents, buffers and antioxidants conventionally used for
parenteral solutions, excipients and other additives
which are compatible with the chelates and which will
not interfere with the manufacture, storage or use of
products.
Viewed from a further aspect the invention provides
a pharmaceutical composition, e.g. an image enhancing or
therapeutic composition, comprising a compound of the
invention together with at least one pharmaceutical
carrier or excipient.
Viewed from a still further aspect the invention
provides the use of a compound according to the
invention or a chelate thereof for the manufacture of an
image enhancing contrast medium or a therapeutic
composition.
Viewed from another aspect the invention provides a
method of generating an image of a human or non-human
animal, especially mammalian, body which method
comprises administering to said body an image enhancing
amount of a compound according to the invention and
thereafter generating an image e.g. an MR, X-ray,
ultrasound or scintigraphic image, of at least a part of
said body.
The present invention will now be further
illustrated by way of the following non-limiting
examples. Unless otherwise indicated, all percentages
given are by weight.
_~,r.,.__........ ... . ~
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Example 1: Asymmetric peptide cluster
Z- [Asp (a, Y-Asp2 (a, Y-Asp4 (a, Y-Aspe (a, Y-Lyslb (a-Reporterl6)
(a) Bis-a,Y-(a,y-(tButyl)-Aspartyl)-N-Cbz-Aspartamide
"Asp3 Cluster" (Compound I)
Into a 500 mL round bottom flask was added 8.5
mmoles N-Cbz-L-Aspartic acid, 10.2 mmoles N-
hydroxybenzotriazole, 25 mL THF:DMF (2:1, v/v), and 10.2
mmoles EDC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide). After stirring at room temperature for
45 minutes, 20.4 mmoles of a,y-(tButyl)-L-Aspartic acid
and 25 mmoles of N,N'-diisopropylethylamine were added
with stirring. After 4 hours, an additional 10.2 mmoles
EDC was added and the reaction continued as above for 3
days. This slurry was worked up by aqueous extraction.
Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 44.5%.
(b) N-Cbz-Aspartamide-((a,y-Aspartyl-(a,y-(tButyl)-
Aspartyl)) "Asp7 Cluster" (Compound II)
Into a 500 mL round bottom flask was added 10
mmoles Compound I, 95 mL chloroform:THF:acetonitrile
(2.5:7:7), 36.4 mmoles N-hydroxybenzotriazole, and 36.5
mmoles DCC (N, N'-dicyclohexylcarbodiimide). After
stirring at room temperature for 20 minutes, 40 mmoles
of a,y-(tButyl)-L-Aspartic acid was added and N,N'-
diisopropylethylamine was added until the pH was
approximately 7. After stirring at room temperature for
16 hours, the reaction was worked up by aqueous
extraction.
Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 12.1%.
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(c) N-Cbz-Aspartamide-{{a,Y-Aspartyl-(a,y-(tButyl)-
Aspartyl)))) "Aspl5 Cluster" (Compound III)
Step 1:
0.85 mmoles of Compound II was stirred in 200 mL of
95% trifluoroacetic acid (aq.) at room temperature for 8
hours. The reaction was evaporated to dryness at 40°C
in vacuo and then re-evaporated to dryness from 200 mL
of toluene and then from THF.
Step 2:
Into a 250 mL round bottom flask was added 0.85
mmoles from Step 1 above, 90 mL DMF:THF (1:1, v/v), 8.12
mmoles N-hydroxybenzotriazole, and 8.12 mmoles EDC (1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide). After
stirring at room temperature for 20 minutes, 16.24
mmoles of a,Y-(tButyl)-L-Aspartic acid and 19.92 mmoles
N,N'-diisopropylethylamine were added. After stirring
at room temperature for 16 hours, the reaction was
worked up by aqueous extraction and ion exchange
chromatography.
Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 82.2%.
(d) N-Cbz-Aspartamide-((a,y-Aspartyl-(a,Y-Aspartyl-
(Aspartyl(a,Y-Lysyl((a-methoxyethylamide, a-amine)))))))
"Aspl5Lys16 Cluster" (Compound IV)
Step 1:
0.7 mmoles of Compound III was stirred in 200 mL of
95~ trifluoroacetic acid (aq.) at room temperature for 8
hours. The reaction was evaporated to dryness at 40°C
in vacuo and then re-evaporated to dryness from 200 mL
__.._..._..~.~._ . _._ ., ...._.....r...._ ...__.._.. . ~
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of toluene and then from THF.
Step 2:
Into a 250 mL round bottom flask was added 0.7
mmoles of compound from Step 1 above, 90 mL DMF:THF
(1:1, v/v), 8.12 mmoles N-hydroxybenzotriazole, and 8.12
mmoles EDC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide). After stirring at room temperature for
20 minutes, 16.24 mmoles of a,y-(tButyl)-L-Aspartic acid
and 19.92 mmoles N,N'-diisopropylethylamine were added.
After stirring at room temperature for 16 hours, the
reaction was worked up by aqueous extraction and ion
exchange chromatography.
Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 99%.
Ste
Into a 250 mL round bottom flask was added 0.7
mmoles of compound from Step 2 above, 100 mL
DMSO:DMF:THF (1.5:3.5:5, v/v), 27.5 mmoles N-
hydroxybenzotriazole, and 27.5 mmoles EDC (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide). After stirring at
room temperature for 20 minutes, 55 mmoles of a-BOC-L-
Lysine and 68.7 mmoles N,N'-diisopropylethylamine were
added. After stirring at room temperature for 16 hours,
the reaction was worked up by aqueous extraction and Gel
permeation chromatography.
Purity: single spot on TLC.
Step 4:
Into a 250 mL round bottom flask was added compound
from Step 3 above, 40 mL DMF:DCM (2:2, v/v), 23.5 mmoles
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N-hydroxybenzotriazole, and 23.5 mmoles EDC (1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide). After stirring at
room temperature for 30 minutes, 75 mmoles of 2-
methoxyethanolarnine were added. After stirring at room
temperature overnight, the reaction was worked up by
aqueous extraction and Ion Exchange chromatography.
Purity: single spot on TLC. Yield: 90%.
{e) N-Cbz-Aspartamide-((a,y-Aspartyl-(a,Y-Aspartyl-
(Aspartyl(a,Y-Lysyl((a-methoxyethylamide, e-TMT)))))))
"Aspl5Lys16TMT16 Cluster" (Compound V)
Into a 250 mL round bottom flask was added Compound
IV, 1.1 molar equivalents of TMT-NCS and 100 mL of 50 mM
sodium borate at pH 9Ø After stirring at room
temperature for 48 hours, the reaction was worked up by
diafiltration (2000 MW cutoff).
Purity: 80% by RP-HPLC.
Example 2: Symmetric aspartic acid cluster
(a) Bis-(a,y-(tButyl)-Aspartyl)succinamide (Compound I)
Synthetic Route A:
Into a 2 Litre round bottom flask was added 20
mmoles succinic acid, 26 mmoles EDC (1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide), 24 mmoles
triethylamine, 12 mmoles TBTU {2-(I-H-benzotriazolyl-1-
yl)-1,1,3,3-tetramethyluronium tetrafluoroborate), and
150 mL THF:DMF (2:1, v/v), then 20 mmoles a,y-(tButyl)-
L-Aspartic acid. This slurry was allowed to react for 4
days at room temperature and then worked up by aqueous
extraction.
~.._~.-_ . ~
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Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 23.2%.
Synthetic Route B:
Into a 2 Litre round bottom flask was added 10
mmoles succinic acid, 100 mL THF:DMF (2:1 v/v), 60
mmoles triethylamine and 20 mmoles TBTU (2-(1-H-
benzotriazoyl-1-yl)-1,1,3,3-tetramethyluronium
tetrafluoroborate). After 15 minutes of stirring, 22
mmoles a,y-(tButyl)-L-Aspartic acid were added. This
slurry was allowed to react for 21 hours at room
temperature and then worked up by aqueous extraction.
Purity: single spot on TLC, identity confirmed by MS and
NMR. Yield: 64.7%.
(b) (Bis-a,y-Aspartyl-(a,Y-(tButyl)-Aspartyl))-
succinamide (Compound II)
Step 1:
4.6 mmoles of Compound I was stirred into 100 mL of
trifluoroacetic acid/dichloromethane (1:1, v/v) at room
temperature for 45 minutes. The reaction was evaporated
to dryness at 30°C in vacuo and then re-evaporated to
dryness from each of five consecutive 100 mL volumes of
chloroform.
Step 2:
The product from Step 1 was dissolved in 250 mL
THF:DMF (1:1, v/v) with 60 mmoles of triethylamine and
40 mmoles of L-aspartic acid-(a,Y-(tButyl)ester. To
this solution was added 60 mmoles of TBTU. After 16
hours, an additional 20 mmoles of L-aspartic acid-(a,y-
(tButyl)ester was added and the reaction continued
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overnight.
Aqueous workup and ion-exchange chromatography
yielded a single major spot on TLC which was identified
as the desired compound by MS and NMR. Yield: 900.
Example 3: X-ray contrast agent
(a? Synthesis of iodinated monomer (Compound I):
NO~ H
O ~ O _ _.
(i H ~)a
HN O_~-
OH~ H O NH i
aft-BOC-L-Lysinc
lohntd Interntedute'ABA'
EDC
~A
1C1
HO~ H O
VII 1
NHS
OH~H O 1 H
~)a
NHS
(C'p
(b) Compound I may be coupled to any one of the Aspx
clusters described in Examples 1 and 2 to form an
iodinated X-ray contrast agent.
