Contrast agents

The invention provides a composition of matter of the formula (I): V-L-R where V is a non-peptidic organic group having binding affinity for an endothelin receptor site, L is a linker moiety or a bond, and R is a moiety detectable in in vivo imaging of a human or animal body. This composition of matter may be used to image diseases and disorders, particularly of the cardiovascular system.

This invention relates to diagnostic imaging techniques in which a disease 
state may be imaged using a targeted contrast agent and to targeted 
contrast agents suitable for use in such techniques. More particularly the 
invention relates to the use of such contrast agents in which the 
targeting vector binds to endothelin receptors. 
The lining of the blood vessels, the endothelium, secretes various agents 
which can cause relaxation and contraction of the vessel, thereby 
controlling blood flow and pressure. Many cardiovascular diseases are 
associated with imbalances in the secretion of such agents, leading for 
example to long term structural damage. 
The endothelins (ETs), are a family of related oligopeptides which are 
generated as a result of stimuli such as hypoxia, shear stress, 
circulating hormones, growth factors and cytokines. The endothelins are 
vasoconstrictors and indeed the dominant isoform, ET-1, is one of the most 
highly potent known mammalian vasoconstrictors and is associated with 
various pathological effects in several diseases and disorders. 
ET-1 is a 21-amino acid peptide which has vasoconstrictor, 
bronchoconstrictor and mitogenic activities and is found in many cell 
types, eg. neurons, glia, vascular smooth muscle, endothelium and gastric 
mucosal cells. 
The ability of ET-1 to elevate blood pressure is related to its induction 
of vascular contraction through the activation of various hormones. This 
effect however is preceded by a transient vasodilatory effect. ET-1 can 
also act as a mitogen to induce smooth muscle cell proliferation and thus 
contributes to vascular proliferative disorders such as restenosis. Two 
types of ET receptors have been identified, ET.sub.A and ET.sub.B. The ETA 
receptors found on smooth muscle cells have high affinity for ET-1 and 
ET-2 while the ET.sub.B receptors found in endothelial cells have affinity 
for all ET isoforms. The levels of ET.sub.B receptors are higher than 
those of ET.sub.A receptors in organs such as kidney and brain, while 
ET.sub.A receptors predominate in the heart and vasculature. 
ET.sub.A and ET.sub.B receptors both contribute to vasoconstriction while 
vasodilation appears to be soley dependent on ET.sub.B receptors in 
endothelial cells. 
In view of the range of potentially harmful effects of the endothelins, 
various ET receptor antagonists have been developed, eg. for use in 
improving haemodynamics (eg. restoring blood flow), reducing inflammation, 
preventing mitogenesis, etc. Thus such antagonists have been proposed for 
use in a wide variety of treatments, eg. of unstable and variant angina, 
asthma, atherosclerosis, cerebral ischemia, myocardial isochemia, renal 
isochemia, stroke, cerebral vasospasm, congestive heart failure, vascular 
complications of diabetics, gastric cancer, gastric mucosal injury, 
hypertension (eg. pulmonary hypertension), idiopathic pulmonary fibrosis, 
migraine, nephropathy, Reynaud's disease, subarachnoid hemorrhage, and 
restenosis and other hyperproliferative vascular diseases. 
It has now been found that it is possible to image endothelin receptor 
sites in vivo using targeted contrast agents in which the targeting vector 
has affinity for endothelin-receptor sites. The endothelin receptors are 
generally located within the cardiovascular system and the 
gastrointestinal tract and are accessible to such contrast agents when 
they are administered into the blood stream or the gi tract. Accordingly, 
using such targeted contrast agents it is possible to detect diseases and 
disorders such as heart failure, atherosclerosis, restricted blood flow, 
gi tract surface damage as well as other vascular and gastric diseases and 
disorders, and also to monitor the progression of treatment for such 
diseases and disorders. 
Viewed from one aspect therefore the invention provides a composition of 
matter of formula I 
EQU V--L--R (I) 
where V is a non-peptidic organic group having binding affinity for an 
endothelin receptor site, L is a linker moiety or a bond, and R is a 
moiety detectable in in vivo imaging of a human or animal body. 
Preferably where R is a radionuclide it is an iodine bonded directly or 
indirectly to V or it is a metal ion chelated by a chelant group in L, 
and/or it is separable from V by biodegradation of an organic linker group 
L. (It should be noted that hydrogen radionuclides, while detectable in 
vitro, may not satisfy the requirement that R be detectable in in vivo 
imaging). 
In many instances, the composition of matter of formula I will be a 
characterisable compound. In others it may be a combination of compounds 
bonded or otherwise associated, eg. conjugated, with each other. For 
convenience sake, the composition of matter will be referred to 
hereinafter as an agent. 
Viewed from a further aspect the invention provides a pharmaceutical 
composition comprising an effective amount (eg. an amount effective to 
enhance image contrast in in vivo imaging) of an agent of formula I 
together with at least one pharmaceutically effective carrier or 
excipient. 
Viewed from a still further aspect the invention provides the use of an 
agent of formula I for the manufacture of a contrast medium for use in a 
method of diagnosis involving administration of said contrast medium to an 
animate subject and generation of an image of at least part of said 
subject. 
Viewed from a still further aspect the invention provides a method of 
generating an image of an animate human or non-human (preferably mammalian 
or avian) animal subject involving administering a contrast agent to said 
subject, eg. into the vascular system or the gi tract, and generating an 
image of at least a part of said subject to which said contrast agent has 
distributed, eg. by X-ray, MR, ultrasound, scintigraphic, PET, SPECT, 
electrical impedance, light or magnetometric imaging modalities, 
characterised in that as said contrast agent is used an agent of formula 
I. 
Viewed from a further aspect the invention provides a method of monitoring 
the effect of treatment of a human or non-human animal subject with a drug 
to combat or provoke effects associated with endothelin, in particular 
ET-1, said method involving administering to said subject an agent of 
formula I and detecting the uptake of said agent by endothelin receptors, 
eg. ET.sub.A and/or ET.sub.B receptors, said administration and detection 
optionally but preferably being effected repeatedly, eg. before, during 
and after treatment with said drug. 
Viewed from a yet further aspect the invention provides a process for the 
preparation of an agent of formula I, said process comprising conjugating 
(i) an organic non-peptidic compound having binding affinity for an 
endothelin receptor to (ii) a compound detectable in a diagnostic imaging 
procedure or a chelant compound and if necessary metallating chelant 
groups in the resultant conjugate with a metal ion detectable in a 
diagnostic imaging procedure. 
The agents of formula I have three characteristic components: a vector (V); 
a linker (L); and a reporter (R). The vector must have the ability to 
target the compound to endothelin receptors, the reporter must be 
detectable in an in vivo diagnostic imaging procedure; and the linker must 
couple vector to reporter, at least until the imaging procedure has been 
completed. 
Vectors 
As the vector, one may use any non-peptidic compound having affinity for 
endothelin receptors. 
Non-peptidic compounds are used as peptidic vectors will generally have 
poor biological stability and may provoke undesired ET-like responses by 
the body. By the term non-peptidic, compounds are excluded which possess a 
--CHR--CO--NH--CHR--CO--NH-- entity. Moreover, the vector may be a 
compound (such as BMS-182874) which has more pronounced affinity for one 
type of endothelin receptor than for the other type, or a compound (such 
as bosentan) which has affinity for both types of endothelin receptors. 
Compounds having more pronounced affinity for a particular type of 
endothelin receptors will generally be preferred. 
Preferably the agent is a compound which does not elicit any unacceptable 
biological response, particularly compounds which act as endothelin 
receptor antagonists and do not elicit the responses associated with 
endothelin itself, especially the blood pressure modifying and mitogenic 
responses. However, biological responses may if desired be modified by 
administration of a therapeutic agent, eg. before, at the same time or 
after administration of the agent of formula I. 
Among non-peptidic vectors, bosentan, Shionogi's 97-139, Shionogi's 50-235, 
BMS-182874, Merck's L-744453, PD-155719, PD-155080, PD-156707, Fujisawa's 
FR 901366, FR 901367, PD-160672, PD-160874, SB 209670, SB 217242, 
L-749329, L-751281, IRL 2500, IRL 2659, IRL 2796, RO 468443, RO 46-2005, 
RO 470203, BMS 193884, TBC 11251, CGS-26303, CGS-26393, CGS-27830, 
L-751281, L-747844 and L-754142 are preferred. 
Examples of suitable endothelin receptor antagonists are given in 
WO-95/03295, US-A-5420123, WO-94/27979, WO-95/03044, and EP-A-405421 and 
by Sakurawi et al. in Chem. Pharm. Bull. 44: 343-351 (1996). The 
development of appropriate non-peptidic endothelin receptor antagonists is 
discussed for example by Ferro et al. in Drugs 51: 12-27 (1996), 
Battistini et al. in TiPS 16: 217-222 (1995), and Doherty, Chapter 4 in 
"Chemical and Structural Approaches to Rational Drug Design", Ed. Weiner 
et al., CRC Press, 1995. 
Suitable non-peptidic vectors will generally be polycyclic compounds 
containing a phenyl group linked to a 5- or 6-membered carbocyclic or 
heterocyclic ring by one or two bridging groups, one of which contains or 
has pendant therefrom a heteroatom interrupted, oxo-substituted moiety 
(eg. a --SO.sub.2 --NH-- or --CO--S-- or --CO--CH.sub.2 --O-- group). 
Thus the vector compound may for example be a compound of formula II 
EQU .O slashed.--Bd--Rg (II) 
where .O slashed. is an optionally substituted phenyl group, optionally 
part of a fused polycyclic structure containing up to 4 fused rings; Bd is 
a 1 to 5 atom long optionally unsaturated organic bridge containing 
backbone atoms selected from C, N, S and O optionally carrying side chain 
substituents and optionally forming part of a fused ring, and containing a 
##STR1## 
moiety where X is C or SO, n is 0 or 1 and Y is O, S or an amine nitrogen; 
and Rg is a 5- or 6-membered saturated or unsaturated optionally 
substituted carbocyclic or heterocyclic ring, optionally part of a fused 
polycyclic structure containing up to 5 fused rings; wherein the optional 
substituents are selected from halogen and oxygen atoms, carbon and 
sulphur oxyacid groups, hydroxy, alkyl, aryl, aralkyl, acyl, alkoxy, 
alkylenedioxy, alkylthio, alkylamino, acyloxy, aralkylamino, aralkyloxy, 
acylthio, amino, and acylamino groups and combinations thereof (eg. 
haloalkyl), and wherein ring heteroatoms are selected from O, N and S, 
aryl groups are mono or bicyclic carbocyclic or heterocyclic rings 
containing up to 10 ring atoms, and no more than 7 rings and no more than 
6 fused rings are present. 
In the compounds of formula II, alkyl or alkylene groups preferably contain 
1 to 6 carbon atoms, aryl moieties preferably are phenyl or naphthyl 
groups or 5-membered heterocycles optionally carrying a fused benzene or 6 
membered aromatic heterocyclic ring, and halo atoms are preferably F, Cl, 
Br or I. 
In such compounds, the amino, hydroxy, carbonyl or oxyacid functions may 
readily be used to conjugate the vector to the reporter. 
Thus examples of such polycyclic vectors include triterpenoids of formula 
III 
##STR2## 
wherein R.sup.1 and R.sup.2, which may be the same or different, are 
hydroxy, sulphur oxyacid, acylamino, or carboxyacylamino groups (wherein 
the acyl moiety conveniently contains 1 to 6 carbons). 
Examples of such triterpenoids include compounds in which the pendant 
phenyl group is of formula 
##STR3## 
In these compounds the hydroxyl and/or OSO.sub.3 H groups on the pendant 
phenyl groups may conveniently be used for vector attachment. 
Further examples of polycyclic vectors of formula II include anthraquinone 
compounds of formula IV 
##STR4## 
(wherein each R.sup.3 which may be the same or different is hydrogen or an 
acyl group; R.sup.4 is a carboxy or acyl group; and R.sup.5 is hydrogen, 
hydroxy or an acyloxy group, the acyl groups being for example C.sub.1-6 
groups such as formyl, acetyl or propionyl). 
In these compounds, the bridging group Bg is part of the fused tetracyclic 
structure. 
Examples of vectors of formula IV include compounds wherein R.sup.3 on the 
tetracyclic ring structure are all hydrogen, the pendant alkylthio group 
is 2-carboxy-2-acetylamino-ethylthio and R.sup.5 is hydrogen (compound FR 
901366) or hydroxy (FR 901367). 
In these compounds the amino and/or carbonyl functions on the pendant 
alkylthio groups may conveniently be used for vector attachment. 
Still further examples of the polycyclic vectors include dibenzodiazepines 
of formula V 
##STR5## 
(where R.sup.6 is hydrogen or alkyl; R.sup.7 is aryl, aralkyl, 
arylcarbonyloxy or aralkylcarbonyl (eg. where the aryl moiety is a phenyl, 
naphthyl or indolyl, eg. indol-3-yl group and any alkyl moiety may have 1 
to 6 carbons); Z is an optionally oxa substituted C.sub.1-6 alkylene group 
optionally with a CH.sub.2 moiety replaced by an amine nitrogen or an 
oxygen atom (eg. a --CH.sub.2 --, --OCH.sub.2 --, 
##STR6## 
hydrogen or a group X.sub.1 as defined in U.S. Pat. No. 5,420,123; and 
R.sup.8 is hydrogen or optionally substituted C.sub.1-6 alkyl, eg. 
carboxyalkyl, aminoalkyl, sulphonyloxyalkyl, etc. group). 
Examples of R.sup.7 groups thus include phenyl, naphthyl, indol-3-yl, 
benzyl, naphthylmethyl, indole-2-carboxyl, indole-3-carboxyl and 
3-indolyl-acetyl. Examples of these dibenzodiazepines of formula V include 
compounds wherein R.sup.6 is hydrogen or propyl, R.sup.8 is carboxymethyl 
and ZR.sup.7 is indol-3yl-methyl, indol(2 or 3)yl-methylcarbonyloxy, 
indol(2 or 3)ylcarbonylamino, indol-3-yl-methylcarbonyloxy, 
indol-3-ylmethylcarbonylamino, 1-naphthyl-methylamino, 1-naphthylmethoxy, 
benzyloxy, and benzylamino. 
With such compounds one may conveniently use the R.sup.8 group for vector 
attachment. 
Still further examples of polycyclic vectors of formula I include 
N-isoxazolyl-sulphonamides, in particular sulphonamides of formula VI 
##STR7## 
(where the isoxazoyl group is attached at the 5 or 3 positions; R.sup.9 is 
an optionally substituted, optionally fused benzene ring (eg. amine or 
oxyacid substituted), a fused pyridine ring, a phenyl group, or a carboxyl 
or amino group where the ring to which it is attached is unsaturated or a 
fused 2,2-propylidene group or an oxygen atom where the ring to which it 
is attached is saturated; and R.sup.10 is a straight chain or branched 
C.sub.1-6 alkyl group or a halogen atom). 
Examples of vectors of formula VI include compounds of formula 
##STR8## 
With such compounds, vector attachment may be affected conveniently using 
active substituents in R.sup.10 or R.sup.9, eg. amine or oxyacid groups. 
Further examples of polycyclic vectors of formula II include 
phenoxyphenylacetic acid substituted benzimidazolinones of formula VII 
##STR9## 
(where R.sup.11 is hydrogen or C.sub.1-6 alkyl optionally substituted by 
oxyacid or other reactive substituents for vector linkage (eg. carboxyl); 
R.sup.12 is C.sub.1-6 alkyl optionally substituted by oxyacid or other 
reactive substituents for vector linkage; R.sup.13 is hydrogen, halogen or 
C.sub.1-6 alkyl or a group R.sup.10 as defined in WO95/03044; and R.sup.14 
is hydrogen, C.sub.1-6 alkoxy or methylenedioxy or a group R.sup.1, 
R.sup.2, R.sup.3a, R.sup.3b or R.sup.10 as defined in WO95/03044) 
Thus examples of vectors of formula VII include 
##STR10## 
Vector attachment of such compounds to linker-reporter moieties may be 
achieved using the carboxyl or other reactive substituents. 
Yet still further examples of polycyclic vector compounds of formula II 
include 1,2-diphenylethylene compounds of the formula I described in WO 
95/03295, eg. compounds of formula VIII 
##STR11## 
(where Z" is selected from alkylsulphonylaminocarbonyl, 
arylsulphonylaminocarbonyl, aryl, arylaminocarbonyl, and carboxy; R.sup.15 
is hydrogen, alkyl, oxyacid or another reactive group for vector 
attachment; R.sup.16 is hydrogen, halogen, carboxyl, C.sub.1-6 
alkoxy-carbonyl, or C.sub.1-6 alkyl, alkoxyl, hydroxy-alkyl or haloalkyl; 
and R.sup.17 is hydrogen, halogen, nitro, carboxyl, aminocarbonyl, 
acetylamino, alkoxycarbonylamino, alkoxy or alkylenedioxy). Where Z" 
contains an aryl function this may be carbocyclic (eq. phenyl) or 
heterocyclic (eq. tetrazolyl) and may be optionally substituted, eg. by 
halogen atoms, carboxy groups or C.sub.1-6 alkyl or haloalkyl groups. 
Examples of vector compounds of formula VIII include 
##STR12## 
(where each R.sup.151 independently is hydrogen, halgen or carboxy or 
C.sub.1-6 alkyl, alkoxy, hydroxy-alkyl or haloalkyl, or alkoxy-carbonyl, 
or C.sub.3-7 cycloalkyl; R.sup.16 is hydrogen or C.sub.1-6 alkyl; and each 
R.sup.171 independently is hydrogen, halogen, nitro, alkyl, alkoxy, 
aminocarbonyl, alkoxycarbonyl, alkoxycarbonylamino or acetylamino). 
Thus for example such compounds include compounds of formulae 
##STR13## 
(where each R.sup.151 is independently halogen, n-propyl or i-butyl; Z" is 
4-i-propyl-phenyl-SO.sub.2 NHCO; R.sup.16 is hydrogen or methyl; and each 
R.sup.171 is independently hydrogen, chlorine or methoxy), 
##STR14## 
(where R.sup.152 is hydrogen, carboxyl or hydroxymethyl; each R.sup.151 is 
independently hydrogen or n-propyl; Z" is carboxy, 
4-i-propyl-phenyl-SO.sub.2 NHCO, 4-t-butyl-phenyl-SO.sub.2 NHCO, 
tetrazol-5-yl or tetrazol-5-yl-NHCO; R.sup.16 is hydrogen or methyl; and 
R.sup.171 is hydrogen, chlorine or methoxy), 
##STR15## 
(where R.sup.152 is carboxy or hydroxymethyl; each R.sup.151 is 
independently hydrogen or n-propyl; Z" is carboxy, 
4-i-propyl-phenyl-SO.sub.2 NHCO, 4-i-butyl-phenyl-SO.sub.2 NHCO, 
4-bromo-phenyl-SO.sub.2 NHCO, tetrazol-5-yl, tetrazol-5-yl-NHCO or 
i-propyl-SO.sub.2 -NHCO; and R.sup.16 is hydrogen or methyl), and 
##STR16## 
(where R.sup.152 is carboxyl or hydroxymethyl; R.sup.151 is hydrogen or 
n-propyl; R.sup.16 is hydrogen or methyl; and Z" is carboxy, 
4-i-propyl-phenyl-SO.sub.2 NHCO, 4-i-butyl-phenyl-SO.sub.2 NHCO, 
4-bromo-phenyl-SO.sub.2 NHCO, tetrazol-5-yl, tetrazol-5-yl-NHCO or 
i-propyl-SO.sub.2 -NHCO; and R.sup.16 is hydrogen or methyl). 
Further examples of polycyclic vectors of formula II include 
3-benzoyl-2-phenyl -3-(benzyl or cyclohexylmethyl) propeneoic acids and 
the furan analogs of formulae IX and X 
##STR17## 
(where each R.sup.18 which may be the same or different is an alkoxy or 
alkylenedioxy group), eg. the compounds 
##STR18## 
Yet further examples of polycyclic vector compounds of formula II include 
compounds of formula XI 
##STR19## 
(where each R.sup.18 which may be the same or different is an alkoxy or 
alkylenedioxy group), eg. the compounds 
##STR20## 
Still further examples of vector compounds of formula II include 
phenylsulphonamides of formulae XII and XIII 
##STR21## 
(where R.sup.19 is C.sub.1-6 alkyl; R.sup.18 is as defined above; Z"' is O 
or CH.sub.2 ; and R.sup.20 is alkoxyaryloxy, hydroxyalkoxy, or 
alkoxyaryl), eg. compounds of formulae 
##STR22## 
Still further examples of the polycyclic vectors of formula II include the 
compounds of formula XIV 
##STR23## 
(where R.sup.19 is as defined above; R.sup.21 is a 5 or 6 ring membered 
aryl group, eg. a phenyl, thiophenyl or isoxazolyl group; and each 
R.sup.22, which may be the same or different is a hydrogen or C.sub.1-6 
alkyl group), eg. compounds of formulae 
##STR24## 
Still further examples of polycyclic vector compounds of formula II include 
the phenoxy-phenyl acetic acids of formula XV 
##STR25## 
(where R.sup.18 is as defined above; R.sup.23 is an aralkyl group, eg. 
3,6-diazaindol-1-yl-methyl group; and R.sup.24 is an aralkyl), eg. a 
compound of formula 
##STR26## 
Still further examples of polycyclic vectors of formula I include compounds 
of formula XVI 
##STR27## 
(where R.sup.25 is an oxyacid or an ester thereof, eg. an alkoxy carbonyl 
group; R.sup.18 is as defined above; and R.sup.26 is a hydrogen atom or an 
optionally substituted C.sub.1-6 alkyl group, eg. carrying a reactive 
functional group capable of use for vector attachment), eg. a compound of 
formula 
##STR28## 
Other compounds suitable for use as vectors include compounds such as those 
described in WO 95/03295, WO 95/03044, WO 94/27979, U.S. Pat. No. 
5,420,123, EP-A-405421, and WO 95/08550, and by Schin-ichi et al. in Eur. 
J. Pharmacol. -Mol. Pharmacol. 247: 219-221 (1993) and by Sakurawi et al. 
in Chem. Pharm. Bull. 44: 343-351 (1996). 
The vectors of formulae II to XVI may be prepared by the techniques 
described in the patent and other publications referred to herein, the 
disclosures of which are incorporated herein by reference. 
CAM-D and other candidate identification and evaluation techniques as 
mentioned above can also be used to find or assess further candidate 
non-peptide vectors. 
Thus it is also possible to obtain molecules that bind specifically to 
endothelin receptors by direct screening of molecular libraries. Screening 
of peptidic libraries may also be used to identify generally effective 
peptidic structures of which non-peptidic analogs may be generated by 
conventional or combinatorial chemistry. Binding moieties identified in 
this way may be coupled to a linker molecule, constituting a general tool 
for attaching any vector molecule (or molecules) to the reporter. 
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. 
As mentioned above, the agents of formula I comprise vector, linker and 
reporter moieties. A linker moiety may serve to link one vector to one 
reporter; alternatively it may link together more than vector and/or more 
than one reporter. Likewise a reporter or a vector may be linked to more 
than one linker. Use in this way of a plurality of reporters (eg. several 
linker-reporter moieties attached to one vector or several reporters 
attached to one linker itself attached to one vector) may enable the 
detectability of the contrast agent to be increased (eg. by increasing its 
radioopacity, echogenicity or relaxivity) or may enable it to be detected 
in more than one imaging modality. Use in this way of a plurality of 
vectors may increase the targeting efficiency of the contrast agent or may 
make the contrast agent able to target more than one site, eg. different 
receptors for an agent which has receptor heterogeneity. Thus for example 
the agent of formula I may include vector moieties with affinity sites 
other than endothelin receptors, eg. with affinities for cell surfaces on 
body duct wall surfaces. Accordingly, the agent may include vectors such 
as antibody fragments and oligopeptides, eg. containing RGD or analogous 
cell surface binding peptide motifs (for example as described in 
EP-A-422937 and EP-A-422938 (Merck)) or other vectors as described in GB 
9700699.3. Such extra vectors may also be selected from 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 can include amino acids, oligopeptides (e.g. hexapeptides), 
molecular recognition units (MRU's), single chain antibodies (SCA'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, clotting factors (such as PF4), polymerized 
fibrin fragments (e.g., E.sub.1), 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 particular intended target site for 
the agent. Monoclonal antibodies are preferred over polyclonal antibodies. 
Preparation of antibodies that react with a desired antigen is well known. 
Antibody preparations are available commercially from a variety of 
sources. Fibrin fragment E.sub.1 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 Beer et al. in J. Immunol. Methods 50:17 
(1982). The above described articles are incorporated herein by reference 
in their entirety. 
It is especially preferred that such extra vectors should bind so as to 
slow but not prevent the motion of the agent in the blood stream and to 
anchor it in place when it is bound to an endothelin receptor site. 
Linker 
The linker component of the contrast agent is at its simplest a bond 
between the vector and reporter moieties. More generally however the 
linker will provide a mono- or multi-molecular skeleton covalently or 
non-covalently linking one or more vectors to one or more reporters, eg. a 
linear, cyclic, branched or reticulate molecular skeleton, or a molecular 
aggregate, with in-built or pendant groups which bind covalently or 
non-covalently, eg. coordinatively, with the vector and reporter moieties 
or which encapsulate, entrap or anchor such moieties. 
Thus linking of a reporter unit to a desired vector 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. 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. 
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.2 CO-- (where X.dbd.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. 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). 
Examples of reactive moieties capable of reaction with amino groups include 
alkylating and acylating agents. Representative alkylating agents include: 
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.2 CO--(where X.dbd.Cl, Br or I), e.g. as described by Wong, 
Y-H.H. in Biochemistry (1979) 24, 5337; 
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; 
iii) arkyl halides such as reactive nitrohaloaromatic compounds; 
iv) alkyl halides as described by McKenzie, J. A. et al. in J. Protein 
Chem. (1988) 7, 581; 
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; 
vi) epoxide derivatives such as epichlorohydrin and bisoxiranes, which may 
react with amino, sulfhydryl or phenolic hydroxyl groups; 
vii) chlorine-containing derivatives of s-triazines, which are very 
reactive towards nucleophiles such as amino, sufhydryl and hydroxy groups; 
viii) aziridines based on s-triazine compounds dET.sub.A iled 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; 
ix) squaric acid diethyl esters as described by Tietze, L. F. in Chem. Ber. 
(1991) 124, 1215; and 
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. 
Representative amino-reactive acylating agents include: 
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; 
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; 
iii) Acid halides; iv) Active esters such as nitrophenylesters or 
N-hydroxysuccinimidyl esters; 
v) acid anhydrides such as mixed, symmetrical or N-carboxyanhydrides; 
vi) other useful reagents for amide bond formation as described by 
Bodansky, M. et al. in Principles of Peptide Synthesis` (1984) 
Springer-Verlag; 
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; 
viii) azlactones attached to polymers such as bisacrylamide, e.g. as 
described by Rasmussen, J. K. in Reactive Polymers (1991) 16, 199; and 
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. 
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. 
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. 
Examples of reactive moieties capable of reaction with carboxyl groups 
include diazo compounds such as diazoacetate esters and diazoacetamides, 
which react with high specificity to generate ester groups, e.g. as 
described by Herriot R. M. in Adv. Protein Chem. (1947) 3, 169. Carboxylic 
acid modifying reagents such as carbodiimides, which react through 
o-acylurea formation followed by amide bond formation, may also usefully 
be employed; linking may be facilitated through addition of an amine or 
may result in direct vector-receptor coupling. Useful water soluble 
carbodiimides include 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide 
(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. 
Other potentially useful reactive moieties include vicinal diones such as 
p-phenylenediglyoxal, which may be used to react with guanidinyl groups, 
e.g. as described by Wagner et al. in Nucleic acid Res. (1978) 5, 4065; 
and diazonium salts, which may undergo electrophilic substitution 
reactions, e.g. as described by Ishizaka, K. and Ishizaka T. in J. 
Immunol. (1960) 85, 163. Bis-diazonium compounds are readily prepared by 
treatment of aryl diamines with sodium nitrite in acidic solutions. It 
will be appreciated that functional groups in the reporter and/or vector 
may if desired be converted to other functional groups prior to reaction, 
e.g. to confer additional reactivity or selectivity. Examples of methods 
useful for this purpose include conversion of amines to carboxylic acids 
using reagents such as dicarboxylic anhydrides; conversion of amines to 
thiols using reagents such as N-acetylhomocysteine thiolactone, 
S-acetylmercaptosuccinic anhydride, 2-iminothiolane or thiol-containing 
succinimidyl derivatives; conversion of thiols to carboxylic acids using 
reagents such as a-haloacetates; conversion of thiols to amines using 
reagents such as ethylenimine or 2-bromoethylamine; conversion of 
carboxylic acids to amines using reagents such as carbodiimides followed 
by diamines; and conversion of alcohols to thiols using reagents such as 
tosyl chloride followed by transesterification with thioacetate and 
hydrolysis to the thiol with sodium acetate. 
Vector-reporter coupling may also be effected using enzymes as zero-length 
crosslinking agents; thus, for example, transglutaminase, peroxidase and 
xanthine oxidase have been used to produce crosslinked products. Reverse 
proteolysis may also be used for crosslinking through amide bond 
formation. 
Non-covalent vector-reporter coupling may, for example, be effected by 
electrostatic charge interactions, through chelation in the form of stable 
metal complexes or through high affinity binding interaction. 
A vector which is coupled to a peptide, lipooligosaccharide 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. 
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 association of the 
agent and consequent potentially increased efficacy. Alternatively, avidin 
or streptavidin may be attached directly to the surface of reporter 
particles. 
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) '.sub.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 cross-linked assemblies of 
agents of formula I of potentially increased efficacy. 
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 electrostatic 
interactions. 
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. 
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 the stability of the agent. The spacer may also contain reactive 
moieties, e.g. as described above to enhance surface crosslinking. 
Spacer elements may typically consist of aliphatic chains which effectively 
separate the reactive moieties of the linker by distances of between 5 and 
30 .ANG.. They may also comprise macromolecular structures such as 
poly(ethylene glycols). Such polymeric structures, hereinafter referred to 
as PEGS, are simple, neutral polyethers which have been given much 
attention in biotechnical and biomedical applications (see e.g. Milton 
Harris, J. (ed) "Poly(ethylene glycol) chemistry, biotechnical and 
biomedical applications" Plenum Press, New York, 1992). PEGs are soluble 
in most solvents, including water, and are highly hydrated in aqueous 
environments, with two or three water molecules bound to each ethylene 
glycol segment; this has the effect of preventing adsorption either of 
other polymers or of proteins onto PEG-modified surfaces. PEGs are known 
to be nontoxic and not to harm active proteins or cells, whilst covalently 
linked PEGs are known to be non-immunogenic and non-antigenic. 
Furthermore, PEGs may readily be modified and bound to other molecules 
with only little effect on their chemistry. Their advantageous solubility 
and biological properties are apparent from the many possible uses of PEGs 
and copolymers thereof, including block copolymers such as 
PEG-polyurethanes and PEG-polypropylenes. 
Appropriate molecular weights for PEG spacers used in accordance with the 
invention may, for example, be between 120 Daltons and 20 kDaltons. 
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 the circulation time of the agent 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). 
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. 
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 
EQU --(Z).sub.m.Y.X.C(R.sup.1 R.sup.2).X.Y.(Z).sub.n-- 
[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-92/17436 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. 
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.). 
Other potentially useful poymeric spacer materials include: 
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; 
ii) block copolymers of polymethacrylates with biodegradable polyesters 
(see e.g. San Roman, J. and Guillen-Garcia, P. in Biomaterials (1991) 12, 
236-241); 
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); 
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); 
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); 
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); 
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); 
viii) polyesters consisting of alternating units of ethylene glycol and 
terephthalic acid, e.g. Dacron.sup.R, which are non-degradable but highly 
biocompatible; 
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. Artiff. 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); 
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); 
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); 
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; 
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 
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). 
The following tables list linking agents which may be useful in targetable 
agents in accordance with the invention. 
______________________________________ 
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.2 C.sub.2 H 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 
______________________________________ 
Homobifunctional linking agents 
Linking agent 
Reactivity Comments 
______________________________________ 
BS --NH.sub.2 
BMH --SH 
BASED (1) photoreactive iodinable disulphide linker 
BSCOES --NH.sub.2 
sulfo-BSCOES --NH.sub.2 water-soluble 
DFDNB --NH.sub.2 
DMA --NH.sub.2 
DMP --NH.sub.2 
DMS --NH.sub.2 
DPDPB --SH disulphide linker 
DSG --NH.sub.2 
DSP --NH.sub.2 disulphide linker 
DSS --NH.sub.2 
DST --NH.sub.2 
sulfo-DST --NH.sub.2 water-soluble 
DTBP --NH.sub.2 disulphide linker 
DTSSP --NH.sub.2 disulphide linker 
EGS --NH.sub.2 
sulfo-EGS --NH.sub.2 water-soluble 
SPBP --NH.sub.2 
______________________________________ 
______________________________________ 
Biotinylation agents 
Agent Reactivity Comments 
______________________________________ 
biotin-BMCC --SH 
biotin-DPPE* preparation of 
biotinylated liposomes 
biotin-LC-DPPE* preparation of 
biotinylated liposomes 
biotin-HPDP --SH disulphide linker 
biotin-hydrazide carbohydrate 
biotin-LC-hydrazide carbohydrate 
iodoacetyl-LC-biotin --NH.sub.2 
NHS-iminobiotin --NH.sub.2 reduced affinity for 
avidin 
NHS-SS-biotin --NH.sub.2 disulphide linker 
photoactivatable biotin nucleic 
acids 
sulfo-NHS-biotin --NH.sub.2 water-soluble 
sulfo-NHS-LC-biotin --NH.sub.2 
______________________________________ 
Notes: DPPE = dipalmitoylphosphatidylethanolamine; LC = long chain 
Notes: DPPE=dipalmitoylphosphatidylethanolamine; LC=long chain 
______________________________________ 
Agents for protein modification 
Agent Reactivity Function 
______________________________________ 
Ellman's reagent 
--SH quantifies/detects/protects 
DTT --S.S-- reduction 
2-mercaptoethanol --S.S-- reduction 
2-mercaptylamine --S.S-- reduction 
Traut's reagent --NH.sub.2 introduces --SH 
SATA --NH.sub.2 introduces protected --SH 
AMCA-NHS --NH.sub.2 fluorescent labelling 
AMCA-hydrazide carbohydrate fluorescent labelling 
AMCA-HPDP --S.S-- fluorescent labelling 
SBF-chloride --S.S-- fluorescent detection of --SH 
N-ethylmaleimide --S.S-- blocks --SH 
NHS-acET.sub.A te --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 
______________________________________ 
In addition to the already contemplated straight chain and branched 
PEG-like linkers (e.g polyethylene glycols and other containing 2 to 100 
recurring units of ethylene oxide), linkers in the VLR system can be 
independently a chemical bond or the residue of a linking group. The 
phrase "residue of a linking group" as used herein refers to a moiety that 
remains, results, or is derived from the reaction of a vector reactive 
group with a reactive site on a vector. The phrase "vector reactive group" 
as used herein refers to any group which can react with functional groups 
typically found on vectors, the derivatization of which only minimally 
effects the ability of the vector to bind to its receptor. However, it is 
specifically contemplated that such vector reactive groups can also react 
with functional groups typically found on relevant protein molecules. 
Thus, in one aspect the linkers useful in the practice of this invention 
derive from those groups which can react with any relevant molecule which 
comprises a vector as described above containing a reactive group, whether 
or not such relevant molecule is a protein, to form a linking group. 
Preferred linking groups are derived from vector reactive groups selected 
from but not limited to: 
a group that will react directly with carboxy, aldehyde, amine (NHR), 
alcohols, sulfhydryl groups, activated methylenes and the like, on the 
vector, for example, active halogen containing groups including, for 
example, chloromethylphenyl groups and chloroacetyl [ClCH.sub.2 
C(.dbd.O)--] groups, activated 2-(leaving group substituted)-ethylsulfonyl 
and ethylcarbonyl groups such as 2-chloroethylsulfonyl and 
2-chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl; epoxy; isocyanato; 
isothiocyanato; aldehyde; aziridine; succinimidoxycarbonyl; activated acyl 
groups such as carboxylic acid halides; mixed anhydrides and the like. 
A group that can react readily with modified vector molecules containing a 
vector reactive group, i.e., vectors containing a reactive group modified 
to contain reactive groups such as those mentioned in (1) above, for 
example, by oxidation of the vector to an aldehyde or a carboxylic acid, 
in which case the "linking group"can be derived from reactive groups 
selected from amino, alkylamino, arylamino, hydrazino, alkylhydrazino, 
arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, 
sulfhydryl, sulfhydrylalkyl, sulfhydrylaryl, hydroxy, carboxy, 
carboxyalkyl and carboxyaryl. The alkyl portions of said linking groups 
can contain from 1 to about 20 carbon atoms. The aryl portions of said 
linking groups can contain from about 6 to about 20 carbon atoms; and 
a group that can be linked to the vector containing a reactive group, or to 
the modified vector as noted above by use of a crosslinking agent. The 
residues of certain useful crosslinking agents, such as, for example, 
homobifunctional and heterobifunctional gelatin hardeners, bisepoxides, 
and bisisocyanates can become a part of a linking group during the 
crosslinking reaction. Other useful crosslinking agents, however, can 
facilitate the crosslinking, for example, as consumable catalysts, and are 
not present in the final conjugate. Examples of such crosslinking agents 
are carbodiimide and carbamoylonium crosslinking agents as disclosed in 
U.S. Pat. No. 4,421,847 and the ethers of U.S. Pat. No. 4,877,724. With 
these crosslinking agents, one of the reactants such as the vector must 
have a carboxyl group and the other such as a long chain spacer must have 
a reactive amine, alcohol, or sulfhydryl group. In amide bond formation, 
the crosslinking agent first reacts selectively with the carboxyl group, 
then is split out during reaction of the thus "activated" carboxyl group 
with an amine to form an amide linkage between thus covalently bonding the 
two moieties. An advantage of this approach is that crosslinking of like 
molecules, e.g.,vector to vector is avoided, whereas the reaction of, for 
example, homo-bifunctional crosslinking agents is nonselective and 
unwanted crosslinked molecules are obtained. 
Preferred useful linking groups are derived from various heterobifunctional 
cross-linking reagents such as those listed in the Pierce Chemical Company 
Immunotechnology Catalog--Protein Modification Section, (1995 and 1996). 
Useful non-limiting examples of such reagents include: 
Sulfo-SMCC Sulfosuccinimidyl 4-(N-maleimidomethyl) 
cyclohexane-1-carboxylate. 
Sulfo-SIAB Sulfosuccinimidyl (4-iodoacetyl) aminobenzoate. 
Sulfo-SMPB Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate. 
2-IT 2-Iminothiolane. 
SATA N-Succinimidyl S-acetylthioacetate. 
In addition to the foregoing description, the linking groups, in whole or 
in part, can also be comprised of and derived from complementary sequences 
of nucleotides and residues of nucleotides, both naturally occurring and 
modified, preferably non-self-associating oligonucleotide sequences. 
Particularly useful, non-limiting reagents for incorporation of modified 
nucleotide moieties containing reactive functional groups, such as amine 
and sulfhydryl groups, into an oligonucleotide sequence are commercially 
available from, for example, Clontech Laboratories Inc. (Palo Alto Calif.) 
and include Uni-Link AminoModifier (Catalog #5190), Biotin-ON 
phosphoramidite (Catalog #5191), N-MNT-C6-AminoModifier (Catalog #5202), 
AminoModifier II (Catalog #5203), DMT-C6-3'Amine-ON (Catalog #5222), 
C6-ThiolModifier (Catalog #5211), and the like. In one aspect, linking 
groups of this invention are derived from the reaction of a reactive 
functional group such as an amine or sulfhydryl group as are available in 
the above Clontech reagents, one or more of which has been incorporated 
into an oligonucleotide sequence, with, for example, one or more of the 
previously described vector reactive groups such as a heterobifunctional 
group on the vector. 
By attaching two complementary oligonucleotide sequences one to the vector 
and the other to the reporter the resulting double-stranded hybridized 
oligonucleotide then comprises the linking group between the vector and 
reporter. 
Other polymer systems that serve as linkers include: 
Poly(L or D or DL-amino acids)=proteins and peptides; naturally occuring or 
synthetic 
Pseudo Poly(amino acids)=(amino acids linked by non-amide bonds) 
Poly (L or D or DL-lactide) and the co-polymers e.g Poly 
(L-lactide/DL-lactide)Poly (glycolide) 
L-lactide/glycolide co-polymers 
Poly-caprolactone and its co-polymers 
Polyanhydrides 
Poly (ortho esters) 
Polyphosphazenes 
Long-chain straight or branched lipids (& phospholipids) Sugars and 
carbohydrates 
Oligonucleotides (see above) as well as mixtures of the above. 
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. 
The present invention accordingly 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. 
Therapeutic compounds used in accordance with the present invention may be 
encapsulated in the interior of a molecular aggregate or particulate 
linker or attached to or incorporated in the encapsulating walls of a 
vesicular linker. Thus, the therapeutic compound may be linked to a part 
of the surface, for example through covalent or ionic bonds, or may be 
physically mixed into an encapsulating material, particularly if the drug 
has similar polarity or solubility to the 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 where the linker-reporter is a gas containing vesicle. 
The therapeutic substance may be covalently linked to the encapsulating 
membrane surface of a vesicular linker using a suitable linking agent, 
e.g. as described herein. Thus, for example, one may initially prepare a 
phospholipid 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 vesicle membrane, as described above. 
Alternatively, the agent may initially be prepared without the 
therapeutic, which may then be coupled to or coated onto particulate (eg. 
vesicular) agents prior to use. Thus, for example, a therapeutic could be 
added to a suspension of microbubbles in aqueous media and shaken in order 
to attach or adhere the therapeutic to the microbubbles. 
The therapeutic may for example be a drug or prodrug known for use in 
combatting restenosis, hypertension, stroke, congestive heart failure or 
other cadiovascular therapy, or any other drug or prodrug having 
ET-combatting effects. 
By targeting an agent according to the invention containing a 
prodrug-activating enzyme to areas of pathology one may image targeting of 
the enzyme, making it possible to visualise when the agent is targeted 
properly and when the agent has disappeared from non-target areas. In this 
way one can determine the optimal time for injection of prodrug into 
individual patients. 
Another alternative is to incorporate a prodrug, a prodrug-activating 
enzyme and a vector in the same particulate linker reporter in such a way 
that the prodrug will only be activated after some external stimulus. Such 
a stimulus may, for example, be a bursting of vesicles by external 
ultrasound, light stimulation of a chromophoric reporter, or magnetic 
heating of a superparamagnetic reporter after the desired targeting has 
been achieved. 
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 agent of the invention; 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. 
Therapeutics may easily be delivered in accordance with the invention to 
the heart and vasculature in general, and to the liver, spleen and kidneys 
and other regions such as the lymph system, body cavities or 
gastrointestinal system. 
By way of example, where the reporter is a chelated metal species (eg. a 
paramagnetic metal ion or a metal radionuclide), the linker may comprise a 
chain attached to a metal chelating group, a polymeric chain with a 
plurality of metal chelating groups pendant from the molecular backbone or 
incorporated in the molecular backbone, a branched polymer with metal 
chelating groups at branch termini (eg. a dendrimeric polychelant), etc. 
What is required of the linker is simply that it bind the vector and 
reporter moieties together for an adequate period. By adequate period is 
meant a period sufficient for the contrast agent to exert its desired 
effects, eg. to enhance contrast in vivo during a diagnostic imaging 
procedure. 
Thus, in certain circumstances, it may be desirable that the linker 
biodegrade after administration. By selecting an appropriately 
biodegradable linker it is possible to modify the biodistribution and 
bioelimination patterns for the vector and/or reporter. Where vector 
and/or reporter are biologically active or are capable of exerting 
undesired effects if retained after the imaging procedure is over, it may 
be desirable to design in linker biodegradability which ensures 
appropriate bioelimination or metabolic breakdown of the vector and/or 
reporter moieties. Thus a linker may contain a biodegradable function 
which on breakdown yields breakdown products with modified biodistribution 
patterns which result from the release of the reporter from the vector or 
from fragmentation of a macromolecular structure. By way of example for 
linkers which carry chelated metal ion reporters it is possible to have 
the linker incorporate a biodegradable function which on breakdown 
releases an excretable chelate compound containing the reporter. 
Accordingly, biodegradable functions may if desired be incorporated within 
the linker structure, preferably at sites which are (a) branching sites, 
(b) at or near attachment sites for vectors or reporters, or (c) such that 
biodegradation yields physiologically tolerable or rapidly excretable 
fragments. 
Examples of suitable biodegradable functions include ester, amide, double 
ester, phosphoester, ether, thioether, guanidyl, acetal and ketal 
functions. 
As discussed above, the linker group may if desired have built into its 
molecular backbone groups which affect the biodistribution of the contrast 
agent or which ensure appropriate spatial conformation for the contrast 
agent, eg. to allow water access to chelated paramagnetic metal ion 
reporters. By way of example the linker backbone may consist in part or 
essentially totally of one or more polyalkylene oxide chains. 
Thus the linker may be viewed as being a composite of optionally 
biodegradable vector binding (V.sub.b) and reporter binding (R.sub.b) 
groups joined via linker backbone (L.sub.b) groups, which linker backbone 
groups may carry linker side chain (L.sub.sc) groups to modify 
biodistribution etc. and may themselves incorporate biodegradable 
functions. The R.sub.b and V.sub.b inding groups may be pendant from the 
linker backbone or may be at linker backbone termini, for example with one 
R.sub.b or V.sub.b group at one L.sub.b terminus, with R.sub.b or V.sub.b 
groups linking together two L.sub.b termini or with one L.sub.b terminus 
carrying two or more R.sub.b or V.sub.b groups. The L.sub.b and L.sub.sc 
groups will conveniently be oligomeric or polymeric structures (eg. 
polyesters, polyamides, polyethers, polyamines, oligopeptides, 
polypeptides, oligo and polysaccharides, oligonucleotides, etc.), 
preferably structures having at least in part a hydrophilic or lipophilic 
nature, eg. hydrophilic, amphiphilic or lipophilic structures. 
The linker may be low, medium or high molecular weight, eg. up to 2MD. 
Generally higher molecular weight linkers will be preferred if they are to 
be loaded with a multiplicity of vectors or reporters or if it is 
necessary to space vector and reporter apart, or if the linker is itself 
to serve a role in the modification of biodistribution. In general however 
linkers will be from 100 to 100,000 D, especially 120 D to 20 kD in 
molecular weight. 
Conjugation of linker to vector and linker to reporter may be by any 
appropriate chemical conjugation technique, eg. covalent bonding (for 
example ester or amide formation), metal chelation or other metal 
coordinative or ionic bonding, again as described above. 
Examples of suitable linker systems include the magnifier polychelant 
structures of U.S. Pat. No. 5,364,613 and WO90/12050, polyaminoacids (eg. 
polylysine), functionalised PEG, polysaccharides, glycosaminoglycans, 
dendritic polymers such as described in WO93/06868 and by Tomalia et al. 
in Angew. Chem. Int. Ed. Engl. 29:138-175 (1990), PEG-chelant polymers 
such as described in W94/08629, WO94/09056 and WO96/26754, etc. 
Where the reporter is a chelated metal ion, the linker group will generally 
incorporate the chelant moiety. Alternatively, the chelated metal may be 
carried on or in a particulate reporter. In either case, conventional 
metal chelating groups such as are well known in the fields of 
radiopharmaceuticals and MRI contrast media may be used, eg. linear, 
cyclic and branched polyaminopolycarboxylic acids and phosphorus oxyacid 
equivalents, and other sulphur and/or nitrogen ligands known in the art, 
eg. DTPA, DTPA-BMA, EDTA, DO3A, TMT (see for example U.S. Pat. No. 
5,367,080), BAT and analogs (see for example Ohmono et al., J. Med. Chem. 
35: 157-162 (1992) and Kung et al. J. Nucl. Med. 25: 326-332 (1984)), the 
N.sub.2 S.sub.2 chelant ECD of Neurolite, MAG (see Jurisson et al. Chem. 
Rev. 93: 1137-1156 (1993)), HIDA, DOXA 
(1-oxa-4,7,10-triazacyclododecanetriacetic acid), NOTA 
(1,4,7-triazacyclononanetriacetic acid), TETA 
(1,4,8,11-tetraazacyclotetradecanetetraacetic acid), THT 
4'-(3-amino-4-methoxy-phenyl)-6,6"-bis(N',N'-dicarboxymethyl-N-methylhydra 
zino)-2,2':6',2"-terpyridine), etc. In this regard, the reader is referred 
to the patent literature of Sterling Winthrop, Nycomed (including Nycomed 
Imaging and Nycomed Salutar), Schering, Mallinckrodt, Bracco and Squibb 
relating to chelating agents for diagnostic metals, eg. in MR, X-ray and 
radiodiagnostic agents. See for example U.S. Pat. No. 4,647,447, 
EP-A-71564, U.S. Pat. No. 4,687,659, WO89/00557, U.S. Pat. No. 4,885,363, 
and EP-A-232751. 
Reporters 
The reporter moieties in the contrast agents of the invention may be any 
moiety capable of detection either directly or indirectly in an in vivo 
diagnostic imaging procedure, eg. moieties which emit or may be caused to 
emit detectable radiation (eg. by radioactive decay, fluore scence 
excitation, spin resonance excitation, etc.), moieties which affect local 
elec tromagnetic fields (eg. paramagnetic, superparamagnetic, 
ferrimagnetic or ferromagnetic species), moieties which absorb or scatter 
radiation energy (eg. chromophores, particles (including gas or liquid 
containing vesicles), heavy elements and compounds thereof, etc.), and 
moieties which generate a detectable substance (eg. gas microbubble 
generators), etc. 
A very wide range of materials detectable by diagnostic imaging modalities 
is known from the art and the reporter will be selected according to the 
imaging modality to be used. Thus for example for ultrasound imaging an 
echogenic material, or a material capable of generating an echogenic 
material will normally be selected, for X-ray imaging the reporter will 
generally be or contain a heavy atom (eg. of atomic weight 38 or above), 
for MR imaging the reporter will either be a non zero nuclear spin isotope 
(such as .sup.19 F) or a material having unpaired electron spins and hence 
paramagnetic, superparamagnetic, ferrimagnetic or ferromagnetic 
properties, for light imaging the reporter will be a light scatterer (eg. 
a coloured or uncoloured particle), a light absorber or a light emitter, 
for magnetometric imaging the reporter will have detectable magnetic 
properties, for electrical impedance imaging the reporter will affect 
electrical impedance and for scintigraphy, SPECT, PET etc. the reporter 
will be a radionuclide. 
Examples of suitable reporters are widely known from the diagnostic imaging 
literature, eg. magnetic iron oxide particles, gas-containing vesicles, 
chelated paramagnetic metals (such as Gd, Dy, Mn, Fe etc.). See for 
example U.S. Pat. No. 4,647,447, WO97/25073, U.S. Pat. No. 4,863,715, U.S. 
Pat. No. 4,770,183, WO96/09840, WO85/02772, WO92/17212, WO97/29783, 
EP-A-554213, U.S. Pat. No. 5,228,446, WO91/15243, WO93/05818, WO96/23524, 
WO96/17628, U.S. Pat. No. 5,387,080, WO95/26205, GB9624918.0, etc. 
Particularly preferred as reporters are: chelated para magnetic metal ions 
such as Gd, Dy, Fe, and Mn, especially when chelated by macrocyclic 
chelant groups (eg. tetraazacyclododecane chelants such as DOTA, DO3A, 
HP-DO3A an d analogues thereof) or by linker chelant groups such as DTPA, 
DTPA-BMA, EDTA, DPDP, etc; metal radionuclide such as .sup.90 Y, .sup.99m 
Tc, .sup.111 In, .sup.47 Sc, .sup.67 /Ga , .sup.51 Cr, .sup.177m Sn, 
.sup.67 Cu, .sup.167 Tm, .sup.97 Ru, .sup.188 Re, .sup.177 Lu, .sup.199 
Au, .sup.203 Pb and .sup.141 Ce; superparamagnetic iron oxide crystals; 
chromophores and fluorophores having absorption and/or emission maxima in 
the range 300-1400 nm, especially 600 nm to 1200 nm, in particular 650 to 
1000 nm; vesicles containing fluorinated gases (ie. containing mater ials 
in the gas phase at 37.degree. C. which are fluorine containing, eg. 
SF.sub.6 or perfluorinated C.sub.1-6 hydrocarbons or other gases and gas 
precursors listed in WO97/29783); chelated heavy metal cluster ions (eg. W 
or Mo polyoxoanions or the sulphur or mixed oxygen/sulphur analogs); 
covalently bonded non-metal atoms which are either high atomic number (eg. 
iodine) or are radioactive, eg .sup.123 I, .sup.131 I, etc. atoms; 
iodinated compound containing vesicles; etc. 
Stated generally, the reporter may be (1) a chelatable metal or polyatomic 
metal-containing ion (ie. TcO, etc), w here the metal is a high atomic 
number metal (eg. atomic number greater than 37), a paramagentic species 
(eg. a transition metal or lanthanide), or a radioactive isotope, (2) a 
covalently bound non-metal species which is an unpaired electron site (eg. 
an oxygen or carbon in a persistent free radical), a high atomic number 
non-metal, or a radioisotope, (3) a polyatomic cluster or crystal 
containing high atomic number atoms, displaying cooperative magnetic 
behaviour (eg. superparamagnetism, ferrimagnetism or ferromagnetism) or 
containing radionuclides, (4) a gas or a gas precursor (ie. a material or 
mixture of materials which is gaseous at 37.degree. C.), (5) a chromophore 
(by which term species which are fluorescent or phosphorescent are 
included), eg. an inorganic or organic structure, particularly a complexed 
metal ion or an organic group having an extensive delocalized electron 
system, or (6) a structure or group having electrical impedance varying 
characteristics, eg. by virtue of an extensive delocalized electron 
system. 
Examples of particular preferred reporter groups are described in more 
detail below. 
Chelated metal reporters: metal radionuclides, paramagnetic metal ions, 
fluorescent metal ions, heavy metal ions and cluster ions 
Preferred metal radionuclides include .sup.90 y, .sup.99m Tc, .sup.111 In, 
.sup.47 Sc, .sup.67 Ga, .sup.51 Cr, .sup.117m Sn, .sup.67 Cu, .sup.167 Tm, 
.sup.97 Ru, .sup.188 Re, .sup.177 Lu, .sup.199 Au, .sup.203 Pb and 
.sup.141 Ce. 
Preferred paramagnetic metal ions include ions of transition and lanthanide 
metals (eg. metals having atomic numbers of 6 to 9, 21-29, 42, 43, 44, or 
57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, 
Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, especially of Mn, Cr, Fe, 
Gd and Dy, more especially Gd. 
Preferred fluorescent metal ions include lanthanides, in particular La, Ce, 
Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu. Eu is especially 
preferred. 
Preferred heavy metal-containing reporters may include atoms of Mo, Bi, Si, 
and W, and in particular may be polyatomic cluster ions (eg. Bi compounds 
and W and Mo oxides) as described in WO91/14460, WO92/17215, WO96/40287, 
and WO96/22914. 
The metal ions are desirably chelated by chelant groups on the linker 
moiety or in or on a p article, (eg. a vesicle or a porous or non-porous 
inorganic or organic solid), in particular linear, macrocyclic, 
terpyridine and N.sub.2 S.sub.2 chelants, such as for example DTPA, 
DTPA-BMA, EDTA, D03A, TaT. Further examples of suitable chelant groups are 
disclosed in U.S. Pat. No. 4,647,447, WO89/00557, U.S. Pat. No. 5,367,080, 
U.S. Pat. No. 5,364,613, etc. 
The linker moiety or the particle may contain one or more such chelant 
groups, if desired metallated by more than one metal species (eg. so as to 
provide reporters detectable in different imaging modalities). 
Particularly where the metal is non-radioactive, it is preferred that a 
polychelant linker or particulate reporter be used. 
A chelant or chelating group as referred to herein may comprise the residue 
of one or more of a wide variety of cheating agents that can complex a 
metal ion or a polyatomic ion (eg. TcO). 
As is well known, a chelating a gent is a compound containing donor atoms 
that can combine by coordinate bonding with a metal atom to form a cyclic 
structure called a chelation complex or chelate. This class of compounds 
is described in the Kirk-Other Encyclopedia of Chemical Technology, Vol. 
5, 339-368. 
The residue of a suitable chelating agent can be selected from 
polyphosphates, such as sodium tripolyphosphate and hexametaphosphoric 
acid; aminocarboxylic acids, such as ethylenediaminetetraacetic acid, 
N-(2-hydroxy)ethylene-diaminetriacetic acid, nitrilotriacetic acid, 
N,N-di(2-hydroxyethyl)glycine, ethylenebis(hydroxyphenylglycine) and 
diethylenetriamine pentacetic acid; 1,3-diketones, such as acetylacetone, 
trifluoroacetylacetone, and thenoyltrifluoroacetone; hydroxycarboxylic 
acids, such as tartaric acid, citric acid, gluconic acid, and 
5-sulfosalicyclic acid; polyamines, such as ethylenediamine, 
diethylenetriamine, triethylenetetraamine, and triaminotriethylamine; 
aminoalcohols, such as triethanolamine and 
N-(2-hydroxyethyl)ethylenediamine; aromatic heterocyclic bases, such as 
2,2'-diimidazole, picoline amine, dipicoline amine and 
1,10-phenanthroline; phenols, such as salicylaldehyde, 
disulfopyrocatechol, and chromotropic acid; aminophenols, such as 
8-hydroxyquinoline and oximesulfonic acid; oximes, such as 
dimethylglyoxime and salicylaldoxime; peptides containing proximal 
chelating functionality such as polycysteine, polyhistidine, polyaspartic 
acid, polyglutamic acid, or combinations of such amino acids; Schiff 
bases, such as disalicylaldehyde 1,2-propylenediimine; tetrapyrroles, such 
as tetraphenylporphin and phthalocyanine; sulfur compounds, such as 
toluenedithiol, meso-2,3-dimercaptosuccinic acid, dimercaptopropanol, 
thioglycolic acid, potassium ethyl xanthate, sodium 
diethyldithiocarbamate, dithizone, diethyl dithiophosphoric acid, and 
thiourea; synthetic macrocyclic compounds, such as dibenzo[18]crown-6, 
(CH.sub.3).sub.6 -[14]-4,11]-diene-N.sub.4, and (2.2.2-cryptate); 
phosphonic acids, such as nitrilotrimethylene-phosphonic acid, 
ethylenediaminetetra(methylenephosphonic acid), and 
hydroxyethylidenediphosphonic acid, or combinations of two or more of the 
above agents. The residue of a suitable chelating agent preferably 
comprises a polycarboxylic acid group and preferred examples include: 
ethylenediamine-N,N,N',N'-tetraacetic acid (EDTA); 
N,N,N',N",N"-diethylene-triaminepentaacetic acid (DTPA); 
1,4,7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetic acid (DOTA); 
1,4,7,10-tetraazacyclododecane-N,N',N"-triacetic acid (DO3A); 
1-oxa-4,7,10-triazacyclododecane-N,N',N"-triacetic acid (OTTA); 
trans(1,2)-cyclohexanodiethylene-triamine-pentaacetic acid (CDTPA). 
Other suitable residues of chelating agents comprise proteins modified for 
the chelation of metals such as technetium and rhenium as described in 
U.S. Pat. No. 5,078,985, the disclosure of which is hereby incorporated by 
reference. 
Suitable residues of chelating agents may also derive from N3S and N2S2 
containing compounds, as for example, those disclosed in U.S. Pat. Nos. 
4,444,690; 4,670,545; 4,673,562; 4,897,255; 4,965,392; 4,980,147; 
4,988,496; 5,021,556 and U.S. Pat. No. 5,075,099. 
Other suitable residues of chelating are described in PCT/US91/08253, the 
disclosure of which is hereby incorporated by reference. 
Preferred chelating groups are selected from the group consisting of 
2-amiomethylpyridine, iminoacetic acid, iminodiacetic acid, 
ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 
(DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), 
carbonyliminodiacetic acid, methyleneiminoacetic acid, 
methyleneiminodiacetic acid, ethylenethioethylene-iminoacetic acid, 
ethylenethioethyleneiminodiacetic acid, TMT, a terpyridinyl group, a 
chelating agent comprising a terpyridyl group and a carboxymethylamino 
group, or a salt of any of the foregoing acids. Especially preferred 
chelating groups are DTPA, DTPA-BMA, DPDP, TMT, DOTA and HPDO3A. 
Representative chelating groups are also described in U.S. Pat. No. 
5,559,214 A, WO 95/26754, WO 94/08624, WO 94/09056, WO 94/29333, WO 
94/08624, WO 94/08629 A1, WO 94/13327 A1 and WO 94/12216 A1. 
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. 
Thus it is desirable that the metal ion be easily complexed to the 
chelating agent, for example, by merely exposing or mixing an aqueous 
solution of the chelating agent-containing moiety with a metal salt in an 
aqueous solution preferably having a pH in the range of about 4 to about 
11. The salt can be any salt, but preferably the salt is a water soluble 
salt of the metal such as a halogen salt, and more preferably such salts 
are selected so as not to interfere with the binding of the metal ion with 
the chelating agent. The chelating agent-containing moiety is preferrably 
in aqueous solution at a pH of between about 5 and about 9, more 
preferably between pH about 6 to about 8. The chelating agent-containing 
moiety can be mixed with buffer salts such as citrate, acetate, phosphate 
and borate to produce the optimum pH. Preferably, the buffer salts are 
selected so as not to interfere with the subsequent binding of the metal 
ion to the chelating agent. 
In diagnostic imaging, the vector-linker-reporter (VLR) construct 
preferably contains a ratio of metal radionuclide ion to chelating agent 
that is effective in such diagnostic imaging applications. In preferred 
embodiments, the mole ratio of metal ion per chelating agent is from about 
1:1,000 to about 1:1. 
In radiotherapeutic applications, the VLR preferably contains a ratio of 
metal radionuclide ion to chelating agent that is effective in such 
therapeutic applications. In preferred embodiments, the mole ratio of 
metal ion per chelating agent is from about 1:100 to about 1:1. The 
radionuclide can be selected, for example, from radioisotopes of Sc, Fe, 
Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Ru, Sn, Sr, Sm, Lu, Sb, W, Re, Po, 
Ta and Tl. Preferred radionuclides include .sup.44 Sc, .sup.64 Cu, .sup.67 
Cu, .sup.212 Pb, .sup.68 Ga, .sup.90 Y, .sup.153 Sm, .sup.212 Bi , 
.sup.186 Re and .sup.188 Re. Of these, especially preferred is 90Y. These 
radioisotopes can be atomic or preferably ionic. 
The following isotopes or isotope pairs can be used for both imaging and 
therapy without having to change the radiolabeling methodology or 
chelator: .sup.47 Sc.sub.21 ; .sup.141 Ce.sub.58 ; .sup.188 Re.sub.75 ; 
.sup.177 Lu.sub.71 ; .sup.199 Au.sub.79 ; .sup.47 Sc.sub.21 ; .sup.131 
I.sub.53 ; .sup.67 Cu.sub.29 ; .sup.131 I.sub.53 and .sup.123 I.sub.53 ; 
.sup.188 Re.sub.75 and .sup.99m Tc.sub.43 ; .sup.90 Y.sub.39 and .sup.87 
Y.sub.39; .sup.47 Sc.sub.21, and .sup.44 Sc.sub.21 ; .sup.90 Y.sub.39 and 
.sup.123 I.sub.53; .sup.146 Sm.sub.62 and .sup.153 Sm.sub.62 ; and .sup.90 
Y.sub.39 and .sup.111 In.sub.49. 
Where the linker moiety contains a single chelant, that chelant may be 
attached directly to the vector moiety, eg. via one of the metal 
coordinating groups of the chelant which may form an ester, amide, 
thioester or thioamide bond with an amine, thiol or hydroxyl group on the 
vector. Alternatively the vector and chelant may be directly linked via a 
functionality attached to the chelant backbone, eg. a CH.sub.2 -phenyl-NCS 
group attached to a ring carbon of DOTA as proposed by Meares et al. in 
JACS 110:6266-6267(1988), or indirectly via a homo or hetero-bifunctional 
linker, eg. a bis amine, bis epoxide, diol, diacid, difunctionalised PEG, 
etc. In that event, the bifunctional linker will conveniently provide a 
chain of 1 to 200, preferably 3 to 30 atoms between vector and chelant 
residue. 
Where the linker moiety contains a plurality of chelant groups, the linker 
preferably is or contains portions of formula 
##STR29## 
where Ch is a chelant moiety and Li is a linker backbone component, ie. 
the linker preferably has pendant chelants, in-backbone chelants or 
terminal chelants or a combination thereof. The pendant and in-backbone 
polymeric structures may be branched but more preferably are linear and 
the repeat units (LiCh) or other repeat units in the polymer may have 
in-backbone or pendant biodistribution modifying groups, eg. polyalkylene 
groups as in WO94/08629, WO94/09056, and WO96/20754. The terminal chelant 
structures Li(Ch).sub.n, which may be dendritic polymers as in WO93/06868, 
may have biodistribution modifying groups attached to termini not occupied 
by chelants and may have biodegradation enhancing sites within the linker 
structure as in WO95/28966. 
The chelant moieties within the polychelant linker may be attached via 
backbone functionalization of the chelant or by utilization of one or more 
of the metal coordinating groups of the chelant or by amide or ether bond 
formation between acid chelant and an amine or hydroxyl carrying linker 
backbone, eg. as in polylysine-polyDTPA, polylysine-polyDOTA and in the 
so-called magnifier polychelants, of WO90/12050. Such polychelant linkers 
may be conjugated to one or more vector groups either directly (eg. 
utilizing amine, acid or hydroxyl groups in the polychelant linker) or via 
a bifunctional linker compound as discussed above for monochelant linkers. 
Where the chelated species is carried by a particulate (or molecular 
aggregate, eg. vesicular) linker, the chelate may for example be an 
unattached mono or polychelate (such as Gd DTPA-BMA or Gd HP-DO3A) 
enclosed within the particle or it may be a mono or polychelate conjugated 
to the particle either by covalent bonding or by interaction of an anchor 
group (eg. a lipophilic group) on the mono/polychelate with the membrane 
of a vesicle (see for example WO96/11023). 
Non-metal Atomic Reporters 
Preferred non-metal atomic reporters include radioisotopes such as .sup.123 
I and .sup.131 I as well as non zero nuclear spin atoms such as .sup.18 F, 
and heavy atoms such as I. 
Such reporters, preferably a plurality thereof, eg. 2 to 200, may be 
covalently bonded to a linker backbone, either directly using conventional 
chemical synthesis techniques or via a supporting group, eg. a 
triiodophenyl group. 
In an embodiment of this invention, the use of radioisotopes of iodine is 
specifically contemplated. For example, if the vector or linker is 
comprised of substituents that can be chemically substituted by iodine in 
a covalent bond forming reaction, such as, for example, substituents 
containing hydroxyphenyl functionality, such substituents can be labeled 
by methods well known in the art with a radioisotope of iodine. The iodine 
species can be used in therapeutic and diagnostic imaging applications. 
While, at the same time, a metal in a chelating agent on the same 
vector-linker can also be used in either therapeutic or diagnostic imaging 
applications. 
As with the metal chelants discussed above, such metal atomic reporters may 
be linked to the linker or carried in or on a particulate linker, eg. in a 
vesicle (see WO95/26205 and GB9624918.0). 
Linkers of the type described above in connection with the metal reporters 
may be used for non-metal atomic reporters with the non-metal atomic 
reporter or groups carrying such reporters taking the place of some or all 
of the chelant groups. 
Organic Chromophoric or Fluorophoric Reporters 
Preferred organic chromophoric and fluorophoric reporters include groups 
having an extensive delocalized electron system, eg. cyanines, 
merocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, 
porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconium 
dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, 
benzothiaphenothiazinium dyes, anthraquinones, napthoquinones, 
indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, 
intramolecular and intermolecular charge-transfer dyes and dye complexes, 
tropones, tetrazines, bis(dithiolene) complexes, bis(benzene-dithiolate) 
complexes, iodoaniline dyes, bis(S,O-dithiolene) complexes, etc. Examples 
of suitable organic or metallated organic chromophores may be found in 
"Topics in Applied Chemistry: Infrared absorbing dyes" Ed. M. Matsuoka, 
Plenum, NY 1990, "Topics in Applied Chemistry: The Chemistry and 
Application of Dyes", Waring et al., Plenum, NY, 1990, "Handbook of 
Fluorescent Probes and Research Chemicals" Haugland, Molecular Probes Inc, 
1996, DE-A-4445065, DE-A-4326466, JP-A-3/228046, 
Narayanan et al. J. Org. Chem. 60: 2391-2395 (1995), Lipowska et al. 
Heterocyclic Comm. 1: 427-430 (1995), Fabian et al. Chem. Rev. 92: 1197 
(1992), WO96/23525, Strekowska et al. J. Org. Chem. 57: 4578-4580 (1992), 
and WO96/17628. Particular examples of chromophores which may be used 
include xylene cyanole, fluorescein, dansyl, NBD, indocyanine green, 
DODCI, DTDCI, DOTCI and DDTCI. 
Particularly preferred are groups which have absorption maxima between 600 
and 1000 nm to avoid interference with haemoglobin absorption (eg. xylene 
cyanole). 
Further such examples include: 
cyanine dyes: such as heptamethinecyanine dyes, e.g. compounds 4a to 4g 
Table II on page 26 of Matsuoka (supra) 
______________________________________ 
#STR30## 
4a: where Y = S, X = I, R = Et 
4b: where Y = S, X = ClO.sub.4, R = Et 
4c: where Y = Cme.sub.2, X = I, R = Me 
4d: where Y = CMe.sub.2, X = ClO.sub.4, R = Me 
4e: where Y = CH.dbd.CH, X = I, R = Et 
4f: where Y = CH.dbd.CH, X = Br, R = Et 
4g: where Y = CH.dbd.CH, X = ClO.sub.4, R = Et 
______________________________________ 
and in Table III on page 28 of Matsuoka (supra), i.e. 
______________________________________ 
#STR31## 
where Y = C, X = I, R = Me 
where Y = CMe.sub.2, X = I, R = Me 
where Y = S, X = Br R = Et; 
______________________________________ 
chalcogenopyrylomethine dyes, e.g., compounds 12 on page 31 of Matsuoka 
(supra), i.e. 
##STR32## 
monochalcogenopyrylomethine dyes, e.g. compounds 13 on page 31, of 
Matsuoka (supra) i.e. 
##STR33## 
pyrilium dyes, e.g., compounds 14 (X.dbd.O) on page 32 of Matsuoka 
(supra), i.e. 
##STR34## 
thiapyrilium dyes, e.g. compounds 15 on page 32, and compound I on page 
167 of Matsuoka (supra), i.e. 
##STR35## 
squarylium dyes, e.g. compound 10 and Table IV on page 30 of Matsuoka 
(supra), i.e. 
##STR36## 
and compound 6, page 26, of Matsuoka (supra), i.e. 
##STR37## 
croconium dyes, e.g. compound 9 and Table IV on page 30 of Matusoka 
(supra), i.e. 
##STR38## 
and compound 7, page 26, of Matsuoka (supra), i.e. 
##STR39## 
azulenium dyes, e.g. compound 8 on page 27 of Matsuoka (supra), i.e. 
##STR40## 
merocyanine dyes, e.g. compound 16, R=Me, on page 32 of Matsuoka (supra), 
i.e. 
##STR41## 
indoaniline dyes such as copper and nickel complexes of indoaniline dyes, 
e.g. compound 6 on page 63 of Matsuoka (supra), i.e 
##STR42## 
benzo[a]phenoxazinium dyes and benzo[a]phenothiazinium dyes, e.g. as shown 
on page 201 of Matusoka (supra), i.e. 
##STR43## 
1,4-diaminoanthraquinone(N-alkyl)-3'-thioxo-2,3-dicarboximides, e.g. 
compound 20, on page 41 of Matusoka (supra) 
##STR44## 
indanthrene pigments, e.g. 
##STR45## 
see compound 21 on page 41 of Matsuoka (supra); 
2-arylamino-3,4-phthaloylacridone dyes, e.g. compound 22 on page 41 of 
Matsuoka (supra) 
##STR46## 
trisphenoquinone dyes, e.g. compound 23 on page 41 of Matsuoka (supra) 
##STR47## 
azo dyes, e.g. the monoazo dye, compound 2 on page 90 of Matsuoka (supra), 
i.e. 
##STR48## 
and Y=C.dbd.O, R.sub.1 =R.sub.2 =Et, R.sub.3 =R.sub.1 =H, 
or Y=SO.sub.2, R.sub.1 =H, R.sub.2 =CH(Me)nBu, R.sub.3 =OMe, and R.sub.4 
=NHAc; 
azo dyes, e.g. the polyazo dye, compound 5 on page 91 of Matsuoka (supra), 
i.e. 
##STR49## 
intramolecular charge transfer donor-acceptor infrared dyes, e.g. 
compounds 6 and 7 on page 91 of Matsuoka (supra), i.e. 
##STR50## 
and 
##STR51## 
nonbenzenoid aromatic dyes, e.g. compound 8, a tropone, on page 92, of 
Matsuoka (supra), i.e. 
##STR52## 
tetrazine radical dyes, e.g. compound 9 on page 92 of Matsuoka (supra), 
i.e. 
##STR53## 
in which, X=p-phenylene or X=p-terphenylene as well as compound 10 on 
page 92 of Matsuoka (supra), i.e. 
##STR54## 
in which X=p-biphenyl; cationic salts of tetrazine radical dyes, e.g. 
compound 11 on page 92 of Matsuoka (supra) 
##STR55## 
in which X=p-phenylene; donor-acceptor intermolecular charge transfer 
dyes, e.g. CT complexes of compounds 13b and 14a to 14c on page 93 of 
Matsuoka (supra), i.e. 
##STR56## 
where X=CH.dbd.N--N(Ph).sub.2 in the donor and a) Y=CN, Z=NO.sub.2 
b) Y=CN, Z=H or 
a) Y=Cl, Z=NO.sub.2 in the acceptor; 
anthraquinone dyes, e.g. compounds 12 (X=S or Se) on page 38 of Matsuoka 
(supra), i.e. 
##STR57## 
wherein X=S or Se and Y=tetrachloro, tetrabromo, 2,3-dicarboxylic acid, 
2,3-dicarboxylic anhydride, or 
2,3-dicarboxylic acid N-phenyl imide; 
naphthoquinone dyes, e.g. compounds 2, 3, and 4 on page 37, of Matsuoka 
(supra), i.e. 
##STR58## 
metallated azo dyes such as azo dyes containing nickel, cobalt, copper, 
iron, and manganese; 
phthalocyanine dyes, e.g. compound 1 in Table II on page 51 of Matsuoka 
(supra), e.g. 
##STR59## 
naphthalocyanine dyes, e.g. compound 3 in Table II on page 51 of Matsuoka 
(supra), e.g. 
##STR60## 
metal phthalocyanines such as phthalocyanines containing aluminum, 
silicon, nickel, zinc, lead, cadmium, magnesium, vanadium, cobalt, copper, 
and iron, e.g. compound 1 in Table III on page 52 of Matsuoka (supra), 
e.g. 
##STR61## 
in which, for example, M=Mg; metal naphthalocyanines such as 
naphthalocyanines containing aluminum, zinc, cobalt, magnesium, cadmium, 
silicon, nickel, vanadium, lead, copper, and iron, see compound 3 in Table 
III on page 52 of Matsuoka (supra), e.g. 
##STR62## 
in which, for example, M=Mg; bis(dithiolene) metal complexes comprising a 
metal ion such as nickel, cobalt, copper, and iron coordinated to four 
sulfur atoms in a bis(S,S'-bidentate) ligand complex, e.g. see Table I on 
page 59 of Matsuoka (supra) 
______________________________________ 
#STR63## 
where 
R.sub.1 = R.sub.2 = CF.sub.3, M = Ni, 
R.sub.1 = R.sub.2 = phenyl, M = Pd, 
R.sub.1 = R.sub.2 = phenyl, M = Pt, 
R.sub.1 = C4 to C10 alkyl, R.sub.2 = H, M = Ni, 
R.sub.1 = C4 to C10 alkyl, R.sub.2 = H, M = Pd, 
R.sub.1 = C4 to C10 alkyl, R.sub.2 = H, M = Pt, 
R.sub.1 = R.sub.2 = phenyl, M = Ni, 
R.sub.1 = R.sub.2 = p-CH.sub.3 -phenyl, M = Ni, 
R.sub.1 = R.sub.2 = p-CH.sub.3 O-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = p-Cl-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = p-CF.sub.3 -phenyl, M = Ni, 
R.sub.1 = R.sub.2 = 3,4,-diCl-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = o-Cl-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = o-Br-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = 3,4,-diCl-phenyl, M = Ni, 
R.sub.1 = R.sub.2 = p-CH.sub.3, M = Ni, 
R.sub.1 = R.sub.2 = 2-thienyl, M = Ni, 
R.sub.1 = p-(CH.sub.3).sub.2 N-phenyl, R.sub.2 = phenyl, M = Ni, and 
R.sub.1 = p-(CH.sub.3).sub.2 N-phenyl, R.sub.2 = p-H.sub.2 N-phenyl, M = 
Ni; 
______________________________________ 
bis(benzenedithiolate) metal complexes comprising a metal ion such as 
nickel, cobalt, copper, and iron coordinated to four sulfur atoms in a 
ligand complex, e.g. see Table III on page 62 of Matsuoka (supra), i.e. 
______________________________________ 
#STR64## 
where 
X = tetramethyl, M = Ni, 
X = 4,5-dimethyl, M = Ni, 
X = 4-methyl, M = Ni, 
X = tetrachloro, M = Ni, 
X = H, M = Ni, 
X = 4-methyl, M = Co, 
X = 4-methyl, M = Cu, and 
X = 4-methyl, M = Fe; 
______________________________________ 
N,O-bidentate indoaniline dyes comprising a metal ion such as nickel, 
cobalt, copper, and iron coordinated to two nitrogen and two oxygen atoms 
of two N,O-bidentate indoaniline ligands, e.g. compound 6 in Table IV on 
page 63 of Matsuoka (supra) e.g. 
______________________________________ 
#STR65## 
where R = Et, R' = Me, M = Cu, 
R = Et, R' = Me, M = Ni, 
R = Me, R' = H, M = Cu, and 
R = Me, R' = H, M = Ni, 
______________________________________ 
bis(S,O-dithiolene) metal complexes comprising a metal ion such as nickel, 
cobalt, copper, and iron coordinated to two sulfur atoms and two oxygen 
atoms in a bis(S,O-bidentate) ligand complex, e.g. see U.S. Pat. No. 
3,806,462, e.g. 
##STR66## 
a-diimine-dithiolene complexes comprising a metal ion such as nickel, 
cobalt, copper, and iron coordinated to two sulfur atoms and two 
imino-nitrogen atoms in a mixed S,S- and N,N-bidentate diligand complex, 
e.g. see Table II on page 180, second from bottom, of Matsuoka (supra) 
(also see Japanese patents: 62/39,682, 63/126,889 and 63/139,303), e.g. 
##STR67## 
and tris(a-diimine) complexes comprising a metal ion coordinated to six 
nitrogen atoms in a triligand complex, e.g. see Table II on page 180 of 
Matsuoka (supra), last compound, (also see Japanese Patents 61/20,002 and 
61/73,902), e.g. 
##STR68## 
Representative examples of visible dyes include fluorescein derivatives, 
rhodamine derivatives, coumarins, azo dyes, metalizable dyes, 
anthraquinone dyes, benzodifuranone dyes, polycyclic aromatic carbonyl 
dyes, indigoid dyes, polymethine dyes, azacarbocyanine dyes, hemicyanine 
dyes, barbituates, diazahemicyanine dyes, stryrl dyes, diaryl carbonium 
dyes, triaryl carbonium dyes, phthalocyanine dyes, quinophthalone dyes, 
triphenodioxazine dyes, formazan dyes, phenothiazine dyes such as 
methylene blue, azure A, azure B, and azure C, oxazine dyes, thiazine 
dyes, naphtholactam dyes, diazahemicyanine dyes, azopyridone dyes, 
azobenzene dyes, mordant dyes, acid dyes, basic dyes, metallized and 
premetallized dyes, xanthene dyes, direct dyes, leuco dyes which can be 
oxidized to produce dyes with hues bathochromically shifted from those of 
the precursor leuco dyes, and other dyes such as those listed by Waring, 
D. R. and Hallas, G., in "The Chemistry and Application of Dyes", Topics 
in Applied Chemistry, Plenum Press, New York, N.Y., 1990. Additonal dyes 
can be found listed in Haugland, R. P., "Handbook of Fluorescent Probes 
and Research Chemicals", Sixth Edition, Molecular Probes, Inc., Eugene OR, 
1996. 
Such chormophores and fluorophores may be covalently linked either directly 
to the vector or to or within a linker structure. Once again linkers of 
the type described above in connection with the metal reporters may be 
used for organic chromophores or fluorophores with the 
chromophores/fluorophores taking the place of some or all of the chelant 
groups. 
As with the metal chelants discussed above chromophores/fluorophores may be 
carried in or on a particulate linker-moieties, eg. in or on a vesicle or 
covalently bonded to inert matrix particles that can also function as a 
light scattering reporter. 
Particulate Reporters or Linker-Reporters 
The particulate reporters and linker-reporters generally fall into two 
categories--those where the particle comprises a matrix or shell which 
carries or contains the reporter and those where the particle matrix is 
itself the reporter. Examples of the first category are: vesicles (eg. 
micelles, liposomes, microballoons and microbubbles) containing a liquid, 
gas or solid phase which contains the contrast effective reporter, eg. an 
echogenic gas or a precursor therefor (see for example GB 9700699.3), a 
chelated paramagnetic metal or radionuclide, or a water-soluble iodinated 
X-ray contrast agent; porous particles loaded with the reporter, eg. 
paramagnetic metal loaded molecular sieve particles; and solid particles, 
eg. of an inert biotolerable polymer, onto which the reporter is bound or 
coated, eg. dye-loaded polymer particles. 
Examples of the second category are: light scattering organic or inorganic 
particles; magnetic particles (ie. superparamagnetic, ferromagnetic or 
ferrimagnetic particles); and dye particles. 
Preferred particulate reporters or reporter-linkers include 
superparamagnetic particles (see U.S. Pat. No. 4,770,183, WO97/25073, 
WO96/09840, etc.), echogenic vesicles (see WO92/17212, WO97/29783, etc.), 
iodine-containing vesicles (see WO95/26205 and GB9624918.0), and 
dye-loaded polymer particles (see WO96/23524). 
The particulate reporters may have one or more vectors attached directly or 
indirectly to their surfaces. Generally it will be preferred to attach a 
plurality (eg. 2 to 50) of vector moieties per particle. Particularly 
conveniently, besides the desired targeting vector, one will also attached 
flow decelerating vectors to the particles, ie. vectors which have an 
affinity for the capillary lumen or other organ surfaces which is 
sufficient to slow the passage of the contrast agent through the 
capillaries or the target organ but not sufficient on its own to 
immobilise the contrast agent. Such flow decelerating vectors (described 
for example in GB9700699.3) may moreover serve to anchor the contrast 
agent once it has bound to its target site. 
The means by which vector to particle attachment is achieved will depend on 
the nature of the particle surface. For inorganic particles, the linkage 
to the particle may be for example by way of interaction between a metal 
binding group (eg. a phosphate, phosphonate or oligo or polyphosphate 
group) on the vector or on a linker attached to the vector. For organic 
(eg. polymeric) particles, vector attachment may be by way of direct 
covalent bonding between groups on the particle surface and reactive 
groups in the vector, eg. amide or ester bonding, or by covalent 
attachment of vector and particle to a linker. Linkers of the type 
discussed above in connection with chelated metal reporters may be used 
although in general the linkers will not be used to couple particles 
together. 
For non-solid particles, eg. droplets (for example of water insoluble 
iodinated liquids as described in U.S. Pat. No. 5,318,767, U.S. Pat. No. 
5,451,393, U.S. Pat. No. 5,352,459 and U.S. Pat. No. 5,569,448) and 
vesicles, the linker may conveniently contain hydrophobic "anchor" groups, 
for example saturated or unsaturated C.sub.12-30 chains, which will 
penetrate the particle surface and bind vector to particle. Thus for 
phospholipid vesicles, the linker may serve to bind the vector covalently 
to a phospholipid compatible with the vesicle membrane. Examples of linker 
binding to vesicles and inorganic particles are described in GB9622368.0 
and WO97/25073. 
Besides the vectors, other groups may be bound to the particle surface, eg. 
stabilisers (to prevent aggregation) and biodistribution modifiers such as 
PEG. Such groups are discussed for example in WO97/25073, WO96/09840, 
EP-A-284549 and U.S. Pat. No. 4,904,479. 
Preferably the V-L-R agents of the invention will have the non-peptidic 
endothelin receptor targetting vectors (such as bosentan or BMS 182874) 
coupled directly or indirectly to a reporter, eg. with covalently bound 
iodine radioisotopes, with metal chelates attached directly or via an 
organic linker group or coupled to a particulate reporter or 
linker-reporter, eg. a superparamagnetic crystals (optionally coated, eg. 
as in WO97/25073), or a vesicle, eg. a gas containing or iodinated 
contrast agent containing micelle, liposome or microballoon. 
All of the publications referred to herein are incorporated herein by 
reference. 
The agents of the invention may be administered to patients for imaging in 
amounts sufficient to yield the desired contrast with the particular 
imaging technique. 
Where the reporter is a metal, 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 0.05 to 2.0 mmoles/kg are generally effective to achieve 
X-ray attenuation. Preferred dosages for most X-ray applications are from 
0.1 to 1.2 mmoles of the lanthanide or heavy metal compound/kg bodyweight. 
Where the reporter is a radionuclide, dosages of 0.01 to 100 mCi, 
preferably 0.1 to 50 mCi will normally be sufficient per 70 kg bodyweight. 
Where the reporter is a superparamagnetic particle, the dosage will 
normally be 0.5 to 30 mg Fe/kg bodyweight. Where the reporter is a gas or 
gas generator, eg. in a microballoon, the dosage will normally be 0.5 to 
100 .mu.L gas per 70 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 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, suppositori es 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 imaging of some portions of the body the most preferred mode for 
administering 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 osmoiality 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 
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. 
The agents of formula I may be therapeutically effective in the treatment 
effective in the treatment of disease states as well as detectable in in 
vivo imaging. Thus for example the vector on the reporter moieites may 
have therapeutic efficacy, eg. by virtue of the radiotherapeutic effect of 
a radionuclide reporter, the efficacy in photodynamic therapy of a 
chromophore (or fluorophore) reporter or the chemotherapeutic effect of 
the vector moiety. 
Use of the agents of formula I in the manufacture of therapeutic 
compositions and in methods of therapeutic or prophylactic treatment of 
the human or non-human animal body are thus considered to represent 
further aspects of the invention. 
In one embodiment, the contrast agent of the invention conveniently 
comprises a gas-containing or gas-generating material, preferably in 
suspension in an aqueous carrier material and conjugated to one or more 
vectors of which at least one is a vector V as defined above, eg. an 
endothelin antagonist. The gas may take the form of microbubbles 
stabilised by a monolayer of a film-forming surfactant, or stabilised by a 
matrix material other than a surfactant. The vectors may be for example 
coupled to such surfactant or matrix and may be bioactive or 
non-bioactive. The vectors may have different targeting specificities and 
in one preferred embodiment are such as to interact with their receptors 
but not to fixedly bind the gas-containing vesicles.

The present invention will now be further illustrated by way of the 
following non-limiting examples. Unless otherwise indicated, all 
percentages qiven are by weight. 
EXAMPLE 1 
Endothelin Receptor Binding Contrast Agent for MR Imaging 
Compound 1 
Lysine (0.1g, 0.7 mmol) is added to a solution of 
27-0-3-[2-(3-carboxy-acryloylamino)-5-hydroxyphenyl]-acryloyloxy mycerone 
(prepared in accordance with EP 628569, 0.5 9, 0.7 mmol) and DCC 
(N,N'-dicyclohexylcarbodiimide) in dry DMF (N,N-dimethylformamide). The 
reaction mixture is stirred at ambient temperature and is followed by TLC. 
The dispersion is left overnight at +4.degree. C. The dispersion is 
filtered and the solvent rotary evaporated before the substance is 
purified by chromatography. 
Compound 2 
Diethylenetriamine-pentaacetic acid dianhydride (17.9 9, 50 mmol) is 
dissolved in dry DMF and compound 1 (0.45 g, 0.5 mmol) dissolved in dry 
DMF is added. The reaction mixture is stirred at elevated temperature 
under nitrogen atmosphere. The reaction is followed by TLC. The solvent is 
rotary evaporated and the substance purified by chromatography. 
Gd(III)Chelate of Compound 2 
To a solution of compound 2 (0.5 9, 0.4 mmol) in water is added gadolinium 
oxide Gd.sub.2 O.sub.3 (0.1 g, 0.2 mmol) and the mixture is heated at 
95.degree. C. After filtration the solution is evaporated and dried in 
vacuo at 50.degree. C. 
EXAMPLE 2 
Endothelin Receptor Binding Contrast Agent for Nuclear Medicine 
.sup.99m Tc Chelate of compound 2 
Compound 2 from Example 1 (1 mg) is dissolved in 0.1 N NaOH. 
SnCl.sub.2.2H.sub.2 O (100 .mu.g) dissolved in 0.05 N HCl and a solution 
of 10-100 mCi .sup.99m Tc in the form of sodium pertechnetate in saline is 
added. The pH of the solution is adjusted to pH 7-8 by addition of 0.5 M 
phosphate buffer (pH 5) after less than one minute. The reaction is 
followed by TLC and the substance is purified by chromatography. 
EXAMPLE 3 
Endothelin Receptor Binding Contrast Agent for Nuclear Medicine 
An aqueous solution of .sup.131 I.sub.2 (2 equivalents) and sodium 
perchlorate (1 equivalent) is added to an aqueous solution of 
2-benzo(1,3)dioxol-5-yl-3-benzyl-4-(4-methoxyphenyl)-4-oxobut-2-enoate 
(prepared in accordance with Example 351 of WO 95/05376, 1 equivalent). 
The solvent is rotary evaporated and the substance is purified by 
chromatography. 
EXAMPLE 4 
Endothelin Receptor Binding Contrast Agent for Ultrasound and Scattering 
Light Imaging 
Compound 3 
DSPE (distearoylphosphatidylethanolamine) (0.5 9, 0.7 mmol) is added to a 
solution of 
27-0-3-[2-(3-carboxy-acryloylamino)5-hydroxyphenyl]-acryloyloxy mycerone 
(prepared in accordance with EP 628569, 0.5 g, 0.7 mmol) and DCC in dry 
DMF. The reaction mixture is stirred at ambient temperature and followed 
by TLC. The dispersion is left overnight at +4.degree. C. The dispersion 
is filtered and the solvent rotary evaporated before the substance is 
purified by chromatography. 
Gas Containing Microparticles Comprising Phosphatidylserine and Compound 3 
To phosphatidylserine (5 mg) is added 5% propylenglycol-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 compound 1 (0.1-1.0 mg) is added. The 
vial is put on a roller table for some hours. The head space of the vial 
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. The washing 
procedure is repeated 2-3 times. 
EXAMPLE 5 
Gas-filled Microbubbles of Distearoyl Phosphatidylserine Comprising a 
Lipopeptide Containing a Vector with Affinity for Endothelin Receptors for 
Targeted Ultrasound Imaging 
a) Synthesis of 4'-[3.4-dimethyl-5-isoxazolyl)sulfamoyl] Succinanilic Acid 
To a solution of sulfisoxazole (Aldrich, 267 mg, 1.00 mmol) in DMF (10 ml) 
was added succinic anhydride (1.00 9, 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 the 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 a white solid. The 
structure was verified by .sup.1 H (300 MHz) and .sup.13 C (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. 
b) Synthesis of a Lipopeptide Functionalised with Sulfisoxazole 
##STR69## 
The structure shown above was synthesised on a manual nitrogen bubbler 
apparatus starting with Fmoc protected Rink Amide MBHA resin (Novabiochem) 
on a 0.125 mmol scale, using amino acids from Novabiochem, palmitic acid 
from Fluka and the compound from a) above. 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 min, A=0.1% TFA/water and 
B=0.1% TFA/acetonitrile, flow rate 1 ml/min, column Vydac 218TP54, 
detection UV 214 nm, retention time 27 min). Further characterisation was 
carried out using MALDI mass spectrometry, giving a m+H at m/z 1359, 
expected 1356. 
c) Preparation of Gas-filled Microbubbles Comprising the Compound from b) 
above 
A solution of 1.4% propylene glycol/2.4% glycerol (1.0 ml) was added to a 
mixture of DSPS (Avanti, 4.5 mg) and the product from b) above (0.5 mg) in 
a vial. The mixture was sonicated for 5 minutes and then heated at 
80.degree. C. for 5 minutes (the 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) above in the final wash solution. 
Incorporation of isoxazole-containing lipopeptide into the bubbles was 
confirmed by MALDI-MS as follows. About 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). 
EXAMPLE 6 
.sup.131 I Labelleled Sulfisoxasole (Compound 4): 
Sulfisoxasole was labelled with .sup.131 I according to the procedure 
supplied with the iodination agent IODO-BEADS iodination agent from 
Pierce. Labelling yield: 91%. 
EXAMPLE 7 
Tc Chelate of N-(MAG-3)-Sulfisoxazole 
The title compound was prepared as outlined in Scheme 1 below according to 
the description in Nuclear Medicine Communications 16 (1995) 942-957. 
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