WO1999056788A1

WO1999056788A1 - Selection of targeted contrast agents from a combinatorial library

Info

Publication number: WO1999056788A1
Application number: PCT/GB1999/001446
Authority: WO
Inventors: Jo Klaveness; Peter Martin Fischer
Original assignee: Nycomed Imaging As
Priority date: 1998-05-07
Filing date: 1999-05-07
Publication date: 1999-11-11
Also published as: AU3837799A; GB9809780D0

Abstract

The invention provides a method for the selection of a candidate targeted contrast agent, comprising the steps of: (i) obtaining a library of vector: reporter conjugates in which conjugates where the vector is an oligomer (and optionally also where vectors are non-oligomeric) the conjugate contains a heterologous reporter; and (ii) testing the members of said library to identify a said member having a binding affinity for a biological target.

Description

SELECTION OF TARGETED CONTRAST AGENTS FROM A COMBINATORIAL LIBRARY

This invention relates to the preparation and use of combinational libraries of vector-reporter conjugates, to such libraries as such and to contrast agents selected from such libraries.

In medical imaging modalities, the use of contrast agents (materials which enhance image contrast, for example between different organs or tissues or between normal and abnormal tissue) is well established. By way of example, X-ray contrast agents are commonly compounds which contain heavy atoms, such as iodine, and therefore increase X-ray opacity in the body sites into which they distribute, ultrasound contrast agents are commonly echogenic gas-containing structures such as microbubbles stabilized by encapsulation (e.g. by a film or membrane) which enhance the ultrasound signal for body sites into which they distribute, MR contrast agents commonly contain superparamagnetic or paramagnetic structures or centres which alter the MR signal from body sites into which they distribute by affecting the relaxation rates for neighbouring water protons, contrast agents for magnetic source imaging commonly contain paramagnetic or more generally superparamagnetic, ferrimagnetic or ferromagnetic structures or centres, light imaging contrast agents commonly contain chromophores or fluorophores, and nuclear imaging contrast agents are usually radionuclide-containing materials which emit detectable radiation on radioactive decay.

Many commercial contrast agents are essentially general purpose agents which distribute into the extracellular space, are retained within the vasculature or the gastrointestinal tract or which are cleared through the liver or kidneys and thus enhance image contrast in those organs or in the bladder. Agents which clear through a particular organ or are retained within a particular body duct or cavity are generally considered to passively target such organs, ducts or cavities. Examples of such contrast agents thus include the MR contrast agents Omniscan^®, Magnevist^® and ProHance^®, the X-ray contrast agents Omnipaque^®, Imagopaque^®, Visipaque^®, Iopamiro^®, Isovist^®, Optiray^® and Ultravist^®, the ultrasound agents Echovist^®, Levovist^®, Albunex^®, FS069, MRX115, NC 100100 and Echogen^® and the nuclear imaging agents ⁹⁹Tc- succinimer, ^99mTc HIDA and ^99mTc-DTPA.

There has recently been an increasing interest in contrast agents which distribute preferentially to particular body sites, e.g. tumour cells, by virtue of active rather than passive targeting. Such active targeting is achieved by the direct or indirect conjugation to a contrast enhancing moiety (a "reporter") of a moiety (a "vector") which binds to or promotes cellular uptake at cell surfaces or other surfaces at the target site of interest. These vector- reporter conjugates (or vector-linker-reporter conjugates) may also be referred to as targeted contrast agents and offer the possibility for detection or imaging of disease sites as well as sites of tissue or organ malfunction and hence also provide the possibility of imaging tissue or organ function. In turn, such images may be used to direct or monitor the success of surgical or chemical treatments.

The vector moieties used to target such contrast agents are typically moieties which have affinity for cell surface receptors, eg antibodies, antibody fragments, oligopeptides , oligonucleotides and oligosaccharides as well as peptide, peptoid and nonpeptidic drug compounds known to accumulate at particular disease or malfunction sites . Examples of appropriate vectors and vector targets may be found in the literature, e.g. PCT/GB97/0295 , PCT/GB97/02955, PCT/GB97/02958 , PCT/GB97/02953 , PCT/GB97/02956 and PCT/GB97/02957 published 7 May 1998 and PCT/GB98/01197 ( 098/47541 filed 24.4.98) in the name of Nycomed Imaging AS, entitled "Contrast Agents". Selection of appropriate vectors has thus tended to involve selection of a material known to target the desired site or alternatively identification of a new material capable of targeting the desired site. An example of this latter approach is that adopted by Gold et al in WO96/40273 and O96/02274 where a combinational library approach (the "SELEX" system) is used to identify a targeting oligonucleotide which is then conjugated to a reporter moiety which is effective as a contrast agent in the desired imaging modality.

Where an oligomer is used as a vector however, its binding affinity for the target receptors is affected by its secondary structure, ie the three-dimensional structural (and functional) conformation, of the vector. This conformation, and hence binding affinity can accordingly be affected by conjugation of the vector to a bulky heterologous reporter moiety, especially where, for the contrast enhancement at the target site to be discernible in the particular imaging modality used, a plurality of reporters must be attached.

The present invention is based on the finding that an improved selection of a vector, linker or reporter in a vector-reporter or vector-linker-reporter conjugate (both referred to hereinafter simply as vector : reporter conjugates) can be made in a combinatorial approach to selection where the combinatorial library used is instead a library of vector : reporter conjugates which, where the vector is oligomeric, contain heterologous reporters .

By heterologous, it is meant that the reporter moiety is not of the same chemical nature as the monomers of the oligomeric vector moiety, e.g. where the reporter is a particulate or is a metallated polyoxyacid and/or polyamine and/or polythiol chelating agent or is an organic moiety with an extended π-bonding system (e.g. a chromophore or fluorophore) and the vector is a biopolyτner such as an oligopeptide, oligonucleotide or oligosaccharide . Heterologous reporters would thus not include reporter moieties which comprise a monomer or a string of monomer residues within the oligomer vector, e.g. complexed to a detectable metal or with naturally occurring monomer atoms replaced by radioisotopes of such atoms, for example H, C, N, 0, P or S radioisotopes .

Thus viewed from one aspect the invention provides a method for the selection of a candidate targeted contrast agent, comprising the steps of: (i) obtaining a library of vector : reporter conjugates in which conjugates where the vector is an oligomer (and optionally also where the vector is non-oligomeric) the conjugate contains a heterologous reporter; and

(ii) testing the members of said library to identify a said member having a binding affinity for a biological target. Binding affinity will be tested for the target of interest; this may for example be a known receptor, e.g. using a cell culture, or it may be an ex vivo tissue sample. Examples of suitable targets include serotonin receptors, histamine receptors, erythrocytes, vasculature lumen cells, endometriosis tissue, tumor tissues, myocardial cells, etc.

By a library is typically meant a collection of at least 3, conveniently at least 10, preferably at least 10², more preferably at least 10³, eg lO⁴ to 10⁷ vector : reporter conjugates. In the library there may be a single type of reporter moiety; however preferably a plurality of reporter types, eg 2 to 100 may be present. The different vector : reporter conjugates in the library may differ typically in one or more of the following characteristics :

(i) vector : reporter conjugation sites on the vector; (ii) vector : reporter conjugation sites on the reporter; (iii) the ratio of vector and reporter moieties in the vector : reporter conjugates, eg varying from 10:1 to 1:10, rather than simply 1:1; (iv) the number of different types of reporter moieties ; (v) the number of different types of vector moieties ; (vi) the chemical nature of the vector moieties, eg small organic molecules (for example varied by skeleton decoration) , drug compounds, peptides, peptoids, oligo ers (eg oligopeptides, oligopeptoids, oligonucleotides and oligosaccharides, whether based on natural monomers (such as natural L-amino acids) or synthetic monomers (such as D-amino acids, side-chain length homologs of natural amino acids, side chain substitution homologs of natural amino acids, β-amino acids, etc)), antibodies, antibody fragments, or any functionally or chemically equivalent, or structurally similar derivative, homologue or analog of any of the above types of vector moiety, etc; (vii) the chemical nature of the reporter moieties, eg chromophores , fluorophores, mono-chelates, polychelates, chelated paramagnetic metal ions, superparamagnetic metal, metal oxide or mixed metal oxide particles, persistent free radicals, particulates, iodinated organic compounds, or other organic molecules with covalently bound radionuclides or chelated metal radionuclides , gas containing vesicles optionally tagged with one or more radiolabels, etc; and (viii)the tagging or non-tagging (e.g. with a virus particle, a short nucleic acid sequence or oligonucleotide, or with a radionuclide or set of radionuclides having characteristic emission profiles, e.g. differentiable using multichannel gamma cameras) of the vector : reporter conjugates to assist in deconvolution and to assist in identifying the library members or library subsets. The library used in the method of the invention may be modified during operation of the method, eg to replace a first library by a second (or further) library, the members whereof are designed on the basis of the successes and failures of members of the earlier libraries, eg the first library. Thus, for example, where testing of one library identifies successful attachment sites on vectors or reporters, successive libraries may include larger numbers of different types of vectors or reporters attached at the identified sites or structurally equivalent sites. Such different vectors or reporters may for example be materials which are more complex to prepare or are more expensive to purchase or require more expensive reagents for their preparation. Likewise for oligomeric vectors, library modification may involve utilization of monomers which are structurally or functionally similar to the monomers used in the oligomeric vectors found to be successful in the earlier library and may involve omission of monomers or oligomeric substructures found to be unsuccessful in the earlier library.

The method of the invention may thus involve screening of a plurality of libraries, eg 2 to 20, preferably 2 to 10, with successive libraries being designed on the basis of the results from the earlier - 7 - libraries and iteration being stopped when one or more conjugates having suitable properties are found.

The libraries used in the method of the invention may comprise a mixture of the various library members; alternatively the library members may be separate, ie separately testable. In the former case, which is the preferred case for the operation of the method of the invention, it is necessary to deconvolute to identify which library members are successful in the testing routine (s) e.g. have a binding affinity for a biological target .

The deconvolution techniques for determining the identity of a member of a combinatorial library, eg a member found to bind strongly in a binding affinity strength assay, have been widely described in the recent literature on combinatorial chemistry (see for example Lebl in Biopolymers (Peptide Science) 3_7 :177 (1995), Madsen in Persp . in Drug Discovery and Design 2. : 269 (1994) , Furka in Drug Development Research 3_6 : 1 (1995) , Konnings in J. Med. Chem. 3_£ : 2710 (1996) ,

Freier in J. Med. Chem 3_8 : 344 (1995) , Davis in J. Med. Chem 3_a : 4363 (1995) , Houghton in Nature 354 : 84 (1991), Pop in Eur . J. Med. Chem. 21:87 (1996), Needels in Proc Nat. Acad Sci USA 9.0_. : 10700 (1993), Erb in Proc Nat. Acad Sci USA 9_1 11422 (1994) and Wilson-Lingard in J. Med Chem 3_9_: 2720 (1996)).

Generally speaking, the major techniques for deconvolution involve either "tagging" library members so that the identity of the member or of a subset of the library to which it belongs may be determined by

"reading" the tag, or orthogonal scanning in which sub- libraries are screened to identify the successful library members.

The "tags" used to identify the library members may take a variety of forms. A phage display library contains its own tag in the virus particle to which the displayed oligopeptides are attached. Cells can be - 8 - infected with successful viruses so as to produce the displayed oligopeptides in amounts large enough to be sequenced. An oligonucleotide tag can be amplified by PCR or similar techniques to produce large enough quantities for sequencing and hence identification of the library member (s) to which the particular tag is attached. Radioisotope tags (eg hydrogen, oxygen, halogen, silicon, phosphorus, carbon and nitrogen isotopes or radioactive metals) can be incorporated into components of some or all of the library members and detection of the nature and energy of the radioactive emission can be used to identify the library member or the library sub-set to which it belongs. Where a reporter moiety can be modified to report in a characteristically different manner without affecting its structural or functional conformation, then a range of modified reporters can be used to "tag" the vector : reporter conjugates .

The use of a combinatorial library of vector : reporter conjugates in which different reporter moieties are used to tag the conjugates either to identify library sub-sets, or more preferably as unique identifiers for particular vector moieties, or even more preferably as unique identifiers for particular library members is novel and forms a further aspect of the invention. View from this aspect the invention provides a method for the selection of a candidate targeted contrast agent, comprising the steps of: (i) obtaining a library of vector : reporter conjugates including a plurality of mutually interdistinguishable reporter moieties; (ii) testing the members of said library for binding affinity to a biological target and using the interdistinguishability of said reporters identifying a member of said library exhibiting relatively superior binding affinity or a sub-set of said library containing at least one member of said library exhibiting relatively superior binding affinity; and (iii) if necessary testing sub-sets of said library to identify said member of said library exhibiting relatively superior binding affinity.

By mutually interdistinguishable reporters, there are included for example chelates of different metal radionuclides which are interdistinguishable by the nature and energy of their radiation emissions; chromophores having different characteristic absorption or emission maxima; crystalline particles having different X-ray scattering angles; vesicles containing different gases or having different acoustic properties such as non-linear behaviour, etc.

If desired, a combined tagging system may be used whereby multiple sites on the library members may be tagged so that the combination of tags serves to uniquely identify the library member or to identify a relatively small library sub-set in which it may be found, while yet leaving the overall structural and functional conformation of the conjugate relatively unchanged. Thus sub-units from which the vector might be built up e.g. amino acids, nucleotides, saccharides and the "decorations" of a decorated small molecular framework vector, may contain or not contain radioisotopes of atoms they normally contain, in particular C, N, O, S, P and H, while reporters of a given type may likewise be present in a range of mutually interdistinguishable forms .

Individual library members may be prepared separately, or multiple members (or indeed the full library) may be prepared in parallel. Standard combinatorial library generation techniques may be used, e.g. multi-pin, split-and-mix generation on bead substrates, multiple spots on a substrate, photodeprotection of a masked substrate, construction of a decorated molecular framework from reagents which import the decorations as the framework is constructed, etc. In general, synthesis of the vectors will - 10 - preferably take place on a solid phase, since this makes it particularly straightforward to produce a wide range of structures; however liquid phase preparation is also feasible. Where solid phase synthesis is used, the library can be tested while attached to the solid phase; however preferably the testing will take place with library members in the solution phase so that the solid phase support does not influence the configuration adopted by the library members. Conjugation of vector to reporter may take place at the beginning, during or after synthesis of the vector moiety. Where solid phase synthesis is used, reporters may particularly readily be conjugated to the synthesised vector before it is released from the substrate, or to the substrate before vector synthesis is begun with the reporter in this case acting as the anchor or part of the anchor attaching the growing vector to the solid support . Where the reporter is to be conjugated to a position other than a terminal position of an oligomeric vector, it can either be added during construction of the vector or, more generally, it will be conjugated after vector construction is complete to an attachment site introduced into the vector during construction, e.g. a trifunctional monomer one function of which was protected or otherwise unused during vector growth.

When one or more candidate contrast agents are selected using the methods of the invention, these contrast agents or derivatives thereof may be manufactured, and if desired formulated with at least one pharmaceutical carrier or excipient . The term "derivative" as used herein includes a candidate contrast agent which comprises a modified vector : reporter conjugate wherein the vector moiety is the same as in the selected library member but the reporter moiety is different. In addition the term "derivative" includes a modified vector : reporter conjugate wherein the reporter moiety is replaced by a - 11 - therapeutic moiety.

The vector moieties in the first library used in the method of the invention may be generated on a purely random basis from individual building blocks (e.g. amino acids, saccharides, nucleotides, the "jig-saw" pieces from which a decorated small organic molecule can be constructed) , or may simply be some or all of the members of a drug library. However in a particularly preferred embodiment of the invention the vector moieties are a directed random selection, e.g. a random selection developed from the successes of binding affinity tests on a random combinatorial oligomeric vector library in which the library members are not conjugated to the reporter moieties. In effect this means that a pre-screen takes place in the absence of the reporter so that the size of the vector : reporter conjugate library that has to be generated for the operation of the method of the invention may be reduced by elimination of vector structures which do not show reasonable binding affinities. Such a pre-screen may be performed using any of the conventional combinatorial chemistry techniques, e.g. the SELEX oligonucleotide technique, etc. However it is especially preferred to use a phage display library for a pre-screen of potential oligopeptide vectors as deconvolution is so relatively straightforward when as in cases like this the members carry a "tag" which permits the successful members to be amplified and sequenced.

In general, vectors will be either oligomers, e.g. 4-mers to 20-mers, preferably 6-mers to 15-mers, which may or may not be cyclized, or they will be organic molecules typically having a molecular weight of from 60 to 10000 D, especially 200 to 2000D.

The reporter moieties may be any moieties detectable in an imaging modality, e.g. mr, X-ray (e.g CT) , ultrasound, light imaging, nuclear imaging, magnetotomography, electrical impedance tomography, etc. - 12 -

Suitable MR reporters include persistent free radicals (e.g. nitroxyl or trityl radicals), chelated paramagnetic metal ions (particularly transition metal or lanthanide ions, e.g. Mn, Fe, Cr, Gd, Dy, or Ho ions), and superparamagnetic metal, metal oxide or mixed metal oxide particles (e.g. ferrites) .

Chelating agents for binding such metal ions may be selected from the many known from the literature. These are generally polyoxyacids optionally on a N, 0 or S interrupted carbon skeleton, e.g. linear and cyclic poly amino carboxylic acids such as DTPA, DTPA-BMA, D03A, HP- D03A, DOTA, etc. as well as others such as DPDP, TMT, etc. Many such chelating agents are known from the literature, e.g. the published patent applications of Nycomed, Salutar, Sterling Winthrop, Schering, Bracco,

Guerbet , Squibb and Mallinckrodt . Such chelating agents generally include groups (such as the oxyacid groups) which can readily be conjugated to groups on the vector moieties. Alternatively they may be background derivatised, e.g. as in the SCN-Ph-derivatized DOTA of Meares et al . Mono chelants may be used but it is also feasible to use polychelants , e.g. D03A dimers or polymers such as polylysine-poly DTPA and DOTA- derivatized low generation dendrimers . Particulates, such as superparamagnetic agents, may be conjugated to the vector using bifunctional agents, such as derivatized PEG phosphates, which at one end bind to the particle surface and at the other bind to the vector, or alternatively where the particle has a lipid membrane around it the vector may be bound to a lipophilic group which penetrates into the membrane and serves as an "anchor" .

The conjugation of vectors to reporters, as well as selection of vectors and reporters is discussed for example in the PCT applications published on 7 May 1998 referred to above and in Appendix I hereto.

Suitable X-ray reporters include chelates of heavy - 13 - metal or cluster ions (e.g. W and W oxide/sulphide clusters and Bi cluster ions) or organic molecules with covalently bound heavy (atomic weight ≥ 32) atoms, e.g. triiodophenyl compounds. Again such materials may be bound directly to the vector or may be contained in or attached to a vesicle (e.g. microbubble, microballoon, micelle, microsponge or liposome) which is bonded to the vector or which has the vector anchored in its membrane. Typically, where particulate reporters such as vesicles or crystalline particles are used, particle sizes will be in the range 3 to 20000 nm, especially 10 to 4000 nm, in particular 30 to 2000 nm.

Suitable ultrasound reporters include gas- containing vesicles, in particular encapsulated microbubbles where the encapsulating material comprises a solid lipid or polymeric material or where it comprises a film, e.g. of a film forming phospholipid. The film forming material may be a mono- or multilayer which encapsulates the gas. Suitable gases include nitrogen, nitrous oxide, carbon dioxide, SF₆, low molecular weight hydrocarbons (e.g. with up to 8 carbons) , fluorinated low molecular weight hydrocarbons (e.g. perfluorobutane , perfluoropentane or perfluoropropane) , and mixtures thereof, e.g. mixtures with air or blood gases. Particularly preferred are perfluorocarbons, e.g. perfluoropentane, perfluoropropane and perfluorobutane . By gas is meant a material or mixture which is gaseous at 37°C. The membranes for such vesicles may be synthetic polymers or more preferably phospholipids .

Suitable light imaging reporters typically include chromophores having an extended delocalized electron system. Preferred materials will have absorption and optionally also emission maxima in the wavelength range 600 to 1200 nm, e.g. 600 to 1100 nm. Examples include phthalocyanines , naphthalocyanines, merocyanines, squarylium dyes, azo dyes, triphenylmethane dyes, - 14 - porphyrins, metalloporphyrins, etc. Further examples of suitable chromophores, ie. light imaging reporters, are described in Matsuoka "Topics in Applied Chemistry: Infrared absorbing dyes", Plenum, NY, 1990 and in PCT/GB97/02956 and PCT/GB97/02957 and in Appendix I hereto .

Suitable magnetometric imaging reporters include paramagnetic and superparamagnetic materials as mentioned above. Suitable nuclear imaging reporters include organic molecules with covalently bound radionuclides (e.g. I radioisotopes) as well as chelated metal radionuclides (e.g. Tc, Sm, In, Y and Ga isotopes such as ^99mTc, ^U1ln, ^l3jSm and ⁶⁷Ga) . These can be attached to the vector as described above for the X-ray and MR reporters (see S. Jurisson et al . in Chem. Rev 9_3_: 1137-1156 (1993) .

Viewed from a further aspect the invention also provides a library of vector : reporter conjugates in which where the vector is an oligomer (and optionally also where vectors are non-oligomeric) the conjugate contains a heterologous reporter, preferably a library of vector : reporter conjugates including a plurality of mutually interdistinguishable reporter moieties.

Viewed from a yet further aspect the invention provides a method of contrast agent preparation which comprises performing a method of candidate selection according to the invention and manufacturing the selected candidate, e.g. under pharmaceutical manufacturing conditions. The invention also extends to the production and use of libraries of candidate contrast agents which do not include a targeting (vector) moiety and where selection of successful candidates is made on the basis of successful performance with regard to one or more parameters considered useful for a contrast agent, e.g. biodistribution (for example organ or tissue or cell surface targeting) , viscosity, surface charge, protein - 15 - binding, lipophilicity, hydrophilicity, stability, pressure or temperature sensitivity, echogenicity, magnetization, relaxivity, fluorescence efficiency, toxicity, osmolality, charge distribution, dipole moment, solubility, metabolic breakdown, complement activation, excretion rate, dispersibility, flexibility, pH stability, oxygen stability, etc.

The invention will now be described further with reference to the following non-limiting Examples:

- 16 - EXAMPLE 1

Library Preparation

Synthesis of a library of radiopharmaceutical contrast agents comprising of ^U1lndium chelate of D03A conjugated to endothelial related peptides:

A mixture of endothelin related peptides (commercially available for Advanced ChemTech, USA) is added to a solution of 10- [7- (4-isothiocyanatophenyl) -2-hydroxy-5- oxo-1,4, 7 (carboxymethyl) -1,4,7, 10-tetraazacyclododecane (prepared according to Dinkelborg et al . in WO 96/02274, Example 1) in DMF . The mixture is stirred for 48 hours at ambient temperature followed by evaporation of the solvent and labelling with ^U1lndium (III) in the form of the acetate .

The library consists of ^U1lndium D03A-labelled endothelin related peptides.

EXAMPLE 2

Library Preparation

Synthesis of a library of contrast agents consisting of GdDTPA bisamides :

Diethylenetriamine pentaacetic acid bisanhydride (DTPA- A) is added to a mixture of 40 different primary and secondary amines (in total 2 equivalents relative to DTPA-A, 0.05 equivalents of each amine) in dry chloroform or acetonitrile (depending on the nature of amines) . The mixture is refluxed under nitrogen atmosphere for 24 hours. The solvent is evaporated, the residue is dissolved in distilled water and titrated (arsenazo III) with gadolinium chloride solution at pH 4.5-6. - 17 -

The library contains up to 1560 different GdDTPA- bisamides .

EXAMPLE 3

Library Preparation

Synthesis of X-ray contrast agent libraries consisting of triiodobenzoic acid amides:

Triiodobenzoic acid chlorides prepared according to the patents/patent applications from Schering, Nycomed, Bracco, Guerbet and Mallinckrodt (e.g. 2 , 4 , 6-triiodo- benzoic acid chlorides in which the 3 and 5 positions carry optionally protected hydroxylated C^g alkyl or hydroxylated C^ alkyl-carbonylamino groups, etc.) and primary/secondary alkyl amines with at least one hydrophilic subsituent (for example an OH-group) which are commercially available from Fluka and Aldrich are used to generate two sets of sample mixtures .

In the first set of sample mixtures one pure acid chloride is reacted with a mixture of 10 different amines (equimolar ratio of amine and acid chloride; equimolar amounts of each amine) in DMF with one equivalent of triethylamine .

In the second set of sample mixtures one pure amine is reacted with a mixture of 10 acid chlorides (equimolar ratio of amine and acid chloride, equimolar amounts of each acid chloride) in DMF with one equivalent of triethylamine .

The two sets of sample mixture now each consist of 10 contrast agent libraries each containing 10 different contrast agents (see Scheme 1 below) . The iodine content in each library is the same. DMF is evaporated for each library, the residue is dissolved in water and - 18 - filtered through a mixed ion exchange column to remove triethylamine hydrochloride . The aqueous solutions of libraries (20 libraries each containing 10 contrast agents (100 different contrast agents x 2)) are evaporated and dissolved in a fixed volume of pure water. Each library thus theoretically contains 10 amide contrast agents and each library contains the same concentration of iodine per ml.

Similar libraries can be prepared using triiodoisophthalic acid chlorides or by using bifunctional reagents to make dimers .

Ri

H-N \

R₂

ACi-io Al-10

ACi + Ai-io AC₂ + Ai-io

10 Libraries

ACio + Ai-io

ACi-io + Al ACi-io + A2 10 Libraries

ACi-io + Aio

SCHEME 1 - 19 - EXAMPLE 4

Library Preparation

Synthesis of a library of contrast agents for MRI and/or magnetic source imaging consisting of conjugates from superparamagnetic carboxymethyldextran particles and dipeptides :

Superparamagnetic carboxymethyldextran particles are prepared according Schroder et al . US-A-5720939, Example

5.

The particle suspension is divided into 10 vials. 10 different -amino acid (X1-X10) t-butyl esters (one equivalent for each carboxy group) and l-ethyl-3- (3- dimethylaminopropyl carbodiimide hydrochloride (EDO (one equivalent) are added to the suspensions (see Scheme 2) . The mixtures were stirred at ambient temperature for 24 hours and purified by dialysis. The particle suspensions were mixed and the ester groups cleaved in 0. IN HCl at ambient temperature during 24 hours. The particle suspension was purified by dialysis and divided into 10 different vials. 10 different - amino acids (Y1-Y10) are added and coupled in the same way as described above. The esters are cleaved and the products purified.

The final product is a library consisting of 10 sublibraries, each sublibrary consisting of 10 different superparamagnetic particles coated with 100 different dipeptides .

Libraries of superparamagnetic particles containing other peptides or other organic compounds can be prepared in a similar way using conventional chemistry, e.g. solid support chemistry. 20

fFe₃0₄j— CH₂COOH

Xi X 10 10 vials

[ Fe₃0₄ )— CH₂CONH(RCOOH) XltoXio

Yl ... 10

Fe₃0₄— X,Y, X, 1Y * 10

^Fe₃0₄—X₂Y, Fe₃0₄1 X₂Y 10

(Fe₃O₄—X₁₀Y₁₀

^Fe₃0₄Y-COOH H₂NCRHCOOtBu H₂NCRHCOOtBu Xi to X10 Yi toY,o

SCHEME 2

EXAMPLE 5

Library Preparation

Preparation of libraries of MR contrast agents consisting of paramagnetic liposomes with different surface properties : - 21 -

Multilamellar liposomes containing isotonic GdHPD03A solution are prepared by the thin film method (see Lasic "Liposomes from Physics to Applications" . Elsevier 1993, pages 63-107) using hydrogenated phosphatidylcholine (Lipoid GmbH, Ludwigshafen, Germany) and C-caproylamine-phosphatidylethanolamine (Avanti Polar Lipid, USA) in ratio 7:3 (w:w) . The extruded liposomes are subjected to freeze-thaw cycles and extruded and untrapped metal chelates are removed by dialysis. GdHPD03A containing liposomes with primary amino groups are isolated as a suspension in isotonic glucose solution. The suspension is divided into 5 vials and 5 different maleimido-succinimide esters (XI- X5) are added to the suspensions (see Scheme 3) (The maleimido-succinimide esters are commercially available from Pierce and others and/or are well described in the literature) . The mixtures are stirred at ambient temperature for 24 hours. The particles are mixed and divided into 4 vials and 4 different mercapto compounds (Y1-Y4) are added. The mixture is stirred at ambient temperature for 12 hours .

In this way, 4 different libraries each consisting of 5 different paramagnetic liposomes are prepared.

The mercapto compounds can be any mercapto containing compound including both synthetic and biological compounds like for example peptides, proteins, modified nucleotides, mercapto sugars, unsubstituted and substituted alkyl or aryl mercaptanes . 22

( CAV-NHX, ( _CAVNHX₂ (CA — NHXYCA NHX, ^CA NHX,

f CA j— NHX.Y, ( CA —NHX,Y₂ ( CA )— NHX,Y₃ ( CA )— NHX,Y₄

CA\-NHX₂Y, CCA -NHX₂Y₂ (CA\-NHX₂Y₃ CAV-NHX₂Y₄

(CA — NHX₃Y, ^\__NHX₃Y₂ MCA -NHX₃Y₃ ( CA )— NHX₃Y₄

CA )-NHX4Y, ( CA >-^NHX4^Y2 ( ^CA )-^NHX>^Y3 ( CA V^NHχ,^γ,

( CA j— NHX₅Y, ( CA — NHX₅Y₂ ( CA\— NHX₅Y₃ (CAV-NHXJY_,

R-SH

N-R— π— O-N CA H^NH2 Y, to Y<

X, toX₅

SCHEME 3

EXAMPLE 6

Library Preparation

Preparation of a library of gas-filled microbubbles encapsulated by phosphatidylserine and different peptide-Mal-PEG₃₄₀₀-distearoylphosphatidylethanolamine: Perfluorobutane containing microbubbles coated with - 23 - phosphatidylserine (90% mol) and Mal-PEG₃₄₀₀-DSPE (10 mol%) are prepared according to Example 2e) in PCT/GB97/02954. 2 -mercaptoethylamine is added to a suspension of the microbubbles in phosphate buffer at pH 7.5. The mixture is placed on a roller table at ambient temperature for 24 hours followed by washing.

The suspension is divided into 5 vials (each containing 5 different BOC-protected α-amino acids) and EDC is added. The amino acids are coupled to microbubbles as previously described. (This type of coupling is well described in connection with solid phase peptide synthesis) . The mixtures are purified, mixed and split into 4 vials followed by coupling of 4 other BOC- protected α-amine acids. The mixtures are purified resulting in 4 libraries each containing 5 different gas-filled microbubbles with different peptides on the surface .

A library of gas-filled microbubbles encapsulated by phosphatidylserine and with different surface and charge (zeta potential) can be prepared in a similar manner using other meracapto containing compounds, and if desired, other carboxylic acids.

Other libraries of ultrasound contrast agents can be prepared using the chemical methods described in the PCT applications published 7 May 1998 referred to above and in Appendix I hereto .

EXAMPLE 7

Library Preparation

Preparation of a library of gas- filled microbubbles tagged with radioactive material: - 24 -

Perfluorobutane containing microbubbles (phosphatidylerine (90 mol%) , phosphatidylethanolamine DTPA (5 mol%) (prepared according to Grant et al . in Maσn Res. Med 11 236-243 (1989) and Mal-PEG₃₄₀₀-DSPE (5 mol%) ) are prepared according to Example 2e) in PCT/GB97/02954.

The particles are labelled with In in 3 different concentrations (Tl, T2 and T3) (See Figure 1) . Three different ligands (A, B and C) are coupled to the particles followed by pooling and splitting. The 3 particle suspensions are further tagged with different concentrations of ¹⁵³Sm (Ul, U2 and U3) . Three different ligands (A, B, C) are coupled to the particles and the result is 3 libraries each containing 3 ultrasound contrast agents each with a unique radioactive label.

This can be expanded to very big libraries using combinations of many different isotopes.

Claims

- 25 - Claims :

1. A method for the selection of a candidate targeted contrast agent, comprising the steps of: (i) obtaining a library of vector : reporter conjugates in which conjugates where the vector is an oligomer (and optionally also where vectors are non-oligomeric) the conjugate contains a heterologous reporter; and

(ii) testing the members of said library to identify a said member having a binding affinity for a biological target.

2. A method as claimed in claim 1 wherein a plurality of different types of reporter molecule are present in the vector : reporter conjugates.

3. A method as claimed in claim 1 or claim 2 wherein said biological target is a known receptor.

4. A method as claimed in any one of claims 1 to 3 wherein the library of vector : reporter conjugates comprises a collection of at least 3, preferably at least 10, more preferably at least 10², most preferably at least 10³, vector : reporter conjugates.

5. A method as claimed in any one of claims 1 to 4 wherein the ratio of vector and reporter moieties in said vector : reporter conjugates is from 10:1 to 1:10.

6. A method as claimed in claim 5 wherein said ratio is 1:1.

7. A method as claimed in any one of claims 1 to 6 wherein the library of vector : reporter conjugates comprises a number of different types of vector - 26 - moieties .

8. A method as claimed in any one of the preceding claims wherein the vector moieties of the vector: reporter conjugates are selected from the group comprising small organic molecules, drug compounds, peptides, peptoids, oligomers, antibodies and antibody fragments .

9. A method as claimed in claim 8 wherein said vector moieties are selected from the group consisting of oligopeptides , oligopeptoids , oligonucleotides and oligosaccharides .

10. A method as claimed in claim 8 or claim 9 wherein said oligomers comprise natural or synthetic monomers .

11. A method as claimed in any one of the preceding claims wherein the reporter moiety is detectable in an imaging modality.

12. A method as claimed in claim 11 wherein said imaging modality is selected from the group comprising MR, X-ray, ultrasound, light imaging, nuclear imaging, magnetotomography and electrical impedance tomography.

13. A method as claimed in claim 11 or claim 12 wherein the reporter moiety is selected from the group comprising chromophores, fluorophores, mono-chelates, polychelates, chelated paramagnetic metal ions, superparamagnetic metal, metal oxide or mixed metal oxide particles, persistent free radicals, particulates, iodinated organic compounds or other organic molecules with covalently bound radionuclides and gas containing vesicles optionally tagged with one or more radiolabels.

14. A method as claimed in any one of the preceding - 27 - claims wherein the vector : reporter conjugates are tagged to identify the library members or library sub-sets.

15. A method as claimed in claim 14 wherein said tags are selected from the group comprising virus particles, oligonucleotides, radionuclides, a set of radionuclides and radioisotopes .

16. A method as claimed in any one of the preceding claims wherein the members of said library of vector: reporter conjugates are provided as a mixture.

17. A method as claimed in claim 16 wherein a deconvolution step is carried out to identify members of said library which have a binding affinity for said target, or to identify a subset of the library to which a member having said binding affinity belongs.

18. A method as claimed in claim 17 wherein said deconvolution step is carried out either by reading a tag associated with a library member or sub-set of library members, or by orthogonal scanning.

19. A method as claimed in any one of the preceding claims wherein a plurality of libraries are screened, with successive libraries being designed on the basis of the results from the screening of the earlier libraries.

20. A method for the selection of a candidate targeted contrast agent, comprising the steps of: (i) obtaining a library of vector : reporter conjugates including a plurality of mutually interdistingishable reporter moieties; (ii) testing the members of said library for binding affinity to a biological target and using the interdistinguishability of said reporters identifying a member of said library exhibiting relatively superior binding affinity or a sub-set of said library containing - 28 - at least one member of said library exhibiting relatively superior binding affinity; and (iii) if necessary testing sub-sets of said library to identify said member of said library exhibiting relatively superior binding affinity.

21. A method as claimed in claim 20 wherein said mutally interdistinguishable reporter moieties include chelates of different metal radionuclides which are interdistinguishable by the nature and energy of their radiation emissions, chromophores having different characteristic absorption or emission maxima, crystalline particles having different X-ray scattering angles, and vesicles containing different gases or having different acoustic properties such as non-linear behaviour .

22. A method as claimed in any one of the preceding claims wherein multiple sites on the library members are tagged so that the combination of tags serves to uniquely identify the library member or to identify a relatively small library sub-set in which it may be found.

23. A method as claimed in any one of the preceding claims further comprising the step of manufacturing said selected member or a derivative thereof and optionally formulating it with at least one pharmaceutical carrier or excipient .

24. A method as claimed in any one of the preceding claims wherein the vector moieties in the first library used are generated on a random basis.

25. A method as claimed in any one of the preceding claims wherein vector moieties in the first library used are a directed random selection generated by carrying - 29 - out a pre-screening step, said pre-screening step including obtaining a random combinatorial oligomeric vector library in which the library members are not conjugated to reporter moieties and testing members of this library to eliminate vector structures which do not show reasonable binding affinities for said target.

26. A library of vector : reporter conjugates in which conjugates where the vector is an oligomer (and optionally also where vectors are non-oligomeric) the conjugate contains a heterologous reporter.

27. A library of vector : reporter conjugates including a plurality of mutually interdistinguishable reporter moieties.

28. A library of vector : reporter conjugates wherein the vector : reporter conjugates are as defined in any one of claims 1 to 25.

29. A library of vector : reporter conjugates for use in a method as defined in any one of claims 1 to 25.

30. A method of contrast agent manufacture comprising (i) obtaining a library of vector : reporter conjugates in which conjugates where the vector is an oligomer (and optionally also where vectors are non-oligomeric) the conjugate contains a heterologous reporter; (ii) testing the members of said library to identify a said member having a binding affinity for a biological target; and (iii) manufacturing said identified member, preferably under pharmaceutical manufacturing conditions.