The present invention relates to a class of compounds useful in the diagnosis of sites of thrombosis, embolism or infection, pharmaceutical formulations containing them, their use in the diagnosis of disease and methods for their preparation.
Prior approaches to thrombus imaging radiopharmaceuticals include radiolabelled fibrinogen or plasminogen; radiolabelled fragment E, of human fibrin; radiolabelled plasminogen activators such as tissue plasminogen activator (t-PA) and labelled anti-fibrin antibodies. Methods based on the detection of sites of platelet accumulation such as the administration of radiolabelled platelets (e.g. using 111In oxine) or radiolabelled anti-platelet antibodies have also been described. More recent efforts have focused on radiolabelled peptides or polypeptides such as the cell adhesion motif RGD (where R, G and D are the standard abbreviations for the amino acids arginine, glycine and aspartic acid respectively); platelet factor 4 or fragments thereof or anticoagulant peptides such as disintegrins.
Factor XIII is a plasma glycoprotein which is present in blood and certain tissues in a catalytically inactive (or zymogen) form. Factor XIII is transformed into its active form Factor XIIIa by thrombin in the presence of calcium ions. Factor XIIIa is also known as plasma transglutaminase, fibrinoligase or fibrin-stabilizing factor. The final step in the formation of a blood clot is the covalent crosslinking of the fibrin which is formed by the proteolytic cleavage of fibrinogen by thrombin. Fibrin molecules align and the enzyme Factor XIIIa catalyses covalent crosslinking of the NH2 and CO2NH2 groups of lysyl and glutaminyl residues respectively giving structural rigidity to the blood clot. The crosslinking stabilises the fibrin clot structure and confers resistance to fibrinolysis. The crosslink formation is an important facet of normal blood coagulation and wound healing as well as pathological conditions such as thrombosis. It may also be implicated in atherosclerosis and tumour growth and metastasis. WO 91/16931 discloses that radiolabelled analogues of Factor XIII (in which the active site has been inactivated by amino acid substitution) are useful as thrombus imaging radiopharmaceuticals.
Factor XIIIa is also known to catalyse the incorporation of low molecular weight amines into the xcex3-glutamine sites of proteins. Thus such low molecular weight amines function as competitive inhibitors of the Factor XIIIa-induced glutaminyl crosslinking of proteins. A range of synthetic amines have been described which are competitive inhibitors of the uptake of labelled putrescine (1,4-butanediamine) into N,Nxe2x80x2-dimethylcasein catalysed by pig liver transglutaminase [L. Lorand et al., Biochem., 18, 1756(1979)].
The possible use of radiolabelled diamines of formula H2N(CH2)nNHR* (n and R* undefined) as potential clot imaging agents was disclosed by Rhodes et al (Chapter 54, p.521 in xe2x80x9cRadiopharmaceuticalsxe2x80x9d, G. Subramanian, B. A. Rhodes, J. F. Cooper and V. J. Sodd [Eds], Society of Nuclear Medicine Inc., 1975). They envisaged that a radiolabelled amine which was an inhibitor of the crosslinking of fibrin could form a substrate for Factor XIIIa and hence become attached to the fibrin of blood clots. U.S. Pat. No. 4,406,075 (Mallinckrodt) discloses radiolabelled aliphatic amines for blood clot imaging of formula:
Y(CH2)2xe2x80x94Xxe2x80x94(CH2)2NH2
where X is O, S, Se*, Te* or lower alkylene.
When X is Se* or Te*, Y is a hydrocarbylamino group, and
when X is O, S or lower alkylene, Y is a radioiodinated hydrocarbylamino group (or salt thereof).
(* denotes a radioactive atom).
WO 89/00051 (Cytrx Biopool Ltd.) claims a method for targeting fibrin deposits using a labelled compound which is covalently bound to fibrin by Factor XIIIa. The fibrin binding compound is stated to be xe2x80x9cany peptide that is a substrate for the blood enzyme commonly known as Factor XIIIaxe2x80x9d.
It has now been discovered that metal complexes with suitable pendant functional groups can also function as substrates for the enzyme Factor XIIIa. Since Factor XIIIa is only released at pathological sites from an inactive precursor, targeting this enzyme provides a means of targeting a diagnostic imaging agent to the site of Factor XIIIa release.
The present invention provides in one aspect a metal complexing agent having attached thereto at least one substituent of formula
xe2x80x94(Y)mxe2x80x94Axe2x80x94NHR,
where:
Y is the same or different at different locations within the molecule and is independently chosen from: an A group, a C4-8 cycloheteroalkylene group, a C4-8 cycloalkylene group, a C5-12 arylene group, a C3-12 heteroarylene group, or a polyalkyleneglycol, polyactic acid or polyglycolic acid moiety,
m is an integer of value 0-20,
A is a 3-10 atom chain of units selected from xe2x80x94CR2xe2x80x94, xe2x80x94CRxe2x95x90CRxe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, xe2x80x94NRCOxe2x80x94, xe2x80x94CONRxe2x80x94, xe2x80x94SO2NRxe2x80x94, xe2x80x94NRSO2xe2x80x94, or xe2x80x94CR2ZCR2xe2x80x94 where Z is xe2x80x94CH2xe2x80x94, O, S, Se or xe2x80x94NRxe2x80x94,
R is the same or different at different locations within the molecule and is independently chosen from H, C1-4 alkyl, C1-4 alkenyl, C1-4 alkynyl, C1-4alkoxyalkyl or C1-4 hydroxyalkyl, with the proviso that the complexing agent does not also have attached thereto a hypoxia localising moiety.
The invention also provides a metal complex of one or more radiometal or paramagnetic metal ions with the metal complexing agent as defined; both as a new compound per se and also for use in the diagnosis or therapy of thrombosis, embolism, atherosclerosis, inflammation or cancer.
Preferably the metal complexing agent is a metal chelating agent, for example a diaminedioxime. In preferred substituents, A is
xe2x80x94NHCO(CH2)2Z(CH2)2xe2x80x94, or
xe2x80x94SO2NH(CH2)2Z(CH2)2xe2x80x94, or
xe2x80x94(CH2)2Z(CH2)2xe2x80x94.
Preferably Z is CH2. Particularly preferred substituents have the formula
xe2x80x94(Y)bxe2x80x94Arxe2x80x94SO2NH(CH2)5NH2
where b is an integer of value 0 to 19 and Ar is an arylene or heteroarylene group.
The complexing agents of the present invention preferably only have a single type of targeting molecule attached, i.e. the xe2x80x94(Y)mxe2x80x94Axe2x80x94NHR substituent. Other substituents on the complexing agent may be present, but the xe2x80x94(Y)mxe2x80x94Axe2x80x94NHR substituent is the one which is expected to be primarily responsible for the biolocalisation properties. The xe2x80x94(Y)mxe2x80x94Axe2x80x94NHR substituent may be attached to either the backbone which connects metal donor atoms of a metal complexing or chelating agent, or to a metal donor atom of the metal complexing or chelating agent.
When Y includes a biocompatible, hydrophilic polymer such as a polyalkyleneglycol, polylactic acid or polyglycolic acid, this polymeric linking group may be useful to prolong the residence time of the metal complex in the bloodstream, ie. to slow down the rate of clearance from the blood following administration. The hydrophilic polymer is preferably a polyalkyleneglycol, most preferably polyethyleneglycol (PEG). The hydrophilic polymer preferably has a molecular weight of 2,000 to 20,000 Daltons.
The metal complex of the present invention may contain one or more metal ions which may be the same or different. Thus in some circumstances polynuclear complexes may have advantageous properties such as certain metal clusters which have superparamagnetic properties and are hence particularly useful as MRI contrast agents. Preferred metal complexes of the present invention involve only a single metal ion. When the metal of the metal complex is a radiometal, it can be either a positron emitter (such as 68Ga or 64Cu) or a xcex3-emitter such as 99mTc, 111In, 113mIn or 67Ga. Suitable metal ions for use in MRI are paramagnetic metal ions such as gadolinium(III) or manganese(II). Most preferred radiometals for diagnostic imaging are xcex3-emitters, especially 99mTc. Metal complexes of certain radionuclides may be useful as radiopharmaceuticals for the radiotherapy of various diseases such as cancer or the treatment of thrombosis or restenosis. Useful radioisotopes for such radiotherapeutic applications include: 90Y, 89Sr, 67Cu, 186Re, 188Re, 169Er, 153Sm and 198Au. Whichever metal complex is chosen, it is strongly preferred that it is bound to the Factor XIIIa substrate in such a way that it does not undergo facile metabolism in blood with the result that the metal complex is cleaved from the Factor XIIIa substrate before the labelled Factor XIIIa substrate reaches the desired in vivo site to be imaged. The Factor XIIIa substrate is therefore preferably covalently bound to the metal complexes of the present invention.
The metal ions of the present invention are complexed using a metal complexing agent or more preferably a metal chelating agent. The chelating agents comprise 2-10 metal donor atoms covalently linked together by a non-coordinating backbone. Preferred chelating agents have 4-8 metal donor atoms and have the metal donor atoms in either an open chain or macrocyclic arrangement or combinations thereof. Most preferred chelating agents have 4-6 metal donor atoms and form 5- or 6-membered chelate rings when coordinated to the metal centre. Such polydentate and/or macrocyclic chelating agents form stable metal complexes which can survive challenge by endogenous competing ligands for the metal in vivo such as transferrin or plasma proteins. Alternatively it is possible to use monodentate complexing agents that form strong stable complexes with the desired metal ions. Examples of known complexing agents of this kind, which are particularly suitable for use with 99mTc, are hydrazines, phosphines, arsines and isonitriles. Unlike chelating agents, complexing agents do not necessarily occupy all the co-ordination centres of the metal ion.
The metal complex should also preferably be of low lipophilicity (since high lipophilicity is often related to non-specific uptake), and exhibit low plasma protein binding (PPB) since plasma-bound label again contributes to undesirable high, non-specific blood background for the imaging agent.
Examples of suitable chelating agents are diaminedioximes (U.S. Pat. No. 4,615,876) or such ligands incorporating amide donors (WO 94/08949); the tetradentate ligands of WO 94/22816; N2S2 diaminedithiols, diamidedithiols or amideaminedithiols; N3S thioltriamides; N4 ligands such as tetraamines, macrocyclic amine or amide ligands such as cyclam, oxocyclam (which forms a neutral technetium complex) or dioxocyclam; or dithiosemicarbazones. The above described ligands are particularly suitable for technetium, but are useful for other metals also. Other suitable ligands are described in Sandoz WO 91/01144, which includes ligands which are particularly suitable for indium, yttrium and gadolinium, especially macrocyclic aminocarboxylate and aminophosphonic acid ligands. Ligands which form non-ionic (i.e. neutral) metal complexes of gadolinium are known and are described in U.S. Pat. No. 4,885,363. The ligand may also comprise a short sequence of amino acids such as the Cys/amino acid/Cys tripeptide of WO 92/13572 or the peptide ligands described in EP 0719790 A2
It is well known to prepare chelating agents which have attached thereto a functional group (xe2x80x9cbifunctional chelatesxe2x80x9d). Functional groups which have been attached to chelating agents include: amine, carboxylic acid, cyanate, thiocyanate, maleimide and active ester such as N-hydroxysuccinimide. Examples of chelate-amine conjugates for diaminedioxime ligands are given in WO 95/19187. The ligands of the present invention can be prepared by reaction of a bifunctional compound which contains both an amine group (preferably protected by use of suitable protecting groups known to those skilled in the art), and a reactive group such as a sulphonyl chloride, acid chloride, active ester or an alkyl/benzyl halide. The reactive group can then be coupled to either the pendant amine group of a bifunctional chelate, or used to derivatise one or more of the amine donor atoms of a N-containing ligand. Alternatively, a mono-protected diamine could be reacted with a bifunctional chelate with a pendant active ester or carboxyl group to give the protected amine group linked to the ligand system via an amide bond. In both synthetic routes outlined above, the resulting ligand-protected amine conjugate is then deprotected under suitable conditions to give the desired amine-functionalised ligand.
The metal complexes of the present invention may be prepared by reacting a solution of the metal in the appropriate oxidation state with the ligand at the appropriate pH. The solution may preferably contain a ligand which complexes weakly to the metal (such as chloride, gluconate or citrate) i.e. the metal complex is prepared by ligand exchange or transchelation. Such conditions are useful to suppress undesirable side reactions such as hydrolysis of the metal ion. When the metal ion is 99mTc, the usual starting material is sodium pertechnetate from a 99Mo generator. Technetium is present in 99mTc-pertechnetate in the Tc(VII) oxidation state, which is relatively unreactive. The preparation of technetium complexes of lower oxidation state Tc(I) to Tc(V) therefore usually requires the addition of a suitable reducing agent such as stannous ion to facilitate complexation. Further suitable reductants are described below.
Thus the present invention relates to diagnostic agents for imaging sites in the mammalian body where the enzyme Factor XIIIa is up-regulated and fibrin is deposited. The present agents are particularly useful for the diagnostic imaging of the human body. The agents comprise substrates for the enzyme Factor XIIIa which are labelled with a metal complex suitable for external imaging such as a radiometal (for scintigraphy) or a paramagnetic metal ion (for MRI). The metal complex of the present invention has a pendant amino functional group which is available for covalent linking to protein glutamyl carboxamide groups by the enzyme Factor XIIIa. The intimate relationship of fibrin and Factor XIIIa highlights the potential use of the agents of the present invention for the diagnosis of disease states where there is both fibrin deposition or accumulation and up-regulation of Factor XIIIa. Increased fibrin deposition is known to be characteristic of diseases such as thrombosis, atherosclerosis, fibrotic liver, and disseminated intravascular coagulation. Fibrin is also deposited at sites of tissue inflammation associated with many disease processes, such as infection, autoimmune disease or cancer. Factor XIIIa and tissue transglutaminase are up regulated during known physiological conditions. During apoptosis and generation of new matrix protein structures elevated levels of the enzymes are seen. The present agents may thus also be used for the detection of apoptosis and diseases states such as arthritis where increased matrix protein deposition occurs. Since Factor XIIIa is up-regulated at the site of interest in vivo (i.e. thrombus, embolism etc.) this provides a localisation mechanism for the metal complexes of the present invention. The covalently linked metal complexes can then be imaged externally by radionuclide scintigraphy or magnetic resonance imaging (MRI) hence providing a non-invasive means of diagnosing the disease site.
The present invention also relates to kits for the preparation of metal complexes linked to Factor XIIIa substrates. The kits are designed to give sterile products suitable for human administration, e.g. via injection into the bloodstream. Possible embodiments are discussed below. When the detectable moiety is 99mTc, the kit would comprise a vial containing the free ligand or chelating agent for the metal together with a pharmaceutically acceptable reducing agent such as sodium dithionite, sodium bisulphite, ascorbic acid, formamidine sulphinic acid, stannous ion, Fe(II) or Cu(I), preferably a stannous salt such as stannous chloride or stannous tartrate. Alternatively, the kit could contain a metal complex which, upon addition of the radiometal or paramagnetic metal, undergoes transmetallation (i.e. ligand exchange) giving the desired product. For 99mTc, the kit is preferably lyophilised and is designed to be reconstituted with sterile 99mTc-pertechnetate (TcO4xe2x88x92) from a 99mTc radioisotope generator to give a solution suitable for human administration without further manipulation.
The agents of the present invention may also be provided in a unit dose form ready for human injection and could for example be supplied in a pre-filled sterile syringe. When the detectable moiety is a radioactive isotope such as 99mTc, the syringe containing the unit dose would also be supplied within a syringe shield (to protect the operator from potential radioactive dose).
The above kits or pre-filled syringes may optionally contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g. cyclodextrins or surfactants such as Pluronic, Tween or phospholipids); pharmaceutically acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or para-aminobenzoic acid) or bulking agents for lyophilisation (such as sodium chloride or mannitol).