Labelled factor XIIIa substrates

Details Labelled factor XIIIa substrates Labelled factor XIIIa substrates

1999-10-04

2003-11-25

O'Sullivan, Peter (Department: 1621)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Heterocyclic carbon compounds containing a hetero ring...

C514S255010, C514S353000, C514S492000, C514S603000, C514S626000, C540S474000, C544S390000, C546S306000, C556S037000, C564S086000

Reexamination Certificate

active

06653299

ABSTRACT:

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
111
In 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 NH
2
and CO
2
NH
2
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 &ggr;-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,N′-dimethylcasein catalysed by pig liver transglutaminase [L. Lorand et al., Biochem., 18, 1756(1979)].
The possible use of radiolabelled diamines of formula H
2
N(CH
2
)
n
NHR* (n and R* undefined) as potential clot imaging agents was disclosed by Rhodes et al (Chapter 54, p.521 in “Radiopharmaceuticals”, G. Subramanian, B. A. Rhodes, J. F. Cooper & 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(CH
2
)
2
—X—(CH
2
)
2
NH
2
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 “any peptide that is a substrate for the blood enzyme commonly known as Factor XIIIa”.
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
—(Y)
m
—A—NHR,
where:
Y is the same or different at different locations within the molecule and is independently chosen from: an A group, a C
4-8
cycloheteroalkylene group, a C
4-8
cycloalkylene group, a C
5-12
arylene group, a C
3-12
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 —CR
2
—, —CR═CR—, —C≡C—, —NRCO—, —CONR—, —SO
2
NR—, —NRSO
2
—, or —CR
2
ZCR
2
— where Z is —CH
2
—, O, S, Se or —NR—,
R is the same or different at different locations within the molecule and is independently chosen from H, C
1-4
alkyl, C
1-4
alkenyl, C
1-4
alkynyl, C
1-4
alkoxyalkyl or C
1-4
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
—NHCO(CH
2
)
2
Z(CH
2
)
2
—, or
—SO
2
NH(CH
2
)
2
Z(CH
2
)
2
—, or
—(CH
2
)
2
Z(CH
2
)
2
—.
Preferably Z is CH
2
. Particularly preferred substituents have the formula
—(Y)
b
—Ar—SO
2
NH(CH
2
)
5
NH
2
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 —(Y)
m
—A—NHR substituent. Other substituents on the complexing agent may be present, but the —(Y)
m
—A—NHR substituent is the one which is expected to be primarily responsible for the biolocalisation properties. The —(Y)
m
—A—NHR 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
68
Ga or
64
Cu) or a &ggr;-emitter such as
99m
Tc,
111
In,
113m
In or
67
Ga. 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 &ggr;-emitters, especially
99m
Tc. 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:
90
Y,
89
Sr,
67
Cu,
186
Re,
188
Re,
169
Er,
153
Sm

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