U.S. patent application number 11/410177 was filed with the patent office on 2006-12-28 for labelled glutamine and lysine analogues.
Invention is credited to Susan Champion, Alex Gibson, Benedicte Guilbert, Peter Knox, Marivi Mendizabal, Anthony Eamon Storey, Ian Andrew Wilson.
Application Number | 20060292075 11/410177 |
Document ID | / |
Family ID | 8234827 |
Filed Date | 2006-12-28 |
United States Patent
Application |
20060292075 |
Kind Code |
A1 |
Storey; Anthony Eamon ; et
al. |
December 28, 2006 |
Labelled glutamine and lysine analogues
Abstract
Synthetic analogues of lysine and glutamine are provided which
function as substrates for the fibrin-stabilising enzyme Factor
XIIIa even when labelled with a detectable moiety. The use of
suitable protecting groups provides compounds which possess reduced
susceptibility to in vivo metabolism especially by peptidases, and
are hence useful agents for the diagnosis of thrombosis, embolism,
atherosclerosis, inflammation or cancer.
Inventors: |
Storey; Anthony Eamon;
(Grantham, GB) ; Mendizabal; Marivi; (London,
GB) ; Champion; Susan; (Tring, GB) ; Gibson;
Alex; (Oxford, GB) ; Guilbert; Benedicte;
(Watford, GB) ; Wilson; Ian Andrew; (Hitchin,
GB) ; Knox; Peter; (Chalfont St. Giles, GB) |
Correspondence
Address: |
GE HEALTHCARE, INC.
IP DEPARTMENT
101 CARNEGIE CENTER
PRINCETON
NJ
08540-6231
US
|
Family ID: |
8234827 |
Appl. No.: |
11/410177 |
Filed: |
April 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09674616 |
Dec 19, 2000 |
7049289 |
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PCT/GB99/01550 |
May 14, 1999 |
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11410177 |
Apr 24, 2006 |
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Current U.S.
Class: |
424/1.69 ;
514/1.9; 514/13.6; 514/14.2; 514/14.9; 530/317; 530/324; 530/325;
530/326; 530/327; 530/328 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 49/14 20130101; A61K 51/088 20130101; A61P 29/00 20180101;
A61P 9/10 20180101; A61P 7/02 20180101 |
Class at
Publication: |
424/001.69 ;
514/009; 514/012; 514/013; 514/014; 514/015; 514/016; 530/324;
530/325; 530/326; 530/327; 530/328; 530/317 |
International
Class: |
A61K 51/00 20060101
A61K051/00; A61K 38/12 20060101 A61K038/12; A61K 38/08 20060101
A61K038/08; C07K 7/64 20060101 C07K007/64; C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
May 15, 1998 |
EP |
98303872.0 |
Claims
1-16. (canceled)
17. A compound of formula: Y--(CR.sub.2).sub.n--X--NHJ where: X is
C.dbd.O or --CR.sub.2--; n is an integer of value 1 to 6; Y is
R.sup.1R.sup.2CR-- where one of R.sup.1 and R.sup.2 is
--NH(B).sub.pZ.sup.1 and the other is --CO(B).sub.qZ.sup.2 where p
and q are integers of value 0 to 45, and each B is independently
chosen from Q or an amino acid residue, where Q is a cyclic
peptide; Z.sup.1 and Z.sup.2 are metal complexing agents (L) or
other protecting groups which are biocompatible groups which
inhibit or suppress in vivo metabolism of the peptide; J and each R
group are independently chosen from H, C.sub.1-4 alkyl, C.sub.1-4
alkenyl, C.sub.1-4 alkynyl, C.sub.1-4 alkoxyalkyl or C.sub.1-4
hydroxyalkyl; with the provisos that: (i) the total number of amino
acid residues in the R.sup.1 and R.sup.2 groups does not exceed 45;
(ii) when X is CR.sub.2 then Z.sup.2 is a metal complexing agent
(L); (iii) at least one of R.sup.1 and R.sup.2 bears at least one
detectable moiety suitable for diagnostic imaging of the human body
chosen from a non-metallic radioisotope or a radiometal; (iv)
R.sup.1 or R.sup.2 includes one or more peptide fragments of
.alpha..sub.2-antiplasmin, fibronectin, beta-casein, tetanus,
amyloid, trappin and polyglutamine residues, said peptide fragment
containing at least three amino acid residues.
18. The compound of claim 17 where the peptide fragment of proviso
(iv) is from .alpha..sub.2-antiplasmin.
19. The compound of claim 18 where the amino acid in the 2-position
from the peptide N-terminus is glutamine.
20. The compound of claim 17 where J is H.
21. The compound of claim 20 of formula:
Y--(CR.sub.2).sub.x--(CH.sub.2).sub.2CONH.sub.2 or
Y--(CR.sub.2).sub.y--(CH.sub.2).sub.4NH.sub.2 where x is an integer
of value 0 to 4, and y is an integer of value 0 to 3.
22. The compound of claim 17 where at least one of Z.sup.1 and
Z.sup.2 is a metal complexing agent.
23. The compound of claim 22 where Z.sup.2 is a metal complexing
agent and Z.sup.1 is not a metal complexing agent.
24. A metal complex of the compound of claim 22.
25. The metal complex of claim 24 where the metal is a
radiometal.
26. The radiometal complex of claim 25 where the radiometal is
.sup.99mTc.
27. A preparation for human administration comprising the compound
of claim 17.
28. A kit comprising the compound of claim 17 for use in the
preparation of the metal complex of compound of claim 17 where at
least one of Z.sup.1 and Z.sup.2 is a metal complexing agent.
29. Use for the diagnosis of sites of thrombosis or embolism of a
compound of formula: Y--(CR.sub.2).sub.n--X--NHJ where: Y, X, R n
and J are as defined in claim 17.
30. Use for the diagnosis of sites of thrombosis or embolism of a
radiometal complex of the compound defined in claim 26, wherein at
least one of Z.sup.1 and Z.sup.2 is a metal complexing agent.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to a class of compounds useful
in the diagnosis of sites of venous and arterial thrombosis,
embolism or infection, pharmaceutical formulations containing them,
their use in the diagnosis of disease and methods for their
preparation.
BACKGROUND OF THE INVENTION
[0002] Prior approaches to thrombus imaging radiopharmaceuticals
include radiolabelled fibrinogen or plasminogen; radiolabelled
fragment E.sub.1 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 .sup.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.
[0003] 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-stabilising 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.sub.2 and CONH.sub.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. As atherothrombotic brain infarctions are a
common sub-type of stroke, Factor XIIIa substrates may allow
diagnosis of stroke. It may also be implicated in atherosclerosis,
inflammatory processes, 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.
[0004] Factor XIIIa is also known to catalyse the incorporation of
low molecular weight amines into the .gamma.-glutamine sites of
proteins. Similarly Factor XIIIa also catalyses the incorporation
of low molecular weight glutamine analogues into lysyl residues.
Thus such low molecular weight amines (or glutamine analogues)
function as competitive inhibitors of the Factor XIIIa-induced
lysyl/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)].
[0005] 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". Preferred peptides
are said to include the tetrapeptide sequence -Asn-Gln-Glu-Gln- (or
NQEQ in standard amino acid abbreviation notation). Also disclosed
is the 12-mer peptide sequence from the NH.sub.2 terminus of the
alpha-2 antiplasmin enzyme:
NH.sub.2-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-OH
together with a synthetic analogue:
NH.sub.2-Asn-Gln-Glu-Gln-Val-Ser-Pro-Tyr-Thr-Leu-Thr-Leu-Leu-Lys-OH,
(denoted NQEQVSPLTLTLLK and NQEQVSPYTLTLLK respectively). The
latter was radiolabelled with .sup.125I and shown to be taken up in
thrombin clots in vitro.
[0006] It has now been discovered that synthetic analogues of
lysine and glutamine labelled with a suitable detectable moiety can
also function as substrates for the enzyme Factor XIIIa. The use of
suitable protecting groups provides compounds which are less
susceptible to in vivo metabolism especially by peptidases, and are
hence more useful targeting agents.
SUMMARY OF THE INVENTION
[0007] The present invention provides the following compounds:
Y--(CR.sub.2).sub.n--X--NHJ where:
[0008] X is C.dbd.O or CR.sub.2;
[0009] n is an integer of value 1 to 6;
[0010] Y is L(A).sub.m- or R.sup.1R.sup.2CR-- where L is a metal
complexing agent, A is --CR.sub.2--, --CR.dbd.CR--, --C.dbd.--C--,
--NRCO--, --CONR--, --SO.sub.2NR--, --NRSO.sub.2--,
--CR.sub.2OCR.sub.2--, --CR.sub.2SCR.sub.2--,
--CR.sub.2NRCR.sub.2--, a C.sub.4-8 cycloheteroalkylene group, a
C.sub.4-8 cycloalkylene group, a C.sub.5-12 arylene group, a
C.sub.3-12 heteroarylene group or a polyalkyleneglycol, polylactic
acid or polyglycolic acid moiety;
[0011] m is an integer of value 0 to 10;
[0012] where one of R.sup.1 and R.sup.2 is --NH(B).sub.pZ.sup.1 and
the other is --CO(B).sub.qZ.sup.2 where
[0013] p and q are integers of value 0 to 30, and
[0014] each B is independently chosen from Q or an amino acid,
[0015] where Q is a cyclic peptide;
[0016] Z.sup.1 and Z.sup.2 are protecting groups;
[0017] J and each R group are independently chosen from H,
C.sub.1-4 alkyl, C.sub.1-4 alkenyl, C.sub.1-4 alkynyl, C.sub.1-4
alkoxyalkyl or C.sub.1-4 hydroxyalkyl; with the provisos that:
[0018] (i) the total number of amino acid residues in the R.sup.1
and R.sup.2 groups does not exceed 30; [0019] (ii) when X is
CR.sub.2, then Y is --CRR.sup.1R.sup.2 and Z.sup.2 is a metal
complexing agent; [0020] (iii) when Y is --CRR.sup.1R.sup.2then at
least one of R.sup.1 and R.sup.2 bears at least one detectable
moiety.
[0021] The invention also includes kits for the preparation of the
above compounds labelled with a detectable moiety, and the use of
these and related compounds in the diagnosis or therapy of
thrombosis, embolism, atherosclerosis, inflammation or cancer.
BRIEF DESCRIPTION OF THE FIGURES
[0022] FIG. 1 shows imaging with .sup.99mTc-Compound 5 at different
dissection time points.
[0023] FIG. 2 shows imaging with .sup.99mTc-Compound 5 at 180 min
at head, clot and thorax.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The present invention provides the following compounds:
Y--(CR.sub.2).sub.n--X--NHJ where:
[0025] X is C.dbd.O or CR.sub.2;
[0026] n is an integer of value 1 to 6;
[0027] Y is L(A).sub.m- or R.sup.1R.sup.2CR-- where L is a metal
complexing agent, A is --CR.sub.2--, --CR.dbd.CR--, --C.dbd.C--,
--NRCO--, --CONR--, --SO.sub.2NR--, --NRSO.sub.2--,
--CR.sub.2OCR.sub.2--, --CR.sub.2SCR.sub.2--,
--CR.sub.2NRCR.sub.2--, a C.sub.4-8 cycloheteroalkylene group, a
C.sub.4-8 cycloalkylene group, a C.sub.5-12 arylene group, a
C.sub.3-12 heteroarylene group or a polyalkyleneglycol, polylactic
acid or polyglycolic acid moiety;
[0028] m is an integer of value 0 to 10;
[0029] where one of R.sup.1 and R.sup.2 is --NH(B).sub.pZ.sup.1 and
the other is --CO(B).sub.qZ.sup.2 where
[0030] p and q are integers of value 0 to 30, and
[0031] each B is independently chosen from Q or an amino acid,
[0032] where Q is a cyclic peptide;
[0033] Z.sup.1 and Z.sup.2 are protecting groups;
[0034] J and each R group are independently chosen from H,
C.sub.1-4 alkyl, C.sub.1-4 alkenyl, C.sub.1-4 alkynyl, C.sub.1-4
alkoxyalkyl or C.sub.1-4 hydroxyalkyl; with the provisos that:
[0035] (iv) the total number of amino acid residues in the R.sup.1
and R.sup.2 groups does not exceed 30; [0036] (v) when X is
CR.sub.2, then Y is --CRR.sup.1R.sup.2 and Z.sup.2 is a metal
complexing agent; [0037] (vi) when Y is --CRR.sup.1R.sup.2 then at
least one of R.sup.1 and R.sup.2 bears at least one detectable
moiety.
[0038] The invention also includes kits for the preparation of the
above compounds labelled with a detectable moiety, and the use of
these and related compounds in the diagnosis or therapy of
thrombosis, embolism, atherosclerosis, inflammation or cancer.
[0039] By the term "cyclic peptide" is meant a sequence of 5 to 15
amino acids in which the two terminal amino acids are bonded
together by a covalent bond which may be a peptide or disulphide
bond or a synthetic non-peptide bond such as a thioether,
phosphodiester, disiloxane or urethane bond.
[0040] By the term "amino acid" is meant an L- or D-amino acid,
amino acid analogue or amino acid mimetic which may be naturally
occurring or of purely synthetic origin, and may be optically pure,
i.e. a single enantiomer and hence chiral, or a mixture of
enantiomers. Preferably the amino acids of the present invention
are optically pure. By the term "amino acid mimetic" is meant
synthetic analogues of naturally occurring amino acids which are
isosteres, i.e. have been designed to mimic the steric and
electronic structure of the natural compound. Such isosteres are
well known to those skilled in the art and include but are not
limited to depsipeptides, retro-inverso peptides, thioamides,
cycloalkanes or 1,5-disubstituted tetrazoles [see M. Goodman,
Biopolymers, 24, 137, (1985)].
[0041] By the term "protecting group" is meant a biocompatible
group which inhibits or suppresses in vivo metabolism of the
peptide or amino acid at the amino or carboxyl terminus. Such
groups are well known to those skilled in the art and are suitably
chosen from, for the amine terminus (Z.sup.1): acetyl, Boc (where
Boc is tert-butyloxycarbonyl), Fmoc (where Fmoc is
fluorenylmethoxycarbonyl), benzyloxycarbonyl, trifluoroacetyl,
allyloxycarbonyl, Dde [i.e.
1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl], Npys (i.e.
3-nitro-2-pyridine sulfenyl), or a metal complexing group; and for
the carboxyl terminus (Z.sup.2): a carboxamide, tert-butyl ester,
benzyl ester, cyclohexyl ester, amino alcohol or a metal complexing
group. Preferably the protecting group is a metal complexing group,
most preferably it is a metal complexing group bound to a metal
i.e. a metal complex. The carboxyl terminus of peptides is
particularly susceptible to in vivo cleavage by carboxypeptidase
enzymes. Consequently, the metal complexing group or metal complex
is preferably attached at the carboxyl terminus. When either
R.sup.1 is --NH(B).sub.p-1QZ.sup.1 or R.sup.2 is
--CO(B).sub.q-1QZ.sup.2 then the protecting group may be the
covalent bond which closes the cyclic peptide (Q) ring.
[0042] A "detectable moiety" is a moiety which emits a signal or is
suitable for diagnostic imaging of the human body and may be a
radioisotope for radiopharmaceutical imaging or therapy, a
paramagnetic metal or species for MRI contrast imaging, a
radiopaque group or metal for X-ray contrast imaging, a gas
microbubble ultrasound contrast agent or a suitable dye for
detection by external light imaging. Preferably, the imaging moiety
is a metal ion, most preferably it is a radiometal.
[0043] When Y is --CRR.sup.1R.sup.2, then preferably either one or
both of R.sup.1 and R.sup.2 comprises one or more peptide fragments
of .alpha..sub.2-antiplasmin, fibronectin or beta-casein,
fibrinogen or thrombospondin. Such peptide fragments comprise at
least 3 and preferably 4-20 amino acid residues. When Y is
--CRR.sup.1R.sup.2 and --(CR.sub.2)--X--NHJ is
--(CH.sub.2).sub.4NH.sub.2 (i.e. the amino acid side chain of
lysine), then preferably either one or both of R.sup.1 and R.sup.2
comprises one or more such peptide fragments of
.alpha..sub.2-antiplasmin, fibronectin or beta-casein. The amino
acid sequences of .alpha..sub.2-antiplasmin, fibronectin,
beta-casein, fibrinogen and thrombospondin can be found in the
following references: .alpha..sub.2-antiplasmin precursor [M. Tone
et al., J. Biochem, 102,1033, (1987)]; beta-casein [L. Hansson et
al, Gene, 139, 193, (1994)]; fibronectin [A. Gutman et al, FEBS
Lett., 207, 145, (1996)]; thrombospondin-1 precursor [V. Dixit et
al, Proc. Natl. Acad. Sci., USA, 83, 5449, (1986)]; R. F.
Doolittle, Ann. Rev. Biochem., 53, 195, (1984).
[0044] Preferably the amino acid sequence is taken from the
N-terminus of: TABLE-US-00001 (i) .alpha..sub.2-antiplasmin, i.e.
NH.sub.2-Asn-Gln-Glu-Gln-Val-
Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu-Lys-OH or variants of this in which
one or more amino acids have been exchanged, added or removed such
as: NH.sub.2-Asn-Gln-Glu-Gln-Val-Ser-Pro-Leu-
Thr-Leu-Thr-Leu-Leu-Lys-Gly-OH, NH.sub.2-Asn-Gln-
Glu-Ala-Val-Ser-Pro-Leu-Thr-Leu-Thr-Leu-Leu- Lys-Gly-OH,
NH.sub.2-Asn-Gln-Glu-Gln-Val-Gly-OH; or (ii) casein ie.
Ac-Leu-Gly-Pro-Gly-Gln-Ser-Lys- Val-Ile-Gly.
[0045] When the compound of the present invention is a peptide,
i.e. Y is R.sup.1R.sup.2CR-- the number of amino acid residues is
preferably 2 to 30, most preferably 3 to 20, especially 3 to
15.
[0046] Preferred compounds have J equal H, i.e. terminate in an
NH.sub.2 group. X is preferably C.dbd.O, i.e. compounds of formula
Y--(CR.sub.2).sub.n--CONH.sub.2 are preferred. Most preferred
compounds are of formula
Y--(CR.sub.2).sub.x--(CH.sub.2).sub.2CONH.sub.2 or
Y--(CR.sub.2).sub.y--(CH.sub.2).sub.4NH.sub.2 where x is an integer
of value 0 to 4, and y is an integer of value 0 to 3. Compounds
having the same side chain as glutamine, i.e. glutamine analogues
of formula Y--(CR.sub.2).sub.x--(CH.sub.2).sub.2CONH.sub.2 are
especially preferred.
[0047] Suitable non-metallic radioisotopes for use in the present
invention include but are not limited to: radioiodine such as
.sup.123I, .sup.125I, .sup.131I, preferably .sup.123I; positron
emitters such as .sup.18F, .sup.11C or .sup.75Br and isotopes for
therapy e.g. .sup.211At.
[0048] The compounds of this invention which comprise metal
complexing agents preferably have only a single type of targeting
molecule attached, i.e. the --(CR.sub.2).sub.n--X--NHJ substituent.
Other substituents on the complexing agent may be present, but the
--(CR.sub.2).sub.n--X--NHJ substituent is the one which is expected
to be primarily responsible for the biolocalisation properties.
Metal complexes 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 .sup.68Ga or .sup.64Cu) or a .gamma.-emitter such as
.sup.99mTc, .sup.111In, .sup.113mIn or .sup.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 .gamma.-emitters, especially .sup.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:
.sup.90Y, .sup.89Sr, .sup.67Cu, .sup.186Re, .sup.188Re, .sup.169Er,
.sup.153Sm and .sup.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.
[0049] These metal ions are complexed using a metal complexing
agent, or more preferably a 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 stable complexes with the desired metal
ion even though they do not form chelate rings upon metal
coordination. Examples of known complexing agents of this kind,
which are particularly suitable for use with .sup.99mTc, are
hydrazines, phosphines, arsines, or isonitriles.
[0050] Examples of suitable chelating agents are bidentate such as
diamines or diphosphines, tridentate such as monoaminedithiols, or
tetradentate such as diaminedioximes (U.S. Pat. No. 4,615,876) or
such ligands incorporating amide donors (WO 94/08949 ); the
tetradentate ligands of WO 94/22816 ; N.sub.2S.sub.2
diaminedithiols, diamidedithiols or amideaminedithiols; N.sub.3S
thioltriamides; N.sub.4 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.
[0051] It is well known to prepare chelating agents which have
attached thereto a functional group ("bifunctional chelates").
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. When the desired Factor XIIIa substrate functionality is
an amine, 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. When the desired Factor XIIIa substrate functionality is a
carboxamide group, the desired ligands can be prepared e.g. by
reaction of a omega-haloalkyl carboxamide of suitable chain length
with a bifunctional chelate with a pendant amine group, giving the
desired carboxamide-linked ligand.
[0052] 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 .sup.99mTc, the usual starting
material is sodium pertechnetate from a .sup.99Mo generator.
Technetium is present in .sup.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.
[0053] The metal complex should also preferably exhibit low
non-specific blood background.
[0054] Thus the present invention relates mainly to diagnostic
agents for imaging sites in the mammalian body where the enzyme
Factor XIII is activated and blood proteins such as fibrin or
collagen are 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 or carboxamide
functional group which is available for covalent linking to protein
glutamyl carboxamide or lysyl amine groups respectively 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 activation of Factor XIII.
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
XIII and tissue transglutaminase are activated 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 XIII is activated 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.
[0055] So far as the therapeutic aspects of this invention are
concerned, the inventors have preliminary in vivo data (not
reported in detail herein) indicating that clots produced in the
presence of labelled peptides of the present invention (as per
Example 17 below) are smaller than those produced in the absence of
the labelled peptide. On this basis it is proposed that the
peptides herein defined, typically containing 4-30 e.g. about 10
amino acid residues, are effective as drugs for increasing the rate
of clot lysis e.g. by acting as potent inhibitors of fibrin cross
linking in clots. Thus the compounds disclosed have possible
pharmaceutical uses as thrombolytic or anticoagulant drugs for
therapy.
[0056] 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
.sup.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 .sup.99mTc, the
kit is preferably lyophilised and is designed to be reconstituted
with sterile .sup.99mTc-pertechnetate (TcO.sub.4.sup.-) from a
.sup.99mTc radioisotope generator to give a solution suitable for
human administration without further manipulation.
[0057] 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 .sup.99mTc, the syringe
containing the unit dose would also be supplied within a syringe
shield (to protect the operator from potential radioactive
dose).
[0058] 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).
[0059] The following examples illustrate the preparation of
compounds of the present invention and their use in imaging. The
syntheses of particular compounds of the present invention are
given in Examples 1-9, their radiolabelling with .sup.123I or
.sup.99mTc in Examples 10-12. Compound 1 (prior art) is included as
a comparative example. Evidence for increased plasma stability in
vitro is given in Example 13. Evidence for uptake in blood clots in
vitro and in vivo is given in Examples 15 and 17 respectively, with
normal rat biodistribution of the radiolabelled compounds reported
in Example 16.
[0060] The in vitro plasma stability of .sup.123I-Compound 1 is
poor (see Example 13), presumably due to protease activity. The
introduction of protecting groups at both the carboxy and amino
termini as for radiolabelled Compounds 2-49 confers a substantial
increase in plasma stability.
[0061] The majority of compounds tested exhibit high in vitro clot
uptake and hence avidity for the clot. The other compounds are of
lower potency with compounds 14, 16, 18, 31, 34, 36, 46 and 48
showing significant reduction in uptake. Removal of the Gln residue
from position 2 of the .alpha..sub.2-antiplasmin derived sequences,
as in compound 14, causes a large drop in uptake of this tracer,
thus strongly suggesting that Gln-2 is an essential amino acid in
this sequence type.
[0062] Details of the biodistribution in normal rats and in the
fresh and aged clot models are given in Examples 16 and 17. The
blood clearance rate of these compounds is relatively fast with
biological half lives between 1-2 hours. The biodistribution of
.sup.99mTc-Compound 3 is given as a representative example, in this
case the t.sub.1/2 of 2 h is estimated. The rapid clearance from
background tissues such as blood, lung, heart and muscle shows that
the agent of the present invention possess favourable
pharmacokinetics for imaging and shows their potential as
radiodiagnostics. Although some hepatobiliary excretion is seen for
these compounds, the main route of excretion is via the urinary
tract.
[0063] Uptake into fresh and aged clots in the rat models for many
of the radiolabelled compounds is very good (relative concentration
or RC=5-15), with clot to background tissue ratios very favourable
for imaging (>5). Example 18 shows that .sup.99mTc-Compound 5 is
suitable for imaging clots in the rat model.
[0064] .sup.99mTc-Compounds 2-49 have improved plasma stability
compared with .sup.123I-Compound 1 (RC =1.5), which may be
responsible for the improved in vivo clot uptake seen with these
compounds.
[0065] Comparison of the clot uptake results of Example 17 for
fresh and aged clots, shows that the present agents exhibit uptake
which is constant and independent of the age of the clot. Thus such
agents will have improved imaging capability for pre-existing
clots, such as those found with pulmonary embolism.
EXPERIMENTAL
[0066] In the following table: [0067] Z is benzyloxycarbonyl,
[0068] Fmoc is fluorenylmethoxycarbonyl,
[0069] Ac is acetyl, TABLE-US-00002 ##STR1## ##STR2## ##STR3##
Peptide Compound MS Theor. MS Expt. NQEQVSPYTLLK 1 1419.6
1419.7.sup.a Ac-NQEQVSPYTLLKG-NH.sub.2 2 1517.7 1517.7
Ac-NQEQVSPYTLLKG-Pn44 3 1816.1 1816.2 Ac-NQEQVSPYTLLKG-Hynic 4
1709.7 1710.0 Ac-NQEQVSPYTLLKG-Pn216 5 1845.2 1845.2 ##STR4##
##STR5## ##STR6## ##STR7## Z-NQEQVSPYTLLKG-Pn44 7 1908.2 1908.1
Z-NQEQVSPYTLLKG-Pn216 8 1937.2 1937.2 Fmoc-NQEQVSPYTLLKG-Pn216 9
2025.4 2025.4 (CF.sub.3).sub.2(C.sub.6H.sub.3)-NQEQVSPYTLLKG-Pn216
10 2044.2 2045.2 Adamtoyl-NQEQVSPYTLLKG-Pn216 11 1966.0 1966.5
Ac-NQEAVSPYTLLKG-Pn44 12 1759.1 1759.0 Ac-NQEAVSPYTLLKG-Pn216 13
1788.1 1788.0 Ac-NAEAVSPYTLLKG-Pn216 14 1766.1 1765.8
Ac-NQQQVSPYTLLKG-Pn216 15 1844.2 1844.1 Ac-NQG-Pn44 16 656.8 656.6
Ac-NQEQVG-Pn44 17 1908.2 1908.1 Z-NQEQVSPYG-Pn216 18 1481.7 1481.7
Ac-NQEQVSPLTLLKG-Pn216 19 1795.2 1795.1
Ac-NQEQVSP-Nal(2)-TLLKG-Pn216 20 1879.3 1879.3
Ac-NQEQVSP(pBr-F)TLLKG-Pn216 21 1908.1 1908.9
Ac-NQEQVSP(I-Y)TLLKG-Pn216 22 1971.2 1972.4
Ac-NQEQVSP(I.sub.2-Y)TLLKG-Pn216 23 2098.2 2099.5
Z-NQEQVSP(I-Y)TLLKG-Pn216 24 2063.2 2064.4
Z-NQEQVSP(I.sub.2-Y)TLLKG-Pn216 25 2189.2 2190.5
Fmoc-NQEQVSP(I-Y)TLLKG-Pn216 26 2152.4 2153.4
Fmoc-NQEQVSP(I.sub.2-Y)TLLKG-Pn216 27 2278.4 2279.3
Ac-NQEQVSPYTLL(D-K)G-Pn216 28 1845.2 1845.2
Ac-NQEQVSP(D-Y)TLL(D-K)G-Pn216 29 1845.2 1845.1
Ac-NQEQV(D-S)P(D-Y)TLL(D-K)G-Pn216 30 1845.2 1845.2
Ac-NQEQ(D-V)(D-S)P(D-Y)TLL(D-K)G-Pn216 31 1845.2 1845.1
Ac-(D-N)QEQVSP(D-Y)TLL(D-K)G-Pn216 32 1845.2 1845.0
Ac-NQEQVSP(D-Y)TLL(D-K).beta.Ala-Pn216 33 1859.2 1859.0
Ac-N-CH.sub.2NH.sub.2-QEQVSP(D-Y)TLL(D-K)G-Pn216 34 1831.2 1831.1
Ac-NQEQ(D-V)(D-S)(D-P)(D-Y)(D-T)(D-L)(D-L)(D-K)G-Pn44 35 1816.1
1815.8
Pn216-CO(CH.sub.2).sub.3CO-G(D-K)(D-L)(D-L)(D-T)(D-Y)(D-P)(D-S)(D-
36 1916.2 1916.0 Fmoc-NQQQ(D-V)S(OMe)PLG-Pn216 37 1532.8 1532.7
Pn44-CO(CH.sub.2).sub.3CO-NQEQVSPYTLLKG-NH.sub.2 38 1887.7 1887.3
Pn216-CO(CH.sub.2).sub.3CO-NQEQVSPYTLLKG-NH.sub.2 39 1916.2 1916.3
Ac-NQEQVSPYTLLKG-(PEG).sub.3.4k-Pn44 40 5200-5600 5401.sup.b
Ac-NQEQVSPYTLLKG-(PEG).sub.10k-Pn216 41 12400-12800 12630.sup.b
Z-NQEQVSPYAAAAG-Pn216 42 1766.0 1765.9
Z-NQEQVSPYG(CH.sub.2).sub.11(CO)-Pn216 43 1677.7 1679.8
Cyclo-[NQEQVSPYTLLKG] 44 1458.6 1458.3 Ac-LGPGQSKVIG-Pn44 45 1294.6
1294.4 p-EAQIVG-Pn44 46 1023.2 1023.0 Ac-LEFDTQSKNILG-Pn216 47
1733.0 1732.9 Ac-GQDPVKG-Pn216 48 1068.3 1068.0
Ac-YEVHHQKLVFFG-Pn216 49 1872.2 1872.3
All compounds except those denoted were analysed using ES+ mass
spectrometry. Those compounds denoted by .sup.a were analysed by
FAB and .sup.b by MALDI-TOF mass spectrometry.
EXAMPLE 1
Syntheses of Compounds 1 and 2
[0070] The protected peptide
Ac-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Gln(Trt)-Val-Ser(tBu)-Pro-Tyr(tBu)-Thr(tBu-
)-Leu-Leu-Lys(Boc)-Gly-OH was assembled on a 2-chlorotrityl resin
by anchoring Fmoc-Lys(Boc) to the resin, and then successive
deprotections/coupling cycles with the appropriate protected amino
acids (as described in P. Lloyd-Williams, F. Albericio and E.
Girald; Chemical Approaches to the Synthesis of Peptides and
Proteins, CRC Press, 1997). The title compound was obtained by
cleavage using 0.1% TFA in dichloromethane, deprotection and
purification by RP-HPLC (System A).
EXAMPLE 2
Syntheses of Compounds 3-9, 12-21, 28-33, 35, 37, 42 and 45-49
[0071] The appropriate protected peptide was assembled as in
Example 1 with the appropriate protected amino acids. The protected
fragment was cleaved from the resin and then coupled with
6-aminomethyl-3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dione
dioxime (prepared as described in WO 95/19187),
3,3,11,11-tetramethyl-7-aminoethyl-4,7,10-triazatridecane-2,12-dionedioxi-
me (prepared as described in WO 98/31399) or
6-Boc-hydrazinopyridine-3-carboxylic acid N-hydroxysuccinimide
ester (prepared as described in U.S. Pat. No. 5,206,370) in
solution using BOP as a coupling agent. The title compounds were
obtained by deprotection in TFA/water/triethylsilane (90/5/5) and
purified by RP-HPLC (System A).
EXAMPLE 3
Syntheses of Compounds 22-27
[0072] In an Eppendorf vial compound 5, 8 or 9 (1 mg), ammonium
acetate buffer (400 .mu.l, 0.2M, pH4), sodium iodide (0.5 eq, 15
mg/10 ml in 0.1M NaOH) and peracetic acid (1.5 eq, 0.1M solution)
were added. The reaction mixture was thoroughly mixed for 1 minute
and the mono- and di-iodo products were separated and collected by
preparative HPLC. The procedure was repeated to give sufficient
quantities of the isolated products.
EXAMPLE 4
Syntheses of Compounds 10 and 11
[0073]
Fmoc-Asn(Trt)-Gln(Trt)-Glu(tBu)-Gln(Trt)-Val-Ser(tBu)-Pro-Tyr(tBu)-
-Thr(tBu)-Leu-Leu-Lys(Boc)-Gly (200 mg, 0.73 mmol), Pn216 (30 mg,
0.87 mmol) and HBTU (33 mg, 0.87 mmol) were dissolved in anhydrous
DMF (2.5 ml). To the solution was added diisopropylethylamine (20
ml, 1.15 mmol) and the reaction mixture was stirred at room for
1.75 hours. The reaction mixture was then treated with piperidine
(0.5 ml) and the mixture stirred at room temperature for 2 hours.
The product was purified by semi-preparative HPLC to give a white
solid (171 mg, 82%); ES.sup.+-MS: m/z 952.40 (M+3H.sup.+).
1-Adamantanecarboxylic acid or 3,5-bis(trifluoromethyl)benzoic acid
(1.5 molar equivalent), the protected peptide
Asn(Trt)-Gln(Trt)-Glu(tBu)-Gln(Trt)-Val-Ser(tBu)-Pro-Tyr(tBu)-Thr(tBu)-Le-
u-Leu-Lys(Boc)-Gly-Pn216 (1 molar equivalent) and HBTU (1.2-1.5
molar equivalent) were dissolved in anhydrous DMF (1 ml).
Diisopropylethylamine (11 molar equivalents) was added and the
reaction mixture was stirred at room temperature until the reaction
was judged complete by HPLC. The protected peptide fragment was
then treated with 95% trifluoroacetic acid in CH.sub.2Cl.sub.2. The
reaction mixture was stirred at room temperature for 2 to 4 hrs.
The product was purified from the reaction mixture by reversed
phase HPLC.
EXAMPLE 5
Synthesis of Compound 34
[0074] The protected peptide was synthesised as for example with
the exception of the Fmoc-Asn(Trt)-.PSI.(CH.sub.2NH)-Gln(Trt)-OH
which was obtained according to the classical methodology for the
synthesis of reduced peptide bonds, by reductive amination of
Gln(Trt) with Fmoc-Asn(Trt) derived aldehyde (see G. Guichard et
al., Peptide Res., 6(3), 121, (1993) and references therein.
[0075] The resulting compound was prepared and purified as in
example 2.
EXAMPLE 6
Syntheses of Compounds 36, 38 and 39
[0076] The protected peptide was synthesised as for example 1 but
on a Rink resin. After removal of the glycine N-protection, the
peptide was reacted with glutaric anhydride whilst still on the
resin. Activation with BOP/HOBt of the glutarate carboxylic acid
and coupling with
6-aminomethyl-3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dione
dioxime or
3,3,11,11-tetramethyl-7-aminoethyl-4,7,10-triazatridecane-2,12-dionedi-
oxime on the resin gave the protected product. Cleavage with
TFA/water (95/5) afforded the crude material which was purified
using RP-HPLC (System A).
EXAMPLE 7
Syntheses of Compounds 40 and 41
[0077] A solution of the required molecular weight
.alpha.-N-(tert-butoxycarbonyl)-poly(ethylene glycol)
amino-.omega.-succinimidyl carbonate and one molar equivalent of
6-aminomethyl-3,3,6,9,9-pentamethyl-4,8-diazaundecane-2,10-dione
dioxime or
3,3,11,11-tetramethyl-7-aminoethyl-4,7,10-triazatridecane-2,12-dionedi-
oxime in anhydrous tetrahydrofuran (5 ml) was refluxed for 5 hours
under nitrogen. The reaction mixture was reduced in vacuo leading
to a white solid which was purified by flash chromatography using
isopropanol/ammonia/water 10:1:1 to give the title compound as a
white solid. The Boc protecting group was removed using 37% HCl
added dropwise to an ice-cold solution in methanol. The solution
was then stirred at room temperature for 4 hours. The reaction
mixture was basified to pH.about.10 by addition of 4M NaOH (4.18
ml). The product was isolated by semi-preparative HPLC (system
D).
[0078] The solid from above was dissolved in DMF and to it was
added the protected peptide fragment. Diisopropylethylamine and
HBTU were added and the reaction mixture was stirred at room
temperature till completion of the reaction. The product was
purified by HPLC (system E) to give a colourless gum.
[0079] This gum was dissolved in dichloromethane (2 ml) and the
solution treated with TFA (0.2 ml) for 5 hours. The reaction was
basified with 1M NaOH (2 ml) and volatiles removed in vacuo. MeOH
(2.5 ml) was added to the residue and the mixture filtered using an
Acrodisc filter (LC13 PVDF 0.45 .quadrature.m). The product was
isolated from the methanolic solution by HPLC (system E).
EXAMPLE 8
Synthesis of Compound 43
[0080] 12-N-Fmoc-aminododecanoic acid was coupled to,
3,11,11-tetramethyl-7-aminoethyl-4,7,10-triazatridecane-2,12-dionedioxime
as described earlier. The Fmoc protecting group was removed by 20%
piperidine in DMF and the product purified by HPLC (system F).
[0081] The above product was coupled with the protected peptide
fragment and subsequently deprotected as described above. The
product was purified by HPLC (system G).
EXAMPLE 9
Synthesis of Compound 44
[0082] The partially deprotected peptide
H-Asn(Trt)-Gln(Trt)-Glu(OtBu)-Gln(Trt)-Val-Ser(tBu)-Pro-Tyr(tBu)-Leu-Leu--
Lys(Boc)-Gly-OH was assembled on a 2-chlorotrtyl chloride resin, by
a Fmoc based strategy, by stepwise elongation with BOP/HOBt. The
N-terminal protection was removed by piperidine treatment and
partially protected peptide cleaved from the solid support by a
0.5% TFA in dichloromethane solution.
[0083] Cyclisation was carried out in solution at a 10 mM
concentration in DMF, with BOP as a condensation reagent, in the
presence of solid sodium bicarbonate according to a known
methodology (see for instance M. Rodriguez et al., Int. J. Pept.
Protein Res., 35, 441, 1 990).
[0084] Final deprotection in a mixture of TFA/water/ethane dithiol
(90/5/5) afforded the crude title compound which was purified by
RP-HPLC (System A).
EXAMPLE 10
I-123 Labelling of Compounds 1-2 and Compound 44
[0085] Ammonium acetate buffer (200 .mu.l, 0.2M, pH 4.0) was added
to the ligand solution (20 .mu.l, 20 .mu.g), and Na .sup.127I (10
.mu.l, 1.5 .mu.g) in an Eppendorf tube. The solution was mixed
thoroughly and Na .sup.123I (5-50 .mu.l, 111 MBq) was then added.
The solution was mixed thoroughly prior to addition of PAA solution
(10 .mu.l, 0.01M), further mixing followed. The activity of the
preparation was measured. In all cases the required product was
separated from reaction by-products and unlabelled substrates by
HPLC.
EXAMPLE 11
Tc-99m Labelling of Compounds 3, 5-43, 45-49
[0086] A 0.1 ml aliquot of the compound dissolved in H.sub.2O (1
mg/ml) was transferred to a nitrogen-filled 1 Oml glass vial
together with deoxygenated saline (0.9% w/v, 1 ml) and 0.035 ml
aqueous NaOH (0.1M). To this solution was added technetium
generator eluate (1 ml, approx. 0.4 GBq) and then aqueous stannous
chloride solution (0.1 ml, ca. 10 .mu.g). The labelling pH was
9.0-10.0. Vials were incubated at ambient laboratory temperature
(15-25.degree. C.) for 30 minutes to effect labelling. The
resulting preparation was either diluted to the desired radioactive
concentration or HPLC purification was performed (System B) to
remove unlabelled starting material and radioactive impurities
prior to testing. After purification the organic solvent was
removed under vacuum and the sample was redissolved in about 5 ml
0.1M phosphate buffer pH 7.4 to give a working concentration of 6-9
MBq/ml. Radiochemical purity was assessed before use by the thin
layer chromatography (TLC) system described below: [0087] i) ITLC
SG 2 cm.times.20 cm eluted with 0.9% w/v saline [0088] ii) Whatman
No. 1 2 cm.times.20 cm eluted with 50:50 v/v acetonitrile :
H.sub.2O
[0089] The labelled substrates remain at, or close to, the origin
in TLC system (i) and move close to the solvent front in system
(ii). When analysed by appropriate detection equipment the
radiochemical purity is typically in excess of 85% labelled
compound.
EXAMPLE 12
Tc-99m Labelling of Compound 4
[0090] A 0.1 ml aliquot of the compound dissolved in water (1
mg/ml) was transferred to nitrogen-filled 10 ml glass vial together
with tricine dissolved in water (0.5 ml, 37.5 mg) and
phosphinedynetris(benzene sulphonic acid)tris sodium salt dissolved
in water (0.1 ml, 10 mg). To this solution was added technetium
generator eluate (1 ml, approx 0.4 GBq) and then a solution of
stannous chloride in 0.1M HCl (0.02 ml, ca 2 .mu.g). The labelling
pH was 4.5-5.5. Vials were incubated at 60.degree. C. for 30
minutes to effect labelling. Purification and assessment of
radiochemical purity was carried out as in Example 10.
EXAMPLE 13
In Vitro Plasma Stability
[0091] To a portion of compound (50 .mu.l, 10 MBq/ml) was added an
equal volume of plasma (rat or human) or saline. The mixtures were
incubated at 37.degree. C. and the stability measured by HPLC
(system C) at 0, 30 and 120 minutes. The saline dilution acted as a
control. TABLE-US-00003 % intact at Compound Species 120 mins
.sup.123I-Cmpd 1 (prior art) Human 0 .sup.123I-Cmpd 2 Human 98 Rat
99 .sup.99mTc-Cmpds 3, 4, 5, 7, 12, Human >90 16, 17;
.sup.123I-Cmpd 44 Rat >90 .sup.99mTc-Cmpds 6, 8-11, 13-15, Rat
>90 19-21, 28-36, 38, 39, 45-49 .sup.99mTc-Cmpds 22-27 Rat
>60
EXAMPLE 14
HPLC Systems
[0092] TABLE-US-00004 Flow Rate: 1 ml/min in all systems. System A
Column Waters C18 250 .times. 4.5 mm. Particle size 4 microns
Gradient: Elution Profile 10-60% B in 25 min. Eluent A: 0.1%
aqueous TFA Eluent B: 0.1% TFA in acetonitrile System B Column
Waters C18 150 .times. 3.9 mm. Particle size 4 microns Gradient:
Elution Profile 0-100% B in 22 min. Eluent A: 0.1% aqueous TFA
Eluent B: 0.1% TFA in acetonitrile System C Column Waters C18 150
.times. 3.9 mm. Particle size 4 microns Gradient: Elution Profile
0-100% B in 20 min. Eluent A: 50 mM NH.sub.4OAc buffer (pH 5.6)
Eluent B: acetonitrile System D Column Hamilton PRP-1, 305 mm
.times. 7.0 mm; Gradient: Elution Profile 0-65% B in 10 min. Eluent
A: 5% aqueous ammonia Eluent B: acetonitrile System E Column
Hamilton PRP-1, 150 mm .times. 4.1 mm; Gradient: Elution Profile
0-100% B in 15 min. Eluent A: 5% aqueous ammonia Eluent B:
acetonitrile System F Column Polymer Laboratories PLRP-S, 150 mm
.times. 2.5 mm; Gradient: Elution Profile 0-100% B in 15 min.
Eluent A: 5% aqueous ammonia Eluent B: acetonitrile System G Column
Hamilton PRP-1, 150 mm .times. 4.1 mm; Gradient: Elution Profile
0-100% B in 15 min. Eluent A: 0.1% aqueous TFA Eluent B: 0.1% TFA
in acetonitrile
EXAMPLE 15
Incorporation Into Human Plasma Clots
[0093] Incorporation of radiolabelled substrates into fibrin was
investigated by induction of an in vitro human plasma clot in the
following manner. To a siliconised 5 ml glass vial was added, (a)
800 .mu.l of Tris(hydroxymethyl)aminomethane buffered saline pH 7.5
containing calcium chloride (50 mM Tris, 150 mM sodium chloride,
4mM calcium chloride.), (b) about 40 .mu.l of physiological salt
solution containing 100 units of thrombin per ml, (c) about 400
.mu.l of human plasma containing the radiolabelled substrate at a
concentration of typically 10 kBq/ml. To aid induction of clot a
roughened glass rod was added to the reaction vial. Control vials
were prepared similarly but with the omission of thrombin and
calcium chloride.
[0094] After incubation of the test solution at ambient laboratory
temperature (ca. 20.degree. C.) for 60 minutes the reaction was
discontinued with the addition of about 400 .mu.l of a cold
solution of 33.5 mM ethylenediaminetetra-acetic acid disodium salt.
Clots were separated from serum by vacuum filtration onto 0.45
.mu.M nitrocellulose filters (pre-soaked in 1.5%
BSA/tris(hydroxymethyl)aminoethane buffered saline pH 7.5
containing 0.1% Tween 20) and washed with about 2.times.10 ml of
tris(hydroxymethyl)aminomethane buffered saline pH 7.5 containing
Tween 20 to a final concentration of 0.1% v/v. The proportion of
total radioactivity was calculated by counting in suitable
detection apparatus.
[0095] The fraction of radioactivity retained on the filter, after
subtraction of the non-specific binding determined from the
control, is a measure of incorporation into the filtered clots.
TABLE-US-00005 % retained % retained % specific Compound (with
thrombin) (no thrombin) uptake* .sup.123I-Cmpd 1 14.6 2.0 12.6
.sup.123I-Cmpd 2 38.6 0.2 38.4 .sup.99mTc-Cmpd 3 39.3 0.3 39.0
.sup.99mTc-Cmpd 4 22.0 0.1 21.0 .sup.99mTc-Cmpd 5 35.5 0.2 35.3
.sup.99mTc-Cmpd 6 68.6 4.3 64.3 .sup.99mTc-Cmpd 7 41.3 0.7 40.6
.sup.99mTc-Cmpd 8 25.9 1.4 24.5 .sup.99mTc-Cmpd 9 44.5 2.7 42.8
.sup.99mTc-Cmpd 10 65.9 0.2 65.7 .sup.99mTc-Cmpd 11 68.1 1.2 66.9
.sup.99mTc-Cmpd 12 34.8 0.3 34.5 .sup.99mTc-Cmpd 13 57.0 0.2 56.8
.sup.99mTc-Cmpd 14 3.3 0.5 2.8 .sup.99mTc-Cmpd 15 55.6 0.4 55.2
.sup.99mTc-Cmpd 16 12.5 0.1 12.4 .sup.99mTc-Cmpd 17 41.3 0.7 40.6
.sup.99mTc-Cmpd 18 9.8 0.1 9.7 .sup.99mTc-Cmpd 19 48.2 0.2 48.0
.sup.99mTc-Cmpd 20 65.0 2.6 62.4 .sup.99mTc-Cmpd 21 60.6 0.5 60.1
.sup.99mTc-Cmpd 22 59.0 0.3 58.7 .sup.99mTc-Cmpd 23 63.8 0.1 63.7
.sup.99mTc-Cmpd 24 56.8 0.4 56.4 .sup.99mTc-Cmpd 25 68.0 4.3 63.7
.sup.99mTc-Cmpd 26 63.3 8.2 55.1 .sup.99mTc-Cmpd 27 Nd Nd nd
.sup.99mTc-Cmpd 28 42.1 0.2 41.9 .sup.99mTc-Cmpd 29 21.1 0.1 21.0
.sup.99mTc-Cmpd 30 14.9 0.2 14.7 .sup.99mTc-Cmpd 31 7.7 0.1 7.6
.sup.99mTc-Cmpd 32 20.2 0.4 19.8 .sup.99mTc-Cmpd 33 63.9 0.1 63.8
.sup.99mTc-Cmpd 34 5.5 0.1 5.4 .sup.99mTc-Cmpd 35 15.9 0.6 15.3
.sup.99mTc-Cmpd 36 8.7 0.1 8.6 .sup.99mTc-Cmpd 37 13.0 0.2 12.8
.sup.99mTc-Cmpd 38 21.0 0.7 20.3 .sup.99mTc-Cmpd 39 23.1 0.1 23.0
.sup.99mTc-Cmpd 40 13.3 0.1 13.2 .sup.99mTc-Cmpd 41 44.5 7.1 37.4
.sup.99mTc-Cmpd 42 14.2 0.2 14.0 .sup.99mTc-Cmpd 43 .sup.123I-Cmpd
44 14.1 0.3 13.8 .sup.99mTc-Cmpd 45 28.7 0.1 28.6 .sup.99mTc-Cmpd
46 6.3 0.2 6.1 .sup.99mTc-Cmpd 47 49.9 0.1 49.8 .sup.99mTc-Cmpd 48
10.6 0.1 10.5 .sup.99mTc-Cmpd 49 33.1 1.6 31.5 *% retained in
plasma clot assay (with thrombin) - % retained in plasma clot assay
(no thrombin)
EXAMPLE 16
Normal Rat Biodistribution
[0096] The resolution of a clot image is dependant on the
combination of rate of incorporation of the radiopharmaceutical and
its blood/tissues clearance rate. For this reason the
biodistribution of several compounds has been determined in rats.
Male Wistar (100-150 g) rats were injected i.v. with 0.1-0.2 ml of
radiolabelled tracer solution (8 MBq/ml) and dissected at different
times post-injection. The % ID in each of the selected tissues was
measured. Some animals were kept in metabolism cages to be able to
determine the % ID excreted in urine and faeces. The dissection
times used for the agent were 15, 30, 60, 240 min. Data are shown
as mean of % ID (n=3). .sup.99mTc-Compound 3 TABLE-US-00006 15 min
30 min 60 min 240 min Muscle 14.7 10.4 5.1 2.9 Blood 5.3 1.8 1.6
0.6 Kidney 7.6 4.9 4.6 3.4 Urine 14.9 34.3 37.0 42.0 Lung 0.7 0.4
0.3 0.3 Liver 6.2 4.1 4.0 3.0 GI Tract 13.5 15.1 18.0 20.7 Heart
0.2 0.1 0.1 0.04
EXAMPLE 17
Incorporation Into Clots Induced in a Rat Model
Rat Inferior Vena Cava Model (IVC)
[0097] The rats (Male Wistar, 250-350 g) were anaesthetised with
15% urethane. After laparotomy, the vena cava was isolated and
freed of surrounding fat tissue. A platinum wire (1.5 cm.times.0.5
mm) was inserted into the inferior vena cava and 5 min post surgery
0.4 ml of ellagic acid (1.2.times.10.sup.-4 M) was injected
intravenously through the femoral vein previously canulated, and
the clot was allowed to form. The average weight of the clots
formed in this model was around 27 mg, n=32, (5-50 mg range). The
compounds were injected 5 min (fresh clot) and 60 min (aged clot)
post-induction. After 60 min the animals were sacrificed and the
clot removed, weighed and counted. Other tissues e.g. blood, lung,
heart, were also dissected and counted. The uptake of tracer into
the clot was determined as the relative concentration (cpm/g of
clot by dose/g animal ) and clot to background tissue.
Results:
[0098] Fresh Clots TABLE-US-00007 Rel. Compound % id/g Conc.
Clot/blood Clot/lung Clot/heart Clot/liver .sup.123I-Cmpd 1 0.5
.+-. 0.1 1.6 .+-. 0.4 1 nd Nd nd .sup.123I-Cmpd 2 4.9 .+-. 0.8 14.5
.+-. 2.2 7 10 19 21 .sup.99mTc-Cmpd 3 1.4 .+-. 0.4 5.1 .+-. 1.2 10
8 17 6 .sup.99mTc-Cmpd 4 2.0 .+-. 0.6 6.5 .+-. 2.0 6 8 15 20
.sup.99mTc-Cmpd 5 6.0 .+-. 2.1 16 .+-. 5.8 21 23 51 43
.sup.99mTc-Cmpd 7 4.8 .+-. 1.3 14.5 .+-. 4.4 15 11 21 6
.sup.99mTc-Cmpd 8 6.7 .+-. 1.7 21.0 .+-. 6.8 14 18 41 21
.sup.99mTc-Cmpd 9 8.8 .+-. 3.6 24.8 .+-. 9.6 13 12 32 13
.sup.99mTc-Cmpd 10 2.9 .+-. 0.5 6.5 .+-. 1.2 4 5 10 5
.sup.99mTc-Cmpd 11 1.1 .+-. 0.4 3.3 .+-. 1.1 10 11 24 11
.sup.99mTc-Cmpd 12 7.3 .+-. 4.1 19.8 .+-. 11.0 20 17 31 8
.sup.99mTc-Cmpd 13 3.4 .+-. 0.7 7.9 .+-. 1.6 7 8 18 18
.sup.99mTc-Cmpd 14 0.5 .+-. 0.1 1.2 .+-. 0.2 2 1.5 3 0.7
.sup.99mTc-Cmpd 15 3.6 .+-. 1.0 8.3 .+-. 2.2 4 6 12 11
.sup.99mTc-Cmpd 16 0.3 .+-. 0.1 1.0 .+-. 0.2 3 3 8 0.8
.sup.99mTc-Cmpd 17 1.2 .+-. 0.7 3.7 .+-. 2.3 7 12 18 5
.sup.99mTc-Cmpd 19 2.5 .+-. 0.3 7.1 .+-. 1.2 3 7 29 17
.sup.99mTc-Cmpd 20 2.3 .+-. 0.6 6.5 .+-. 1.3 12 11 30 18
.sup.99mTc-Cmpd 21 2.3 .+-. 1.3 7.4 .+-. 4.0 11 16 28 13
.sup.99mTc-Cmpd 22 3.8 .+-. 1.6 13.0 .+-. 5.5 11 13 28 25
.sup.99mTc-Cmpd 23 7.6 .+-. 1.8 18.2 .+-. 4.1 6 6 17 21
.sup.99mTc-Cmpd 24 4.0 .+-. 2.2 11.1 .+-. 6.7 10 10 21 11
.sup.99mTc-Cmpd 26 14.1 .+-. 10 41.4 .+-. 28 16 11 38 16
.sup.99mTc-Cmpd 28 3.5 .+-. 2.0 9.7 .+-. 5.0 13 19 31 35
.sup.99mTc-Cmpd 29 4.7 .+-. 1.9 13.0 .+-. 4.9 14 20 44 44
.sup.99mTc-Cmpd 31 0.3 .+-. 0.2 0.9 .+-. 0.6 0.6 1 2 3
.sup.99mTc-Cmpd 32 1.7 .+-. 0.7 4.6 .+-. 1.9 10 11 26 27
.sup.99mTc-Cmpd 33 2.7 .+-. 0.2 7.8 .+-. 0.6 11 16 32 28
.sup.99mTc-Cmpd 34 0.5 .+-. 0.2 1.8 .+-. 0.5 2 3 6 8
.sup.99mTc-Cmpd 35 1.2 .+-. 0.2 3.7 .+-. 0.8 2 2 4 2
.sup.99mTc-Cmpd 36 0.2 .+-. 0.1 0.7 .+-. 0.3 1 1 3 4
.sup.99mTc-Cmpd 37 0.4 .+-. 0.2 0.9 .+-. 0.4 0.6 0.7 1 1
.sup.99mTc-Cmpd 38 1.9 .+-. 0.5 5.4 .+-. 1.5 5 6 15 3
.sup.99mTc-Cmpd 39 2.5 .+-. 0.3 9.0 .+-. 1.1 9 14 29 31
.sup.99mTc-Cmpd 41 0.7 .+-. 0.1 2.2 .+-. 0.3 3 4 6 8
.sup.99mTc-Cmpd 42 0.3 .+-. 0.1 0.3 .+-. 0.2 2 0.3 1 63
.sup.123I-Cmpd 44 0.4 .+-. 0.1 1.1 .+-. 0.2 0.9 1.5 1 2
.sup.99mTc-Cmpd 45 2.0 .+-. 0.5 5.5 .+-. 1.3 6 5 10 3
.sup.99mTc-Cmpd 46 0.8 .+-. 0.4 2.0 .+-. 0.7 5 3 6 1
.sup.99mTc-Cmpd 47 0.8 .+-. 0.7 2.2 .+-. 2.0 0.8 1 3 1
.sup.99mTc-Cmpd 48 0.3 .+-. 0.2 1.0 .+-. 0.5 0.6 0.9 2 3
.sup.99mTc-Cmpd 49 2.6 .+-. 1.1 8.5 .+-. 3.7 4 6 16 4 Rel . .times.
Conc . .times. ( RC ) = % .times. .times. id .times. / .times. g
.times. .times. of .times. .times. clot % .times. .times. id
.times. / .times. g .times. .times. in .times. .times. rest .times.
.times. of .times. .times. body ##EQU1##
[0099] Aged Clots TABLE-US-00008 Clot/ Clot/ Clot/ Clot/ Compound %
id/g Rel. Conc. blood lung heart liver .sup.123I-Cmpd 2 5.5 .+-.
1.7 14.5 .+-. 3.9 24 12 23 20 .sup.99mTc-Cmpd 3 2.1 .+-. 0.8 6.2
.+-. 2.2 8 11 23 5 .sup.99mTc-Cmpd 4 1.2 .+-. 1.1 4.1 .+-. 4.0 8 10
19 19 .sup.99mTc-Cmpd 5 3.6 .+-. 1.7 11 .+-. 5.1 13 31 33 24
.sup.99mTc-Cmpd 7 2.1 .+-. 0.3 6.5 .+-. 1.3 9 7 15 3
.sup.99mTc-Cmpd 8 5.3 .+-. 1.3 16.4 .+-. 4.3 10 9 24 16
.sup.99mTc-Cmpd 9 3.7 .+-. 0.4 11.4 .+-. 1.5 8 7 21 6
.sup.99mTc-Cmpd 2.9 .+-. 1.4 6.3 .+-. 2.7 6 6 16 5 10
.sup.99mTc-Cmpd 0.4 .+-. 0.3 1.2 .+-. 0.8 5 6 13 4 11
.sup.99mTc-Cmpd 4.1 .+-. 1.2 11.2 .+-. 3.4 19 15 75 7 12
.sup.99mTc-Cmpd 4.5 .+-. 1.5 10.4 .+-. 3.0 6 9 29 19 13
.sup.99mTc-Cmpd 0.6 .+-. 0.4 1.4 .+-. 0.8 1.4 1.3 2 0.7 14
.sup.99mTc-Cmpd 3.4 .+-. 0.6 7.6 .+-. 1.6 3 0.6 1 nd 15
.sup.99mTc-Cmpd 0.8 .+-. 0.1 2.7 .+-. 0.6 3 5 7 3 17
.sup.99mTc-Cmpd 3.2 .+-. 2.3 9.1 .+-. 7.1 14 5 13 6 19
.sup.99mTc-Cmpd 1.7 .+-. 0.5 4.9 .+-. 1.4 10 14 36 23 20
.sup.99mTc-Cmpd 2.7 .+-. 2.3 8.5 .+-. 6.8 31 13 32 31 21
.sup.99mTc-Cmpd 6.9 .+-. 2.5 22.7 .+-. 7.4 11 1 3 4 22
.sup.99mTc-Cmpd 7.5 .+-. 2.5 17.6 .+-. 4.6 13 1 1 1 23
.sup.99mTc-Cmpd 4.1 .+-. 2.5 11.9 .+-. 8.0 15 20 40 37 24
.sup.99mTc-Cmpd 4.0 .+-. 0.9 12.9 .+-. 3.5 7 12 31 54 26
.sup.99mTc-Cmpd 4.5 .+-. 1.1 13.1 .+-. 3.4 17 2 3 1 28
.sup.99mTc-Cmpd 3.8 .+-. 0.4 10.0 .+-. 1.7 11 30 67 29 29
.sup.99mTc-Cmpd 0.2 .+-. 0.1 0.6 .+-. 0.2 0.6 4 9 2 31
.sup.99mTc-Cmpd 0.7 .+-. 0.1 2.0 .+-. 0.4 3 15 43 21 32
.sup.99mTc-Cmpd 2.2 .+-. 0.6 6.7 .+-. 1.6 16 9 24 16 33
.sup.99mTc-Cmpd 0.4 .+-. 0.1 1.4 .+-. 0.4 3 -- -- 6 34
.sup.99mTc-Cmpd 0.6 .+-. 0.1 1.5 .+-. 0.4 1 0.5 1 2 35
.sup.99mTc-Cmpd 0.1 .+-. 0.02 0.2 .+-. 0.1 0.6 10 18 4 36
.sup.99mTc-Cmpd 0.2 .+-. 0.03 0.5 .+-. 0.1 0.4 1 2 9 37
.sup.99mTc-Cmpd 0.8 .+-. 0.3 2.4 .+-. 0.7 3 0.5 1 2 38
.sup.99mTc-Cmpd 2.4 .+-. 0.3 8.2 .+-. 1.1 15 12 43 11 39
.sup.99mTc-Cmpd 0.4 .+-. 0.1 1.1 .+-. 0.3 1 6 13 4 41
.sup.99mTc-Cmpd 0.4 .+-. 0.1 1.3 .+-. 0.3 5 7 21 6 42
.sup.123I-Cmpd 0.5 .+-. 0.03 1.2 .+-. 0.1 0.9 2 1 1 44
.sup.99mTc-Cmpd 2.0 .+-. 0.8 5.7 .+-. 2.3 8 5 11 3 45
.sup.99mTc-Cmpd 0.5 .+-. 0.2 1.3 .+-. 0.6 4 3 5 1 46
.sup.99mTc-Cmpd 0.5 .+-. 0.3 1.5 .+-. 0.8 1 24 46 44 47
.sup.99mTc-Cmpd 0.2 .+-. 0.04 0.8 .+-. 0.2 1 109 32 26 48
.sup.99mTc-Cmpd 1.2 .+-. 0.5 3.9 .+-. 1.6 4 9 29 19 49
EXAMPLE 18
Imaging of Clots Induced in a Rat Model
[0100] Clots were induced in male Wistar rats (250-350 g) as
described in Example 17, with the exception that for these
experiments the platinum wire was located in the jugular vein. The
compounds were injected 60 min post injection and planar images
acquired between 15-180 min p.i. A Park medical Isocam I gamma
camera was used for these experiments, 300K or 150K counts were of
the thorax were collected using a LEUHR or a LEPH collimator. Clots
were visualised from 15 min p.i. Reaching the highest clot to
background ratio at 180 min p.i. due to rapid clearance of the
compound.
Abbreviations
[0101] Ac Acetyl [0102] Boc tert-butyloxycarbonyl [0103] Cmpd
compound [0104] DMF dimethylformamide [0105] ES electrospray [0106]
FAB fast atom bombardment [0107] Fmoc fluorenylmethoxycarbonyl
[0108] HPLC high performance liquid chromatography [0109] MALDI-TOF
matrix assisted laser desorption ionisation--time of flight [0110]
Nal napthylalanine [0111] RCP radiochemical purity [0112] RP-HPLC
reverse phase high performance liquid chromatography [0113] TFA
trifluoroacetic acid [0114] TLC thin layer chromatography
Sequence CWU 1
1
38 1 12 PRT Artificial Sequence fragment of human antiplasmin 1 Asn
Gln Glu Gln Val Ser Pro Tyr Thr Leu Leu Lys 1 5 10 2 13 PRT
Artificial Sequence fragment of human antiplasmin MOD_RES (1)..(1)
ACETYLATION MOD_RES (13)..(13) AMIDATION 2 Asn Gln Glu Gln Val Ser
Pro Tyr Thr Leu Leu Lys Gly 1 5 10 3 13 PRT Artificial Sequence
derivatised antiplasmin sequence MOD_RES (1)..(1) ACETYLATION
MOD_RES (13)..(13) BLOCKED 3 Asn Gln Glu Gln Val Ser Pro Tyr Thr
Leu Leu Lys Gly 1 5 10 4 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (13)..(13) BLOCKED 4 Asn Gln Glu Gln
Val Ser Pro Tyr Thr Leu Leu Lys Gly 1 5 10 5 13 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1) BLOCKED
MOD_RES (13)..(13) BLOCKED 5 Asn Gln Glu Gln Val Ser Pro Tyr Thr
Leu Leu Lys Gly 1 5 10 6 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) ACETYLATION MOD_RES
(13)..(13) BLOCKED 6 Asn Gln Glu Ala Val Ser Pro Tyr Thr Leu Leu
Lys Gly 1 5 10 7 13 PRT Artificial Sequence derivatised antiplasmin
sequence MOD_RES (1)..(1) ACETYLATION MOD_RES (13)..(13) BLOCKED 7
Asn Ala Glu Ala Val Ser Pro Tyr Thr Leu Leu Lys Gly 1 5 10 8 13 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MOD_RES (13)..(13) BLOCKED 8 Asn Gln Gln Gln
Val Ser Pro Tyr Thr Leu Leu Lys Gly 1 5 10 9 3 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1)
ACETYLATION MOD_RES (3)..(3) BLOCKED 9 Asn Gln Gly 1 10 6 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MOD_RES (6)..(6) BLOCKED 10 Asn Gln Glu Gln
Val Gly 1 5 11 9 PRT Artificial Sequence derivatised antiplasmin
sequence MOD_RES (1)..(1) BLOCKED MOD_RES (9)..(9) BLOCKED 11 Asn
Gln Glu Gln Val Ser Pro Tyr Gly 1 5 12 13 PRT Artificial Sequence
derivatised antiplasmin sequence MOD_RES (1)..(1) ACETYLATION
MOD_RES (13)..(13) BLOCKED 12 Asn Gln Glu Gln Val Ser Pro Leu Thr
Leu Leu Lys Gly 1 5 10 13 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) ACETYLATION MISC_FEATURE
(8)..(8) MISC_FEATURE "Xaa" = 2-napthylalanine MOD_RES (13)..(13)
BLOCKED 13 Asn Gln Glu Gln Val Ser Pro Xaa Thr Leu Leu Lys Gly 1 5
10 14 13 PRT Artificial Sequence derivatised antiplasmin sequence
MOD_RES (1)..(1) ACETYLATION MISC_FEATURE (8)..(8) MISC_FEATURE
"Xaa" = pBr-Phe MOD_RES (13)..(13) BLOCKED 14 Asn Gln Glu Gln Val
Ser Pro Xaa Thr Leu Leu Lys Gly 1 5 10 15 13 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1)
ACETYLATION MISC_FEATURE (8)..(8) MISC_FEATURE "Xaa" = I-Tyr
MOD_RES (13)..(13) BLOCKED 15 Asn Gln Glu Gln Val Ser Pro Xaa Thr
Leu Leu Lys Gly 1 5 10 16 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) ACETYLATION MISC_FEATURE
(8)..(8) MISC_FEATURE "Xaa" = diiodo-Tyr MOD_RES (13)..(13) BLOCKED
16 Asn Gln Glu Gln Val Ser Pro Xaa Thr Leu Leu Lys Gly 1 5 10 17 13
PRT Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) BLOCKED MISC_FEATURE (8)..(8) Xaa = iodo-tyrosine MOD_RES
(13)..(13) BLOCKED 17 Asn Gln Glu Gln Val Ser Pro Xaa Thr Leu Leu
Lys Gly 1 5 10 18 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) BLOCKED MISC_FEATURE (8)..(8)
Xaa = diiodo-Tyr MOD_RES (13)..(13) BLOCKED 18 Asn Gln Glu Gln Val
Ser Pro Xaa Thr Leu Leu Lys Gly 1 5 10 19 13 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1)
ACETYLATION MISC_FEATURE (12)..(12) MISC_FEATURE "Xaa" = D-Lys
MOD_RES (13)..(13) BLOCKED 19 Asn Gln Glu Gln Val Ser Pro Tyr Thr
Leu Leu Xaa Gly 1 5 10 20 13 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) ACETYLATION MISC_FEATURE
(8)..(8) MISC_FEATURE "Xaa" = D-Tyr MISC_FEATURE (12)..(12)
MISC_FEATURE "Xaa" = D-Lys MOD_RES (13)..(13) BLOCKED 20 Asn Gln
Glu Gln Val Ser Pro Xaa Thr Leu Leu Xaa Gly 1 5 10 21 13 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MISC_FEATURE (6)..(6) MISC_FEATURE "Xaa" =
D-Ser MISC_FEATURE (8)..(8) MISC_FEATURE "Xaa" = D-Tyr MISC_FEATURE
(12)..(12) MISC_FEATURE "Xaa" = D-Lys MOD_RES (13)..(13) BLOCKED 21
Asn Gln Glu Gln Val Xaa Pro Xaa Thr Leu Leu Xaa Gly 1 5 10 22 13
PRT Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MISC_FEATURE (5)..(5) MISC_FEATURE"Xaa" =
D-Val MISC_FEATURE (6)..(6) MISC_FEATURE "Xaa" = D-Ser MISC_FEATURE
(8)..(8) MISC_FEATURE "Xaa" = D-Tyr MISC_FEATURE (12)..(12)
MISC_FEATURE "Xaa" = D-Lys MOD_RES (13)..(13) BLOCKED 22 Asn Gln
Glu Gln Xaa Xaa Pro Xaa Thr Leu Leu Xaa Gly 1 5 10 23 13 PRT
Artificial Sequence derivatised antiplasmin sequence MISC_FEATURE
(1)..(1) MISC_FEATURE "Xaa" = D-Asn MOD_RES (1)..(1) ACETYLATION
MISC_FEATURE (8)..(8) MISC_FEATURE "Xaa" = D-Tyr MISC_FEATURE
(12)..(12) MISC_FEATURE "Xaa" = D-Lys MOD_RES (13)..(13) BLOCKED 23
Xaa Gln Glu Gln Val Ser Pro Xaa Thr Leu Leu Xaa Gly 1 5 10 24 13
PRT Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MISC_FEATURE (8)..(8) MISC_FEATURE "Xaa" =
D-Tyr MISC_FEATURE (12)..(12) MISC_FEATURE "Xaa" = D-Lys MOD_RES
(13)..(13) bAla MOD_RES (13)..(13) BLOCKED 24 Asn Gln Glu Gln Val
Ser Pro Xaa Thr Leu Leu Xaa Ala 1 5 10 25 12 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1) BLOCKED
MISC_FEATURE (7)..(7) MISC_FEATURE "Xaa" = D-Tyr MISC_FEATURE
(11)..(11) MISC_FEATURE "Xaa" = D-Lys MOD_RES (12)..(12) BLOCKED 25
Gln Glu Gln Val Ser Pro Xaa Thr Leu Leu Xaa Gly 1 5 10 26 13 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MISC_FEATURE (5)..(12) MISC_FEATURE = D-amino
acids MOD_RES (13)..(13) BLOCKED 26 Asn Gln Glu Gln Val Ser Pro Tyr
Thr Leu Leu Lys Gly 1 5 10 27 9 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) BLOCKED MISC_FEATURE (2)..(9)
MISC_FEATURE = D-amino acids MOD_RES (9)..(9) AMIDATION 27 Gly Lys
Leu Leu Thr Tyr Pro Ser Val 1 5 28 9 PRT Artificial Sequence
derivatised antiplasmin sequence MOD_RES (1)..(1) BLOCKED
MISC_FEATURE (5)..(5) MISC_FEATURE "Xaa" = D-Val MISC_FEATURE
(6)..(6) MISC_FEATURE "Xaa" = O-methyl serine MOD_RES (9)..(9)
BLOCKED 28 Asn Gln Gln Gln Xaa Xaa Pro Leu Gly 1 5 29 13 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) BLOCKED MOD_RES (13)..(13) AMIDATION 29 Asn Gln Glu Gln
Val Ser Pro Tyr Thr Leu Leu Lys Gly 1 5 10 30 13 PRT Artificial
Sequence derivatised antiplasmin sequence MOD_RES (1)..(1)
ACETYLATION MOD_RES (13)..(13) BLOCKED 30 Asn Gln Glu Gln Val Ser
Pro Tyr Thr Leu Leu Lys Gly 1 5 10 31 13 PRT Artificial Sequence
derivatised antiplasmin sequence MOD_RES (1)..(1) BLOCKED MOD_RES
(13)..(13) BLOCKED 31 Asn Gln Glu Gln Val Ser Pro Tyr Ala Ala Ala
Ala Gly 1 5 10 32 9 PRT Artificial Sequence derivatised antiplasmin
sequence MOD_RES (1)..(1) BLOCKED MOD_RES (9)..(9) BLOCKED 32 Asn
Gln Glu Gln Val Ser Pro Tyr Gly 1 5 33 13 PRT Artificial Sequence
derivatised antiplasmin sequence MISC_FEATURE (1)..(1) Xaa = Asn,
which is cyclised with the Gly at residue 13 MISC_FEATURE
(13)..(13) Xaa = Gly which is cyclised with the Asn at residue 1 33
Xaa Gln Glu Gln Val Ser Pro Tyr Thr Leu Leu Lys Xaa 1 5 10 34 10
PRT Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MOD_RES (10)..(10) BLOCKED 34 Leu Gly Pro Gly
Gln Ser Lys Val Ile Gly 1 5 10 35 6 PRT Artificial Sequence
derivatised antiplasmin sequence MISC_FEATURE (1)..(1) MISC_FEATURE
"Xaa" = pyro-Glu MOD_RES (6)..(6) BLOCKED 35 Xaa Ala Gln Ile Val
Gly 1 5 36 12 PRT Artificial Sequence derivatised antiplasmin
sequence MOD_RES (1)..(1) ACETYLATION MOD_RES (12)..(12) BLOCKED 36
Leu Glu Phe Asp Thr Gln Ser Lys Asn Ile Leu Gly 1 5 10 37 7 PRT
Artificial Sequence derivatised antiplasmin sequence MOD_RES
(1)..(1) ACETYLATION MOD_RES (7)..(7) BLOCKED 37 Gly Gln Asp Pro
Val Lys Gly 1 5 38 12 PRT Artificial Sequence derivatised
antiplasmin sequence MOD_RES (1)..(1) ACETYLATION MOD_RES
(12)..(12) BLOCKED 38 Tyr Glu Val His His Gln Lys Leu Val Phe Phe
Gly 1 5 10
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