Technetium Labelled Peptides .
Field of the Invention.
The present invention relates to technetium imaging agents comprising radiolabelled
c-Met binding peptides suitable for SPECT or PET imaging in vivo. The c-Met
binding peptides are labelled via chelator conjugates. Also disclosed are
pharmaceutical compositions, methods of preparation of the agents and compositions,
plus methods of in vivo imaging using the compositions, especially for use in the
diagnosis of cancer.
Background to the Invention.
Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a growth factor
which is involved in various physiological processes, such as wound healing and
angiogenesis. The high affinity interaction of HGF interaction with its receptor (c-
Met) is implicated in tumour growth, invasion and metastasis.
Knudsen et al have reviewed the role of HGF and c-Met in prostate cancer, with
possible implications for imaging and therapy [Adv.Cancer Res., 9J_, 31-67 (2004)].
Labelled anti-met antibodies for diagnosis and therapy are described in WO
03/057155.
c-Met has been shown to be involved in tumour growth, invasion and metastasis in
many human cancers of epithelial origin. c-Met is expressed by most carcinomas and
its elevated expression relative to normal tissue has been detected in many cancers,
including: lung, breast, colorectal, pancreatic, head and neck, gastric, hepatocellular,
ovarian, renal, glioma, melanoma and a number of sarcomas. In colorectal carcinoma
(CRC), over-expression of c-Met has been detected in dysplastic aberrant crypt foci,
the earliest pre-neoplastic lesions of the disease. In head and neck squamous cell
cancer, c-Met is reportedly expressed or overexpressed in roughly 80% of primary
tumours. In prostate cancer metastasis to bone, c-Met was reported overexpressed in
over 80% of bone metastasis.
Under normal conditions, c-Met is expressed on epithelial cells and activated in a
paracrine fashion, by mesenchymally derived HGF. The activation of c-Met in
normal cells is a transient event and is tightly regulated. In tumour cells, however, c-
Met can be constitutively active. In cancer, aberrant c-Met stimulation can be
achieved through c-Met amplification/over-expression, activating c-Met mutations
(e.g. structural alterations) and acquisition of autonomous growth control through
creation of autocrine signalling loops. In addition, a defective down-regulation of the
c-Met receptor will also contribute to aberrant c-Met expression in the cell membrane.
While the over-expression of c-Met is HGF dependent (autocrine/paracrine),
structural alterations caused by mutations are HGF independent (e.g. loss of
extracellular domain).
WO 2004/078778 discloses polypeptides or multimeric peptide constructs which bind
c-Met or a complex comprising c-Met and HGF. Approximately 10 different
structural classes of peptide are described. WO 2004/078778 discloses that the
peptides can be labelled with a detectable label for in vitro and in vivo applications, or
with a drug for therapeutic applications. The detectable label can be: an enzyme, a
fluorescent compound, an optical dye, a paramagnetic metal ion, an ultrasound
contrast agent or a radionuclide. Preferred labels of WO 2004/078778 are stated to be
radioactive or paramagnetic, and most preferably comprise a metal which is chelated
by a metal chelator. WO 2004/078778 states that the radionuclides therein can be
selected from: 1 F, 124I, 125I, 131 I, 123I, 77Br, 76Br, Tc, 1Cr, 67Ga, 6 Ga, 47Sc, 167Tm,
141Ce, In, 168Yb, 175Yb, 140La, 0Y, Y, 1 Sm, 166Ho, 165Dy, 166Dy, 62Cu, 64Cu, 67Cu,
7Ru, 103Ru, 186Re, 203Pb, 211Bi, 212Bi, 213Bi, 214Bi, 105Rh, 10 Pd, 117mSn, 149Pm, 161Tb,
1 Lu, 1 Au and 1 Au. WO 2004/078778 states (page 62) that the preferred
radionuclides for diagnostic purposes are: 64Cu, 6 Ga, 6 Ga, mTc and 1ln, with
mTc being particularly preferred.
WO 2004/078778 teaches at Examples 14-17 methods of increasing the serum
residence time of the c-Met binding peptides: conjugation to a moiety which binds
non-covalently to human serum albumin; conjugation to PEG; fusion to serum protein
and conjugation to maleimide.
WO 2009/016180 discloses imaging agents which comprises a conjugate of Formula I :
(I)
where:
Z1 is attached to the N-terminus of cMBP, and is H or MIG;
Z2 is attached to the C-terminus of cMBP and is OH, OB , or MIG,
where B is a biocompatible cation;
cMBP is a cMet binding cyclic peptide of 17 to 30 amino acids which
comprises the amino acid sequence (SEQ-1):
Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X 6;
wherein X1 is Asn, His or Tyr;
X2 is Gly, Ser, Thr or Asn;
X3 is Thr or Arg;
X4 is Ala, Asp, Glu, Gly or Ser;
X5 is Ser or Thr;
X6 is Asp or Glu;
and Cysa d are each cysteine residues such that residues a and b as well
as c and d are cyclised to form two separate disulfide bonds;
M is a metabolism inhibiting group;
L is a synthetic linker group;
BzpM is a benzopyrylium dye.
WO 2008/139207 discloses cMet binding cyclic peptide similar to those of WO
2009/016180, in this case labelled with a particular class of cyanine dyes.
The optical imaging agents of WO 2009/016180 and WO 2008/139207 are said to be
useful for optical imaging to obtain images of sites of c-Met over-expression or
localisation in vivo, particularly for imaging colorectal cancer.
The Present Invention.
The present invention relates to imaging agent compositions comprising radioactive
technetium ( mTc or 4mTc) labelled c-Met binding peptides suitable for positron
emission tomography (PET) or single photon emission tomography (SPECT) imaging
in vivo. The c-Met binding peptides are labelled via a chelator conjugate of the
peptide with a diaminedioxime ligand. The conjugates are radiolabelled with
technetium under mild, room temperature conditions giving the desired radiometal
complex in high yield and radiochemical purity. The mTc imaging agents can be
readily prepared form a sterile, non-radioactive kit which is reconstituted with
[ mTc]-pertechnetate from a commercial mTc generator.
The imaging agents exhibit good, target-specific tumour uptake in vivo. The imaging
agents also appear in addition to be well tolerated in preclinical models.
Detailed Description of the Invention.
In a first aspect, the present invention provides an imaging agent which comprises a
radioactive Tc complex of a chelator conjugate of a c-Met binding peptide, said
chelator conjugate being of Formula I :
(I)
where:
Tc is the radioisotope 94mTc or 99mTc;
cMBP is an 18 to 30-mer c-Met binding cyclic peptide of Formula II:
-(A) -Q-(A') -
(P)
where Q is the amino acid sequence (SEQ-1):
-Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X 6-
wherein X is Asn, His or Tyr;
X2 is Gly, Ser, Thr or Asn;
X3 is Thr or Arg;
X4 is Ala, Asp, Glu, Gly or
X5 is Ser or Thr;
X6 is Asp or Glu;
and Cysa d are each cysteine residues such that residues a and b as well
as c and d are cyclised to form two separate disulfide bonds;
A and A' are independently any amino acid other than Cys, or one of A
and A' is Lys(s-Z 3);
x and y are independently integers of value 0 to 13, and are chosen
such that [x + y] = 1 to 13;
Z1 is attached to the N-terminus of cMBP, and isMIG or Z3;
Z2 is attached to the C-terminus of cMBP and isMIG or Z ;
wherein each M is independently a metabolism inhibiting group
which is a biocompatible group which inhibits or suppresses in vivo
metabolism of the cMBP peptide;
Z3 is a chelator of Formula (III):
(III)
wherein E^E 6 are each independently an R' group;
each R' is independently H or Ci-4 alkyl, C 3-7 alkylaryl, C2-7 alkoxyalkyl, C1-4
hydroxyalkyl, C1-4 fluoroalkyl, C2-7 carboxyalkyl or C1-4 aminoalkyl, or two or more
R' groups together with the atoms to which they are attached form a carbocyclic,
heterocyclic, saturated or unsaturated ring;
L is a synthetic linker group of formula -(A)m- wherein each A is
independently -CR2- , -CR=CR- , -CºC- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO R- , - R(C=0 ) R-, - R(C=S) R-, -S0 2 R- , - RS0 2- , -CR2OCR2- ,
-CR2SCR2- , -CR2 RCR2- , a C4-8 cycloheteroalkylene group, a C4-8
cycloalkylene group, a C5-12 arylene group, or a C3-12 heteroarylene group, or a
monodisperse polyethyleneglycol (PEG) building block;
each R is independently chosen from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl,
Ci-4 alkoxyalkyl or C1-4 hydroxyalkyl;
m is an integer of value 1 to 10;
with the proviso that the conjugate of Formula I comprises one Z3 group and said Z3
group forms a metal complex with the Tc.
By the term "imaging agent" is meant a compound suitable for imaging the
mammalian body. Preferably, the mammal is an intact mammalian body in vivo, and
is more preferably a human subject. The imaging agent is typically administered in a
non-pharmacologic amount, i.e. at a dosage designed to have a minimal biological
effect on the mammalian subject. Preferably, the imaging agent can be administered
to the mammalian body in a minimally invasive manner, i.e. without a substantial
health risk to the mammalian subject when carried out under professional medical
expertise. Such minimally invasive administration is preferably intravenous
administration into a peripheral vein of said subject, without the need for local or
general anaesthetic.
The term "in vivo imaging" as used herein refers to those techniques that noninvasively
produce images of all or part of an internal aspect of a mammalian subject.
By the term "chelator conjugate" is meant that chelator is covalently bonded to the
cMBP peptide.
By the term "c-Met binding cyclic peptide" is meant a peptide which binds to the
hepatocyte growth factor receptor, also known as c-Met (or simply MET). Suitable
such peptides of the present invention are cyclic peptides of 18 to 30 amino acids of
Formula I . Such peptides have an apparent K for c-Met of less than about 20 nM.
The cMBP sequence of said peptides comprises proline residues, and it is known that
such residues can exhibit cis/trans isomerisation of the backbone amide bond. The
cMBP peptides of the present invention include any such isomers. The cMBP of the
present invention is suitably used as a monomer, i.e. dimers and/or heterodimers of
the cMBP are outside the scope.
The Z1 group substitutes the amine group of the last amino acid residue of the cMBP,
i.e., the amino- or N- terminus. The Z2 group substitutes the carbonyl group of the
last amino acid residue of the cMBP - i.e. the carboxy or C- terminus.
By the term "metabolism inhibiting group" (M ) is meant a biocompatible group
which inhibits or suppresses in vivo metabolism of the cMBP peptide at either the
amino terminus (Z1) or carboxy terminus (Z2) . For the imaging agents of the present
invention, the MIG includes the Z3 group - i.e. the chelator of Formula III. In that case,
the technetium complex is covalently attached at the cMBP N- or C- terminus (as Z1
or Z2 respectively), and the technetium complex serves to block metabolism of the
cMBP. Other such M groups are well known to those skilled in the art and are
suitably chosen from, for the peptide amine terminus:
N-acylated groups -NH(C=0)R where the acyl group -(C=0)R has R chosen
from: Ci_6 alkyl, or C3-10 aryl groups or comprises a polyethyleneglycol (PEG)
building block. Preferred such PEG groups are the biomodifiers of Formula IA or IB:
(IA)
17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula IA
wherein p is an integer from 1 to 10. Alternatively, a PEG-like structure based on a
propionic acid derivative of Formula IB can be used:
(IB)
where p is as defined for Formula IA and
q is an integer from 3 to 15.
In Formula IB, p is preferably 1 or 2, and q is preferably 5 to 12.
Preferred such amino terminus M groups are acetyl, benzyloxycarbonyl or
trifluoroacetyl, most preferably acetyl.
By the term "amino acid" is meant an L - or D- amino acid, amino acid analogue (eg.
naphthyl alanine) 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. Conventional 3-letter or single letter
abbreviations for amino acids are used herein. 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 isostere amino acids are known to be used within peptides, 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)].
By the term "peptide" is meant a compound comprising two or more amino acids, as
defined above, linked by a peptide bond (i.e. an amide bond linking the amine of one
amino acid to the carboxyl of another).
When A and A' are "any amino acid other than Cys" that means that the additional
amino acids of the A and A' groups lack free thiol groups, in particular Cys residues.
That is because an additional Cys residue would risk disulfide bridge scrambing with
the Cysa-Cysb and Cys -Cysd disulfide bridges of the Q sequence, with consequent
loss of c-Met binding affinity.
In Formula (I), the Z group (i.e. the chelator of Formula III) is suitably attached at
one of the following locations (i)-(iii):
(i) amino terminus of cMBP, as a Z1 group. Such conjugation would
use a carboxy-functionalised chelator of Formula III;
(ii) carboxy terminus of cMBP, as a Z2 group. Such attachment at the
C-terminus directly can be achieved using an amine-functionalised
chelator of Formula III. For such conjugation, an additional Lys
residue to permit chelator conjugation is therefore unnecessary. In
addition, the attached technetium complex can function as a M
group;
(iii) attachment to a Lys (epsilon) amine side chain of a Lys residue
located in the A or A' groups.
When x is 94m, the technetium radioisotope is 4mTc, which is particularly suitable for
positron emission tomography (PET) imaging in vivo. When x is 99m, the technetium
radioisotope is mTc, which is particularly suitable for single photon emission
tomography (SPECT) imaging in vivo. Preferably, Tc is mTc.
By the term "radioactive Tc complex of a chelator" is meant a radiometal complex of
a chelating agent. By the term "radiometal complex" is meant a coordination metal
complex of the radiometal with the chelator of Formula (III), wherein said chelator is
covalently bonded to the cMBP peptide via the linker group (L) of Formula I . The
term "chelator" or "chelating agent" has its conventional meaning and refers to 2 or
more metal donor atoms arranged such that chelate rings result upon metal
coordination. The chelator of Formula (III) has a diaminedioxime donor set. Thus,
the Tc complex of the present invention is believed to be of Formula (IV):
(IV)
where E1 to E6 and L are as defined for Formula (III).
The terms "comprising" or "comprises" have their conventional meaning throughout
this application and imply that the components listed must be present, but that other,
unspecified compounds or species may be present in addition. The term 'comprising'
includes as a preferred subset "consisting essentially of which means that the
composition has the components listed without other compounds or species being
present.
Preferred features.
Preferred cMBP peptides of the present invention have a K for binding of c-Met to
c-Met/HGF complex of less than about 10 nM (based on fluorescence polarisation
assay measurements), most preferably in the range 1 to 5 nM, with less than 3nM
being the ideal.
The imaging agents of the first aspect are preferably provided in a form suitable for
mammalian administration. By the phrase "in a form suitable for mammalian
administration" is meant a composition which is sterile, pyrogen-free, lacks
compounds which produce toxic or adverse effects, and is formulated at a
biocompatible pH (approximately pH 6.5 to 8.5) and physiologically compatible
osmolality. Such compositions lack particulates which could risk causing emboli in
vivo, and are formulated so that precipitation does not occur on contact with
biological fluids (eg. blood). Such compositions also contain only biologically
compatible excipients, and are preferably isotonic. A preferred such form is the
radiopharmaceutical composition of the fourth aspect (see below).
The imaging agents of the present invention suitably have both cMBP peptide termini
protected by M groups, which will usually be different. Having both peptide termini
protected in this way is important for in vivo imaging applications, since otherwise
rapid peptide metabolism would be expected with consequent loss of selective binding
affinity for c-Met. When both Z1 and Z2 are MIG, preferably Z1 is acetyl and Z2 is a
primary amide. Most preferably, Z1 is acetyl and Z2 is a primary amide and the Tc
moiety is attached to the epsilon amine side chain of a lysine residue of cMBP.
Q preferably comprises the amino acid sequence of either SEQ-2 or SEQ-3:
Ser-Cysa-X1-Cys -X2-Gly-Pro-Pro-X -Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X -X6
(SEQ-2);
Ala-Gly-Ser-Cys a-X1-Cys -X2-Gly-Pro-Pro-X -Phe-Glu-Cysd-Trp-Cysb-Tyr-
X4-X -X6-Gly-Thr (SEQ-3).
In SEQ-1, SEQ-2 and SEQ-3, X3 is preferably Arg. The cMBP peptide of the first
aspect preferably has the amino acid sequence (SEQ-7):
Ala-Gly-Ser-Cys a-Tyr-Cys -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys d-Trp-Cysb-
Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.
The imaging agent of Formula (I) is preferably chosen such that one of A and A' is
Lys(s-Z3) and cMBP comprises only one such Lys residue. More preferably, the
single Lys(s-Z3) moiety is at the carboxy terminus, so that the cMBP is of Formula
IIA:
-(A) -Q-(A') Z-Lys( -Z3)-
(IIA)
wherein:
z is an integer of value 0 to 12, and [x + z] = 0 to 12.
In Formula I and Formula II, the -(A)x- or -(A') - groups preferably comprise a linker
peptide which is chosen from:
-Gly-Gly-Gly-Lys- (SEQ-4),
-Gly-Ser-Gly-Lys- (SEQ-5) or
-Gly-Ser-Gly-Ser-Lys- (SEQ-6),
and the Z group is attached to the epsilon amine group of the Lys residue of said
linker peptide.
The chelator of Formula (III) is preferably attached at either the C-terminus (Z2
group), or as a Lys(s-Z3) moiety. More preferably it is attached as a Lys(s-Z3) moiety,
and most preferably when the Lys(s-Z3) moiety is at the carboxy terminus as for
Formula (IIA) above.
In the imaging agent of Formula (I), the chelator is preferably of Formula IIIA:
(IIIA)
where q is an integer of value 1 to 6, and A is as defined for Formula
(III).
In Formula (IIIA), (A)q is preferably -(NH)(A) t- where t is an integer of value 0 to 5 .
The imaging agents of the first aspect can be prepared as described in the third aspect
(below).
In a second aspect, the present invention provides a chelator conjugate of Formula I,
as defined in the first aspect. Preferred aspects of the cMBP peptide of Formula (II)
and chelator of Formula (III) in the second aspect are as described in the first aspect
(above).
The chelator conjugates of the second aspect can be obtained by the bifunctional
chelate approach. The term "bifunctional chelate" has its conventional meaning, and
refers to a chelating agent having covalently attached thereto a pendant functional
group. The functional group is used as a reactive site to attach the chelator to the
cMBP peptide. The bifunctional chelate approach and associated syntheses have been
described by Bartholoma et al [Chem.Rev., 110(5), 2903-2920 (2010)]; Chakraborty
et al [Curr.Top.Med.Chem., 10(1 1), 1113-1 134 (2010)] and Brechbiel et al
[Quart.J.Nucl.Med.Mol.Imaging, 52(2), 166-173 (2008)]. The functional group of the
present invention is preferably an amine, carboxylic acid or activated ester, more
preferably a primary amine or an activated ester. Bifunctional chelators having a
pendant amine functional group can be conjugated to the carboxyl group of a peptide.
Bifunctional chelators having a carboxyl or activated ester functional group can be
conjugated to an amine group of a peptide.
By the term "activated ester" or "active ester" is meant an ester derivative of the
associated carboxylic acid which is designed to be a better leaving group, and hence
permit more facile reaction with nucleophile, such as amines. Examples of suitable
active esters are: N-hydroxysuccinimide (NHS); sulfo-succinimidyl ester;
pentafluorophenol; pentafluorothiophenol; /?ara-nitrophenol; hydroxybenzotriazole
and PyBOP (i.e. benzotriazol-l-yl-oxytripyrrolidinophosphonium
hexafluorophosphate). Preferred active esters are N-hydroxysuccinimide or
pentafluorophenol esters, especially N-hydroxysuccinimide esters.
c-Met binding peptides of formula cMBP of the present invention may be obtained by
a method of preparation which comprises:
(i) solid phase peptide synthesis of a linear peptide which has the same peptide
sequence as the desired cMBP peptide and in which the Cysa and Cysb are
unprotected, and the Cys and Cysd residues have thiol-protecting groups;
(ii) treatment of the peptide from step (i) with aqueous base in solution to give
a monocyclic peptide with a first disulphide bond linking Cysa and Cysb;
(iii) removal of the Cys and Cysd thiol-protecting groups and cyclisation to
give a second disulphide bond linking Cys and Cysd, which is the desired
bicyclic peptide product Z^fcMBPJ-Z 2.
By the term "protecting group" is meant a group which inhibits or suppresses
undesirable chemical reactions, but which is designed to be sufficiently reactive that it
may be cleaved from the functional group in question under mild enough conditions
that do not modify the rest of the molecule. After deprotection the desired product is
obtained. Amine protecting groups are well known to those skilled in the art and are
suitably chosen from: Boc (where Boc is t r t-butyloxycarbonyl), Fmoc (where Fmoc
is fluorenylmethoxycarbonyl), trifluoroacetyl, allyloxycarbonyl, Dde [i.e. l-(4,4-
dimethyl-2,6-dioxocyclohexylidene)ethyl] or Npys (i.e. 3-nitro-2-pyridine sulfenyl).
Suitable thiol protecting groups are Trt (Trityl), Acm (acetamidomethyl), t-Bu (tertbutyl),
t r t-Butylthio, methoxybenzyl, methylbenzyl or Npys (3-nitro-2-pyridine
sulfenyl). The use of further protecting groups are described in Protective Groups in
Organic Synthesis, 4th Edition, Theorodora W. Greene and Peter G. M. Wuts, [Wiley
Blackwell, (2006)]. Preferred amine protecting groups are Boc and Fmoc, most
preferably Boc. Preferred thiol protecting groups are Trt and Acm.
Examples 1 and 2 provide further specific details. Further details of solid phase
peptide synthesis are described in P. Lloyd-Williams, F. Albericio and E. Girald;
Chemical Approaches to the Synthesis of Peptides and Proteins, CRC Press, 1997.
The cMBP peptides are best stored under inert atmosphere and kept in a freezer.
When used in solution, it is best to avoid pH above 7 since that risks scrambling of the
disulfide bridges.
A preferred bifunctional diaminedioxime chelator is Chelator 1, which has the
Formula:
(Chelator 1)
wherein the bridgehead primary amine group is conjugated to L (i.e. the linker group)
and/or cMBP peptide.
The diaminedioxime chelator of Formula III can be prepared by reaction of the
appropriate diamine with either:
(i) the appropriate chloronitroso derivative Cl-C(E2E )-CH(NO)E 1;
(ii) an alpha-chloro oxime of formula Cl-C(E2E )-C(=NOH)E 1;
(iii) an alpha-bromoketone of formula Br-C(E 2E )-C(=0)E 1 followed by
conversion of the diaminediketone product to the diaminedioxime with
hydroxylamine.
Route (i) is described by S. Jurisson et al [Inorg. Chem., 26, 3576-82 (1987)].
Chloronitroso compounds can be obtained by treatment of the appropriate alkene with
nitrosyl chloride (NOC1) as is known in the art. Further synthetic details of
chloronitroso compounds are given by: Ramalingam [Synth. Commun., 25(5), 743-
752 (1995)]; Glaser [J. Org. Chem., 61(3), 1047-48 (1996)]; Clapp [J. Org Chem.,
36(8) 1169-70 (1971)]; Saito [Shizen Kagaku, 47, 41-49 (1995)] and Schulz [Z.
Chem., 21(1 1), 404-405 (1981)] Route (iii) is described in broad terms by Nowotnik
et al [Tetrahedron, 50(29), p .8617-8632 (1994)]. Alpha-chloro-oximes can be
obtained by oximation of the corresponding alpha-chloro-ketone or aldehyde, which
are commercially available. Alpha-bromoketones are commercially available.
Further details of specific carboxy-functionalised and amine-functionalised chelators
of the invention, and their conjugation to cMBP peptides, are provided in the
supporting Examples.
In a third aspect, the present invention provides a method of preparation of the
imaging agent of the first aspect, which comprises reaction of the chelator conjugate
of Formula I of the first aspect with a supply of Tc as defined in the first aspect, in a
suitable solvent.
Preferred aspects of the cMBP peptide and chelator in the chelator conjugate, and Tc
in the third aspect are as described in the first aspect (above).
The suitable solvent is typically aqueous in nature, and is preferably a biocompatible
carrier solvent as defined in the fourth aspect (below).
mTc is commercially available from radioisotope generators, which provide [ mTc]-
pertechnetate in sterile form . Methods of preparing technetium complexes are well
known in the art [see eg. I.Zolle (Ed) Technetium-99mPharmaceuticals, Springer,
New York (2007)]. 4mTc can be prepared and processed by the method of Bigott et
al [Nucl.Med.Biol, 33(7), 923-933 (2006)].
In a fourth aspect, the present invention provides a radiopharmaceutical composition
which comprises the imaging agent of the first aspect together with a biocompatible
carrier, in a form suitable for mammalian administration.
Preferred aspects of the imaging agent in the fourth aspect are as defined in the first
aspect. The term "in a form suitable for mammalian administration" is as defined
above.
The "biocompatible carrier" is a fluid, especially a liquid, in which the imaging agent
can be suspended or preferably dissolved, such that the composition is physiologically
tolerable, i.e. can be administered to the mammalian body without toxicity or undue
discomfort. The biocompatible carrier is suitably an injectable carrier liquid such as
sterile, pyrogen-free water for injection; an aqueous solution such as saline (which
may advantageously be balanced so that the final product for injection is isotonic); an
aqueous buffer solution comprising a biocompatible buffering agent (e.g. phosphate
buffer); an aqueous solution of one or more tonicity-adjusting substances (eg. salts of
plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (eg. sorbitol or mannitol), glycols (eg. glycerol), or other non-ionic
polyol materials (eg. polyethyleneglycols, propylene glycols and the like). Preferably
the biocompatible carrier is pyrogen- free water for injection, isotonic saline or
phosphate buffer.
The imaging agents and biocompatible carrier are each supplied in suitable vials or
vessels which comprise a sealed container which permits maintenance of sterile
integrity and/or radioactive safety, plus optionally an inert headspace gas (eg. nitrogen
or argon), whilst permitting addition and withdrawal of solutions by syringe or
cannula. A preferred such container is a septum-sealed vial, wherein the gas-tight
closure is crimped on with an overseal (typically of aluminium). The closure is
suitable for single or multiple puncturing with a hypodermic needle (e.g. a crimped-on
septum seal closure) whilst maintaining sterile integrity. Such containers have the
additional advantage that the closure can withstand vacuum if desired (eg. to change
the headspace gas or degas solutions), and withstand pressure changes such as
reductions in pressure without permitting ingress of external atmospheric gases, such
as oxygen or water vapour.
Preferred multiple dose containers comprise a single bulk vial (e.g. of 10 to 30 cm3
volume) which contains multiple patient doses, whereby single patient doses can thus
be withdrawn into clinical grade syringes at various time intervals during the viable
lifetime of the preparation to suit the clinical situation. Pre-filled syringes are
designed to contain a single human dose, or "unit dose" and are therefore preferably a
disposable or other syringe suitable for clinical use. The pharmaceutical compositions
of the present invention preferably have a dosage suitable for a single patient and are
provided in a suitable syringe or container, as described above.
The pharmaceutical composition may contain additional optional excipients such as:
an antimicrobial preservative, pH-adjusting agent, filler, radioprotectant, solubiliser or
osmolality adjusting agent.
By the term "antimicrobial preservative" is meant an agent which inhibits the growth
of potentially harmful micro-organisms such as bacteria, yeasts or moulds. The
antimicrobial preservative may also exhibit some bactericidal properties, depending on
the dosage employed. The main role of the antimicrobial preservative(s) of the present
invention is to inhibit the growth of any such micro-organism in the pharmaceutical
composition. The antimicrobial preservative may, however, also optionally be used to
inhibit the growth of potentially harmful micro-organisms in one or more components
of kits used to prepare said composition prior to administration. Suitable antimicrobial
preservative(s) include: the parabens, i.e. methyl, ethyl, propyl or butyl paraben or
mixtures thereof; benzyl alcohol; phenol; cresol; cetrimide and thiomersal. Preferred
antimicrobial preservative(s) are the parabens.
The term "pH-adjusting agent" means a compound or mixture of compounds useful to
ensure that the pH of the composition is within acceptable limits (approximately pH
4.0 to 10.5, preferably 6.5 to 8.5 for the agents of the present invention) for human or
mammalian administration. Suitable such pH-adjusting agents include
pharmaceutically acceptable buffers, such as tricine, phosphate or TRIS [i.e.
tm(hydroxymethyl)aminomethane], and pharmaceutically acceptable bases such as
sodium carbonate, sodium bicarbonate or mixtures thereof. When the composition is
employed in kit form, the pH adjusting agent may optionally be provided in a separate
vial or container, so that the user of the kit can adjust the pH as part of a multi-step
procedure.
By the term "filler" is meant a pharmaceutically acceptable bulking agent which may
facilitate material handling during production and lyophilisation. Suitable fillers
include inorganic salts such as sodium chloride, and water soluble sugars or sugar
alcohols such as sucrose, maltose, mannitol or trehalose.
By the term "radioprotectant" is meant a compound which inhibits degradation
reactions, such as redox processes, by trapping highly-reactive free radicals, such as
oxygen-containing free radicals arising from the radiolysis of water. A combination of
two or more different radioprotectants may be used. The radioprotectants of the
present invention are suitably chosen from: ethanol; ascorbic acid; paraaminobenzoic
acid (i.e. 4-aminobenzoic acid); gentisic acid (i.e. 2,5-dihydroxybenzoic
acid), and where applicable salts of such acids with a biocompatible cation. By the
term "biocompatible cation" (B ) is meant a positively charged counterion which
forms a salt with an ionised, negatively charged group, where said positively charged
counterion is also non-toxic and hence suitable for administration to the mammalian
body, especially the human body. Examples of suitable biocompatible cations include:
the alkali metals sodium or potassium; the alkaline earth metals calcium and
magnesium; and the ammonium ion. Preferred biocompatible cations are sodium and
potassium, most preferably sodium. The radioprotectant of the present invention
preferably comprises ascorbic acid or sodium ascorbate.
By the term "solubiliser" is meant an additive present in the composition which
increases the solubility of the imaging agent in the solvent. A preferred such solvent
is aqueous media, and hence the solubiliser preferably improves solubility in water.
Suitable such solubilisers include: C1-4 alcohols; glycerine; polyethylene glycol
(PEG); propylene glycol; polyoxyethylene sorbitan monooleate; sorbitan monooloeate;
polysorbates; poly(oxyethylene)poly(oxypropylene)poly(oxyethylene) block
copolymers (Pluronics™); cyclodextrins (e.g. alpha, beta or gamma cyclodextrin,
hydroxypropyl -P-cyclodextrin or hydroxypropyl-y-cyclodextrin) and lecithin.
Preferred solubilisers are cyclodextrins, C1-4 alcohols and Pluronics™, more
preferably cyclodextrins and C2-4 alcohols. When the solubiliser is an alcohol, it is
preferably ethanol or propanol, more preferably ethanol. Ethanol has a potential dual
role, since it can also function as a radioprotectant. When the solubiliser is a
cyclodextrin, it is preferably a gamma cyclodextrin, more preferably hydroxypropyl-
b-cyclodextrin (HPCD). The concentration of cyclodextrin can be from about 0.1 to
about 40 mg/mL, preferably between about 5 and about 35 mg/mL, more preferably
20 to 30 mg/ml, most preferably around 25 mg/ml.
The radiopharmaceutical compositions of the present invention may be prepared by
various methods:
(i) aseptic manufacture techniques in which the radiometal complex
formation is carried out in a clean room environment;
(ii) terminal sterilisation, in which the radiometal complex formation is carried
out without using aseptic manufacture and then sterilised at the last step
[eg. by gamma irradiation, autoclaving dry heat or chemical treatment (e.g.
with ethylene oxide)];
(iii) kit methodology in which a sterile, non-radioactive kit formulation
comprising the chelator conjugate of Formula I and optional excipients is
reacted with a supply of the desired technetium radiometal.
Method (iii) is preferred, and kits for use in this method are described in the fifth
embodiment (below).
Preferably, the imaging agent composition is chosen such that any unlabelled cMBP
peptide is present in said composition at no more than 50 times the molar amount of
the Tc-labelled cMBP peptide. Thus, the chemical excess of chelator conjugate used
for Tc radiolabelling can be as high as a 1000-fold excess over the Tc, and the
unlabelled conjugate may compete for c-Met sites in vivo. Low concentrations of
chelator conjugate can, however, affect the RCP. Thus, for mTc, RCP values of
>90% can be achieved using 45 nanomoles of Compound 1, but varied between 70
and 95% when using 15-30 nanomoles. Any excess unlabelled peptide can be
removed by HPLC or SPE.
By the term "unlabelled" is meant that the c-Met binding cyclic peptide is non
radioactive, i.e. is not radiolabelled with Tc, or any other radioisotope. Such
unlabelled peptides primarily include the non-radioactive chelator conjugates of the
second aspect (above). The term 'unlabelled' excludes the c-Met binding cyclic
peptide labelled with Tc, where said Tc is present in the mTc used to radiolabel
said c-Met binding cyclic peptide and is thus a product of the same radiolabelling
reaction. Preferably, the unlabelled c-Met binding cyclic peptide is present in said
composition at less than 20, more preferably less than 10, most preferably less than 5
times the molar amount of the corresponding Tc-labelled peptide.
In a fifth aspect, the present invention provides a kit for the preparation of the
radiopharmaceutical composition of the fourth aspect, which comprises the chelator
conjugate of the second aspect in sterile, solid form such that upon reconstitution with
a sterile supply of the radiometal in a biocompatible carrier, dissolution occurs to give
the desired radiopharmaceutical composition.
Preferred aspects of the chelator conjugate in the fifth aspect are as described in the
second aspect of the present invention (above).
By the term "kit" is meant one or more non-radioactive pharmaceutical grade
containers, comprising the necessary chemicals to prepare the desired
radiopharmaceutical composition, together with operating instructions. The kit is
designed to be reconstituted with the desired radiometal to give a solution suitable for
human administration with the minimum of manipulation.
The sterile, solid form is preferably a lyophilised solid.
For mTc, the kit is preferably lyophilised and is designed to be reconstituted with
sterile mTc-pertechnetate (Tc(V) from a mTc radioisotope generator to give a
solution suitable for human administration without further manipulation. Suitable kits
comprise a container (eg. a septum-sealed vial) containing the chelator conjugate in
either free base or acid salt form, together with a biocompatible reductant such as
sodium dithionite, sodium bisulfite, ascorbic acid, formamidine sulfinic acid, stannous
ion, Fe(II) or Cu(I). The biocompatible reductant is preferably a stannous salt such as
stannous chloride or stannous tartrate. Alternatively, the kit may optionally contain a
non-radioactive metal complex which, upon addition of the technetium, undergoes
transmetallation (i.e. metal exchange) giving the desired product. The non-radioactive
kits may optionally further comprise additional components such as a transchelator,
radioprotectant, antimicrobial preservative, pH-adjusting agent or filler - as defined
above.
In a sixth aspect, the present invention provides a method of imaging the human or
animal body which comprises generating an image of at least a part of said body to
which the imaging agent of the first aspect, or the radiopharmaceutical composition of
the second aspect has distributed using PET or SPECT, wherein said imaging agent or
composition has been previously administered to said body.
Preferred aspects of the imaging agent or composition in the sixth aspect are as
described in the first and fourth aspects respectively of the present invention (above).
Preferably, the radiopharmaceutical composition of the fourth aspect is used.
Preferably, the mammal is an intact mammalian body in vivo, and is more preferably a
human subject. Preferably, the imaging agent can be administered to the mammalian
body in a minimally invasive manner, i.e. without a substantial health risk to the
mammalian subject even when carried out under professional medical expertise. Such
minimally invasive administration is preferably intravenous administration into a
peripheral vein of said subject, without the need for local or general anaesthetic.
The imaging of the sixth aspect is preferably of sites of c-Met over-expression or
localisation. The site of c-Met over-expression or localisation is preferably a cancer
or a precancerous lesion.
The imaging method of the sixth aspect may optionally be carried out repeatedly to
monitor the effect of treatment of a human or animal body with a drug or radiation,
said imaging being effected before and after said treatment, and optionally also during
said treatment. Of particular interest is early monitoring of the efficacy of cancer
therapy to ensure that malignant growth is controlled before the condition becomes
terminal.
Included in this aspect is a method of diagnosis of sites of c-Met over-expression or
localisation of the mammalian body in vivo, which comprises the imaging method of
the sixth aspect.
In a seventh aspect, the present invention provides the use of the imaging agent of the
first aspect, the radiopharmaceutical composition of the fourth aspect, or the kit of the
fifth aspect, in a method of diagnosis of the human or animal body.
Preferred aspects of the imaging agent or composition in the seventh aspect are as
described in the first and fourth aspects respectively of the present invention (above).
The method of diagnosis of the seventh aspect preferably comprises the imaging
method of the sixth aspect and preferred aspects thereof.
The invention is illustrated by the non-limiting Examples detailed below. Example 1
provides the synthesis of a cMBP peptide of the invention having metabolism
inhibiting groups (Z1 = Z2 = MIG) at both termini (Peptide 1). Examples 2 to 4
provide the synthesis of a bifunctional amine-functionalised chelator of the invention
(Chelator 1). Example 5 provides the synthesis of a bifunctional active esterfunctionalised
chelator of the invention (Chelator 1A). Example 6 provides the
synthesis of a chelator conjugate of a peptide of the invention (Peptide 1) with
Chelator 1 ("Compound 1"). Example 7 provides the preparation of a mTc complex
of the invention ( mTc-Compound 1), and shows that the complexation proceeds
efficiently in high radiochemical yield at room temperature. Example 8 provides the
biodistribution of mTc-Compound 1, and shows that the technetium complex
exhibits useful tumour uptake, with good tumour:background ratios.
Abbreviations.
Conventional single letter or 3-letter amino acid abbreviations are used.
%id: percentage injected dose
Ac: Acetyl
Acm: Acetamidomethyl
ACN: Acetonitrile
Boc: t r t-Butyloxycarbonyl
tBu: tertiary-butyl
DCM: Dichloromethane
DMF: Dimethylformamide
DMSO: Dimethyl sulfoxide
EDC: N- -dimethylaminopropy -V-ethylcarbodiimide.
Fmoc: 9-Fluorenylmethoxycarbonyl
HATU: 0-(7-Azabenzotriazol-l-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate.
HBTU: 0-Benzotriazol- 1-yl-N,N,N,N 4etramethyluronium hexafluorophosphate
HPLC: High performance liquid chromatography
HSPyU 0-(N-succinimidyl)-N,N,N ,N'-tetramethyleneuronium hexafluorophosphate
NHS: N-hydroxy-succinimide
NMM: N-Methylmorpholine
NMP: l-Methyl-2-pyrrolidinone
Pbf: 2,2,4, 6,7-Pentamethyldihydrobenzofuran-5-sulfonyl
PBS: Phosphate-buffered saline
PyBOP: benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
s.c: sub-cutaneously,
tBu: t r t-butyl
TFA: Trifluoroacetic acid
THF: Tetrahydrofuran
TIS: Triisopropylsilane
Trt: Trityl.
Compounds of the Invention .
where:
Compound 1 is functionalised at the epsilon amine group of the carboxy terminal Lys of
Peptide 1.
Example 1: Synthesis of Peptide 1.
Step (a): synthesis of protected precursor linear peptide.
The precursor linear peptide has the structure:
Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys(Acm)-Trp-
Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH 2
The peptidyl resin H-Ala-Gly-Ser(tBu)-Cys(Trt)-Tyr(tBu)-Cys(Acm)-Ser(tBu)-Gly-
Pro-Pro-Arg(Pbf)-Phe-Glu(OtBu)-Cys(Acm)-Trp(Boc)-Cys(Trt)-Tyr(tBu)-
Glu(OtBu)-Thr(^ eMepro)-Glu(OtBu)-Gly-Thr(tBu)-Gly-Gly-Gly-Lys(Boc)-Polymer
was assembled on an Applied Biosystems 433A peptide synthesizer using Fmoc
chemistry starting with 0.1 mmol Rink Amide Novagel resin. An excess of 1 mmol
pre-activated amino acids (using HBTU) was applied in the coupling steps. Glu-Thr
pseudoproline (Novabiochem 05-20-1 122) was incorporated in the sequence. The
resin was transferred to a nitrogen bubbler apparatus and treated with a solution of
acetic anhydride ( 1 mmol) and NMM ( 1 mmol) dissolved in DCM (5 mL) for 60 min.
The anhydride solution was removed by filtration and the resin washed with DCM
and dried under a stream of nitrogen.
The simultaneous removal of the side-chain protecting groups and cleavage of the
peptide from the resin was carried out in TFA (10 mL) containing 2.5 % TIS, 2.5 % 4-
thiocresol and 2.5 % water for 2 hours and 30 min. The resin was removed by
filtration, TFA removed in vacuo and diethyl ether added to the residue. The formed
precipitate was washed with diethyl ether and air-dried affording 264 mg of crude
peptide.
Purification by preparative HPLC (gradient: 20-30 % B over 40 min where A =
H2O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 10 mL/min, column:
Phenomenex Luna 5m C18 (2) 250 x 21.20 mm, detection: UV 214 nm, product
retention time: 30 min) of the crude peptide afforded 100 mg of pure Peptide 1 linear
precursor. The pure product was analysed by analytical FIPLC (gradient: 10-40 % B
over 10 min where A = H2O/0.1 % TFA and B = ACN/0.1 % TFA, flow rate: 0.3
mL/min, column: Phenomenex Luna 3m C18 (2) 50 x 2 mm, detection: UV 214 nm,
product retention time: 6.54 min). Further product characterisation was carried out
using electrospray mass spectrometry (MH2
2+ calculated: 1464.6, MH2
2+ found: 1465.1).
Step (b): Formation of Monocyclic Cys4-16 disulfide bridge.
Cys4-16; Ac-Ala-Gly-Ser-Cys-Tyr-Cys(Acm)-Ser-Gly-Pro-Pro-Arg-Phe-Glu-
Cys(Acm)-Trp-Cys-Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys-NH 2.
The linear precursor from step (a) (100 mg) was dissolved in 5 % DMSO/water (200
mL) and the solution adjusted to pH 6 using ammonia. The reaction mixture was
stirred for 5 days. The solution was then adjusted to pH 2 using TFA and most of the
solvent removed by evaporation in vacuo. The residue (40 mL) was injected in
portions onto a preparative FIPLC column for product purification.
Purification by preparative HPLC (gradient: 0 % B for 10 min, then 0-40 % B over 40
min where A = H2O/0. 1% TFA and B = ACN/0. 1% TFA, flow rate: 10 mL/min,
column: Phenomenex Luna 5m C18 (2) 250 x 21.20 mm, detection: UV 214 nm,
product retention time: 44 min) of the residue afforded 72 mg of pure Peptide 1
monocyclic precursor. The pure product (as a mixture of isomers P I to P3) was
analysed by analytical HPLC (gradient: 10-40 % B over 10 min where A = H2O/0. 1%
TFA and B = ACN/0.1 % TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3
C18 (2) 50 x 2 mm, detection: UV 214 nm, product retention time: 5.37 min (PI);
5.61 min (P2); 6.05 min (P3)). Further product characterisation was carried out using
electrospray mass spectrometry (MH2
2+ calculated: 1463.6, MH2
2+ found: 1464.1 (PI);
1464.4 (P2); 1464.3 (P3)).
Step (c): Formation of Second Cys6-14 disulfide bridge (Peptide 1).
The monocyclic precursor from step (b) (72 mg) was dissolved in 75 % AcOH/water
(72 mL) under a blanket of nitrogen. 1M HC1 (7.2 mL) and 0.05 M I2 in AcOH (4.8
mL) were added in that order and the mixture stirred for 45 min. 1M ascorbic acid ( 1
mL) was added giving a colourless mixture. Most of the solvents were evaporated in
vacuo and the residue (18 mL) diluted with water/0. 1% TFA (4 mL) and the product
purified using preparative HPLC. Purification by preparative HPLC (gradient: 0 % B
for 10 min, then 20-30 % B over 40 min where A = H2O/0. 1% TFA and B =
ACN/0.1 % TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5m C18 (2) 250
x 21.20 mm, detection: UV 214 nm, product retention time: 43-53 min) of the residue
afforded 52 mg of pure Peptide 1. The pure product was analysed by analytical
HPLC (gradient: 10-40 % B over 10 min where A = H2O/0.1 % TFA and B =
ACN/0. 1% TFA, flow rate: 0.3 mL/min, column: Phenomenex Luna 3m C18 (2) 50 x
2 mm, detection: UV 214 nm, product retention time: 6.54 min). Further product
characterisation was carried out using electrospray mass spectrometry (MH2
2+
calculated: 1391.5, MH2
2+ found: 1392.5).
Example 2 : Synthesis of l,l,l-?m{2-aminoethyl)methane.
Step 1(a): 3(methoxycarbonylmethylene)glutaric acid dimethylester.
Carbomethoxymethylenetriphenylphosphorane (167g, 0.5mol) in toluene (600ml) was
treated with dimethyl 3-oxoglutarate (87g, 0.5mol) and the reaction heated to 100°C
on an oil bath at 120°C under an atmosphere of nitrogen for 36h. The reaction was
then concentrated in vacuo and the oily residue triturated with 40/60 petrol
ether/diethylether 1:1, 600ml. Triphenylphosphine oxide precipitated out and the
supernatant liquid was decanted/filtered off. The residue on evaporation in vacuo was
Kugelrohr distilled under high vacuum Bpt (oven temperature 180-200°C at 0.2torr)
to give 3-(methoxycarbonylmethylene)glutaric acid dimethylester (89.08g, 53%).
NMR ^(CDCl,): d 3.31 (2H, s, CH2), 3.7(9H, s, 3xOCH 3), 3.87 (2H, s, CH2), 5.79 (1H, s,
=CH, ) ppm.
NMR 1 C(CDC13), d 36.56,CH3, 4 8 .7, 2xCH 3, 52.09 and 52.5 (2xCH 2) ; 122.3 and 146.16
C=CH; 165.9, 170.0 and 170.5 3xCOO ppm.
Step Kb): Hydrogenation of 3-(methoxycarbonylmethylene)glutaric acid
dimethylester.
3-(Methoxycarbonylmethylene)glutaric acid dimethylester (89g, 267mmol) in
methanol (200ml) was shaken with (10% palladium on charcoal: 50% water) (9 g)
under an atmosphere of hydrogen gas (3.5 bar) for (30h). The solution was filtered
through kieselguhr and concentrated in vacuo to give 3-
(methoxycarbonylmethyl)glutaric acid dimethylester as an oil, yield (84. 9g, 94 %).
NMR ^(CDCy, d 2.48 (6H, d, J=8Hz, 3xCH2), 2.78 (1H, hextet, J=8Hz CH, ) 3.7 (9H, s,
3xCH ) .
NMR 1 C(CDC1 ), d 2 8 .6, CH; 37.50, 3xCH ; 51.6, 3xCH 2; 172.28,3xCOO.
Step 1(c): Reduction and esterification of trimethyl ester to the triacetate.
Under an atmosphere of nitrogen in a 3 necked 2L round bottomed flask lithium
aluminium hydride (20g, 588mmol) in THF (400ml) was treated cautiously with
tra(methyloxycarbonylmethyl)methane (40g, 212mmol) in THF (200ml) over lh. A
strongly exothermic reaction occurred, causing the solvent to reflux strongly. The
reaction was heated on an oil bath at 90°C at reflux for 3 days. The reaction was
quenched by the cautious dropwise addition of acetic acid (100ml) until the evolution
of hydrogen ceased. The stirred reaction mixture was cautiously treated with acetic
anhydride solution (500ml) at such a rate as to cause gentle reflux. The flask was
equipped for distillation and stirred and then heating at 90°C (oil bath temperature) to
distil out the THF. A further portion of acetic anhydride (300ml) was added, the
reaction returned to reflux configuration and stirred and heated in an oil bath at 140°C
for 5h. The reaction was allowed to cool and filtered. The aluminium oxide precipitate
was washed with ethyl acetate and the combined filtrates concentrated on a rotary
evaporator at a water bath temperature of 50°C in vacuo (5 mmHg) to afford an oil.
The oil was taken up in ethyl acetate (500ml) and washed with saturated aqueous
potassium carbonate solution. The ethyl acetate solution was separated, dried over
sodium sulfate, and concentrated in vacuo to afford an oil. The oil was Kugelrohr
distilled in high vacuum to give tm(2-acetoxyethyl)methane (45. 3g, 95.9%) as an oil.
Bp. 220 °C at 0.1 mmHg.
NMR^CDCy, d 1.66(7H, m, 3xCH2 CH), 2.08(1H, s, 3xCH3); 4.1(6H, t, 3xCH20).
NMR 1 C(CDC13), d 20.9, CH3; 29.34, CH; 32.17, CH2; 62.15, CH20 ; 171, CO.
Step 1(d): Removal of Acetate groups from the triacetate.
Jm(2-acetoxyethyl)methane (45. 3g, 165mM) in methanol (200ml) and 880 ammonia
(100ml) was heated on an oil bath at 80°C for 2 days. The reaction was treated with a
further portion of 880 ammonia (50ml) and heated at 80°C in an oil bath for 24h. A
further portion of 880 ammonia (50ml) was added and the reaction heated at 80°C for
24h. The reaction was then concentrated in vacuo to remove all solvents to give an oil.
This was taken up into 880 ammonia (150ml) and heated at 80°C for 24h. The
reaction was then concentrated in vacuo to remove all solvents to give an oil.
Kugelrohr distillation gave acetamide bp 170-180 0.2mm. The bulbs containing the
acetamide were washed clean and the distillation continued. Tris{2-
hydroxyethyl)methane (22.53g, 92%) distilled at bp 220 °C 0.2mm.
NMR ^(CDCy, d 1.45(6H, q, 3xCH2), 2.2(1H, quintet, CH); 3.7(6H, t 3xCH2OH); 5.5(3H,
brs, 3xOH).
NMR 1 C(CDC13), d 22.13, CH; 33.95, 3xCH2; 57.8, 3xCH2OH.
Step 1(e): Conversion of the triol to the tm(methanesulfonate).
To an stirred ice-cooled solution of tm(2-hydroxyethyl)methane (lOg, 0.0676mol) in
dichlorom ethane (50ml) was slowly dripped a solution of methanesulfonyl chloride
(40g, 0.349mol) in dichlorom ethane (50ml) under nitrogen at such a rate that the
temperature did not rise above 15°C. Pyridine (21.4g, 0.27mol, 4eq) dissolved in
dichlorom ethane (50ml) was then added drop-wise at such a rate that the temperature
did not rise above 15°C, exothermic reaction. The reaction was left to stir at room
temperature for 24h and then treated with 5N hydrochloric acid solution (80ml) and
the layers separated. The aqueous layer was extracted with further dichloromethane
(50ml) and the organic extracts combined, dried over sodium sulfate, filtered and
concentrated in vacuo to give tra[2-(methylsulfonyloxy)ethyl]methane contaminated
with excess methanesulfonyl chloride. The theoretical yield was 25. 8g.
NMR ^(CDCy, d 4.3 (6H, t, 2xCH2), 3.0 (9H, s, 3xCH3), 2 (1H, hextet, CH), 1.85 (6H, q,
3xCH2) .
Step 1(f): Preparation of 1,1,1 - tm(2-azidoethyl)methane.
A stirred solution of tm[2-(methylsulfonyloxy)ethyl]methane [from Step 1(e),
contaminated with excess methyl sulfonyl chloride] (25. 8g, 67mmol, theoretical) in dry
DMF (250ml) under nitrogen was treated with sodium azide (30. 7g, 0.47mol) portionwise
over 15 minutes. An exotherm was observed and the reaction was cooled on an
ice bath. After 30 minutes, the reaction mixture was heated on an oil bath at 50°C for
24h. The reaction became brown in colour. The reaction was allowed to cool, treated
with dilute potassium carbonate solution (200ml) and extracted three times with 40/60
petrol ether/diethylether 10:1 (3x150ml). The organic extracts were washed with
water (2x1 50ml), dried over sodium sulfate and filtered. Ethanol (200ml) was added
to the petrol/ether solution to keep the triazide in solution and the volume reduced in
vacuo to no less than 200ml. Ethanol (200ml) was added and reconcentrated in vacuo
to remove the last traces of petrol leaving no less than 200ml of ethanolic solution.
The ethanol solution of triazide was used directly in Step 1(g).
CARE: DO NOT REMOVE ALL THE SOLVENT AS THE AZIDE IS
POTENTIALLY EXPLOSIVE AND SHOULD BE KEPT IN DILUTE SOLUTION
AT ALL TIMES.
Less than 0.2ml of the solution was evaporated in vacuum to remove the ethanol and
an NMR run on this small sample: NMR ^(CDC^), d 3.35 (6H, t, 3xCH2), 1.8 (1H, septet,
CH, ), 1.6 (6H, q, 3xCH2) .
Step Kg): Preparation of l,l,l -tm(2-aminoethyl)m ethane.
rm(2-azidoethyl)methane (15.06g, 0.0676 mol), (assuming 100% yield from previous
reaction) in ethanol (200ml) was treated with 10% palladium on charcoal (2g, 50%
water) and hydrogenated for 12h. The reaction vessel was evacuated every 2 hours to
remove nitrogen evolved from the reaction and refilled with hydrogen. A sample was
taken for NMR analysis to confirm complete conversion of the triazide to the triamine.
Caution: unreduced azide could explode on distillation. The reaction was filtered
through a celite pad to remove the catalyst and concentrated in vacuo to give tris{2-
aminoethyl)methane as an oil. This was further purified by Kugelrohr distillation
bp.l80-200°C at 0.4mm/Hg to give a colourless oil (8.1g, 82.7% overall yield from
the triol).
NMR ^(CDCy, d 2.72 (6H, t, 3xCH2N), 1.41 (H, septet, CH), 1.39 (6H, q, 3xCH2) .
NMR 1 C(CDC13), d 39.8 (CH2NH2), 38.2 (CH2 ), 31.0 (CH).
Example 3 : Preparation of 3-chloro-3-methyl-2-nitrosobutane.
A mixture of 2-methylbut-2-ene (147ml, 1.4mol) and isoamyl nitrite (156ml, 1.16mol)
was cooled to -30 °C in a bath of cardice and methanol and vigorously stirred with an
overhead air stirrer and treated dropwise with concentrated hydrochloric acid (140ml,
1.68mol ) at such a rate that the temperature was maintained below -20°C. This
requires about l h as there is a significant exotherm and care must be taken to prevent
overheating. Ethanol (100ml) was added to reduce the viscosity of the slurry that had
formed at the end of the addition and the reaction stirred at -20 to -10°C for a further
2h to complete the reaction. The precipitate was collected by filtration under vacuum
and washed with 4x3 0ml of cold (-20°C) ethanol and 100ml of ice cold water, and
dried in vacuo to give 3-chloro-3-methyl-2-nitrosobutane as a white solid. The ethanol
filtrate and washings were combined and diluted with water (200ml) and cooled and
allowed to stand for l h at -10°C when a further crop of 3-chloro-3-methyl-2-
nitrosobutane crystallised out. The precipitate was collected by filtration and washed
with the minimum of water and dried in vacuo to give a total yield of 3-chloro-3-
methyl-2-nitrosobutane ( 115g 0.85mol, 73%) >98% pure by NMR.
NMR ^(CDCy, As a mixture ofisomers (isomer1, 90%) 1.5 d, (2H, CH3), 1.65 d, (4H, 2
xCH3), 5.85,q, and 5.95,q, together 1H. (isomer2, 10%), 1.76 s, (6H, 2x CH3), 2.07(3H,
CH ) .
Example 4: Synthesis of [N- l -dimethyl-2-N-hvdrox imine propyl)2-
aminoethyll-f2-aminoethyl)methane (Chelator 1).
To a solution of tm(2-aminoethyl)methane (4.047g, 27.9mmol) in dry ethanol (30ml)
was added potassium carbonate anhydrous (7.7g, 55.8mmol, 2eq) at room temperature
with vigorous stirring under a nitrogen atmosphere. A solution of 3-chloro-3-methyl-
2-nitrosobutane (7.56g, 55.8mol, 2eq) was dissolved in dry ethanol (100ml) and 75ml
of this solution was dripped slowly into the reaction mixture. The reaction was
followed by TLC on silica [plates run in dichloromethane, methanol, concentrated
(0.88sg) ammonia; 100/30/5 and the TLC plate developed by spraying with ninhydrin
and heating]. The mono-, di- and tri-alkylated products were seen with RF's
increasing in that order. Analytical HPLC was run using PRP reverse phase column in
a gradient of 7.5-75% acetonitrile in 3% aqueous ammonia. The reaction was
concentrated in vacuo to remove the ethanol and resuspended in water ( 110ml). The
aqueous slurry was extracted with ether (100ml) to remove some of the trialkylated
compound and lipophilic impurities leaving the mono and desired dialkylated product
in the water layer. The aqueous solution was buffered with ammonium acetate (2eq,
4.3g, 55.8mmol) to ensure good chromatography. The aqueous solution was stored at
4°C overnight before purifying by automated preparative HPLC.
Yield (2.2g, 6.4mmol, 23%).
Mass spec; Positive ion 10 V cone voltage. Found: 344; calculated M+H= 344.
NMR ^(CDCy, d 1.24(6H, s, 2xCH3), 1.3(6H, s, 2xCH3), 1.25-1.75(7H, m, 3xCH2CH),
(3H, s, 2xCH2), 2.58 (4H, m, CH2N), 2.88(2H, t CH2N2), 5.0 (6H, s, NH2 , 2xNH, 2xOH).
NMR ¾ ((CD3)2SO) 51.1 4xCH; 1.29, 3xCH2; 2.1 (4H, t, 2xCH2);
NMR 1 C((CD3)2SO), d 9.0 (4xCH ), 25.8 (2xCH ), 31.0 2xCH2, 34.6 CH2, 56.8 2xCH2N;
160.3, C=N.
HPLC conditions: flow rate 8ml/min using a 25mm PRP column [A=3% ammonia
solution (sp.gr = 0.88) /water; B = Acetonitrile].
Load 3ml of aqueous solution per run, and collect in a time window of 12.5-13.5 min.
Example 5: Synthesis of Tetrafluorothiophenyl ester of Chelator 1-glutaric acid
(Chelator 1A).
a) Synthesis of [Chelator l]-glutaric acid intermediate.
Chelator 1 (100 mg, 0.29 mmol) was dissolved in DMF (10 mL) and glutaric
anhydride (33 mg, 0.29 mmol) added by portions with stirring. The reaction was
stirred for 23 hours to afford complete conversion to the desired product. The pure
acid was obtained following RP-HPLC in good yield.
b) Synthesis of Chelator 1A.
Chelator 1A
To [Chelator l]-glutaric acid (from Step a; 300 mg, 0.66 mmol) in DMF (2 mL) was
added HATU (249 mg, 0.66 mmol) and MM (132 , 1.32 mmol). The mixture
was stirred for 5 minutes then tetrafluorothiophenol (0.66 mmol, 119 mg) was added.
The solution was stirred for 10 minutes then the reaction mixture was diluted with 20 %
acetonitrile/water (8 mL) and the product purified by RP- PLC yielding 110 mg of
the desired product following freeze-drying.
Example 6: Synthesis of Peptide 1 Conjugate with Chelator 1 (Compound 1).
Chelator 1A (3.8 mg, Example 5), Peptide 1 (3.4 mg) and sym.-collidine (1.6 )
were dissolved in DMF ( 1 mL) and the solution stirred for 90 min. The reaction
mixture was diluted with water (6 mL) and the product purified using preparative
HPLC.
Purification by preparative HPLC (10-20 % B over 40 min where A = H2O/0.1 %
TFA and B = ACN/0.1 % TFA, flow rate: 10 mL/min, column: Phenomenex Luna 5m
C18 (2) 250 x 21.20 mm, detection: UV 214 nm) of the crude peptide afforded 1.1 mg
(28 %) pure Compound 1. The purified material was analysed by analytical FIPLC
(gradient: 10-40 % B over 5 min where A = H2O/0.1 % TFA and B = ACN/0.1 %
TFA, flow rate: 0.6 mL/min, column: Phenomenex Luna 3 CI8 (2) 20 x 2 mm,
detection: UV 214 nm, tR: 3.04 min). Further product characterisation was carried out
using electrospray mass spectrometry (found m/z: 161 1.2, expected MH2
2+: 161 1.2).
Example 7: mTc-Radiolabelling of Compound 1.
Step (i): Lyophilised Vials.
Compound 1 (45 nmol) was lyophilised in a first vial (Vial 1). A lyophilised kit
containing the following formulation (Vial 2) was prepared separately:
Step (ii): Radiolabelling .
Vial 1 was reconstituted with a watenethanol solution (0.1 mL of a 50:50 mixture),
and the vial sonicated or mixed for 10 minutes. The resulting solution of Compound
1 (0.1 mL) was then added to Vial 2 . mTc-pertechnetate eluate from a Drytec™
generator (GE Healthcare; 0.9 ml, 0.25-2.18 GBq) was then added to the vial, and the
solution allowed to stand at room temperature for 20min before HPLC analysis. The
RCP was 93 to 94%.
Example 8: Biodistribution of mTc-Compound 1 in Tumour-bearing Nude Mice.
CD-I male nude mice (ca. 20g) were housed in individual ventilated cages, with ad
libitum access to food and water. HT-29 cells (ATCC, Cat. no. HTB-38) were grown
in McCoy's 5a medium (Sigma # M8403) supplemented with 10% fetal bovine serum
and penicillin/streptomycin. Cells were split 1:3 two times a week, at 70-80%
confluent using 0.25% trypsin and incubated in 5% C0 2 at 37°C. The mice were
injected s.c under light gas anaesthesia (Isoflurane) with the HT-29 cell suspension at
one site (nape of the neck) with a nominal dose of 106 cells per injections in a volume
of 100 mΐ using a fine bore needle (25 G). The tumours were then allowed to develop
for 20 days, or until at least 200mm3 in volume (for inclusion in the study).
After the 20 day growth time, animals were injected with mTc-Compound 1 (0. 1 ml,
1-5 MBq/animal), as an intravenous bolus via the tail vein. At various times post
injection animals were euthanised, dissected and the following organs and tissues
removed. The results at 120 min p.i. were as follows:
44% of activity retained in the body at 120 min p.i. was present in the tumour.
CLAIMS.
1. An imaging agent which comprises a radioactive Tc complex of a chelator
conjugate of a c-Met binding peptide, said chelator conjugate being of Formula I :
Z^cMBPj-Z 2
(I)
where:
Tc is the radioisotope 4mTc or mTc;
cMBP is an 18 to 30-mer c-Met binding cyclic peptide of Formula II:
-(A) -Q-(A') -
(P)
where Q is the amino acid sequence (SEQ-1):
-Cys^X^Cys^X^Gly-Pro-Pro-X^Phe-Glu-Cys^Trp-Cys^Tyr-X^X^X 6-
wherein X1 is Asn, His or Tyr;
X2 is Gly, Ser, Thr or Asn;
X3 is Thr or Arg;
X4 is Ala, Asp, Glu, Gly or Ser;
X5 is Ser or Thr;
X6 is Asp or Glu;
and Cysa d are each cysteine residues such that residues a and b as well
as c and d are cyclised to form two separate disulfide bonds;
A and A' are independently any amino acid other than Cys, or one of A
and A' is Lys(s-Z 3);
x and y are independently integers of value 0 to 13, and are chosen
such that [x + y] = 1 to 13;
Z1 is attached to the N-terminus of cMBP, and isMIG or Z3;
Z2 is attached to the C-terminus of cMBP and isMIG or Z3;
wherein each M is independently a metabolism inhibiting group
which is a biocompatible group which inhibits or suppresses in vivo
metabolism of the cMBP peptide;
Z3 is a chelator of Formula (III):
(III)
wherein E^E 6 are each independently an R' group;
each R' is independently H or C i -4 alkyl, C 3-7 alkylaryl, C2-7 alkoxyalkyl, C1-4
hydroxyalkyl, C1-4 fluoroalkyl, C2-7 carboxyalkyl or C1-4 aminoalkyl, or two or more
R' groups together with the atoms to which they are attached form a carbocyclic,
heterocyclic, saturated or unsaturated ring;
L is a synthetic linker group of formula -(A)m- wherein each A is
independently -CR2- , -CR=CR- , -CºC- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO R- , - R(C=0 ) R-, - R(C=S) R-, -S0 2 R- , - RS0 2- , -CR2OCR2- ,
-CR2SCR2- , -CR2 RCR2- , a C4-8 cycloheteroalkylene group, a C4-8
cycloalkylene group, a C5-12 arylene group, or a C3-12 heteroarylene group, or a
monodisperse polyethyleneglycol (PEG) building block;
each R is independently chosen from H, C1-4 alkyl, C2-4 alkenyl, C2-4 alkynyl,
C i -4 alkoxyalkyl or C1- hydroxyalkyl;
m is an integer of value 1 to 10;
with the proviso that the conjugate of Formula I comprises one Z3 group, and said Z
group forms a metal complex with the Tc.
2 . The imaging agent of Claim 1, wherein Q comprises the amino acid sequence
of either SEQ-2 or SEQ-3:
Ser-Cysa-X1-Cys -X2-Gly-Pro-Pro-X -Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X -X6
(SEQ-2);
Ala-Gly-Ser-Cysa-X1-Cys -X2-Gly-Pro-Pro-X -Phe-Glu-Cysd-Trp-Cysb-Tyr-
X4-X -X6-Gly-Thr (SEQ-3).
3 . The imaging agent of Claim 1 or Claim 2, wherein X3 is Arg.
4 . The imaging agent of any one of Claims 1 to 3, where one of A and A' is
Lys(s-Z 3) and cMBP comprises only one Lys residue.
5 . The imaging agent of Claim 4, where cMBP is of Formula IIA:
-(A) -Q-(A') Z-Lys(8-Z3)-
(IIA)
wherein:
z is an integer of value 0 to 12, and [x + z] = 0 to 12.
6 . The imaging agent of any one of Claims 1 to 5, wherein either the -(A)x- or
-(A') - groups comprise a linker peptide which is chosen from:
-Gly-Gly-Gly-Lys- (SEQ-4),
-Gly-Ser-Gly-Lys- (SEQ-5) or
-Gly-Ser-Gly-Ser-Lys- (SEQ-6),
and the Z3 group is attached to the epsilon amine group of the Lys
residue of said linker peptide.
7 . The imaging agent of Claim 6, where cMBP has the amino acid sequence
(SEQ-7):
Ala-Gly-Ser-Cys a-Tyr-Cys -Ser-Gly-Pro-Pro-Arg-Phe-Glu-Cys d-Trp-Cysb-
Tyr-Glu-Thr-Glu-Gly-Thr-Gly-Gly-Gly-Lys.
8 . The imaging agent of any one of Claims 1 to 7, where the chelator is of
Formula IIIA:
(IIIA)
where q is an integer of value 1 to 6 .
9 . The imaging agent of any one of claims 1 to 8, where Tc is mTc.
10. The chelator conjugate of Formula I, as defined in any one of claims 1 to 8 .
11. A method of preparation of the imaging agent of any one of claims 1 to 9,
which comprises reaction of the chelator conjugate of claim 10 with a supply of Tc as
defined in claim 1 or claim 9, in a suitable solvent.
12. A radiopharmaceutical composition which comprises the imaging agent of any
one of Claims 1 to 9 together with a biocompatible carrier, in a form suitable for
mammalian administration.
13. A kit for the preparation of the radiopharmaceutical composition of claim 12,
which comprises the chelator conjugate of claim 10 in sterile, solid form such that
upon reconstitution with a sterile supply of radioactive Tc in a biocompatible carrier,
dissolution occurs to give the desired radiopharmaceutical composition;
wherein Tc is as defined in claim 1 or claim 9 .
14. A method of imaging the human or animal body which comprises generating
an image of at least a part of said body to which the imaging agent of any one of
claims 1 to 9, or the radiopharmaceutical composition of claim 12 has distributed
using PET or SPECT, wherein said imaging agent or composition has been previously
administered to said body.
15. The method of claim 14, where the images are of sites of c-Met overexpression
or localisation.
16. The method of claim 15, where the site of c-Met over-expression or
localisation is a cancer or precancerous lesion.
17. The method of any one of claims 14 to 16, which is carried out repeatedly to
monitor the effect of treatment of a human or animal body with a drug or radiation,
said imaging being effected before and after said treatment, and optionally also during
said treatment.
18. The use of the imaging agent of any one of claims 1 to 9, the composition of
claim 12, or the kit of claim 13 or claim 14, in a method of diagnosis of the human or
animal body.