Apoptosis PET Imaging Agents.
Field of the Invention.
The present invention relates to radiopharmaceutical imaging in vivo of apoptosis and
other forms of cell death. The invention provides PET imaging agents which target
apoptotic cells via selective binding to the aminophospholipid
phosphatidylethanolamine (PE), which is exposed on the surface of apoptotic cells.
Also provided are pharmaceutical compositions, kits and methods of in vivo imaging.
Background to the Invention.
Apoptosis or programmed cell death (PCD) is the most prevalent cell death pathway
and proceeds via a highly regulated, energy-conserved mechanism. In the healthy
state, apoptosis plays a pivotal role in controlling cell growth, regulating cell number,
facilitating morphogenesis, and removing harmful or abnormal cells. Dysregulation
of the PCD process has been implicated in a number of disease states, including those
associated with the inhibition of apoptosis, such as cancer and autoimmune disorders,
and those associated with hyperactive apoptosis, including neurodegenerative
diseases, haematologic diseases, AIDS, ischaemia and allograft rejection. The
visualization and quantitation of apoptosis is therefore useful in the diagnosis of such
apoptosis-related pathophysiology.
Therapeutic treatments for these diseases aim to restore balanced apoptosis, either by
stimulating or inhibiting the PCD process as appropriate. Non-invasive imaging of
apoptosis in cells and tissue in vivo is therefore of immense value for early assessment
of a response to therapeutic intervention, and can provide new insight into devastating
pathological processes. Of particular interest is early monitoring of the efficacy of
cancer therapy to ensure that malignant growth is controlled before the condition
becomes terminal.
There has consequently been great interest in developing imaging agents for apoptosis
[see eg. Zeng et al, Anti-cancer Agent Med.Chem., 9(9), 986-995 (2009); Zhao, ibid,
9(9), 1018-1023 (2009) and M. De Saint-Hubert et al, Methods, 48, 178-187 (2009)].
Of the probes available for imaging cell death, radiolabelled Annexin V has receivedthe most attention. Annexin V binds only to negatively charged phospholipids, which
renders it unable to distinguish between apoptosis and necrosis.
The lanthionine-containing antibiotic peptides ("lantibiotics") duramycin and
cinnamycin are two closely related 19-mer peptides with a compact tetracyclic
structure [Zhao, Amino Acids, DOI 10.1007/s00726-009-0386-9, Springer-Verlag
(2009), and references cited therein]. They are crosslinked via four covalent,
intramolecular bridges, and differ by only a single amino acid residue at position 2 .
The structures of duramycin and cinnamycin are shown schematically below, where
the numbering refers to the position of the linked amino acid residues in the 19-mer
sequence:
Pro9 Phe
Duramycin X
Cinnamycin X
Programmed cell death or apoptosis is an intracellular, energy-dependent self-
destruction of the cell. The redistribution of phospholipids across the bilayer of the
cell plasma membrane is an important marker for apoptosis. Thus, in viable cells, the
aminophospholipids phosphatidylethanolamine (PE) and phosphatidyl serine (PS) are
predominantly constituents of the inner leaflet of the cell plasma membrane. In
apoptopic cells, there is a synchronised externalization of PE and PS.
Both duramycin and cinnamycin bind to the neutral aminophospholipid PE with
similar specificity and high affinity, by forming a hydrophobic pocket that fits around
the PE head-group. The binding is stabilised by ionic interaction between the β-
hydroxyaspartic acid residue (HO-Asp15) and the ethanolamine group. Modifications
to this residue are known to inactivate duramycin [Zhao et al, J.Nucl.Med, 49, 1345-1352 (2008)]. Zhao [Amino Acids, DOI 10.1007/s00726-009-0386-9, Springer-
Verlag (2009)] cites earlier work by Wakamatsu et al [Biochemistry, 29, 113-188
(1990)], where NMR studies show that none of the 'HNMR resonances of the 5
terminal amino acids of cinnamycin are shifted on binding to PE - suggesting that
they are not involved in interactions with PE.
US 2004/0147440 Al (University of Texas System) describes labelled anti-
aminophospholipid antibodies, which can be used to detect pre-apoptopic or
apoptopic cells, or in cancer imaging. Also provided are conjugates of duramycin
with biotin, proteins or anti-viral drugs for cancer therapy.
WO 2006/055855 discloses methods of imaging apoptosis using a radiolabelled
compound which comprises a phosphatidyl serine-binding C2 domain of a protein.
WO 2009/1 14549 discloses a radiopharmaceutical made by a process comprising:
(i) providing a polypeptide having at least 70% sequence similarity with
CKQSCSFGPFTFVCDGNTK,
wherein the polypeptide comprises a thioether bond between amino acids
residues 1-18, 4-14, and 5-1 1, and an amide bond between amino acids
residues 6-19, and, wherein one or more distal moieties of structure
are covalently bound to the amino acid at position 1, position 2, or,
positions 1 and 2 of the polypeptide, and wherein R1 and R2 are each
independently a straight or branched, saturated or unsaturated C1-4
alkyl; and
(ii) chelating one or more of the distal moieties with mTc
( mTc=0) +,
( mTc≡N)2+, (0= mTc=0) + or [ mTc(CO)
3]+, wherein x is a redox or
oxidation state selected from the group consisting of +7, +6, +5, +4, +3, +2,
+1, 0 and -1, or, a salt, solvate or hydrate thereof.The 'distal moiety' of WO 2009/1 14549 is a complexing agent for the radioisotope
mTc, which is based on hydrazinonicotinamide (commonly abbreviated "HY C").
HYNIC is well known in the literature [see e.g. Banerjee et al, Nucl.Med.Biol, 32, 1-
20 (2005)], and is a preferred method of labelling peptides and proteins with mTc
[R.Alberto, Chapter 2, pages 19-40 in IAEA Radioisotopes and Radiopharmaceuticals
Series 1 : "Technetium-99m Radiopharmaceuticals Status and Trends" (2009)].
WO 2009/1 14549 discloses specifically mTc-HYMC-duramycin, and suggests that
the radiopharmaceuticals taught therein are useful for imaging apoptosis and/or
necrosis, atherosclerotic plaque or acute myocardial infarct.
Zhao et al [J.Nucl.Med, 49, 1345-1352 (2008)] disclose the preparation of mTc-
HYNIC-duramycin. Zhao et al note that duramycin has 2 amine groups available for
conjugation to HYNIC: at the N-terminus (Cysl residue), and the e psilon- a side
chain of the Lys2 residue. They purified the HYNIC-duramycin conjugate by HPLC
to remove the fos-HYNIC-functionalised duramycin, prior to radiolabelling with
mTc. Zhao et al acknowledge that the mTc-labelled wowo-HYNIC-duramycin
conjugates studied are probably in the form of a mixture of isomers.
Whilst HYNIC forms stable mTc complexes, it requires additional co-ligands to
complete the coordination sphere of the technetium metal complex. The HYNIC may
function as a monodentate ligand or as a bidentate chelator depending on the nature of
the amino acid side chain functional groups in the vicinity [King et al, Dalton Trans.,
4998-5007 (2007); Meszaros et al [Inorg.Chim.Acta, 363, 1059-1069 (2010)]. Thus,
depending on the environment, HYNIC forms metal complexes having 1- or 2- metal
donor atoms. Meszaros et al note that the nature of the co-ligands used with HYNIC
can have a significant effect on the behviour of the system, and state that none of the
co-ligands is ideal.
The Present Invention.
The present invention provides radiopharmaceutical imaging agents, particularly for
imaging disease states of the mammalian body where abnormal apoptosis is involved.
The imaging agents comprise an 1 F-radiolabelled lantibiotic peptide.The invention provides radiotracers which form reproducibly, in high radiochemical
purity (RCP). The present inventors have also established that attachment of the
radiolabel complex at the N-terminus (Cysa residue) of the lantibiotic peptide of
Formula II herein is strongly preferred, since attachment at even the amino acid
adjacent to the N-terminus (Xaa of Formula II) has a deleterious effect on binding to
phosphatidylethanolamine. This effect was not recognized previously in the prior art,
and hence the degree of impact on binding affinity is believed novel.
The 1 F-labelled imaging agents of the present invention are suitable for PET
(Positron Emission Tomography), which has the advantage over the imaging agents of
the prior art of more facile quantitation of the image.
Detailed Description of the Invention.
In a first aspect, the present invention provides an imaging agent which comprises a
compound of Formula I :
(I)
wherein:
LBP is a lantibiotic peptide of Formula II:
'-Xaa-Gln-Serb-Cys -Serd-Phe-Gly-Pro-Phe-Thr -Phe-Val-Cys
(HO-Asp)-Gly-Asn-Thr a-Lysd
(Π)
Xaa is Arg or Lys;
Cysa-Thra, Serb-Cysb and Cys -Thr are covalently linked via thioether
bonds;
Serd-Lysd are covalently linked via a lysinoalanine bond;
HO-Asp is β-hydroxyaspartic acid;
Z - ) - is attached to Cysa and optionally also to Xaa of LBP when Xaa is
Lys, wherein Z1 is either 1 F or 1 F coordinated to the metal of
complex;
Z2 is attached to the C-terminus of LBP and is OH or OB ,
where B is a biocompatible cation;L is a synthetic linker group of formula -(A)
m- wherein each A is
independently -CR2- , -CR=CR- , -C≡C- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO R- , -CR=N-0-, - 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, -Ar-, -NR-Ar-, -O-Ar-, -Ar-(CO)-, an amino acid, a
sugar or a monodisperse polyethyleneglycol (PEG) building block, wherein
each Ar is independently a C5-12
arylene group, or a C3-12
heteroarylene group,
and wherein each R is independently chosen from H, C1-
alkyl, C2-4
alkenyl,
C2-4
alkynyl, C1-
alkoxyalkyl or C1-
hydroxyalkyl;
m is an integer of value 1 to 20;
n is an integer of value 0 or 1.
The imaging agents of the present invention are 1 F-labelled lantibiotic peptides. By
the term "1 F-radiolabelled" or "1 F-labelled" is meant that the lantibiotic peptide has
covalently conjugated thereto the radioisotope 1 F. The 1 F is suitably attached via a
C-F fluoroalkyl or fluoroaryl bond, since such bonds are relatively stable in vivo, and
hence confer resistance to metabolic cleavage of the 1 F radiolabel from the peptide.
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. 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 imaging agents of the first aspect are particularly suitable for
imaging apoptosis and other forms of cell death, as is described in the sixth aspect
(below).
The term "in vivo imaging" as used herein refers to those techniques that non-
invasively produce images of all or part of an internal aspect of a mammalian subject.
A preferred imaging technique of the present invention is positron emission
tomography (PET).By the term "metal complex" is meant a coordination complex of a non-radioactive
metal. Preferred such complexes comprise a chelating agent. Suitable non
radioactive metals of the invention include aluminium, gallium or indium.
By the term "amino acid" is meant an L - or D- amino acid, amino acid analogue (eg.
naphthylalanine) 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 "monodisperse polyethyleneglycol (PEG) building block" is meant PEG
biomodifiers of Formula IA or IB:
(IA)
17-amino-5-oxo-6-aza-3, 9, 12, 15-tetraoxaheptadecanoic acid of Formula IA
wherein q is an integer from 1 to 15 and 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 and q are as defined for Formula
IA and
In Formula IB, p is preferably 1 or 2, and q is preferably 1 to 12.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).
The term "lantibiotic peptide" refers to a peptide containing at least one lanthionine
bond. "Lanthionine" has its conventional meaning, and refers to the sulfide analogue
of cystine, having the chemical structure shown:
Lanthionine
By the term "covalently linked via thioether bonds" is meant that the thiol functional
group of the relevant Cys residue is linked as a thioether bond to the Ser or Thr
residue shown via dehydration of the hydroxyl functional group of the Ser or Thr
residue, to give lanthionine or methyllanthionine linkages. Such linkages are
described by Willey et al [Ann.Rev.Microbiol, 61, 477-501 (2007)].
By the term "lysinoalanine bond" is meant that the epsilon amine group of the Lys
residue is linked as an amine bond to the Ser residue shown via dehydration of the
hydroxyl functional group of the Ser giving a -(CH 2)-NH-(CH2 )4- linkage joining the
two ζ -carbon atoms of the amino acid residues.
When Z1 is attached to Cysa, it is attached to the N-terminus of the LBP peptide.
When Z1 is also attached to Xaa, that means that Xaa is Lys, and Z1 is attached to the
epsilon amino group of the Lys residue.
The Z2 group substitutes the carbonyl group of the last amino acid residue of the LBP
- i.e. the carboxy terminus. Thus, when Z2 is OH, the carboxy terminus of the LBP
terminates in the free C0 2H group of the last amino acid residue, and when Z2 is OB
that terminal carboxy group is ionised as a C0 2B group.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.
Preferred embodiments.
In the imaging agent of the first aspect, Z1 is preferably attached only to Cysa of LBP.
When Xaa is Arg, that means that Z1 is attached to the LBP N-terminus, at the free
amino group of the Cysa residue. When Xaa is Lys, that means that steps are taken to
either:
(i) selectively functionalise the LBP peptide at the Cysa residue in
preference to the epsilon amine group of the Xaa residue; or
(ii) a composition comprising LBP functionalized with Z1 either at Cysa or
at Xaa is prepared, then the Xaa-functionalised species is removed.
In the imaging agent of the first aspect, Xaa is preferably Arg. Z2 is preferably OH or
OB .
In Formula I, n is preferably 1, i.e. the linker group (L) is present. When Z1 is 1 F,
preferred radiofluorinated substituents 1 F-(L) - are of Formula X, wherein -(L) - is
chosen to be-X1-(A)
x- :
1 F-X 1-(A) - (X)
where: x is an integer of value 0 to 5;
X1 is chosen from -Ar-, -Ar- -, -Ar-O-, -Ar-(CO)- or -Si(R a)
2- ;
wherein A, Ar and R are as defined for the L group (above) and each Ra is
independently C1- alkyl.
The Ar group of Ar 1 is preferably a Ci-6 aryl group, wherein the 1 F radiolabel is
covalently bonded to said aryl group. Ar 1 preferably comprises a phenyl ring or a
heterocyclic ring chosen from a triazole, isoxazole or pyridine ring.When X1 is -Si(R a)
2-, Ra can be linear or branched or combinations thereof. Ra is
preferably branched, and is preferably -C(CH 3)
3. More preferably, both Ra groups are
-C(CH 3)
3
In one embodiment, most preferred substituents of Formula X arise from either N-
acylation of the N^-amino group of the Cys residue or the N^amino group of Lys in
LBP with a fluonnated active ester, or condensation of an amino-oxy derivative of the
Cys or Lys amine residue with a radiofluorinated benzaldehyde, and comprise the
following structural elements:
where n = 1 to 6 .
In the above reaction scheme, n is preferably 1 to 3 .In another embodiment, most preferred substituents of Formula X comprise
organosilicon derivatives having 1 F-Si bonds:
When Z1 is 1 F coordinated to the metal of a metal complex, a preferred metal is
aluminium. The aluminium is preferably a metal complex of an aminocarboxylate
ligand. The term "aminocarboxylate ligand" has its conventional meaning, and refers
to a chelating agent where the donor atoms are a mixture of amine (N) donors and
carboxylic acid (O) donors. Such chelators may be open chain (e.g. EDTA, DTPA or
HBED), or macrocyclic (eg. DOTA or NOTA). Suitable such chelators include
DOTA, HBED and NOTA, which are well known in the art. A preferred such
chelator for aluminium isNOTA.
Preferably, the imaging agent is provided in sterile form, i.e. in a form suitable for
mammalian administration as is described in the fourth aspect (below).
The imaging agents of the first aspect can be obtained as described in the third aspect
(below).
a second aspect, the present invention provides a precursor of Formula III:
Z -(L) -[LBP]-Z2
(III)
wherein:
L, n, LBP and Z2 are as defined in the first aspect;
functional group which is chosen from:
(i) an amino-oxy group;
(ii) an azide group;
(iii) an alkyne group;(iv) a nitrile oxide;
(iv) an aluminium, indium or gallium metal complex of an
aminocarboxylate ligand.
Preferred aspects of L, n, LBP, Z2 and the metal complex in the second aspect are as
defined in the first aspect (above).
By the term "amino-oxy group" is meant the LBP peptide of Formula III having
covalently conjugated thereto an amino-oxy functional group. Such groups are of
formula - 0 - H2, preferably -CH 2O- H2 and have the advantage that the amine of
the amino-oxy group is more reactive than a Lys amine group in condensation
reactions with aldehydes to form oxime ethers. Such amino-oxy groups are suitably
attached at the Cys or Lys residue of the LBP.
The precursor of the second aspect is non-radioactive. Preferably, the precursor is
provided in sterile form, to facilitate the preparation of imaging agents in
pharmaceutical composition form - as is described in the fourth aspect (below).
In Formula III, Z3 is preferably attached to Cysa and optionally also Xaa of LBP.
Preferably, Z3 is attached only to Cysa of the LBP.
Amino-oxy functionalised LBP peptides can be prepared by the methods of Poethko
et al [J.Nucl.Med., 45, 892-902 (2004)], Schirrmacher et al [Bioconj.Chem., 18,
2085-2089 (2007)], Solbakken et al [Bioorg.Med.Chem.Lett, 16, 6190-6193 (2006)]
or Glaser et al [Bioconj. Chem., 19, 951-957 (2008)]. The amino-oxy group may
optionally be conjugated in two steps. First, the N-protected amino-oxy carboxylic
acid or N-protected amino-oxy activated ester is conjugated to the LBP peptide.
Second, the intermediate N-protected amino-oxy functionalised LBP peptide is
deprotected to give the desired product [see Solbakken and Glaser papers cited
above]. N-protected amino-oxy carboxylic acids such as Boc-NH-0-CH 2(C=0)OH
and Eei-N-0-CH 2(C=0)OH are commercially available, e.g. from Novabiochem and
IRIS. The term "protected" refers to the use of a protecting group. 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 becleaved 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); Eei (where Eei is
ethoxyethylidene); 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). 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 Eei, most preferably Eei.
Methods of functionalising peptides with azide groups are described by Nwe et al
[Cancer Biother.Radiopharm., 24(3), 289-302 (2009)]. Li et al provide the synthesis
of a compound of the type Ν3-Ι_ 0 2Η, where L1 is -(CH2 )4- and its use to conjugate
to amine-containing biomolecules [Bioconj.Chem., 18(6), 1987-1994 (2007)].
Hausner et al describe related methodology for Ν3-Ι_ 0 2Η, where L1 is -(CH2)
2-
[J.Med.Chem., 51(19), 5901-5904 (2008)]. De Graaf et al [Bioconj.Chem., 20(7),
1281-1295 (2009)] describe non-natural amino acids having azide side chains and
their site-specific incorporation in peptides or proteins for subsequent click
conjugation.
Methods of functionalising peptides with alkyne groups are described by Nwe et al
[Cancer Biother.Radiopharm., 24(3), 289-302 (2009)]. Smith et al provide the
synthesis of alkyne-functionalised isatin precursors, where the isatin compound is
specific for caspase-3 or caspase-7 [J.Med.Chem., 51(24), 8057-8067 (2008)]. De
Graaf et al [Bioconj.Chem., 20(7), 1281-1295 (2009)] describe non-natural amino
acids having alkyne side chains and their site-specific incorporation in peptides or
proteins for subsequent click conjugation.
The term "nitrile oxide" refers to a substituent of formula -C≡N+-0 . Click
cycloaddition with 1 F-labelled alkynes, under the conditions described above, leads
to isoxazole rings. The nitrile oxides can be obtained by the methods described by Ku
et al [Org.Lett, 3(26), 4185-4187 (2001)], and references therein. Thus, they aretypically generated in situ by treatment of an alpha-halo aldoxime with an organic
base such as triethylamine. A preferred method of generation, as well as conditions
for the subsequent click cyclisation to the desired isoxazole are described by Hansen
et al [J.Org.Chem., 70(19), 7761-7764 (2005)]. Hansen et al generate the desired
alpha-halo aldoxime in situ by reaction of the corresponding aldehyde with
chloramine-T trihydrate. See also K.B.G.Torsell "Nitrile Oxides, Nitrones and
Nitronates in Organic Synthesis" [VCH, New York (1988)].
Methods of preparing functionalised NOTA chelators, their conjugation with peptides
and the radiolabelling of the chelator conjugates with 1 F are described by McBride et
al [J.Nucl.Med., 51(3), 454-461 (2009); Bioconj.Chem., 21(7), 1331-1340 (2010)],
and Laverman et al [J.Nucl.Med., 51(3), 454-461 (2010)].
In a third aspect, the present invention provides a method of preparation of the
imaging agent of the first aspect, which comprises reaction of either the precursor of
the second aspect or the LBP peptide as described in the first aspect, with a supply of
1 F in suitable chemical form, in a suitable solvent.
Preferred aspects of the precursor and the LBP peptide in the third aspect are each as
described in the first and second aspects of the present invention (above).
The "suitable solvent" is typically aqueous in nature, and is preferably a
biocompatible carrier solvent as defined in the fourth aspect (below).
The "supply of 1 F in suitable chemical form" is chosen depending on the functional
group of the precursor or LBP peptide. When an amine group of a Lys residue or the
amino group of Cysa of the LBP peptide is used, then the chemical form of the 1 F is
suitably an active ester or an 1 F-labelled carboxylic acid in the presence of an
activating agent. By the term "activating agent" is meant a reagent used to facilitate
coupling between an amine and a carboxylic acid to generate an amide. Suitable such
activating agents are known in the art and include carbodiimides such as EDC [N-(3-
dimethylaminopropy -V-ethylcarbodiimide and ^iV-dialkylcarbodiimides such as
dicyclohexylcarbodiimide or diisopropylcarbodiimide; and triazoles such as HBTU[O-(benzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate], HATU
[0-(7-azabenzotriazol- 1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate], and
PyBOP [benzotriazol- 1-yloxy)tripyrrolidinophosphonium hexafluorophosphate], .
Such activating agents are commercially available. Further details are given in
"March's Advanced Organic Chemistry", 5th Edition, pages 508-510, Wiley
Interscience (2001). A preferred such activating agent is EDC.
1 F-labelled activated esters, such as [1 F]SFB can be prepared by the method of
Glaser et al, and references therein [J.Lab.Comp.Radiopharm., 52, 327-330 (2009)],
or the auto 07)]:
-SFB 1
F-Py-TFP
Olberg et al [J.Med.Chem., 53(4), 1732-1740 (2010)] have reported that F-Py-TFP
(the tetrafluorophenyl ester of fluoronicotinic acid), has advantages over 1 F-SFB for
1 F-labelling of peptides.
1 F-labelled carboxylic acids can be obtained by the method of Marik et al cited
above.
When the precursor comprises an amino-oxy group, the suitable chemical form is an
1 F-fluorinated aldehyde, preferably 1 F-fluorobenzaldehyde or -(di-t r t-butyl- 1 F-
fluorosilyl)benzaldehyde (1 F-SiFA-A), more preferably 1 F-fluorobenzaldehyde.
1 F-labelled aliphatic aldehydes of formula 1 F(CH2)
20[CH 2CH20 ] CH2CHO, where
q is 3, can be obtained by the method of Glaser et al [Bioconj.Chem., 19(4), 951-957
(2008)].
1 F-fluorobenzaldehyde can be obtained by the method of Glaser et al
[J.Lab.Comp.Radiopharm., 52, 327-330 (2009)]. The precursor to 1 F-
fluorobenzaldehyde, i.e. Me3N+-C6H4-CHO. CF3SO3 is obtained by the method of
Haka et al [J.Lab.Comp.Radiopharm., 27, 823-833 (1989)].1 F-SiFA-A, i.e.
1 F-Si(Bu )
2-C6H4-CHO can be obtained by the method of
Schirrmacher et al [Ang.Chem.Int.Ed.Engl., 45(36), 6047-6050 (2006);
Bioconj.Chem., 18(6), 2085-2089 (2007) and Bioconj.Chem., 20(2), 317-321 (2009)].
Schirrmacher et al also disclose methods of 1 F-radiolabelling of amino-oxy
functionalised peptides precursors using 1 F-SiFA-A.
When the precursor comprises an azide-functionalised LBP peptide, the suitable
chemical form is an 1 F-labelled terminal alkyne. Such radiofluorinated alkynes can
be obtained by the method of Kim et al [Appl.Rad.Isotop., 68(2), 329-333 (2010)], or
Marik et al [Tet.Lett, 47, 6681-6684 (2006)].
When the precursor comprises an alkyne-functionalised LBP peptide, the suitable
chemical form is an 1 F-labelled terminal azide. A preferred such compound is 1 F-
fluoroethyl azide as described by Gaeta et al [Bioorg.Med.Chem.Lett., 20(15), 4649-
4652 (2010)] and Glaser et al [Bioconj.Chem., 18(3), 989-993 (2007)].
When the precursor comprises an alkyne-functionalised or azide-functionalised LBP
peptide, the radiofluorination reaction involves click chemistry. A suitable solvent for
such click reactions is, for example acetonitrile, a C1-
alkylalcohol,
dimethylformamide, tetrahydrofuran, or dimethylsulfoxide, or aqueous mixtures of
any thereof, or water. Aqueous buffers can be used in the pH range of 4-8, more
preferably 5-7. The reaction temperature is preferably 5 to 100°C, more preferably at
75 to 85°C, most preferably at ambient temperature (typically 15-37 °C). The click
cycloaddition may optionally be carried out in the presence of an organic base, as is
described by Meldal and Tornoe [Chem. Rev. 08 (2008) 2952, Table 1 (2008)].
The click reactions are carried out in the presence of a click cycloaddition catalyst.
By the term "click cycloaddition catalyst" is meant a catalyst known to catalyse the
click (alkyne plus azide) or click (alkyne plus isonitrile oxide) cycloaddition reaction,
giving triazole and isoxazole rings respectively. Suitable such catalysts are known in
the art for use in click cycloaddition reactions. Preferred such catalysts include Cu(I),
and are described below. Further details of suitable catalysts are described by Wu andFokin [Aldrichim.Acta, 40(1), 7-17 (2007)] and Meldal and Tornoe [Chem. Rev., 108,
2952-3015 (2008)].
A preferred click cycloaddition catalyst comprises Cu(I). The Cu(I) catalyst is present
in an amount sufficient for the reaction to progress, typically either in a catalytic
amount or in excess, such as 0.02 to 1.5 molar equivalents relative to the azide or
isonitrile oxide reactant. Suitable Cu(I) catalysts include Cu(I) salts such as Cul or
[Cu(NCCH3)
4][PF6], but advantageously Cu(II) salts such as copper (II) sulphate may
be used in the presence of a reducing agent to generate Cu(I) in situ. Suitable
reducing agents include: ascorbic acid or a salt thereof for example sodium ascorbate,
hydroquinone, metallic copper, glutathione, cysteine, Fe2+, or Co2+. Cu(I) is also
intrinsically present on the surface of elemental copper particles, thus elemental
copper, for example in the form of powder or granules may also be used as catalyst.
Elemental copper, with a controlled particle size is a preferred source of the Cu(I)
catalyst. A more preferred such catalyst is elemental copper as copper powder,
having a particle size in the range 0.001 to 1mm, preferably 0.1 mm to 0.7 mm, more
preferably around 0.4 mm. Alternatively, coiled copper wire can be used with a
diameter in the range of 0.01 to 1.0 mm, preferably 0.05 to 0.5 mm, and more
preferably with a diameter of 0.1 mm. The Cu(I) catalyst may optionally be used in
the presence of bathophenanthroline, which is used to stabilise Cu(I) in click
chemistry.
Further details of 1 F-labelling of peptides using click, active ester and metal complex
methodology are provided by Olberg et al [J.Med.Chem., 53(4), 1732-1740 (2010)
and Curr.Top.Med.Chem., 10(16), 1669-1679 (2010)].
Certain LBP peptides are commercially available. Thus, cinnamycin and duramycin
are available from Sigma-Aldrich. Duramycin is produced by the strain: D3168
Duramycin from Streptoverticillium cinnamoneus. Cinnamycin can be biochemically
produced by several strains, eg. from Streptomyces cinnamoneus or from
Streptoverticillium griseoverticillatum. See the review by C. Chatterjee et al [Chem.
Rev., 105, 633-683 (2005)].Other peptides can be obtained by solid phase peptide synthesis as described in P.
Lloyd-Williams, F. Albericio and E. Girald; Chemical Approaches to the Synthesis o f
Peptides and Proteins, CRC Press, 1997.
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 described in the first
aspect of the present invention (above).
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 4.0 to
10.5). 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 (e.g. blood). Such compositions also contain only biologically compatible
excipients, and are preferably isotonic.
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 (e.g. salts of
plasma cations with biocompatible counterions), sugars (e.g. glucose or sucrose),
sugar alcohols (e.g. sorbitol or mannitol), glycols (e.g. glycerol), or other non-ionic
polyol materials (e.g. 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 50 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 "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. The radioprotectants of the present invention are suitably chosen from:
ascorbic acid, ^ara-aminobenzoic acid (i.e. 4-aminobenzoic acid), gentisic acid (i.e.
2,5-dihydroxybenzoic acid) and salts thereof with a biocompatible cation as described
above. 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; polyethyleneglycol (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.
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) 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.The radiopharmaceutical compositions of the fourth aspect may be prepared under
aseptic manufacture (i.e. clean room) conditions to give the desired sterile, non-
pyrogenic product. It is preferred that the key components, especially the associated
reagents plus those parts of the apparatus which come into contact with the imaging
agent (eg. vials) are sterile. The components and reagents can be sterilised by
methods known in the art, including: sterile filtration, terminal sterilisation using e.g.
gamma-irradiation, autoclaving, dry heat or chemical treatment (e.g. with ethylene
oxide). It is preferred to sterilise some components in advance, so that the minimum
number of manipulations needs to be carried out. As a precaution, however, it is
preferred to include at least a sterile filtration step as the final step in the preparation
of the pharmaceutical composition.
The radiopharmaceutical compositions of the present invention may be prepared by
various methods:
(i) aseptic manufacture techniques in which the 1 F-radiolabelling step is
carried out in a clean room environment;
(ii) terminal sterilisation, in which the 1 F-radiolabelling 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 a suitable precursor of Formula III and optional excipients is
reacted with a suitable supply of 1 F;
(iv) aseptic manufacture techniques in which the 1 F-radiolabelling step is
carried out using an automated synthesizer apparatus.
Method (iv) is preferred. Kits for use in this method are described in the fifth
embodiment (below).
By the term "automated synthesizer" is meant an automated module based on the
principle of unit operations as described by Satyamurthy et al [Clin.Positr.Imag., 2(5),
233-253 (1999)]. The term 'unit operations' means that complex processes are
reduced to a series of simple operations or reactions, which can be applied to a range
of materials. Such automated synthesizers are preferred for the method of the present
invention especially when a radiopharmaceutical composition is desired. They arecommercially available from a range of suppliers [Satyamurthy et al, above],
including: GE Healthcare; CTI Inc; Ion Beam Applications S.A. (Chemin du
Cyclotron 3, B-1348 Louvain-La-Neuve, Belgium); Raytest (Germany) and Bioscan
(USA).
Commercial automated synthesizers also provide suitable containers for the liquid
radioactive waste generated as a result of the radiopharmaceutical preparation.
Automated synthesizers are not typically provided with radiation shielding, since they
are designed to be employed in a suitably configured radioactive work cell. The
radioactive work cell provides suitable radiation shielding to protect the operator from
potential radiation dose, as well as ventilation to remove chemical and/or radioactive
vapours. The automated synthesizer preferably comprises a cassette. By the term
"cassette" is meant a piece of apparatus designed to fit removably and
interchangeably onto an automated synthesizer apparatus (as defined above), in such a
way that mechanical movement of moving parts of the synthesizer controls the
operation of the cassette from outside the cassette, i.e. externally. Suitable cassettes
comprise a linear array of valves, each linked to a port where reagents or vials can be
attached, by either needle puncture of an inverted septum-sealed vial, or by gas-tight,
marrying joints. Each valve has a male-female joint which interfaces with a
corresponding moving arm of the automated synthesizer. External rotation of the arm
thus controls the opening or closing of the valve when the cassette is attached to the
automated synthesizer. Additional moving parts of the automated synthesizer are
designed to clip onto syringe plunger tips, and thus raise or depress syringe barrels.
The cassette is versatile, typically having several positions where reagents can be
attached, and several suitable for attachment of syringe vials of reagents or
chromatography cartridges (eg. solid phase extraction or SPE). The cassette always
comprises a reaction vessel. Such reaction vessels are preferably 1 to 10 cm3, most
preferably 2 to 5 cm3 in volume and are configured such that 3 or more ports of the
cassette are connected thereto, to permit transfer of reagents or solvents from various
ports on the cassette. Preferably the cassette has 15 to 40 valves in a linear array,
most preferably 20 to 30, with 25 being especially preferred. The valves of the
cassette are preferably each identical, and most preferably are 3-way valves. The
cassettes are designed to be suitable for radiopharmaceutical manufacture and aretherefore manufactured from materials which are of pharmaceutical grade and ideally
also are resistant to radiolysis.
Preferred automated synthesizers of the present invention comprise a disposable or
single use cassette which comprises all the reagents, reaction vessels and apparatus
necessary to carry out the preparation of a given batch of radiofluorinated
radiopharmaceutical. The cassette means that the automated synthesizer has the
flexibility to be capable of making a variety of different radiopharmaceuticals with
minimal risk of cross-contamination, by simply changing the cassette. The cassette
approach also has the advantages of: simplified set-up hence reduced risk of operator
error; improved GMP (Good Manufacturing Practice) compliance; multi-tracer
capability; rapid change between production runs; pre-run automated diagnostic
checking of the cassette and reagents; automated barcode cross-check of chemical
reagents vs the synthesis to be carried out; reagent traceability; single-use and hence
no risk of cross-contamination, tamper and abuse resistance.
Included in this aspect of the invention, is the use of an automated synthesizer
apparatus to prepare the radiopharmaceutical composition of the second aspect.
In a fifth aspect, the present invention provides a kit for the preparation of the
radiopharmaceutical composition of the fourth aspect, which comprises the precursor
of the second aspect or the LBP peptide as defined in the first aspect in sterile, solid
form such that upon reconstitution with a sterile supply of 1 F in suitable chemical
form, dissolution occurs to give the desired radiopharmaceutical composition.
The term "suitable chemical form" is as defined in the third aspect (above).
Preferred aspects of the precursor 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 1 F to give a solution suitable for human
administration with the minimum of manipulation.
The sterile, solid form is preferably a lyophilised solid.
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.
Included in this aspect of the invention, is the use of a cassette which comprises the
kit of the fifth aspect in conjunction with an automated synthesizer apparatus to
prepare the radiopharmaceutical composition of the second aspect.
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 composition of the fourth aspect has
distributed using PET, 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).
The method of the sixth aspect is preferably carried out where the part of the body is
disease state where abnormal apoptosis is involved. By the term "abnormal
apoptosis" is meant dysregulation of the programmed cell death process. Such
dysregulation has been implicated in a number of disease states, including those
associated with the inhibition of apoptosis, such as cancer and autoimmune disorders,
and those associated with hyperactive apoptosis, including neurodegenerative
diseases, haematologic diseases, AIDS, ischaemia and allograft rejection.There is also emerging evidence that apoptosis contributes to the instability of the
atherosclerotic lesions. Plaques vulnerable to rupture typically have a large necrotic
core and an attenuated fibrous cap, which is significantly infiltrated by macrophages
and lymphocytes. Although the consequences of cell death within the advance lesion
are not precisely defined, morphological data suggest that apoptosis of macrophages
contributes substantially to the size of the necrotic core, whereas apoptosis of smooth
muscle cells (SMCs) results in thinning of the fibrous cap. Extensive apoptosis of
macrophages is believed to occur at sites of plaque rupture, and possibly contributes
to the process of rupture. Therefore, detection of apoptosis may help identify
atherosclerotic lesions prone to rupture.
The visualization and quantitation of apoptosis is therefore useful in the diagnosis of
such apoptosis-related pathophysiology.
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, said imaging
being effected before and after treatment with said drug, and optionally also during
treatment with said drug. Therapeutic treatments for these diseases aim to restore
balanced apoptosis, either by stimulating or inhibiting the PCD process as appropriate.
Of particular interest is early monitoring of the efficacy of cancer therapy to ensure
that malignant growth is controlled before the condition becomes terminal.
In a seventh aspect, the present invention provides the use of the imaging agent of the
first aspect, the 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 use of the seventh aspect is preferably where the diagnosis of the human or
animal body is of a disease state where abnormal apoptosis is involved. Such
"abnormal apoptosis" is as described in the sixth aspect (above).
The invention is illustrated by the non-limiting Examples detailed below. Example 1
and Example 2 provide the syntheses of Precursor 1A and Precursor IB respectively,amino-oxy functionalised LBP peptides of the invention protected with two different
amino-protecting groups. Example 3 provides the synthesis of Precursor 2, an amino-
oxy functionalised LBP peptides of the invention. Example 4 provides the synthesis
of Compound 1, a non-radioactive fluorinated compound of the invention where the
fluorine isotope is 1 F. Compound 1 is useful for determining biological binding
properties of the 1 F counterpart (Compound 1A). Example 5 provides a method of
1 F-labelling Precursor 1 using 1 F-benzaldehyde, to give an 1 F-labelled compound of
the invention (Compound 1A). Example 6 provides binding affinity data for
phosphatidylethanolamine and demonstrates that the generation of Compound 1 has
no significant effect on the binding affinity. Compound 1A was assessed by
biodistribution in the EL4 mouse lymphoma xenograft model. The results from this
work is provided in Example 7 .
Abbreviations.
Conventional single letter or 3-letter amino acid abbreviations are used.
Ac: Acetyl.
ACN: Acetonitrile.
Boc: t r t-Butyloxycarbonyl.
DIPEA: Ν,Ν ΏD-diisopropylethylamine.
DMSO: Dimethylsulfoxide.
EOS: End of synthesis.
Fmoc : 9-Fluorenylmethoxy carbonyl .
HATU: ^-(T-Azabenzotriazol-l-y - N^^-tetramethyluronium hexafluorophosphate.
FIPLC: High performance liquid chromatography.
ΜΡ : l-Methyl-2-pyrrolidinone.
PBS: Phosphate-buffered saline.
PyBOP: Benzotriazol-l-yl-oxytripyrrolidinophosphonium hexafluorophosphate.
RAC: radioactive concentration.
RCP: Radiochemical purity.
tBu: rt-Butyl.
TFA: Trifluoroacetic acid.
TFP: Tetrafluorophenyl.
TR: retention time.Table 1: Compounds of the Invention.
Formula II (with bridges as specified in the first aspect):
Cysa-Xaa-Gln-Serb-Cys -Serd-Phe-Gly-Pro-Phe-Thr -Phe-Val-Cysb-
(HO-Asp)-Gly-Asn-Thr a-LysdExample 1: Synthesis of (¾oc-aminooxy)acetyl-Duramvcin (Precursor 1A).
Duramycin (Sigma-Aldrich; 8.0 mg, 4.0 µηιοΐ), (Boc-aminooxy)acetic acid TFP ester
(Invitrogen; 1.3 mg, 3.8 µιηοΐ) and DIPEA (2.1 µΐ 12.5 µιηοΐ) were dissolved in
MP ( 1 mL). The reaction mixture was shaken for 30 min. The mixture was then
diluted with water/0.1% TFA (6 mL) and the product purified using preparative
HPLC.
Purification was by preparative HPLC (Beckman System Gold chromatography
system using the following conditions: solvent A = H2O/0.1% TFA and solvent B =
ACN/0.1% TFA, gradient: 20-50% B over 40 min; flow rate: 10 mL/min; column:
Phenomenex Luna 5 µιη C18 (2) 250 x 21.2 mm; detection: UV 214 nm), afforded 3.8
mg pure Precursor 1A (yield 44%). The purified material was analysed by analytical
LC-MS (gradient: 20-70% B over 5 min, t
R : 1.93 min, found m/z: 1093.7, expected
MH2
2+: 1093.5).
Separation of the Precursor 1 regioisomers could not be achieved under the above
analytical or preparative HPLC conditions. In each case the two regioisomers eluted
as a single peak.
Separation of the Precursor 1A regioisomers can, however, be achieved by analytical
HPLC under more gentle eluting conditions: LC-MS gradient 25-35% B over 5 min,
t
R : 2.0 min, found m/z: 1093.7 and t
R : 2.3 min, found m/z 1093.7, expected MH2
2+:
1093.5. Similar conditions can be used by preparative HPLC to isolate each
regioisomer.xample 2 : Synthesis of (Eei-aminooxy)acetyl-Duramycin (Precursor IB).
Duramycin (Sigma-Aldrich; 50 mg, 25 µιηοΐ), (Eei-aminooxy)acetic acid NHS ester (
Iris Biotech., 5.1 mg, 20 µιηοΐ) and DIPEA (17 , 100 µιηοΐ) were dissolved in
NMP ( 1 mL). The reaction mixture was shaken for 45 min. The mixture was then
diluted with water/0.1% acetic acid (8 mL) and the product purified using preparative
HPLC
Purification by preparative HPLC (as for Example 1with gradient 14-45% B over 40
min where A = water/0.1% acetic acid and B = ACN) afforded 14 mg pure Precursor
IB (yield 26%). The purified material was analysed by LC-MS (gradient: 20-50% B
over 5 min, t : 2.5 and 2.7 min, found m/z 1078.8, expected MH2
2+: 1078.5).
Chromatographic resolution of the (Eei-aminooxy)acetyl-Duramycin regioisomers
could be achieved on analytical HPLC using 0 . 1%> TFA. However, the Eei protecting
group is labile in 0 . 1%> TFA so preparative separation was not feasible. The
regioisomers were not resolved using 0.1%> acetic acid.
Example 3 : Synthesis of Aminooxyacetyl-Duramycin (Precursor 2).
Precursor IB (14 mg) was treated with 2.5% TFA/water (2.8 mL) under argon for 40
min. The reaction mixture was diluted with water (3 1 mL) and the product
lyophilized (frozen under argon using isopropanol/dry-ice) affording 18 mg Precursor2 . The lyophilized product was analysed by LC-MS (gradient: 20-50% B over 5 min,
t
R:2.5 and 2.1 min, found m/z: 1043.8, expected MH2
2+: 1043.5).
Chromatographic resolution of the Precursor 2 regioisomers could be achieved on
analytical HPLC using 0 .1% TFA. However, due to the high reactivity of the free
aminooxy group towards traces of ketones and aldehydes in the solvent and the
atmosphere no attempt was made to separate the regioisomers at this stage.
Example 4: Synthesis of N-(4-Fluorobenzylidene)-aminooxyacetyl-Duramycin
(Compound 1).
Precursor 1A (Example 1; 1.0 mg, 0.46 µιηοΐ) was treated with TFA ( 1 mL) for 30
min. The TFA was removed in vacuo and the residue redissolved in 40% ACN/water
( 1 mL). 4-Fluorobenzaldehyde (1.0 µΐ , 9.2 µιηοΐ) was added and the reaction mixture
shaken for 30 min. The reaction mixture was then diluted with 20% ACN/water/0. 1%
TFA (6 mL) and the product purified by preparative HPLC.
Purification by preparative HPLC (as for Example 1 with gradient: 20-50% B over 40
min) afforded 0.6 mg pure Compound 1 (yield 60%>). The purified material was
analysed by analytical LC-MS (gradient: 20-70%> B over 5 min, ¾: 2.09 min, found
m/z: 1096.5, expected MH2
2+: 1096.5). Separation of the Compound 1 regioisomers
could not be achieved using either analytical or preparative HPLC. In each case the
two regioisomers eluted as a single peak.Example 5: Radiosynthesis of Compound 1A from Precursor 2.
Compound 1A is produced in a two-step procedure using an automated
synthesizer and cassette (FASTlab™, GE Healthcare).
Step (a) synthesis and purification of 1 F-benzaldehyde .
[1 F]fiuoride was produced using a GEMS PETtrace cyclotron with a silver target via
the [1 0 ](p,n) [1 F] nuclear reaction. Total target volumes of 1.5 - 3.5 mL were used.
The radiofluoride was trapped on a Waters QMA cartridge (pre-conditioned with
carbonate), and the fluoride is eluted with a solution of KryptofiX2 .
2.
2.
(4 mg, 10.7 µΜ)
and potassium carbonate (0.56 mg, 4.1 µΜ) in water (80 µ ) and acetonitrile (320
µ .). Nitrogen was used to drive the solution off the QMA cartridge to the reaction
vessel. The [1 F]fluoride was dried for 9 minutes at 120°C under a steady stream of
nitrogen and vacuum. Trimethylammonium benzaldehyde triflate, [Haka et al,
J.Lab.Comp.Radiopharm., 27, 823-833 (1989)] (3.3 mg, 10.5 µΜ), in
dimethylsulfoxide (1.1 mL) was added to the dried [1 F]fluoride, and the mixture
heated at 105°C for 7 minutes to produce 4-[ 1 F]fluorobenzaldehyde.
The crude labelling mixture was then diluted with ammonium hydroxide solution and
loaded onto an MCX+ SPE cartridge (pre-conditioned with water as part of the
FASTlab sequence). The cartridge was washed with water, dried with nitrogen gas
before elution of 4-[ 1 F]fluorobenzaldehyde back to the reaction vessel in ethanol ( 1
mL). 4-7% (decay corrected) of [1 F]fluorobenzaldehyde remained trapped on the
cartridge.
Step (b): Aldehyde Condensation with Amino-oxy derivative (Precursor 2).
Precursor 2 (5 mg) was transferred to the FASTlab reaction vessel prior to elution of
4-[ 1 F]fluorobenzaldehyde from the MCX+ cartridge. The mixture was then heated at
60°C for 5 minutes. The crude reaction material was then diluted with water and
loaded onto a tC2 SPE cartridge. This was then dried with nitrogen and vacuum,
washed with an ethanolic solution and dried again. Compound 1A was then eluted
into a collection vial with ethanol followed by water (6 mL total). The EOS yield was
16-34% (non-decay corrected). Analytical FIPLC confirmed that Compound 1A was
prepared with an RCP of 97% and was stable for at least 180 min (RCP 94%, RAC
150 MBq/mL).HPLC Conditions
Column: Phenomenex, Jupiter 4u, Proteo 90A, 250 x 4.6 mm.
Gradient: 0 min 50%B
5 min 50% B
20 min 90% B
25 min 90% B
Flow rate: 1 mL/min
UV detection: 254 nm.
Mobile phase A: 50 mM ammonium acetate
Mobile phase B : methanol.
Compound 1A (TR) = 22.6 min.
Example 6: Affinity for Phosphatidylethanolamine.
A Biacore 3000 (GE Healthcare, Uppsala) was equipped with an L I chip. Liposomes
made of POPE/POPC (20% PE) were applied for the affinity study using the capture
technique recommended by the manufacturer. Each run consisted of activation of the
chip surface, immobilization of liposomes, binding of peptide and wash off of both
liposomes and peptide (regeneration). Similar applications can be found in Frostell-
Karlsson et al [Pharm. Sciences, V.94 (1), (2005)]. Thorough washing of needle,
tubing and liquid handling system with running buffer was performed after each
cycle.
BIACORE software: The BIACORE control software including all method
instructions was applied. A method with commands was also written in the
BIACORE Method Definition Language (MDL) to have full control over pre
programmed instructions. BIACORE evaluation software was applied for analysing
the sensorgrams.
Compound 1was found to be a good binder to phosphatidyl ethanolamine. The KD
for duramycin and Compound 1was both less than 100 nM. The results are given in
Table 2 :Table 2 :
Example 7: Tumour Uptake Studies..
Compound 1A was assessed by biodistribution in the EL4 mouse lymphoma
xenograft model. Briefly, following establishment of tumour growth in C57/B16
mice, the animals were treated with either:
(i) a saline/DMSO solution; or
(ii) with chemotherapy (67 mg/kg etoposide and 100 mg/kg
cyclophosphamide in 50% saline 50% DMSO).
Twenty four hours after therapy or vehicle treatment, the animals were assessed for
the biodistribution of Compound 1A. In addition, the tumours were extracted and
assessed for levels of apoptosis by measuring caspase activity (capase-Glo assay). An
increase of tumour retention of Compound 1A was observed which followed an
increase in tumour apoptosis.CLAIMS.
1. An imaging agent which comprises a compound of Formula I :
Z L LBP -Z2
(I)
wherein:
LBP is a lantibiotic peptide of Formula II:
Cysa-Xaa-Gln-Serb-Cys -Serd-Phe-Gly-Pro-Phe-Thr -Phe-Val-Cysb-
(HO-Asp)-Gly-Asn-Thr a-Lysd
(Π)
Xaa is Arg or Lys;
Cysa-Thra, Serb-Cysb and Cys -Thr are covalently linked via thioether
bonds;
Serd-Lysd are covalently linked via a lysinoalanine bond;
HO-Asp is β-hydroxyaspartic acid;
Z - ) - is attached to Cysa and optionally also Xaa of LBP, wherein Z1 is
either 1 F or 1 F coordinated to the metal of a metal complex;
Z2 is attached to the C-terminus of LBP and is OH or OB ,
where B is a biocompatible cation; and
L is a synthetic linker group of formula -(A)
m- wherein each A is
independently -CR2- , -CR=CR- , -C≡C- , -CR2C0 2- , -C0 2CR2- , - RCO- ,
-CO R- , -CR=N-0-, - 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, -Ar-, -NR-Ar-, -O-Ar-, -Ar-(CO)-, an amino acid, a
sugar or a monodisperse polyethyleneglycol (PEG) building block, wherein
each Ar is independently a C5-12
arylene group, or a C3-12
heteroarylene group;
each R is independently chosen from H, C1-
alkyl, C2-4
alkenyl, C2-4
alkynyl,
Ci
-4
alkoxyalkyl or C1-
hydroxyalkyl;
m is an integer of value 1 to 20;
n is an integer of value 0 or 1;
2 . The imaging agent of claim 1, where Z1 is attached only to Cysa of LBP.
3 . The imaging agent of claim 1 or claim 2, where Xaa is Arg.4 . The imaging agent of any one of claims 1 to 3, where Z - ) - comprises a
group of Formula X:
^F-X^A) - (X)
where: x is an integer of value 0 to 5;
X1 is chosen from -Ar-, -Ar-NR-, -Ar-O-, -Ar-(CO)- or -Si(R a)
2- ;
wherein A, Ar and R are as defined for the L group in claim 1, and each Ra is
independently C 1- alkyl.
5 . The imaging agent of claim 4, where Ar 1 comprises a phenyl ring or a
heterocyclic ring chosen from a triazole, isoxazole or pyridine ring.
6 . The imaging agent of any one of claims 1 to 3, where Z1 comprises an
aluminium complex of an aminocarboxylate ligand, wherein the 1 F radiolabel is
coordinated to said aluminium of said complex.
7 . A precursor of Formula III:
Z -(L) -[LBP]-Z2
(III)
wherein:
L, n, LBP and Z2 are as defined in any one of claims 1 to 3;
Z is a functional group which is chosen from:
(i) an amino-oxy group;
(ii) an azide group;
(iii) an alkyne group;
(iv) a nitrile oxide;
(v) an aluminium, indium or gallium metal complex of an
aminocarboxylate ligand.
8 . A method of preparation of the imaging agent of any one of claims 1 to 6,
which comprises reaction of either the precursor of claim 8 or the LBP peptide as
defined in any one of claims 1 to 3, with a supply of 1 F in suitable chemical form, in
a suitable solvent.9 . A radiopharmaceutical composition which comprises the imaging agent of any one
of claims 1 to 6, together with a biocompatible carrier, in a form suitable for
mammalian administration.
10. A kit for the preparation of the radiopharmaceutical composition of claim 9,
which comprises the precursor of claim 7 or the LBP peptide as defined in any one of
claims 1 to 3 in sterile, solid form such that upon reconstitution with a sterile supply
of 1 F in suitable chemical form as defined in claim 8, dissolution occurs to give the
desired radiopharmaceutical composition.
11. The kit of claim 10, where the sterile, solid form is a lyophilised solid.
12. 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 6, or the composition of claim 9 has distributed using PET, wherein said imaging
agent or composition has been previously administered to said body.
13. The method of claim 12, where said part of the body is a disease state where
abnormal apoptosis is involved.
14. The method of claim 12 or claim 13, which is carried out repeatedly to monitor
the effect of treatment of a human or animal body with a drug, said imaging being
effected before and after treatment with said drug, and optionally also during
treatment with said drug.
15. The use of the imaging agent of any one of claims 1 to 6, the composition of
claim 9, or the kit of claim 10 in a method of diagnosis of the human or animal body.
16. The use of claim 15, where the diagnosis is of a disease state where abnormal
apoptosis or other forms of cell death are involved.