RADIOLABELLED OCTREOTATE ANALOGUES AS PET TRACERS
Field of the Invention
The present invention describes a novel radiotracer(s) for Positron Emission
Tomography (PET) imaging; specifically imaging of neuroendocrine tumors including
gastroenteropancreatic neuorendocrine tumors. Specifically the present invention describes
novel [ F]Fluoroethyltriazol-[Tyr ]Octreotate analogues. Radiotracers of the present invention
can target somatostatin receptors found on the cell surface of gastroenteropancreatic
neuroendocrine tumors. The present invention also describes intermediate(s), precursor(s),
pharmaceutical composition(s), methods of making, and methods of use of the novel
radiotracer(s).
Description of the Related Art
PET is becoming increasingly important for the early detection of disease in oncology
and neurology. Radio-labelled peptides in particular are being investigated more frequently
for the detection of disease, the monitoring of treatment, and in peptide receptor radiotherapy
(PRRT)(Chen, X. Y., et al., European Journal of Nuclear Medicine and Molecular Imaging
2004, 3 1, (8), 1081-1089; Lei, M., et al., Current Medical Imaging Reviews 6, (1), 33-41).
The peptide [Tyr ]octreotate (TOCA) (Figure 1) has previously been labelled with
various radioisotopes for the purpose of imaging (Nikolopoulou, A., etal., Journal of Peptide
Science 2006, 12, 124-1 3 1 ; Li, W. P., et al., Bioconjugate Chemistry 2002, 13, 721 -728;
Reubi, J. C , et al., European Journal of Nuclear Medicine and Molecular Imaging 2000, 27,
(3) , 273-282) and PRRT of neuroendocrine tumours (Teunissen, J., etal., European Journal
of Nuclear Medicine and Molecular Imaging 2009, 36, (11), 1758-1766) . [Tyr3] Octreotate is a
somatostatin analogue that has a longer biological half life ( 1 .5-2 hours) than somatostatin
and retains receptor specificity (Weiner, R. E., et al., Seminars in Nuclear Medicine 2001 , 3 1,
(4) , 296-31 1) . It has been found that somatostatin receptors, of which there are 5 subtypes
(sstr 1-5), are over expressed on the surface of neuroendocrine tumours (Rufini, V., etal.,
Seminars in Nuclear Medicine 2006, 36, (3), 228-247). This over expression enables
selective targeting of tumours with a radiolabeled octreotate analogue. The first discovered
eight amino acid sequenced peptide to mimic somatostatin was octreotide. It was found that
the cyclic octapeptide contained the important sites for binding to the somatostatin receptor
and was initially used as an opiate antagonist (Maurer, R., etal., PNAS 1982, 79, 4815-4817)
for the treatment of painful conditions such as acute and chronic pancreatitis (Uhl, W., etal.,
Digestion, International Journal of Gastroenterology 1999, 60, 23-31 ). Comparing octreotide
to [Tyr ]octreotate, the latter has been shown to have a higher affinity for the somatostatin
receptors (Reubi, J. C , et al., European Journal of Nuclear Medicine and Molecular Imaging
2000, 27, (3), 273-282; Wild, R., etal., European Journal of Nuclear Medicine and Molecular
Imaging 2003, 30, (10), 1338-1347); it appears that substituting phenylalanine for tyrosine and
threoninol for threonine at the C-terminus increases affinity. Nonetheless, [ ln]-DTPAoctreotide
(Octreoscan™) is still the peptide of choice in the clinic and was approved by the
FDA as an imaging agent for somatostatin receptor positive neuroendocrine tumours in 1994
(Rufini, V., et al., Seminars in Nuclear Medicine 2006, 36, (3), 228-247).
Using prosthetic groups (i.e. small radiolabeled organic molecules which can then be
coupled to the main pharmacophore of interest) is the strategy generally employed when
labelling peptides or other macromolecules to overcome the limitations of F such as basicity
and poor reactivity (Okarvi, S. M., European Journal of Nuclear Medicine 2001 , 28, (7), 929-
38). The approaches used to date all vary in the number of steps involved, the overall reaction
time, isolated yield and method of isolation. Octreotide has previously been labelled with Fmodified
organic prosthetic groups. The initial strategy employed by Hostetler etal. (Journal
of Labelled Compounds & Radiopharmaceuticals 1999, 42, S720-S721) was to directly label
the N-terminus of octreotide with the activated ester of [ F]fluorobenzoic acid ([ F]FBA).
Subsequent biodistribution studies showed that the [ F]fluorobenzoyl-octreotide analogue
was too lipophilic and showed significant uptake in the liver. Octreotide has also been directly
labelled at the N-terminus with 2-[ F]fluoropropionate 4-nitrophenylester, which itself involves
three chemical modification steps to synthesize (Guhlke, S., etal., Applied Radiation and
Isotopes 1994, 45, (6), 715-727). The main drawback to this chemistry is the need to Bocprotect
the lysine side chain of octreotide during conjugation which requires removal in the
final step (Guhlke, S., etal., Nuclear Medicine and Biology 1994, 2 1, (6), 819-825).
Schottelius et al. {Clinical Cancer Research 2004, 10, ( 1 1), 3593-3606) wanted to
develop an F-labelled octreotate analogue with improved tumour uptake and better
pharmacokinetics compared to previous analogues. The authors chose to modify octreotate
with carbohydrate groups in order to reduce lipophilicity, to consequently reduce hepatic
elimination and conversely aid renal elimination. A glucose modified octreotate (Gluc-Lys-
TOCA) was developed and the lysine of the peptide labelled with 2-[ F]fluoropropionate 4-
nitrophenylester. The final product Gluc-Lys([ F]FP)-TOCA was evaluated in patients by
Meisetschlager et al. (Journal of Nuclear Medicine 2006, 47, 566-573) and was found to be
superior to [ ln]-DTPA-octreotide at detection of neuroendocrine tumours. However while
[ ln]-DTPA-octreotide showed improvements in tumour uptake, the lengthy synthesis time (3
hours) and low yields (20-30 %) made it a non-viable option for routine clinical use
(Meisetschlager, G., et al., Journal of Nuclear Medicine 2006, 47, 566-573). Schottelius et al.
{Clinical Cancer Research 2004, 10, ( 1 1), 3593-3606) also labelled two other carbohydrate
analogues, Cel-S-Dpr-[Tyr ]octreotate and Gluc-S-Dpr[Tyr ]octreotate with
[ F]fluorobenzaldehyde to give the oxime-derivatised radiotracers. The Cel-SDpr([
F]FBOA)-[Tyr ]octreotate showed improved tumour uptake compared to GlucLys([
F]FP)-TOCA and had a shorter synthesis time (50 min) with improved yields (65-85 %).
Gluc-S-Dpr[Tyr ]octreotate was labelled with both [ F]fluoropropionate ([ F]FP) and
[ F]fluorobenzaldehyde ([ F]FBOA). The Gluc-S-Dpr-([ F]FP) -Tyr ]octreotate labelled
analogue showed tumor uptake similar to Gluc-Lys([ F]FP)-TOCA but also had high tumour
to organ ratios (blood, liver and muscle). The Gluc-S-Dpr-([ F]FBOA)[Tyr ]octreotate showed
comparable tumour uptake to Cel-S-Dpr([ F]FBOA)-[Tyr ]octreotate, but had a high
tumour/muscle ratio.
The most recently published F-labelled octreotide analogue was [ F]-aluminum
fluoride - 1,4,7-triazacyclononane-1 ,4,7-triacetic acid octreotide ([ F]AIF-NOTA-Octreotide)
(Laverman, P., et al., Journal of Nuclear Medicine 5 1 , (3), 454-461). The advantage of using
the [ F]aluminium fluoride labelling strategy is that the fluorine-18 azeotropic drying step is
not required, meaning shorter overall reaction times. By HPLC, the product was observed as
two isomers, equating to approximately 50 % incorporation of [ F]AIF into the NOTA chelate,
the remainder was stated as non-chelated [ F]AIF. The authors commented that the two
isomers could be separated by HPLC. When re-analysed they saw re-equilibration to the two
isomers. The conformation of these two isomers has not been established to date.
Click chemistry has been utilised previously in fluorine-18 labelling of peptides (Li, Z.
B., etal., Bioconjugate Chemistry 2007', (18), 1987-1994; Ramenda, T., etal., Chemical
Communications 2009, 48, 7521-7523; Hausner etal. J. Med. Chem, 2008, 5901 ; Mamat et
al. Mini-Rev. Org. Chem. 2009, 6, 21). Since the reaction is efficient it can be applied to the
synthesis of radiolabeled tracers and ligands with short lived isotopes (half life F, 109.7 min)
for positron emission tomography (PET)( Glaser, M., and Arstad, E., Bioconjugate Chemistry
2007, 18, (3), 989-993; Glaser, M., etal., Journal of Labelled Compounds &
Radiopharmaceuticals 2009, 52, (9-10), 407-414).
Marik and Sutcliffe (Marik, J., etal., Tetrahedron Letters 2006, (47), 6681-6684) took
the approach of labelling terminal alkynes with fluorine-18 and adding the azide moiety to
various peptides. CuS0 4/Na-ascorbate was initially employed as a catalytic system but the
labelled peptide was only isolated in 10 % yield. Improvements were observed when CuS0 4
was replaced by Cul with addition of , -diisopropylethylamine (DIPEA). Sirion et al.
(Tetrahedron Letters 2007, 48, 3953-3957) found that using Cul gave traces of the 1,5-
substituted triazole by-product. The authors synthesized four mesylate precursors, two
acetylene and two azides all of which were labelled using [ F]TBAF, with fBuOH as solvent
(Kim, D. W., etal., Journal of the American Chemical Society 2006, 128, 16394-16397).
However there still exists a need in the art for radiolabeled octreotate analogues with
improved tumour uptake and pharmacokinetic parameters compared to previously labelled
analogues. There also exists a need for an efficient and effective method to prepare such
radiolabeled octreotate analogues. The present invention, as described below, answers these
needs.
Brief Description of the Drawings
Figure 1 describes the peptide [Tyr ]octreotate (TOCA), five alkyne (1a-5a) and
triazole (1b-5b) octreotate analogues, and the scrambled negative control alkyne (6a) and
triazole (6b).
Figure 1a depicts semi-preparative HPLC trace of the reaction mixture to synthesise
5b showing the by-products formed 1 1 and 12. Semi-preparative HPLC using a Luna C18
100 X 10 mm 5 micron, gradient 25-50 % MeCN/H20 0.1 % TFA. Top trace: Radioactivity
channel 5b (retention time 16.05 min). Bottom trace: UV channel, l 254 nm, the two by
products, 12 (retention time 14.27 min) and 1 1 (retention time 17.06 min).
Figure 2 depicts HPLC Analysis of FET-G-PEG-TOCA (1b) carried out using a
Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of
5-80 % ACN/0.1% TFA over 15 min.
Figure 3 depicts HPLC Analysis of FETE-PEG-TOCA (2b) carried out using a
Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of
5-80 % ACN/0.1% TFA over 15 min.
Figure 4 depicts HPLC Analysis of FET-G-TOCA (3b) carried out using a
Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of
5-80 % ACN/0.1% TFA over 15 min.
Figure 5 depicts HPLC Analysis of FETE-TOCA (4b) carried out using a Phenomenex
Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of 5-80 %
ACN/0.1% TFA over 15 min.
Figure 6 depicts HPLC Analysis of FET-pAG-TOCA (5b) carried out using a
Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of
5-80 % ACN/0.1% TFA over 15 min.
Figure 7 depicts HPLC Analysis of FET-pAG-[W-c(CTFTYC)K] (6b) carried out using a
Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) using a gradient of
5-80 % ACN/0.1% TFA over 15 min.
Figure 8 depicts HPLC Analysis of FET-G-PEG-TOCA (1b) spiked with [ F]-standards
carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min)
using a gradient of 5-80 % ACN/0.1% TFA over 15 min.
Figure 9 depicts HPLC Analysis of FETE-PEG-TOCA (2b) spiked with [ F]-standards
carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min)
using a gradient of 5-80 % ACN/0.1% TFA over 15 min.
Figure 10 depicts HPLC Analysis of FET-G-TOCA (3b) spiked with [ F]-standards
carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min)
using a gradient of 5-80 % ACN/0.1% TFA over 15 min.
Figure 1 1 depicts HPLC Analysis of FETE-TOCA (4b) spiked with [ F]-standards
carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min)
using a gradient of 5-80 % ACN/0.1% TFA over 15 min. This analogue appears to show signs
of degradation overtime, due to being stored in aqueous solution for > 1 month; hence the two
peaks. On initial formation the [ F]FETE-TOCA was seen as one peak only.
Figure 12 depicts HPLC Analysis of FET-3AG-TOCA (5b) spiked with [ F]-standards
carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min)
using a gradient of 5-80 % ACN/0.1% TFAover 15 min.
Figure 13 depicts HPLC Analysis of FET-3AG-[W-c(CTFTYC)K] (6b) spiked with [ F]-
standards carried out using a Phenomenex Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate
1 mL/min) using a gradient of 5-80 % ACN/0.1% TFAover 15 min.
Figure 14 depicts affinity profiles of different [ F]fluoroethyltriazole-[Tyr ]octreotate
analogs for somatostatin receptor subtypes sstr-2, 3 and 4 determined using a calcium flux
fluorometric imaging plate reader (FLIPR) assay (see Examples). The activation of calcium
flux by [ F]fluoroethyltriazole-[Tyr ]octreotate analogs in sstr-2, 3 or 4-expressing cells that
were pre-loaded with a calcium dye was assessed at different concentrations; the assay was
performed in duplicate. Fluorescence output was measured and data expressed as %maximal
fluorescence signal. The half-maximal receptor activation for the various agonist ligands is
summarized. ND = not determined due to lack of activity.
Figure 15. RadioHPLC chromatography of [ F]-FET-pAG-TOCA. (a) Reference
standard showing analyte retention time at 8.47 min and (b) typical plasma extract obtained
30 min after injection of the radiotracer into mice, indicating a stable radiotracer.
Figure 16. PET-CT images showing localization of (a) [ F]-FET-pAG-TOCA and (b)
the scrambled peptide FET-pAG-[W-c-(CTFTYC)K] in tumors, kidney, and bladder of AR42J
tumor bearing mice. Transverse and sagittal static (30-60 min fused; 0.5 mm slice) images are
shown.
Figure 17. Time activity curves comparing the tissue pharmacokinetics of
[ F]fluoroethyltriazole-[Tyr ]octreotate analogs in AR42J tumor, kidney, liver, muscle, and
urine. Dynamic PET/CT imaging was performed for 60 min after i.v. injection of each
radiotracer into tumor bearing mice. For clarity, the liver curves have been expanded (zoom).
Tissue radiotracer uptake values are expressed as %injected dose/mL of tissue. Values
represent the mean ± SEM (n=3-5); upper and lower bars are used for clarity. Symbols are (□)
FET-G-PEG-TOCA, (o) FETE-PEG-TOCA, (·) FET-pAG-TOCA, (■) FET-G-TOCA, and (A)
FETE-TOCA.
Figure 18. Specificity of FET-pAG-TOCA localization in the AR42J xenograft model.
Kinetics of [ F]-FET-pAG-TOCA and effect of saturating receptor binding sites with excess
cold unlabelled octreotide are shown. Blocking studies were carried out by injecting octreotide
(10mg/kg; i.v.) 10 min before i.v. injection of [ F]-FET-pAG-TOCA. Dynamic imaging was
performed over 60 min. Tissue radiotracer uptake values are expressed as %injected dose/mL
of tissue. The graphs also illustrate pharmacokinetics of the scrambled peptide, FET-pAG-[Wc-(
CTFTYC)K] in the same mouse model. Values represent the mean ± SEM (n=3-5); upper
and lower bars are used for clarity. Symbols are (·) FET-pAG-TOCA in octreotide nalve mice,
(A) FET-pAG-TOCA in mice pre-dosed with 10 mg/kg unlabelled octreotide, and (a)FET-pAG-
[W-c-(CTFTYC)K] in octreotide nalve mice.
Summary of the Invention
The present invention provides a triazole linked [Tyr ]octreotate analogue(s).
The present invention provides a 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate
analogue(s). A compound of the present invention combines high specific binding with rapid
target localization and rapid pharmacokinetics for high contrast PET imaging.
The present invention provides a method of making a 2-[ F]fluoroethyl triazole linked
[Tyr ]Octreotate analogue(s) of the invention.
The present invention provides a method of imaging using a 2-[ F]fluoroethyl triazole
linked [Tyr ]Octreotate analogue(s) of the invention.
The present invention provides an alkyne linked [Tyr ]Octreotate analogue(s) and a
method of making the same.
The present invention provides a pharmaceutical composition comprising at least one
2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue of the invention together with a
pharmaceutically acceptable carrier, excipient, or biocompatible carrier.
The present invention further provides a method of detecting somatostatin receptor in
vitro or in vivo comprising administering a 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate
analogue(s) of the invention or a pharmaceutical composition thereof.
The present invention further provides a method of detecting somatostatin receptor in
vitro or in vivo using a 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue(s) of the
invention or a pharmaceutical composition thereof.
Detailed Description of the Invention
The present invention provides a triazole linked [Tyr ]octreotate analogue of Formula
(I) :
FV LINKER-R 2 (I)
wherein :
, wherein Y is a reporter moiety that contains at least one
radioisotope;
R2 has the following structure:
LINKER is a linker group as described in WO20081 39207 or a synthetic linker group of
formula -(A)m- wherein each A is independently -CR , -CR=CR- , = ,
-CR2C0 2- , -C0 2CR2- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0 2NR- , -NRS0 2-
, -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a C4-8cycloheteroalkylene group, a
C4-8cycloalkylene group, a C5 - i2arylene group, or a C3- i2heteroarylene group, an amino acid, a
sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently
chosen from H, d-^alkyl, C2.4alkenyl, C2.4alkynyl, -4alkoxy d-^alkyl or hydroxyd-^alkyl ; m
is an integer of value 1 to 20.
According to the invention, the radioisotope of the reporter moiety Y can be any
radioisotope known in the art. In one embodiment, the radioisotope is any PET radioisotope
known in the art (e.g. , F, 7 Br, 7 Br, 2 1, C, Rb, Ga, 4Cu and Cu; preferably, C or
F; most preferably, F). In one embodiment, the radioisotope is any SPECT radioisotope
known in the art (e.g. , l, 4 l, 1) . The radioisotope may be directly incorporated into the
reporter moiety Y (e.g. -CH 2CH2 F or -CH 2CH2 CH2F) or may be incorporated into a
chelating agent by methods known in the art (see e.g. WO 2006/067376).
The present invention provides a 2-fluoroethyl triazole linked [Tyr ]octreotate analogue
of Formula ( II) :
R LINKER-R 2 ( II)
wherein:
R is ; wherein X is a radioisotope as described
above and wherein LINKER and R2 are each as described for Formula (I).
The present invention provides a 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue of Formula ( I II) :
R LINKER -R2 (II I)
wherein:
R2 has the following structure:
LINKER is a linker group as described in WO20081 39207 or a synthetic linker group of
formula -(A) M- wherein each A is independently -CR , -CR=CR- , C--=C ,
-CR 2C0 2- , -C0 2CR2- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0 2NR- , -NRS0 2-
, -CR 2OCR 2- , -CR 2SCR 2- , -CR 2NRCR 2- , a C4-8cycloheteroalkylene group, a
C4-8cycloalkylene group, a C5 - i2arylene group, or a C3 - i2heteroarylene group, an amino acid, a
sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently
chosen from H, CV^alkyl, C2-4alkenyl, C2-4alkynyl, C1-4alkoxy C _20alkyl or hydroxyC^oalkyl; m
is an integer of value 1 to 20.
The present invention provides a triazole linked [Tyr ]octreotate analogue of Formulae
(I), (II) and/or (III), each as described above, wherein F and R2 are each as described above
for Formulae (I), (II) and (III) and LINKER is
-(polyethylene
wherein n is an integer from 1-20; preferably, n is an integer from 1-10; more preferably, n is
an integer from 1-6.
In one embodiment of the invention, R2 compound of Formulae (I), (II) and (III) each as
described above is:
R2 h
preferably, R2 has the following structure:
The present invention provides 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue, FETE-PEG-TOCA, of Formula (2b):
wherein P and R2 are each as described above for the compound of Formula (lb).
The present invention provides 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue, FET-G-TOCA (3b):
wherein and R2 are each as described above for the compound of Formula (lb).
The present invention provides 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue, FETE-TOCA (4b):
wherein and R2 are each as described above for the compound of Formula (lb).
The present invention provides 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue, FET-3AG-TOCA (5b):
wherein F and R2 are each as described above for the compound of Formula (lb).
The present invention provides 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue, FET-bAQ-[W-c—(CTFTYC)K] (7b):
Pharmaceutical or Radiopharmaceutical Composition
The present invention provides a pharmaceutical composition comprising at least one
triazole linked [Tyr ]octreotate analogue or 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue of the invention, as described herein, together with a pharmaceutically acceptable
carrier, excipient, or biocompatible carrier.
The present invention provides a pharmaceutical composition comprising at least one
triazole linked [Tyr ]octreotate analogue or 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue of the invention, as described herein, together with a pharmaceutically acceptable
carrier, excipient, or biocompatible carrier suitable for mammalian administration.
As would be understood by one of skill in the art, the pharmaceutically acceptable
carrier, excipient, or biocompatible carrier can be any pharmaceutically acceptable carrier,
excipient, or biocompatible carrier known in the art.
The "pharmaceutically acceptable carrier, excipient, or biocompatible carrier" can be
any fluid, especially a liquid, in which a triazole linked [Tyr ]octreotate analogue or a 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention can be suspended or
dissolved, such that the pharmaceutical composition is physiologically tolerable, e.g., 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 either isotonic or not hypotonic) ; 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). The biocompatible carrier may also comprise biocompatible organic solvents such
as ethanol. Such organic solvents are useful to solubilise more lipophilic compounds or
formulations. Preferably the biocompatible carrier is pyrogen-free water for injection, isotonic
saline or an aqueous ethanol solution. The pH of the biocompatible carrier for intravenous
injection is suitably in the range 4.0 to 10.5.
A pharmaceutical composition of the invention may be administered parenterally, i.e.,
by injection, and is most preferably an aqueous solution. Such a composition may optionally
contain further ingredients such as buffers; pharmaceutically acceptable solubilisers (e.g.,
cyclodextrins or surfactants such as Pluronic, Tween or phospholipids) ; pharmaceutically
acceptable stabilisers or antioxidants (such as ascorbic acid, gentisic acid or paraaminobenzoic
acid). A method for preparation of a pharmaceutical composition of the
invention may further comprise the steps required to obtain a pharmaceutical composition
comprising a radiolabeled compound, e.g., removal of organic solvent, addition of a
biocompatible buffer and any optional further ingredients. For parenteral administration, steps
to ensure that the pharmaceutical composition of the invention is sterile and apyrogenic also
need to be taken. Such steps are well-known to those of skill in the art.
Intermediates
The present invention provides an alkyne linked [Tyr ]octreotate analogue of Formula
(IV) :
- LINKER-R 2 (IV)
wherein :
R2 has the following structure:
LINKER is a linker group as described in WO20081 39207 or a synthetic linker group of
formula -(A)m- wherein each A is independently -CR , -CR=CR- , C--=C ,
-CR2C0 2- , -C0 2CR2- , -NRCO- , -CONR- , -NR(C=0)NR-, -NR(C=S)NR-, -S0 2NR- , -NRS0 2-
, -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a C4-8cycloheteroalkylene group, a
C4-8cycloalkylene group, a C5 - i2arylene group, or a C3-i2heteroarylene group, an amino acid, a
sugar or a monodisperse polyethyleneglycol (PEG) building block; each R is independently
chosen from H, d-^alkyl, C2.4alkenyl, C2.4alkynyl, -4alkoxy d-^alkyl or hydroxyd-^alkyl; m
is an integer of value 1 to 20.
The present invention provides an alkyne linked [Tyr ]octreotate analogue of Formula
(IV) as described above wherein and R2 are each as described above for Formula (IV) and
LINKER is -(polyethylene glycol) n- ;
wherein n is an integer from 1-20; preferably, n is an integer from 1-10; more preferably, n is
an integer from 1-6.
In one embodiment of the invention, R2 compound of Formula (IV) as described above
is:
The present invention provides an alkyne linked [Tyr ]octreotate analogue, G-PEGTOCA,
of Formula (la):
wherei
preferably R2 has the following structure:
The present invention provides an alkyne linked [Tyr ]octreotate analogue, E-PEGTOCA,
of Formula (2a):
wherein P and R2 are each as described above for the compound of Formula (1a).
The present invention provides an alkyne linked [Tyr ]octreotate analogue, G-TOCA
(3a):
wherein and R2 are each as described above for the compound of Formula (1a).
The present invention provides an alkyne linked [Tyr ]octreotate analogue, E-TOCA
(4a):
wherein and R2 are each as described above for the compound of Formula (1a).
The present invention provides an alkyne linked [Tyr ]octreotate analogue, 3AG-TOCA
wherein P and R2 are each as described above for the compound of Formula (1a).
The present invention provides an alkyne linked [Tyr ]octreotate analogue, 3AG-[Wc—(
CTFTYC)K] (6a):
wherein F is as
Synthesis
A 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue(s) of the present invention
and an alkyne linked [Tyr ]Octreotate analogue(s) of the present invention can be prepared by
methods known in the art (WO201 0/026388 which is hereby incorporated in its entirety by
reference) and by those methods exemplified below.
The introduction of a PET radioisotope (i .e., any positron-emitting radioisotope) as
described herein may be introduced into a compound of Formulae (I), (II), (III) and a 2-
fluoroethyl triazole linked [Tyr ]Octreotate analogue either prior to formation of the triazole
moiety or after the formation of the triazole moiety by any means known in the art (e.g. WO
2010/026388). A SPECT radioisotope can be introduced into a molecule of Formulae (I) or
(II), each as described herein, in a likewise manner.
According to the present invention, a method of making a 2-[ F]fluoroethyl triazole
linked [Tyr ]Octreotate analogue(s) of the present invention comprises the step of reacting an
alkyne linked [Tyr ]Octreotate analogue(s) with 2-[18F]Fluoroethylazide under copper
catalyzed click chemistry conditions to form the corresponding 2-[ F]fluoroethyl triazole linked
[Tyr ]octreotate analogue, each as described herein.
In one embodiment of the invention, the synthesis of 2-[ F]fluoroethyl triazole linked
[Tyr ]Octreotate analogue is automated. [ F]-radiotracers may be conveniently prepared in
an automated fashion by means of an automated radiosynthesis apparatus. There are
several commercially-available examples of such apparatus, including Tracerlab™ and
FASTlab™ (both from GE Healthcare Ltd.). Such apparatus commonly comprises a
"cassette", often disposable, in which the radiochemistry is performed, which is fitted to the
apparatus in order to perform a radiosynthesis. The cassette normally includes fluid
pathways, a reaction vessel, and ports for receiving reagent vials as well as any solid-phase
extraction cartridges used in post-radiosynthetic clean up steps.
The present invention therefore provides in another aspect a cassette for the
automated synthesis of the PET radiotracer as defined herein comprising:
(i) a vessel containing containing an alkyne-linked [Tyr3]octreotate analogue, as
described herein; and
(ii) a vessel containing an azide capable of undergoing a click chemistry reaction with
an alkyne-linked [Tyr ]octreotate analogue of vessel (iii) (e.g. tosylethyl azide in a solution of
MeCN, DMSO or DMF); and
(iii) adding the contents of said vessel (ii) to a suitable source of F.
According to the present invention, a cassette of the present invention may, optionally,
further comprise one or more of the following:
(iv) a QMA cartridge;
(v) QMA eluent to release the trapped fluorine-1 8 consisting of K222, MeCN, water
and a base (e.g., TBAHC0 3, K2C0 3, Cs2C0 3) ;
(vi) a vessel containing a copper catalyst (e.g., copper (I) catalyst; copper sulphate in
an aqueous solution);
(vii) a vessel containing Na-ascorbate (preferably, in sodium acetate buffer solution at
pH 5.0);
(viii) a vessel containing a copper (I) stabilising ligand (e.g., BPDS);
(ix) a line directed to an HPLC system; and
(ix) an ion-exchange cartridge for removal of excess F.
Examples
Reagents and solvents were purchased from Sigma-Aldrich Co. Ltd. (Gillingham,
United Kingdom) and VWR International Ltd UK, and used without further purification. BPDS
(9) was purchased from Pfaltz & Bauer Inc. Waterbury, USA. [ F]-AIF-NOTA was synthesized
according to Laverman P, et al., J Nucl Med. 201 0;51 :454-461 . [ Ga]-DOTATATE was
purchased from Covidien (UK) Commercial Ltd (Gosport, UK).
MALDI-TOF were measured at The London School of Pharmacy on a Finnigan
Lasermat 2000 instrument. Analytical HPLC was carried out using a Beckman Gold
instrument with Karat32 software or Laura software. The radio HPLC system was a Beckman
System Gold instrument equipped with a g detector (Bioscan Flow-count). A Phenomenex
Luna C18(2) column (50 x 4.6 mm, 3 mih ; flow rate 1 mL/min) was used for analytical HPLC. A
semipreparative column (Phenomenex Luna C 18, 100 x 10 mm, 5mih , 110A; flow rate of 3
mL/min) was used for the final purification of the peptides. The following mobile phase system
was used for analytical HPLC: solvent A, water/TFA (0.1%); solvent B, acetonitrile/TFA
(0.1%); linear gradient of 5-80% solvent ACN/0.1% TFA over 15 min. Non-radioactive
compounds were purified using a preparative HPLC (Agilent1200, column Phenomenex Luna
C 18(2), 75 X30 mm, 5 mih , flow rate 15 mL/min). To separate samples for log D calculations a
MSE Micro Centaur centrifuge apparatus was used, Gamma counts were carried out using a
Wallac 1282 Compugamma Universal gamma counter and results recorded using the
EdenTerm v 1.2 software. [ F]Fluoride was produced by a cyclotron (PET Trace, GE Medical
systems) using the 0(p,n) F nuclear reaction with 16.4 MeV proton irradiation of an
enriched [ 0]H 20 target.
Abbreviations:
ACN: Acetonitrile
DCM: Dichloromethane
DIPEA: Diisopropylethylamine
Fmoc: 9-Fluorenylmethyloxycarbonyl
HBTU: 0-Benzotriazol-1-yl-N,N,W,W-tetramethyluronium hexafluorophosphate
HOBt: 1-Hydroxybenzotriazole
NMP: 1-Methyl-2-pyrrolidinone
PyBOP: (Benzotriazol-l-yloxy)tripyrrolidinophosphonium hexafluorophosphate
SASRIN: Super acid sensitive resin
TFA: Trifluoroacetic acid
TIS: Triisopropylsilane
Example 1. General method for the synthesis of [19Flfluoroethyl triazole peptide
standards
(a) To a Wheaton vial charged with copper powder (20 mg, -40 mesh) was added an alkyne
linked [Tyr ]octreotate analogue (alkyne-peptide) (5 mg) in a solution of DMF/H20 ( 1 :1 v/v (60
m I_)). To this was added [ F]fluoroethyl azide ( 1 .5 eq, 0.354 M in DMF)(Glaser, M.; and
Arstad, E., Bioconjugate Chemistry 2007, 18, (3), 989-993). The reaction was left stirring at
room temperature for 30 minutes and then quenched with 0 .1 ml_ of 20 % MeCN/H20 0 .1 %
TFA before injection for purification using the Agilent preparative HPLC. All analogues were
isolated using a gradient of 20-80 % MeCN/H20 0 .1 % TFA over 30 minutes. Mass
spectrometry result are summarized in Table 1 below.
Table 1: Mass s ectrometry of the F-standards
(b) DMF only. The [ F]standards, [ F]-1 b-6b, were also synthesised using copper powder,
and DMF as solvent. For compounds [ F]-1 a and [ F]-3a, the use of DMF as the sole
solvent proceeded slowly (> 3 hours) . Addition of water (DMF:H20 (3:2)) enhanced the rate of
reaction with reaction completion within 15 minutes.
Example 2a. General Method for the Synthesis of 2-[ 8Flfluoroethyl triazole linked
[Tyr310ctreotate analogue
As illustrated in Scheme 1 below, a radiolabeled [Tyr ]octreotate analogue of the invention
may be prepared by labelling an alkyne linked [Tyr ]octreotate analogues by means of a
copper catalysed azide-alkyne cycloaddition reaction (CuAAC) to form a 1,4-substituted
triazole using the reagent 2-[ F]fluoroethyl azide, i.e. a 2-[ F]fluoroethyl triazole linked
[Tyr ]Octreotate analogue of the invention. An unexpected variability in reactivity during the
CuAAC reaction was observed for each alkyne analogue investigated.
-TOCA
elds
Scheme 1: The reaction pathway to the F-labelled triazoles: 1a, 2a, 3a, 4a, 5a and 6a, each
as described herein.
Example 2b. Alkyne preparation
Five alkyne functionalised octreotate analogues (Figure 1) were synthesized in a manner
analogous to the synthesis of bAB-TOCA (5a) described below. A scrambled peptide (6b)
(Figure 1) was designed as a negative control to show no specificity to the somatostatin
receptor. The linkers between the octreotate and the alkyne functionality were chosen to
complement the peptide and for ease of synthesis. Two analogues were designed containing
polyethylene glycol groups ((1a) and (2a)). The octreotate alkynes (1a-5a) and scrambled
analogue (6a) were labelled using [ F]fluoroethyl azide (8) (Scheme 1).
Example 2b. 1 Synthesis of bAB-TOCA (5a)
2b.1.1 Synthesis of propynoyl-B-Ala-OH
Propiolic acid (3.00 mmol, 184 m I ) was added to a solution of H-b-Ala-OMe HCI salt
(3.00 mmol, 419 mg), PyBOP (3.00 mmol, 1.56 g) and DIPEA (9.00 mmol, 1.53 mL) dissolved
in NMP (5 mL). The reaction mixture was shaken for 30 min then diluted with water/0.1% TFA
and loaded onto a preparative HPLC column for purification affording propynoyl- -Ala-OMe.
Propynoyl- -Ala-OMe was dissolved in ACN/water/0.1% TFA (200 mL) and the
solution adjusted to pH 11 using 0.1 M NaOH. The solution was stirred for 1 hr and then
reduced in vacuo. The residue (50 mL) was injected onto a preparative HPLC column for
product purification.
Purification and characterisation
Purification by preparative HPLC (gradient: 0% ACN/0.1% TFA over 60 min) afforded
260 mg (61% based on 3 mmol starting material) pure propynoyl - -Ala-OH.
The purified material was analysed by analytical LC-MS (gradient: 0-15% ACN/0.1%
TFA over 5 min, fR: 0.29 min, found m/z: 142.3, expected MKT: 142.0).
2D.1 .2 Assembly of H-Gly-[Tyr3l-Octreotate on solid support
The peptidyl resin H-Gly-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-
Cys(Trt)-Thr(tBu)-Polymer was assembled using standard peptide synthesis procedures. The
peptidyl resin H-Gly-D-Phe-Cys(Trt)-Tyr(tBu)-D-Trp(Boc)-Lys(Boc)-Thr(tBu)-Cys(Trt)-Thr(tBu)-
Polymer was assembled on the commercially available CEM Liberty microwave peptide
synthesizer using Fmoc chemistry starting with 0.25 mmol Fmoc-Thr(tBu)-SASRIN resin. 1.0
mmol amino acid was applied in each coupling step (5 min at 80 C) using 0.9 mmol
HBTU/0.9 mmol HOBt/2.0 mmol DIPEA for in situ activation. Fmoc was removed by treatment
of the resin with a solution of 20% piperidine in NMP.
2P.1 .3 Synthesis of propynoyl-B-Ala-Glv-iTyr3 -Octreotate (i.e.. BAB-TOCA (5a))
Propynoyl -Ala-OH (0.50 mmol, 7 1 mg, described above in 2b. 1.1) and PyBOP
(0.500 mmol, 260 mg) were dissolved in NMP (5 mL) and added to the Octreotate resin (0.25
mmol, described above in 2b. 1.2). DIPEA (2.00 mmol, 340 m ) was added and the mixture
shaken for 90 min. The reagents were removed by filtration and the resin washed with NMP,
DCM and diethyl ether and dried.
The simultaneous removal of the side-chain protecting groups and cleavage of the
peptide from the resin was carried out in TFA (100 mL) containing 2.5% TIS and 2.5% water
for 90 min. The resin was removed by filtration, washed with TFA and the combined filtrates
evaporated in vacuo. Diethyl ether was added to the residue, the formed precipitate washed
with diethyl ether and dried. The dry precipitate was dissolved in 50% ACN/water and left over
night in order to remove remaining Trp protecting groups. The solution was then lyophilised
affording 294 mg (96%) crude propynoyl - -Ala-Gly-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-
OH.
2,2'-Dithiodipyridine (0.27 mmol, 59 mg) dissolved in ACN (0.75 ml_) was added in
three equal portions to crude propynoyl - -Ala-Gly-D-Phe-Cys-Tyr-D-Trp-Lys-Thr-Cys-Thr-OH
(294 mg) dissolved in ACN/water/0.1% TFA (300 ml_) and the solution shaken for 30 min. The
reaction mixture was loaded onto a preparative HPLC column for product purification
Purification and characterisation
Purification by preparative HPLC (gradient: 20-40% ACN/0.1% TFA over 60 min)
afforded 140 mg (48%) pure propynoyl - -Ala-Gly-[Tyr ]-Octreotate.
The purified material was analysed by analytical LC-MS (gradient: 20-30% ACN/0.1%
TFA over 5 min, fR: 2.49 min, found m/z: 1229.5, expected MKT: 1229.5).
Radiochemistry
Example 3. Preparation of 2-[ 8FlFluoroethylazide (8)
The method used to synthesise 8 was slightly modified to that employed by Glaser et
al. (Glaser, M., etal., Bioconjugate Chemistry 2007, 18, (3), 989-993; Demko, Z. P., etal.,
Angewandte Chemie-lnternational Edition 2002, 4 1, ( 12), 2 113-21 16). The use of KHC0 3
instead of K2C0 3 during the [ F]fluoride drying step gave more consistent isolated yields after
purification by distillation, due to the enhanced stability of the precursor, 7 using the milder
base.
To a mixture of Kryptofix 222 (5 mg, 13.3 mihoI) , potassium hydrogencarbonate ( 1 .4
mg, 16.7 mihoI , in 100 m I_water), and acetonitrile (0.5 ml_) was added [ F]fluoride (5-15 mCi)
in water (0.1-1 ml_). The solvent was removed by heating at 100 °C under a stream of
nitrogen (100 mL/min). Afterwards, acetonitrile (0.5 ml_) was added, and the distillation was
continued. This procedure was repeated twice more. After cooling to room temperature, a
solution of 2-azidoethyl-4-toluenesulfonate (7) ( 1 .3 m I_, 6.5 mi oΐ I , M.; et al.,
Bioconjugate Chemistry 2007, 18, (3), 989-993; Demko, Z. P.; et al., Angewandte Chemie-
International Edition 2002, 4 1, ( 12), 2 113-21 16) in anhydrous acetonitrile (0.2 ml_) was added.
The reaction mixture was stirred for 15 min at 80 °C. [ F]8 was distilled at 130 °C into a
trapping vial containing acetonitrile (30 mL)(Arstad, E., WO2008/0 15391 A1). Compound [ F]8
was collected with radiochemical yields between 50-55 % (decay-corrected).
Generation of 2-[ F]fluoroethyl azide for the preparation of samples via click
chemistry, subsequently used for in vivo studies, was carried out on a remotely controlled
apparatus built at Hammersmith Imanet. The system enables the use -200-300 mCi of
[ F]fluoride as starting activity. Radiochemical yields (decay corrected) of isolated
[ F]fluoroethyl azide using this system range from 19-26 %. However, the generation of 2-
[ F]fluoroethyl azide can be achieved by other means known in the art (Glaser, M.; et al.,
Bioconjugate Chemistry 2007, 18, (3), 989-993; Demko, Z. P.; et al., Angewandte Chemie-
International Edition 2002, 4 1, (12), 2 113-21 16); WO2008/01 5391 A 1) .
Example 4. General method for labelling
For alkynes 1a, 2a and 4a: To a solution of copper (II) sulfate pentahydrate (4 eq) in
water (25 m I_) was added sodium L-ascorbate (4.4 eq) in 25 m I_ of sodium acetate buffer
solution (pH 5.0, 250mM) under N2 followed by BPDS (9) (5 eq) in 25 m I of water. For
alkynes 3a and 5a, CuS0 4 (2 eq), Na-ascorbate (2.2 eq), and BPDS (9) (4 eq) were used.
A solution of [ F]8 in MeCN (100 m I ) was added followed by the alkyne (2 mg) in DMF
(30 m I_) and the reaction carried out at room temperature (see Table 2 for optimal reaction
time). The reaction was then diluted with H20 0.1 % TFA (100 m I_) and purified by reverse
phase preparative HPLC using the gradient stated in Table 2 . The HPLC fraction was then
diluted with H20 ( 1 5-1 9 mL) and loaded onto a tC1 8 light SPE cartridge. The product was
eluted with ethanol in 100 m fractions and made up to a solution of 10 % ethanol/PBS buffer
solution (pH 7.0) with >98 % RCP.
Table 2 : HPLC solvents MeCN/H 20 0.1 % TFA ("B"), 3mL/min using a Luna C18 5u, 100 X 10
mm, 5micron
Example 5. Octanol/Phosphate buffer partition co-efficient measurements (Log D)
The octanol/PBS partition coefficients were determined using the shake flask method.
Both solvents were presaturated with each other by shaking together for 5 min. To a solution
of 500 m I_of both octanol and PBS was added 20m I_ of radiolabeled ligand in EtOH (n=3).
The solutions mixtures were shaken in a rotamixer for 5 minutes. After equilibration the
mixtures were centrifuged (10 min at 13,000 rpm) to achieve good separation. Samples from
each layer (25m I_) were taken and measured in a g counter and Log D was calculated
according to the formula:
log D = log (cpm in octanol layer/cpm in aqueous layer).
Log D values were measured using the F-labelled triazole. As expected the Log D values
for the analogues 1b and 2b were the lowest due to the PEGylation, and the analogue with
the highest Log D value was 4b (Table 3). The receptor affinities for [ F]1b-6b were
determined using a competitive binding assay in AR42J tumours cells with [ ln]-OctreoScan
as the labeled radiotracer. The half-maximal inhibitory concentration (IC50) values were
calculated and the results of the displacement curves are summarised (Table 3). As a
reference peptide, the IC50 value was measured for Octreotide and was found to be 14.7 ± 7.7
nM. The IC50 values for the octreotate analogues were found to be comparable or lower than
octreotide, indicating high binding affinity for the somatostatin receptor; all showed high
binding affinity to the receptor in the nanomolar range. The introduction of the fluoroethyl
triazole moiety decreased the affinity for [ F]-2b,3b,4b and 5b but not significantly, the values
were still below or comparable to octreotide. In this study the compound [ F]-5b showed the
highest affinity with an IC50 value of 1.6 ± 0.2 nM. PEGylation of the peptides, through the
addition of six sequential ethylene glycol groups, appeared not to significantly affect the
overall affinity of [ F]-1b and [ F]-2b giving IC50 values of 2.9 ± 1.3 nM and 13.2 ± 7.8 nM,
respectively. In comparison [ F]-6b, which contained a scrambled amino acid sequence,
showed low affinity giving an IC50 value > 10mM. This result showed that our analogues are
specifically binding to the somatostatin receptor.
Table 3 : Competitive binding assay IC50 values using 1a-5a and [ F]-1b-6b displacing [ ln]-
DTPA-Octreotide on AR42J tumour cells and Log D values of [ 8F]-1b-6b.
N.B. (n = 4 for each concentration and assay repeated three times). Log D partition co-efficient
measurements 1b-5b (n = 3), 6b (n = 6). IC50 values of AIF-NOTA-Octreotide, Ga-NOTA-Octreotide
found by Laverman, P., et al.(Journal of Nuclear Medicine 51, (3), 454-461) were 3.6 ±0.6, 13.0 ± 3.0
nM respectively.
Example 6. In Vitro Receptor binding determination.
To determine the binding affinity of [ F]-fluoroethyltriazole-[Tyr] -octreotate analogues ( F-
1b-6b), and the alkyne-octreotate analogues (1a-6a) an in vitro assay was done using a
modification of the method previously reported by Hofland and co-workers (Hofland, L. J.; et
al., Endocrinology 1995, 136, (9), 3698-706). AR42J cells (5 X 104) were seeded in 24-well
plates, washed twice in PBS and incubated with increasing concentrations (0, 0.01 , 0.1 , 1,
1.0, 10, 100, 1000 and 10000 nM) of the compound being analysed for 10 min at room
temperature, allowing for sufficient time for binding to occur under these experimental
condition (Scemama, J. L.; et al., Gut 1987, 28 Suppl, 233-6). The plates were then incubated
for a further 30 min with [ ln]OctreoScan (Covidien, Gosport, UK: (50,000 cpm per well). A
final volume of 0.25 ml_ of incubation buffer per well was used. The incubation buffer
consisted of 10 mM HEPES, 5.0 mM, MgCI2 6H20 , 1.0 mM bacitracin, 1% BSA, and the final
pH was adjusted to 7.4. At the end of the incubation, the plates were washed three times in
ice cold incubation buffer and solubilised in 0.2N NaOH solution. The contents of each well
were then transferred to counting tubes and samples counted using a gamma-counter
(Biosoft, Ferguson, MO). Each concentration was performed in quadruple and the experiment
repeated three times. Results are expressed as a percentage of control (first four wells treated
with only labelled [ ln]OctreoScan). The IC50 values were calculated from the fitted sigmoidal
displacement curve using GraphPad Prism software (version 4.00) for Windows, GraphPad
Software, Inc).
Example 7. Use of copper wire as catalyst
The use of copper wire as an alternative source of catalytic Cu(l) was investigated. The
experiments were carried out using 3a due to its enhanced reactivity compared to other
analogues using the CuS0 4/Na-ascorbate method. It was found that pre-mixing the copper
wire and alkyne then heating before addition of 8 showed the reaction to be complete within 5
minutes (Table 4, entry 2). Without pre-mixing the two components, the click reaction took
longer to reach completion (Table 4, entry 1). Other pH buffer systems (Table 4, entries 4,5
and 6) were also applied, but all proved inferior to the sodium acetate buffer solution (pH 5.0,
250 mM) (Table 4, entry 1). CuS0 4 and BPDS (9) were added to the reaction using copper
wire, the rationale behind this approach being that the added CuS0 4 would improve the
comproportionation reaction by increasing the concentration of available Cu(ll) (Gopin, A., et
al., Bioconjugate Chemistry 2006, 17, 1432-1440; Bonnet, D., etal., Bioconjugate Chemistry
2006, 17, 1618-1 623). The ligand (9) as previously mentioned, was added to stabilise the
Cu(l) species. The strategy worked well and showed improvement in yields at room
temperature (Table 4, entry 7). To establish whether this was a tandem effect, reactions were
carried out to investigate both additional reagents separately (Table 4, entries 7,8). It appears
that that both reagents affect the rate to some extent but used together they have a greater
impact on the rate of the reaction. MonoPhos™ (Campbell-Verduyn, L . S., et al., Chemical
Communications 2009, (16), 2139-2141) was investigated as an alternative ligand, but proved
less efficient in the reaction (Table 4, entry 10).
Table 4 : Copper wire catalysed experiments using alkyne 3a (Analytical radiochemical yield
by HPLC)
All buffers are 250mM concentrations. Sodium acetate buffer (AB) pH 5.0; Sodium phosphate buffer
(PB) pH 6.0; Ammonium formate buffer (FB) pH 4.0; Tris buffer (TB) pH 8.0. Each experiment
contained 100-120 mg of a copper wire coil (rt = room temperature).
Results
The alkyne linked [Tyr ]octreotate analogues, G-TOCA (3a) and 3AG-TOCA (5a) have
been identified to be highly reactive in the click reaction showing complete conversion to the
2-[18F]fluoroethyl triazole linked [Tyr ]Octreotate analogues FET-G-TOCA (3b) and FET-3AGTOCA
(5b) under mild conditions and with short synthesis times (5 minutes at 20°C). As well
as ease of synthesis, in vitro binding to the pancreatic tumour AR42J cells showed that both
FET-G-TOCA (3b) and FET-3AG-TOCA (5b) have high affinity for the somatostatin receptor
with IC50 of 4.0 ± 1.4, and 1.6 ± 0.2 nM respectively.
The variability in click reaction rates observed for the alkyne-peptides 1a-5a, can be
attributed to the variation in linker, since the peptide moiety remains unchanged. The
electronic property of the group adjacent to the terminal alkyne centre is believed to neither
enhance nor reduce the rate of reaction (Hein, J. E., et al., Chemical Society Reviews 2010,
(39), 1302-1 3 15). Contrary to this, it has now been found that terminal alkynes directly
substituted with an amide moiety (1a, 3a, 5a, 6a (Figure 1)) had enhanced reactivity
compared to those directly substituted with an ethyl-linked amide (2a and 4a, (Figure 1)).
Similar results were found by Li et al. (Tetrahedron Letters 2004, 45, 3143-3146) during their
investigations of the 1,3-Huisgen cycloaddition reaction and separately by Golas etal.
(Macromolecular Rapid Communications 2008, 29, 1167-1 17 1) who investigated the effect of
electron withdrawing groups and steric hindrance around the azide moiety on the rate of the
CuAAC. Another factor to be taken into consideration along with the electronic nature of the
alkyne is the size of the alkyne-peptide. Kinetically it would be expected that the larger the
alkyne-peptide the slower the rate of reaction, which was found to be the case with the
PEGylated analogues (1a, 2a). It can be seen that 1a reacts more slowly that 3a and 5a
(Table 5, conditions B). Electronically they are similar, all being directly linked amide
substituted terminal alkynes, but 1a contains six sequential ethylene glycol groups. It is
possible that the PEG chain is surrounded by a bulky water cloud which could sterically hinder
the alkyne functionality (Shiftman, M. L , Current Hepatitis Reports 2003, 2, 17-23). Alkynes
2a and 4a are comparable in the same respect (PEGylated vs. non-PEGylated); it was
observed that 2a shows a slower rate of reaction during the CuAAC. Due to the slower rate of
reaction and variation found using 1a and 2a during the click reaction, it was necessary to
increase the concentration of reagents. Improvements were seen in the reproducibility and
rate of reaction (Conditions D, Table 5). Analogues 1b-6b were isolated to give non-decay
corrected yields of 3-7 % (based on starting fluoride activity) after 90-120 minutes. Reaction
rate was increased using higher temperatures with reaction times being reduced, but this led
to significant by-product formation and made purification more difficult (Table 5, conditions C).
A general trend found during the click reaction with analogues 1a-6a were two stable
by-products seen during HPLC monitoring. The two by-products 1 1 and 12 (Scheme 2, Figure
1a) were isolated from the reaction media using 5a (Figure 1) . They were separated,
collected and analysed by collision induced dissociation mass spectrometry (CID-MS). The
more polar compound 12 was elucidated to be the alkyne precursor (5a) during CID-MS
analysis. Although this was found to be the case, when the reaction mixture was admixed
with 5a and assessed chromatographically, by-product 12 did not co-elute on the HPLC trace.
It was found that reactions showing incomplete incorporation of 2-[ F]fluoroethyl azide (8) did
not significantly proceed any further, even in the presence of 12, suggesting insufficient
reactivity of this species in the CuAAC reaction. The by-product 1 1 was analysed and found
to correspond to the 1-vinyl triazole (Scheme 2).
Scheme 2 : Suspected pathway to form by-product 1 1
Presumably 1 1 was formed via the click reaction with azidoethene (10), which is suspected to
be a by-product from an elimination mechanism in the initial labelling step. Kim etal. (Applied
Radiation and Isotopes 2010, 68, 329-333) reported a similar occurrence using 4-tosyloxy-1-
butyne; they observed elimination to form vinyl acetylene, which was then able to react with
the azide in the click reaction. The main issue with by-product 1 1 is the similar retention time
to the radiolabeled product during purification, making it difficult to obtain the highest yield
possible as well as high specific radioactivity.
The experiments carried out using the CuS0 4/Na-ascorbate method were generally
done using sodium acetate buffer (AB) at pH 5.0. When an experiment using 3a was carried
out using distilled water the conversion to radiolabeled product was slower, showing 78 %
conversion at 5 min compared to >98 % conversion using the buffered system.
A one-pot reaction was carried out in which distillation of 8 was avoided. The reaction
was attempted with both 2a and 3a but both showed slower reaction rates presumably due to
competing side reactions. Another variable that can affect the rate of reaction is the volume of
2-[ F]fluoroethyl azide. A reaction carried out using conditions B (Table 5) with 2a and 50 m I
of 2-[ F]fluoroethyl azide (8) gave 84 % conversion to 3b after 30 minutes. When using 100
m I of 8 under the same conditions no reaction was observed (Table 5).
It was found that using BPDS ligand (9) (Scheme 1), a Cu(l) stabilizing ligand (Gill, H.
S., etal., Journal of Medicinal Chemistry 2009, 52, 5816-5825), greatly enhances the rate of
the click reaction. The reaction was evaluated without any ligand. Reactions carried out with
4a without 9 (Table 5, conditions A) showed 14 % conversion to the desired product but on
addition of 9 (conditions B), gave 47 % conversion. Using the same reagent concentrations
but heating the reaction to 80°C gave >98 % conversion to 4b after 5 minutes (Table 5,
conditions C); although the conversion to product at this temperature was excellent, an
increase in by-products was observed, some of which co-eluted with 4b.
Table 5 : Radiochemical Analytical Yields observed using HPLC analysis
Conditions A: Alkyne (2 mg), CuS0 4 (2 eq,) , Na-ascorbate (2.2 eq), phi 5.0, rt
Conditions B: Alkyne (2 mg), CuS0 4 (2 eq), Na-ascorbate (2.2 eq), 9 (4 eq), pH 5.0, rt
Conditions C: Alkyne (2 mg), CuS0 4 (2 eq), Na-ascorbate (2.2 eq), 9 (4 eq), pH 5.0, 80 C
dConditions D: Alkyne (2 mg), CuS0 4 (4 eq), Na-ascorbate (4.4 eq), 9 (5 eq), pH 5.0, rt
e No alkyne remains in the reaction only the by-products are found in the UV trace.
In conclusion, five novel alkyne functionalised octreotate analogues were reacted in the click
reaction with 2-[ F]fluoroethyl azide. The most reactive alkynes were G-TOCA (3a) and 3AGTOCA
(5a), showing complete conversion to the labelled triazole FET-G-TOCA (3b) and FET-
3AG-TOCA (5b) in five minutes at room temperature using optimised conditions. As well as
efficiency in the click reaction both analogues have shown high binding affinities to the
somatostatin receptor.
Example 8. In vitro binding assay. The affinity of a [ F]fluoroethyltriazole-[Tyr ]octreotate
analogue of the present invention for somatostatin receptor subtype sstr-2, versus sstr-3 and
sstr-4 as control low affinity receptor subtypes, was determined using a fluorometric imaging
plate reader (FLIPR) assay. The assay involved measuring the [ F]fluoroethyltriazole-
[Tyr ]octreotate induced activation of a calcium flux in sstr-2, 3 or 4-expressing Chem-1 cells
(Millipore, St Charles, MO, USA) that were pre-loaded with a calcium dye. Briefly, Chem-1
cells expressing specific sstr-subtype were seeded in 96 well plates at 50,000 cells/well and
incubated in a 5% C0 2 incubator for 24 h . Cells were washed and loaded with Fluo-8-No-
Wash Ca + dye in GPCRProfiler™ Assay Buffer (Millipore) for 90 min at 30°C in a 5% C0 2
incubator. Different concentrations of a [ F]fluoroethyltriazole-[Tyr ]octreotate of the present
or somatostatin (Sigma; positive control) were added followed by fluorescence determination.
The assay was performed in agonist mode in duplicate. Antagonistic activity was not
evaluated because of agonist activity. Fluorescence output was measured and data
expressed as %maximal fluorescence signal after baseline correction. The half-maximal
receptor activation for the various ligands was estimated by sigmoid dose response fitting
using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego CA USA).
Example 9. Animals and tumor models. Six to eight-week old female BALB/c nu/nu
athymic mice were obtained from Harlan United Kingdom Ltd (Bicester, UK). High sstr-2
pancreatic tumor cell line AR42J (Taylor J.E., etal., 1994;15:1229-1236) and low sstr-2
human colon cancer cell line HCT1 16 (LGC Standards, Middlesex, UK) were cultured,
respectively, in F12K and RPMI1640 growth medium containing 10% (v/v) fetal bovine serum,
2 mmol/L L-glutamine, 100 units/mL penicillin, and 100 g/mL streptomycin and grown in a 5%
C0 2 incubator at 37 °C. Tumors were established by subcutaneous injection of 100 m I of PBS
containing 1 x 106 cells. All animal experiments were done by licensed investigators in
accordance with the United Kingdom Home Office Guidance on the Operation of the Animal
(Scientific Procedures) Act 1986 and within guidelines set out by the United Kingdom National
Cancer Research Institute Committee on Welfare of Animals in Cancer Research (Workman,
P., etal., Br J Cancer. 2010;102:1555-1577). Tumor dimensions were measured continuously
using a caliper and tumor volumes were calculated by the equation: volume = (tt/6) c a c b c
c, where a, b, and c represent three orthogonal axes of the tumor. Mice were used when their
tumors reached -200 mm3.
Example 10. In vivo plasma stability of FET-pAG-TOCA. FET-pAG-TOCA (~3.7MBq) was
injected via the tail vein into non-tumor bearing BALB/c nu/nu mice. Blood was obtained under
general isofluorane anesthesia at 30 min post injection and plasma samples were prepared
and immediately frozen on ice. For analysis, the samples were thawed and kept at 4°C
immediately prior to use. Plasma (-0.2 mL) was clarified by addition of ice-cold methanol ( 1 .5
mL) followed by centrifugation of the mixture (3 minutes, 20,000 c g 4 qC). The supernatant
was evaporated to dryness using a rotary evaporator (Heidolph Instruments GMBH & Co,
Schwabach, Germany) at a bath temperature of 35 °C. The residue was re-suspended in
HPLC mobile phase ( 1 .2 mL), clarified (0.2 mih filter) and the sample ( 1 mL) injected via a 1
mL sample loop onto the HPLC. Samples were analyzed by radio-HPLC on an Agilent 1100
series HPLC system (Agilent Technologies, Stockport, UK) equipped with a g-RAM Model 3
gamma-detector (IN/US Systems inc., Florida, USA) and Laura 3 software (Lablogic,
Sheffield, UK) and UV (254 nm). A Waters mBondapak C18 reverse-phase column (300 mm c
7.8 mm) stationary phase was eluted with a mobile phase comprising of 67 % water (0.1%
TFA)/33 % acetonitrile (0.1% TFA) delivered isocratically at 3mL/min.
Example 11. PET imaging studies. Dynamic PET imaging scans were carried out on a
dedicated small animal PET scanner, (Siemens Inveon PET module, Siemens Molecular
Imaging Inc, UK) (Workman, P., et al., Br J Cancer. 2010;102:1555-1577; Leyton, J., et al.,
Cancer Res. 2006;66:7621 -7629). Briefly, tail veins were cannulated under general
anesthesia (isofluorane). The animals were placed within a thermostatically controlled
environment within the scanner; heart rate was monitored throughout the study. The mice
were injected with 3.0-3.7 MBq of the different radiolabeled compounds and dynamic PET-CT
scans were acquired in list-mode format over 60 min. In the case of FET-pAG-TOCA blocking
studies were also done whereby radiotracer injection and imaging commenced 10 min after
i.v. injection of 10 mg/kg unlabelled octreotide (Sigma) to mice; this dose of octreotide was
~ 100-fold higher than the equivalent dose of unlabelled FET-pAG-TOCA in the radiotracer
injectate. The acquired data in all cases were sorted into 0.5-mm sinogram bins and 19 time
frames (0.5 c 0.5 c 0.5 mm voxels; 4 c 15s, 4 c 60s, and 11 c 300s) for image reconstruction.
The image data-sets obtained were visualized and quantified using the Siemens Inveon
Research Workplace software. Three-dimensional regions of interest (3D ROIs) were
manually defined on five adjacent tumor, liver, kidney, muscle or urine/bladder regions (each
0.5 mm thickness). Data were averaged for tissues at each of the 19 time points to obtain time
versus radioactivity curves (TACs). Radiotracer uptake for each tissue was normalized to
injected dose and expressed as percent injected activity per mL of tissue (%ID/ml_).
Example 12. Direct counting of tissue radioactivity. After the 60 min PET scan, a part of
the tumor tissue was obtained from mice after exsanguination via cardiac puncture under
general isoflurane anesthesia. All samples were weighed and their radioactivity directly
determined using a Cobra I I Auto-gamma counter (formerly Packard Instruments Meriden CT
USA) applying a decay correction. The results were expressed as a percentage of the injected
dose per gram (%ID/g).
Example 13. Statistics. Statistical analyses were performed using the software GraphPad
Prism, version 4.00 (GraphPad, San Diego, CA). Between-group comparisons were made
using the nonparametric Mann-Whitney test. Two-tailed P value < 0.05 were considered
significant.
Results
Radiotracers. All radiotracers were successfully prepared with >98 % radiochemical purity.
The total synthesis time was ~ 1 .5 h . The lipophilicity (Log D) of the radiotracers was
measured using methods known in the art (Barthel, H., etal., Br J Cancer. 2004;90:2232-
2242) and the measured Log D is shown in Table 6 . Because the animals were scanned on
different days, the mean specific radioactivity for each radiotracer is also presented (Table 6).
Table 6. Comparison of tissue uptake of [ F]-octreotate analogs in AR42J tumor xenografts
growing in nude mice. Imaging data are presented for the 60 min time point together with data
obtained from counting pieces of tissue directly in a g-counter soon after the imaging study. In
vivo FET-pAG-TOCAblocking studies were done after i.v. injection of unlabelled octreotide
(10 mg/kg) to mice followed 10 min later by injection of the radiotracer. Data are mean ± SE,
n=3-6.
Octreotate analog Log D Specific Tumor Tumor Tumor
radio¬ radiotracer radiotracer
activity studied uptake at 60 uptake after
(GBq/ min by 60 min by g-
imaging counting
mitioI)* (%ID/ml) (%ID/g)
FET-G-PEG-TOCA -2.68 4.8 AR42J 5.36 ± 0.45 8.29 ± 1.42
FETE-PEG-TOCA -2.77 5.9 AR42J 5.14 ± 0.40 9.78 ± 2.57
FET-G-TOCA - 1 .82 5.9 AR42J 11.0 ± 1.49 17.04 ± 2.76
FETE-TOCA - 1 .50 8.4 AR42J 6.11 ± 1.46 4.50 ± 1.51
FET-PAG-TOCA -2.06 3.9 AR42J 8.23 ± 2.02 11.58 ± 0.67
FET-PAG-TOCA -2.06 18.7 HCT1 16 2.42 ± 0.35 0.52 ± 0.39
FET-PAG-TOCA blocking -2.06 11.2 AR42J 6.24 ± 0.64 3.93 ± 0.99
FET-PAG-[W-c-(CTFTYC)K] - 1 . 1 4 12.3 AR42J 0.10 ± 0.05 0.22 ± 0.12
[ 8 F]AI F-NOTA-OC ND 36. 1 AR42J 6.43 ± 0.85 12.73 ± 0.05
[ 8Ga]DOTA-TATE ND ND AR42J 2.75 ± 0.11 3.78 ± 0.32
* Determined at the end of synth
ND: Not determined
In vitro sstr-subtype specificity. All the [ F]fluoroethyltriazole-[Tyr ]octreotate analogs
exhibited agonist activity on sstr-2 (Figure 14), with the scrambled peptide, (FET-pAG-[W-c-
(CTFTYC)K]), having expectedly poor affinity. The affinity of the ligands (EC50) ranged
between 4 and 19 nM (versus somatostatin at 5.6 nM) with the polyethylene glycol (PEG)-
TOCA analogs showing the lowest affinity to sstr-2. Antagonistic activity was not evaluated
because the analogs possessed significant agonist activity. None of the compounds exhibited
detectable activity against sstr-3. All [ F]fluoroethyltriazole-[Tyr ]octreotate analogs except the
scrambled peptide showed detectable activity against sstr-4 but the affinity was very poor (³
5.4 mM). In vitro studies revealed that the triazole analogs had high selective affinity to sstr-2
with half-maximal agonist activity in the calcium flux assay for this G-protein coupled receptor
(EC50) ranging from 4 to 19 nM, compared to the scrambled peptide, which had a low affinity.
FET-pAG-TOCA is stable in vivo. The in vivo stability of FET-pAG-TOCA was examined.
Typical radio-chromatograms of dose solution and 30 min mouse plasma are shown in Figure
15. No metabolites of FET-pAG-TOCA were seen; only intact parent tracer was found.
The combined effect of high binding affinity for sstr-receptor and rapid washout from nontarget
tissue produced high-contrast PET images in vivo, demonstrated for FET-pAG-TOCA in
Figure 16. The increasing tumor time versus activity curves of FET-pAG-TOCA derived from
the in vivo dynamic PET image data (Figure 17) reflected the high selective binding of FETPAG-
TOCA. The radiotracer was metabolically stable in mice and had low bone uptake
indicating no significant defluorination.
Pharmacokinetics and in vivo tumor localization of [ 8F]fluoroethyltriazole-
[Tyr3]octreotate analogs. Given the high affinity and systemic stability of FET-pAG-TOCA, it
was reassuring to observe good localization of the radiotracer in tumor. Figure 16a shows
typical transverse and sagittal PET image slices through sstr-2 expressing AR42J tumor
bearing mice demonstrating localization of FET-pAG-TOCA in tumor, kidney and bladder/urine
with high signal-to-background contrast. In contrast, no radiotracer localization was seen in
tumor and kidneys of the mice when the scrambled peptide FET-pAG-[W-c-(CTFTYC)K] was
injected (Figure 16b). In this case, tracer localization was seen mainly in brain, urine, liver and
intestines (data not shown). The comparative pharmacokinetics of all [ F]fluoroethyltriazole-
[Tyr ]octreotate analogs in tumor, kidney, liver, muscle and bladder/urine are shown in Figure
17. Radiotracer uptake in the AR42J tumor was characterized by a rapid increase over the
entire scanning period of 60 min. FET-G-TOCA had the highest tumor uptake followed by
FET-pAG-TOCA, which had higher or comparable uptake as [ F]-AIF-NOTA-OC. These
tracers were superior to the clinical radiotracer, [ Ga]-DOTATATE, with respect to tumor
uptake (Table 6). PEG-linkers, embodied within the structures of FET-G-PEG-TOCA and
FETE-PEG-TOCA, reduced tumor uptake (Figure 17; Table 6). Non-specific uptake in liver
was in general low (<7 %ID/ml_) with FET-pAG-TOCA showing the lowest liver uptake; FETG-
TOCA and FETE-PEG-TOCA showed the highest liver uptake. The PEG-TOCA analogs
had the highest urinary clearance in keeping with their lower lipophilicity. Radiotracer kinetic
profiles in kidney which also expresses sstr- (Bates, CM, etal., Kidney Int. 2003;63:53-63)
were different from those in tumors, however, the magnitude of uptake was highest for the two
radiotracers, FET-pAG-TOCA and FET-G-TOCA; [ F]-AIF-NOTA-octreotide had relatively low
kidney uptake. The mean muscle uptake was < 3 %ID/ml_ for all radiotracers. Radiotracer
uptake in the bone was low for all the analogs indicating little/no defluorination. We compared
the uptake of the radiotracers in the imaging studies to direct tissue counting. The profiles
were generally in agreement, but the magnitude was higher for direct counting, consistent with
partial volume averaging. Direct radioactivity determination (gamma counting) of only a part of
the tissue compared to sampling of the whole tumor in the case of imaging could also have
led to systematic differences.
The uptake of FET-pAG-TOCA is specific. Given the high tumor uptake of the radiotracers,
we next assessed the specificity of uptake in vivo using FET-pAG-TOCA as the prototypical
[ F]fluoroethyltriazole-[Tyr ]octreotate. We demonstrated that radiotracer uptake was specific:
i) In keeping with the poor affinity for sstr-2, the radiolabeled scrambled peptide, (FET-pAG-
[W-c-(CTFTYC)K]), did not show detectable tumor uptake in the AR42J model in vivo (Figure
18).-FET-pAG-[W-c-(CTFTYC)K] uptake was also low in the high sstr-expressing normal
tissue (kidneys), and uptake was higher in liver compared to FET-pAG-TOCA. ii) To show that
the tumor uptake of radiotracer was receptor-mediated, blocking studies were conducted by
pre-injecting mice with excess unlabelled octreotide (100-fold molar equivalent) to saturate
sstr-binding sites. This resulted in a 2-fold (by direct counting) lower uptake of FET-pAGTOCA
in AR42J xenografts (Figure 18; Table 6). Following blocking with unlabelled
octreotide, kidney (early time points only), muscle and to a smaller extent liver radioactivity
concentrations increased and urine radioactivity decreased (Figure 18). iii) Further evidence
for the specificity of FET-pAG-TOCA uptake was provided by the low uptake in low sstrexpressing
HCT1 16 xenografts compared to the AR42J xenografts (Table 6).
Interestingly, the tumor uptake for FET-G-TOCA and FET-pAG-TOCA compounds ranked
amongst the highest reported to date and were higher than that of [ Ga]-DOTA-TATE (Table
6), which is used clinically. Similarly the tumor uptake of the two [ F]-fluoroethyltriazole-[Tyr] 3-
octreotate analogs was significantly higher than those reported for [ ln]-DTPA-octreotide by
Froidevaux et al., (3.03 ± 0.26 %ID/g) in the same tumor model (Froidevaux, S., etal.,
Endocrinology. 2000;141 :3304-3312). The tracers also showed similar or higher uptake
compared to [ F]-AIF-NOTA-OC. In contrast to the high tumor uptake of FET-G-TOCA and
FET-pAG-TOCA, the two PEGylated analogs showed lower tumor (and kidney) uptake. This
was an unexpected finding given that PEGylation of peptides often increases the half-life and
generally reduces the overall clearance from the body (Veronese, F.M., etal., Drug Discov
Today. 2005;10:1451-1458). This finding may be explained in part by the fact that one of the
properties of PEGylation is also to make the molecule more water soluble (Veronese, F.M., et
al., BioDrugs. 2008;22:31 5-329), supporting a faster clearance from the circulation compared
to the less hydrophilic non-PEGylated analogs; supported by high urinary clearance (Figure
17f). The low uptake of the PEG-TOCA analogs could also be explained by their lower in vitro
affinity (Figure 14).
It was predicted that tissues such as liver and muscle that lacked receptor expression
(Reynaert, H., etal., Gut. 2004;53:1 180-1 189) will show low uptake of the radiotracers. The
PEG-TOCA analogs showed lower non-target tissues uptake. It is likely that the higher
hydrophilicity resulting from PEGylation in this series leads to more rapid elimination from nontarget
tissues. The time course from the PET studies allowed this effect to be quantified.
Interestingly, FET-pAG-TOCA with intermediate hydrophilicity compared to the PEGylated
analogs (Table 6), showed similar low uptake in non-target tissues, including liver and muscle.
This is a positive attribute of FET-pAG-TOCA that was not realized in FET-G-TOCA, which
had the highest tumor uptake.
Imaging
A triazole linked [Tyr ]octreotate analogue or a 2-[ F]fluoroethyl triazole linked
[Tyr ]octreotate analogue of the invention can be used as a radiotracer or an imaging agent
for those disease states or tumors that exhibit increased or high levels of somatostatin
receptors.
In one embodiment, a 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the
invention can be used as a PET radiotracer for those disease states or tumors that exhibit
increased or high levels of somatostatin receptors. In one embodiment, a 2-[ F]fluoroethyl
triazole linked [Tyr ]octreotate analogue of the invention can be used as a PET radiotracer
that is useful for the in vivo detection of neuroendocrine tumours which are known to express
increased levels of somatostatin receptors. In one embodiment, a 2-[ F]fluoroethyl triazole
linked [Tyr ]octreotate analogue of the invention can be used as a PET radiotracer that is
useful for the detection of lung tumors that express high levels of somatostatin receptors.
Therefore in one aspect, the present invention provides a PET imaging method to
determine the distribution and/or the extent of a disease state or a tumor that exhibits
increased or high levels of somatostatin receptors, wherein said method comprises:
i) administering to said subject a 2-[ F]fluoroethyl triazole linked
[Tyr ]Octreotate analogue(s) of the invention as described herein;
ii) allowing said 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue(s)
of the invention to bind to the somatostatin receptor(s) in said subject;
iii) detecting signals emitted by the F comprised in said 2-[ F]fluoroethyl
triazole linked [Tyr ]Octreotate analogue(s) of the invention; and
iv) generating an image representative of the location and/or amount of said
signals.
The method optionally further comprises the step of determining the distribution and extent of
the disease state in said subject wherein said distribution and extent of disease state is
directly correlated with said signals.
In another aspect, the present invention provides a PET imaging method to determine
the distribution and/or the extent of a or multiple neuroendocrine tumors in a subject, wherein
said method comprises:
i) administering to said subject a 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate
analogue(s) of the invention as described herein;
ii) allowing said 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue(s) of
the invention to bind to the somatostatin receptor(s) on the surface of said
neuroendocrine tumour(s) in said subject;
iii) detecting signals emitted by the 18F comprised in said 2-[ F]fluoroethyl
triazole linked [Tyr3]Octreotate analogue(s) of the invention; and
iv) generating an image representative of the location and/or amount of said
signals.
The method optionally further comprises the step of determining the distribution and extent of
neuroendocrine tumour(s) in said subject wherein said distribution and extent of
neuroendocrine tumour(s) is directly correlated with said signals.
In another aspect, the present invention provides a PET imaging method to determine
the distribution and/or the extent of a or multiple lung tumors in a subject, wherein said
method comprises:
i) administering to said subject a 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate
analogue(s) of the invention as described herein;
ii) allowing said 2-[ F]fluoroethyl triazole linked [Tyr ]Octreotate analogue(s) of
the invention to bind to the somatostatin receptor(s) on the surface of the lung
tumour(s) in said subject;
iii) detecting signals emitted by the F comprised in said 2-[ F]fluoroethyl triazole
linked [Tyr ]Octreotate analogue(s) of the invention; and
iv) generating an image representative of the location and/or amount of said
signals.
The method optionally further comprises the step of determining the distribution and extent of
lung tumour(s) in said subject wherein said distribution and extent of lung tumour(s) is directly
correlated with said signals.
The step of "administering " the 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue of the invention is preferably carried out parenterally, and most preferably
intravenously. The intravenous route represents the most efficient way to deliver the 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention throughout the body of
the subject. Intravenous administration neither represents a substantial physical intervention
nor a substantial health risk to the subject. The 2-[ F]fluoroethyl triazole linked
[Tyr ]octreotate analogue of the invention is preferably administered as the
radiopharmaceutical composition of the invention, as defined herein. The administration step
is not required for a complete definition of the PET imaging method of the invention. As such,
the PET imaging method of the invention can also be understood as comprising the abovedefined
steps (ii)-(v) carried out on a subject to whom the 2-[ F]fluoroethyl triazole linked
[Tyr ]octreotate analogue of the invention has been pre-administered.
Following the administering step and preceding the detecting step, the 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention is allowed to bind to
somatostatin receptor(s). For example, when the subject is an intact mammal, the 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention will dynamically move
through the mammal's body, coming into contact with various tissues therein. Once the 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention comes into contact
with somatostatin receptor(s), a specific interaction takes place such that clearance of the 2-
[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of the invention from tissue with
somatostatin receptor(s) takes longer than from tissue without, or with less somatostatin
receptor(s). A certain point in time will be reached when detection of 2-[ F]fluoroethyl triazole
linked [Tyr ]octreotate analogue of the invention specifically bound to somatostatin receptor(s)
is enabled as a result of the ratio between PET radiotracer bound to tissue with somatostatin
receptor(s) versus that bound in tissue without, or with less somatostatin receptor(s).
The "detecting " step of the method of the invention involves detection of signals
emitted by the F comprised in the 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue
of the invention by means of a detector sensitive to said signals, i.e. a PET camera. This
detection step can also be understood as the acquisition of signal data.
The "generating " step of the method of the invention is carried out by a computer which
applies a reconstruction algorithm to the acquired signal data to yield a dataset. This dataset
is then manipulated to generate images showing the location and/or amount of signals emitted
by the F. The signals emitted directly correlate with the expression of somatostatin
receptor(s) such that the "determining " step can be made by evaluating the generated image.
The "subject" of the invention can be any human or animal subject. Preferably the
subject of the invention is a mammal. Most preferably, said subject is an intact mammalian
body in vivo. In an especially preferred embodiment, the subject of the invention is a human.
The in vivo imaging method may be used to study somatostatin receptor(s) in healthy
subjects, or in subjects known or suspected to have a pathological condition associated with
abnormal expression of somatostatin receptor(s).
In an alternative embodiment, the PET imaging method of the invention may be carried
out repeatedly during the course of a treatment regimen for said subject, said regimen
comprising administration of a drug to combat a neuroendocrine tumor. For example, the PET
imaging method of the invention can be carried out before, during and after treatment with a
drug to combat a a neuroendocrine tumor. In this way, the effect of said treatment can be
monitored over time. PET is particularly well-suited to this application as it has excellent
sensitivity and resolution, so that even relatively small changes in a lesion can be observed
over time, a particular advantage for treatment monitoring.
All patents, journal articles, publications and other documents discussed and/or cited
above are hereby incorporated by reference.
Claims:
1. A 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue of Formula (III):
R LINKER-R2 (III)
wherei
R2 has the following structure:
LINKER is a linker group of formula -(A)m- wherein each A is independently -CR , -
CR=CR- , Cº C , -CR2C0 2- , -C0 2CR2- , -NRCO- , -CONR- , -NR(C=0)NR-, -
NR(C=S)NR-, -S0 2NR- , -NRS0 2- , -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a d -
scycloheteroalkylene group, a C4-8cycloalkylene group, a C5 - i2arylene group, or a d -
i2heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG)
building block; each R is independently chosen from H, d-^alkyl, C2.4alkenyl, C2.4alkynyl, d
4alkoxy d-^alkyl or hydroxyd-^alkyl; m is an integer of value 1 to 20.
2 . A 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue selected from the group
consisting of:
FET-G-PEG-TOCA, of Formula (lb) :
FETE-PEG-TOCA, of Formula (2b):
FET-G-TOCA (3b):
FETE-TOCA (4b):
- AG-TOCA (5b):
R2 has the following structure:
3 . A 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue according to Claim 1 or 2,
wherein, R2 is:
4 . A 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue, FET-3AG-[W-c—
(CTFTYC)K] (7b):
wherein F is
5 . A 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue according
wherein R3 is:
6 . A pharmaceutical composition comprising at least one compound of claims 1 -5 and a
pharmaceutically acceptable carrier, excipient, or biocompatible carrier.
7 . An alkyne linked [Tyr ]octreotate analogue of Formula (IV):
F LINKER-R2 (IV)
wherein:
R2 has the following structure:
LINKER is a linker group of formula -(A)m- wherein each A is independently -CR , -
CR=CR -C=C , -CR2C0 2- , -C0 2CR2- , -NRCO- , -CONR- , -NR(C=0)NR-,
NR(C=S)NR-, -S0 2NR- , -NRS0 2- , -CR2OCR2- , -CR2SCR2- , -CR2NRCR2- , a d -
scycloheteroalkylene group, a C4-8cycloalkylene group, a C5 - i2arylene group, or a d -
i2heteroarylene group, an amino acid, a sugar or a monodisperse polyethyleneglycol (PEG)
building block; each R is independently chosen from H, d-^alkyl, C2.4alkenyl, C2.4alkynyl, d
4alkoxy d-^alkyl or hydroxyd-^alkyl; m is an integer of value 1 to 20.
8 . An alkyne linked [Tyr ]octreotate analogue selected from the group consisting of:
G-PEG-TOCA, of Formula (la):
E-PEG-TOCA, of Formula (2a):
G-TOCA (3a) :
E-TOCA (4a) :
3AG-TOCA
wherein for each of the compounds above:
R2 has the following structure:
9 . An alkyne linked [Tyr ]octreotate analogue according to Claim 7 or 8, wherein R2 is:
10 . An alkyne linked [Tyr ]octreotate analogue, 3AG-[W-c— (CTFTYC)K] (6a):
H
wherein is and R3 is:
11. An alkyne erein R3 is:
1 . A method of making comprising the step of reacting an alkyne linked [Tyr ]Octreotate
analogue(s) with 2-[18F]Fluoroethylazide under copper catalyzed click chemistry conditions to
form the corresponding 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate analogue.
13. The method of claim 12, wherein said alkyne linked [Tyr ]Octreotate analogue(s) is a
compound of claim 7 and said corresponding 2-[ F]fluoroethyl triazole linked [Tyr ]octreotate
analogue is a compound of claim 1.
14. A method of imaging comprising the steps of administering a compound of claim 1 to a
subject and detecting said compound in said subject.
15 . A method of detecting a disease state or a tumor that exhibits increased or high levels
of somatostatin receptors in vivo in a subject comprising the steps of:
(i) administering to said subject a compound of claim 1 or a pharmaceutical
composition thereof;
(ii) allowing said compound or pharmaceutical composition thereof to bind to
somatostatin receptors found in said subject;
(iii) detecting signals emitted by the radioisotope in said compound or
pharmaceutical composition thereof;
(iv) generating an image representative of the location and/or amount of said
signals; and, optionally,
(v) determining the distribution and extent of said disease state in said subject.
16. The method of detecting neuroendocrine tumor(s) in vivo in a subject comprising the
steps of:
(i) administering to said subject a compound of claim 1 or a pharmaceutical
composition thereof;
(ii) allowing said compound or pharmaceutical composition thereof to bind to
somatostatin receptor(s) found on the surface of the neuroendocrine tumour(s) in
said subject;
(iii) detecting signals emitted by the radioisotope in said compound or
pharmaceutical composition thereof;
(iv) generating an image representative of the location and/or amount of said
signals; and, optionally,
(v) determining the distribution and extent of said neuroendocrine tumour(s) in said
subject.
17. The method of detecting lung tumor(s) in vivo in a subject comprising the steps of:
(i) administering to said subject a compound of claim 1 or a pharmaceutical
composition thereof;
(ii) allowing said compound or pharmaceutical composition thereof to bind to
somatostatin receptor(s) found on the surface of the lung tumour(s) in said subject;
(iii) detecting signals emitted by the radioisotope in said compound or
pharmaceutical composition thereof;
(iv) generating an image representative of the location and/or amount of said
signals; and, optionally,
(v) determining the distribution and extent of said lung tumour(s) in said subject.