ISOTOPIC CARBON CHOLINE ANALOGS
Field of the Invention
The present invention describes a novel radiotracer(s) for Positron Emission
Tomography (PET) or Single Photon Emission Computed Tomography (SPECT)
imaging of disease states related to altered choline metabolism (e.g., tumor imaging of
prostate, breast, brain, esophageal, ovarian, endometrial, lung and prostate cancer -
primary tumor, nodal disease or metastases). 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 Related Art
The biosynthetic product of choline kinase (EC 2.7.1.32) activity, phosphocholine, is
elevated in several cancers and is a precursor for membrane phosphatidylcholine
(Aboagye, E.O., et al., Cancer Res 1999; 59:80-4; Exton, J.H., Biochim Biophys Acta
1994; 1212:26-42; George, T.P., et al, Biochim Biophys Acta 1989; 104:283-91; and
Teegarden, D., et al, J Biol Chem 1990; 265(1 1):6042-7). Over-expression of
choline kinase and increased enzyme activity have been reported in prostate, breast,
lung, ovarian and colon cancers (Aoyama, C , et al, Prog Lipid Res 2004; 43(3):266-
81; Glunde, K., et al, Cancer Res 2004; 64(12):4270-6; Glunde, K., et al, Cancer
Res 2005; 65(23): 11034-43; Iorio, E., et al, Cancer Res 2005; 65(20): 9369-76;
Ramirez de Molina, A., et al, Biochem Biophys Res Commun 2002; 296(3): 580-3;
and Ramirez de Molina, A., et al, Lancet Oncol 2007; 8(10): 889-97) and are largely
responsible for the increased phosphocholine levels with malignant transformation
and progression; the increased phosphocholine levels in cancer cells are also due to
increased breakdown via phospholipase C (Glunde, K., et al, Cancer Res 2004;
64(12):4270-6).
Because of this phenotype, together with reduced urinary excretion, [ C]choline has
become a prominent radiotracer for positron emission tomography (PET) and PETComputed
Tomography (PET-CT) imaging of prostate cancer, and to a lesser extent
imaging of brain, esophageal, and lung cancer (Hara, T., et al, J Nucl Med 2000;
41:1507-13; Hara, T., etal, J Nucl Med 1998; 39:990-5; Hara, T., etal, J Nucl Med
1997; 38:842-7; Kobori, O., et al, Cancer Cell 1999; 86:1638-48; Pieterman, R.M.,
et al. , J Nucl Med 2002; 43(2): 167-72; and Reske, S.N. Eur J Nucl Med Mol Imaging
2008; 35:1741). The specific PET signal is due to transport and phosphorylation of
the radiotracer to [ C]phosphocholine by choline kinase.
Of interest, however, is that [ CJcholine (as well as the fluoro-analog) is oxidized to
[ C]betaine by choline oxidase (see Figure 1 below)(EC 1.1.3. 17) mainly in kidney
and liver tissues, with metabolites detectable in plasma soon after injection of the
radiotracer (Roivainen, A., et al. , European Journal of Nuclear Medicine 2000;
27:25-32). This makes discrimination of the relative contributions of parent
radiotracer and catabolites difficult when a late imaging protocol is used.
Lipid
+ h Phosphorylation + P0 3
2 Incorporation NH,"C
C-Choline ne
Excretion
betaine
Figure 1. Chemical structures of major choline metabolites and their pathways.
[ F]Fluoromethylcholine ([ F]FCH):
FCH
was developed to overcome the short physical half-life of carbon- 11 (20.4 min)
(DeGrado, T.R., et al. , Cancer Res 2001 ; 61(1): 110-7) and a number of PET and
PET-CT studies with this relatively new radiotracer have been published (Beheshti,
M., et al. , Eur J Nucl Med Mol Imaging 2008;35(10): 1766-74; Cimitan, M., et al ,
Eur J Nucl Med Mol Imaging 2006; 33(12): 1387-98; de Jong, I.J., et al , Eur Nucl
Med Mol Imaging 2002; 29: 1283-8; and Price, D.T., et al. , J Urol 2002; 168(1):273-
80). The longer half-life of fluorine-18 (109.8 min) was deemed potentially
advantageous in permitting late imaging of tumors when sufficient clearance of parent
tracer in systemic circulation had occurred (DeGrado, T.R., et al., J Nucl Med 2002;
43(l):92-6).
WO2001/82864 describes 18F-labeled choline analogs, including
[18F]Fluoromethylcholine ([18FJ-FCH) and their use as imaging agents {e.g., PET)
for the non-invasive detection and localization of neoplasms and pathophysiologies
influencing choline processing in the body (Abstract). WO200 1/82864 also describes
18F-labeled di-deuterated choline analogs such as [18F]fluoromethyl-[l- 2H2]choline
([ F]FDC)(hereinafter referred to as "[ F]D2-FCH"):
FDC
The oxidation of choline under various conditions; including the relative oxidative
stability of choline and [l,2- H 4]choline has been studied (Fan, F., et al, Biochemistry
2007, 46, 6402-6408; Fan, F., et a , Journal of the American Chemical Society 2005,
127, 2067-2074; Fan, F., et al., Journal of the American Chemical Society 2005, 127,
17954-17961; Gadda, G. Biochimica et Biophysica Acta 2003, 1646, 112-118; Gadda,
G., Biochimica et Biophysica Acta 2003, 1650, 4-9). Theoretically the effect of the
extra deuterium substitution was found to be neglible in the context of a primary
isotope effect of 8-10 since the b-secondary isotope effect is -1.05 (Fan, F., et al.,
Journal of the American Chemical Society 2005, 127, 17954-17961).
[18FJFluoromethylcholine is now used extensively in the clinic to image tumour status
(Beheshti, M., et al, Radiology 2008, 249, 389-90; Beheshti, M., et al, Eur J Nucl
MedMol Imaging 2008, 35, 1766-74).
The present invention, as described below, provides a novel C-radiolabeled
radiotracer that can be used for PET imaging of choline metabolism and exhibits
increased metabolic stability and a favourable urinary excretion profile.
Brief Description of the Drawings
Figure 1 depicts the chemical structures of major choline metabolites and their
pathways.
Figure 3 shows NMR analysis of tetradeuterated choline precursor. Top, H NMR
spectrum; bottom, C NMR spectrum. Both spectra were acquired in CDCI 3 .
Figure 4 depicts the HPLC profiles for the synthesis of [ F]fluoromethyl tosylate (9)
and [ F]fluoromethyl- [1,2- H4]choline (D4-FCH) showing (A) radio-HPLC profile
for synthesis of (9) after 15 mins; (B) UV (254 nm) profile for synthesis of (9) after
15 mins; (C) radio-HPLC profile for synthesis of (9) after 10 mins; (D) radio-HPLC
profile for crude (9); (E) radio-HPLC profile of formulated (9) for injection; (F)
refractive index profile post formulation (cation detection mode).
Figure 5a is a picture of a fully assembled cassette of the present invention for the
production of [18F]fluoromethyl-[l,2- 2H4]choline (D4-FCH) via an unprotected
precursor.
Figure 5b is a picture of a fully assembled cassette of the present invention for the
production of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) via a PMB-protected
precursor.
Figure 6 depicts representative radio-HPLC analysis of potassium permanganate
oxidation study. Top row are control samples for [ F]fluoromethylcholine
([ F]FCH) and [ F]fluoromethyl-[l,2- H4]choline ([ F]D4-FCH), extracts from the
reaction mixture at time zero (0 min). Bottom row are extracts after treatment for 20
mins. Left hand side are for [18F]fluoromethylcholine ([18F]FCH), right are for [18F]
fluoromethyl- [1,2- H4]choline ([ F]D4-FCH).
Figure 7 shows chemical oxidation potential of [18FJfluoromethylcholine and
[18F]fluoromethyl-[l,2- 2H4]choline in the presence of potassium permanganate.
Figure 8 shows time-course stability assay of [ FJfluoromethylcholine and
[18F]fluoromethyl-[l,2- 2H4]choline in the presence of choline oxidase demonstrating
conversion of parent compounds to their respective betaine analogues.
Figure 9 shows representative radio-HPLC analysis of choline oxidase study. Top
row are control samples for [18FJfluoromethylcholine and [18F]fluoromethyl-[l,2-
H4]choline, extracts from the reaction mixture at time zero (0 min). Bottom row are
extracts after treatment for 40 mins. Left hand side are of [ F]fluoromethylcholine,
right are of [18F]fluoromethyl- [1,2-2H4]choline.
Figure 10. Top: Analysis of the metabolism of [ F]fluoromethylcholine (FCH) to
[ F]FCH-betaine and [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) to [ F]D4-FCHbetaine
by radio-HPLC in mouse plasma samples obtained 15 min after injecting the
tracers i.v. into mice. Bottom: summary of the conversion of parent tracers,
[ F]fluoromethylcholine (FCH) and [ F]fluoromethyl-[l,2- H4]choline (D4-FCH), to
metabolites, [ F]FCH-betaine (FCHB) and [ F]D4-FCH betaine (D4-FCHB), in
plasma.
Figure 11. Biodistribution time course of [ F]fluoromethylcholine (FCH),
[ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2- H4]choline
(D4-FCH) in HCT-116 tumor bearing mice. Inset: the time points selected for
evaluation. A) Biodistribution of [ F]fluoromethylcholine; B) biodistribution of
[ F]fluoromethyl-[l- H 2]choline; C) biodistribution of [ F]fluoromethyl-[l,2-
2H4]choline; D) time course of tumor uptake for [18FJfluoromethylcholine (FCH),
[ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2- H4]choline
(D4-FCH) from charts A-C. Approximately 3.7 MBq of [ F]fluoromethylcholine
(FCH), [ F]fluoromethyl-[l- H2]choline (D2-FCH) and [ F]fluoromethyl-[l,2-
H4]choline (D4-FCH) injected into awake male C3H-Hej mice which were sacrificed
under isofluorane anesthesia at the indicated time points.
Figure 12 shows radio-HPLC chromatograms to show distribution of choline
radiotracer metabolites in tissue harvested from normal white mice at 30 min p.i. Top
row, radiotracer standards; middle row, kidney extracts; bottom row, liver extracts.
On the left is [ F]FCH, on the right [ F]D4-FCH.
Figure 13 show radio-HPLC chromatograms to show metabolite distribution of
choline radiotracers in HCT1 16 tumors 30 min post-injection. Top-row, neat
radiotracer standards; bottom row, 30 min tumor extracts. Left side, [18F]FCH;
middle, [ F]D4-FCH; right, [ C]choline.
Figure 14 shows radio-HPLC chromatograms for phosphocholine HPLC validation
using HCT116 cells. Left, neat [ F]FCH standard; middle, phosphatase enzyme
incubation; right, control incubation.
Figure 15 shows distribution of radiometabolites for [ F]fluoromethylcholine
analogs: F]fluoromethylcholine, [ F]fluoromethyl-[l- H 2]choline and
[18F]fluoromethyl-[l,2- 2H4]choline at selected time points.
Figure 16 shows tissue profile of [ F]FCH and [ F]D4-FCH. (a) Time versus
radioactivity curve for the uptake of [ F]FCH in liver, kidney, urine (bladder) and
muscle derived from PET data, and (b) corresponding data for [18FJD4-FCH. Results
are the mean + SE; n = 4 mice. For clarity upper and lower error bars (SE) have been
used. (Leyton, et al, Cancer Res 2009: 69:(19), pp 7721-7727).
Figure 17 shows tumor profile of [ F]FCH and [ F]D4-FCH in SKMEL28 tumor
xenograft (a) Typical [ F]FCH-PET and [ F]D4-FCH-PET images of SKMEL28
tumor-bearing mice showing 0.5 mm transverse sections through the tumor and
coronal sections through the bladder. For visualization, 30 to 60 min summed image
data are displayed. Arrows point to the tumors (T), liver (L) and bladder (B). (b).
Comparison of time versus radioactivity curves for [ F]FCH and [ F]D4-FCH in
tumors. For each tumor, radioactivity at each of 19 time frames was determined. Data
are mean %ID/vox6omean + SE (n = 4 mice per group) (c) Summary of imaging
variables. Data are mean + SE, n = 4; *P = 0.04. For clarity upper and lower error bars
(SE) have been used.
Figure 18 shows the effect of PD0325901, a mitogenic extracellular kinase inhibitor,
on uptake of [18FJD4-FCH in HCT1 16 tumors and cells (a) Normalized time versus
radioactivity curves in HCT116 tumors following daily treatment for 10 days with
vehicle or 25mg/kg PD0325901. Data are the mean + SE; n = 3 mice (b) Summary of
imaging variables %ID/vox 6o, %ID/vox60max, and AUC. Data are mean + SE; * P =
0.05.(c) Intrinsic cellular effect of PD0325901 (ImM ) on [ F]D4-FCH
18 phosphocholine metabolism after treating HCTl 16 cells for 1 hr with [ FJD4-FCH in
culture. Data are mean + SE; n=3 ; * P = 0.03.
Figure 19 shows expression of choline kinase A in HCTl 16 tumors (a) A typical
Western blot demonstrating the effect of PD0325901 on tumor choline kinase A
(CHKA) protein expression. HCTl 16 tumors from mice that were injected with
PD0325901 (25mg/kg daily for 10 days, orally) or vehicle were analyzed for CHKA
expression by western blotting b-actin was used as the loading control (b) Summary
densitometer measurements for CHKA expression expressed as a ratio to b-actin. The
results are the mean ratios + SE; n = 3, * P = 0.05.
Figure 20 shows biodistribution time course of C-choline, C-D4-choline and FD4-
choline in BALB/c nude mice. Approximately 18.5 MBq of C-labeled tracer or
18 3.7 MBq of F was administered i.v. into anaesthetized animals prior to sacrifice at
indicated time points. Tissues were excised, weighed and counted, with counts
normalized to injected dose/g wet weight tissue. Mean values n = 3) and SEM are
shown.
Figure 21 shows metabolic profile of C-choline, C-D4-choline and F-D4-choline
in the liver (A) and kidney (B) of BALB/c nude mice. Radiolabeled metabolite
profile was assessed at 2, 15, 30 and 60 min after i.v. injection of parent radiotracers
using radio-HPLC. Mean values n = 3) and SEM are shown. Abbreviations: Betaid,
betaine aldehyde; p-Choline, phosphocholine.
Figure 22 shows metabolic profile of C-choline, C-D4-choline and F-D4-choline
in HCTl 16 tumors. Radiolabeled metabolite profile in HCTl 16 tumor xenografts
was assessed at 15 min and 60 min after i.v. injection of parent radiotracers using
radio-HPLC. Mean values (n = 3) and SEM are shown. * P < 0.05; ** P < 0.01; *** P
< 0.001.
Figure 23 depicts C-choline (o), C-D4-choline (A) and F-D4-choline (■ ) PET
image analysis. HCT116 tumor uptake profiles were examined following 60 min
dynamic PET imaging. A, representative axial PET-CT images of HCT116 tumorbearing
mice (30 - 60 min summed activity) for C-choline, C-D4-choline and FD4-
choline. Tumor margins, indicated from CT image, are outlined in red. B, The
tumor time versus radioactivity curve (TAC). Mean + SEM n = 4 mice per group).
Figure 24 shows pharmacokinetics of C-choline, C-D4-choline and F-D4-
choline in HCT116 tumors. A, Modified compartmental modeling analysis, taking
into account plasma metabolites and their flux into the exchangeable space in tumor,
was used to derive K\, a measure of irreversible retention within the tumor. B, The
kinetic parameter, ¾, an indirect measure of choline kinase activity, was calculated
using a two site compartmental model as previously described (29, 30). C, Ratio of
betaine to phosphocholine in tumors. Metabolites were quantified by radio-HPLC at
15 and 60 min post injection of tracer. Mean values n = 4) and SEM are shown. * P
< 0.05; *** P < 0.001. Abbreviations: p-choline, phosphocholine.
Figure 25 shows dynamic uptake and metabolic stability of F-D4-choline in tumors
of different histological origin. A, The tumor time versus radioactivity curve (TAC)
obtained from 60 min dynamic PET imaging. Mean + SEM n = 3-5 mice per group).
B, Metabolic profile of 18F-D4-choline in tumors. Radiolabeled metabolite profile in
HCT116 tumor xenografts was assessed post PET imaging using radio-HPLC. Mean
values n = 3) and SEM are shown. C, Choline kinase expression in malignant
melanoma, prostate adenocarcinoma and colon carcinoma tumors. Representative
western blot from tumor lysates n = 3 xenografts per tumor cell line). Actin was
used as a loading control. Abbreviations: CKa, choline kinase alpha.
Figure 26 shows effect of tumor size on F-D4-choline uptake and retention. Tracer
uptake profiles were examined following 60 min dynamic PET imaging in PC3-M
tumors at 100 mm3 (·) and 200 mm3 (o). A, The tumor time versus radioactivity
curve using average decay-corrected counts. Mean + SEM n = 3-5 mice per group).
B, The tumor time versus radioactivity curve using the maximum voxel decaycorrected
counts. Mean + SEM n = 3-5).
Figure 27 shows analyte identification on radio-chromatograms. Representative
radio-chromatograms of 18F-D4-choline-treated HCT116 cell lysates. A, l h uptake
of 18F-D4-choline into HCT116 cells followed by cell lysis and l h incubation with
vehicle at 37°C. B, l h uptake of F-D4-choline into HCT116 cells followed by cell
lysis and l h incubation with alkaline phosphatase dissolved in vehicle. The labeled
peaks are: 1, 18F-D4-choline; 2, 18F-D4-phosphocholine.
Figure 28 shows choline oxidase treatment of F-D4-choline. A, Representative
radio-chromatogram of 18F-D4-choline. B, 18F-D4-choline chromatogram following
20 min treatment with choline oxidase. C, 18F-D4-choline chromatogram following 40
min treatment. The labelled peaks are: 1, F-D4-betainealdehyde; 2, F-D4-betaine;
3, F-D4-choline.
Figure 29 shows correlation between total kidney activity and % radioactivity
retained as phosphocholine. Data were derived from C-choline, C-D4-choline and
F-D4-choline uptake values and metabolism at 2, 15, 30 and 60 min post tracer
injection.
Figure 30 shows C-choline (o), C-D4-choline (A) and F-D4-choline (■ ) PET
imaging analysis in HCT116 tumors. The tumor time versus radioactivity curve
(TAC) over the initial 14 min of the dynamic PET scans to illustrate subtle variations
in tracer kinetics. Mean + SEM n =4 mice per group).
Figure 31 shows time course of F-D4-choline uptake in vitro in human melanoma
(·), prostate ( ▲ ) and colon (■) cancer cell lines. Uptake was measured in vehicletreated
(closed symbols) and hemicholinium-3-treated cells (5 mM; open symbols).
Mean values + SEM are shown n =3). Insert: representative western blot of choline
kinase-a expression in the three cell lines. Actin was used as a loading control.
Abbreviations: CKa, choline kinase alpha.
Figure 32 shows representative axial PET-CT images of PC3-M tumor-bearing mice
(summed activity 30 - 60 min) at 100 mm3 and 200 mm3 respectively. Tumor
margins, indicated from CT image, are outlined in red.
Summary of the invention
The present invention provides a compound of Formula (III):
(III)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2 ) m R8, -(CD2 ) m R8, -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen
selected from F, CI, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl
group; and
Q is an anionic counterion; with the proviso the compound of Formula (III) is
not C-choline.
Detailed Description of the Invention
The present invention provides a novel radiolabeled choline analog compound
of formula (I):
(I)
wherein:
Ri, R2, R , and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF2)mR , -CH(R )2, or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope; and
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine,
fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline,
fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutylcholine,
fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyldiethanol-
choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, 1,1-
dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-
dideuterofluoromethyl-propyl-choline, or an [ F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
Ri, R2, R , and R4 are each independently hydrogen;
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not fluoromethylcholine,
fluoromethyl-ethyl-choline, fluoromethyl-propyl-choline, fluoromethyl-butyl-choline,
fluoromethyl-pentyl-choline, fluoromethyl-isopropyl-choline, fluoromethyl-isobutylcholine,
fluoromethyl-sec-butyl-choline, fluoromethyl-diethyl-choline, fluoromethyldiethanol-
choline, fluoromethyl-benzyl-choline, fluoromethyl-triethanol-choline, or
an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
R and R2 are each hydrogen;
R and R 4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion;
with the proviso that said compound of formula (I) is not 1,1-
dideuterofluoromethylcholine, 1,1-dideuterofluoromethyl-ethyl-choline, 1,1-
dideuterofluoromethyl-propyl-choline, or an [18F] analog thereof.
In a preferred embodiment of the invention, a compound of Formula (I) is
provided wherein:
Ri, R2, R , and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
X and Y are each independently hydrogen, deuterium (D), or F;
Z is a halogen selected from F, CI, Br, and I or a radioisotope;
Q is an anionic counterion.
According to the present invention, when Z of a compound of Formula (I) as
described herein is a halogen, it can be a halogen selected from F, CI, Br, and I ;
preferably, F.
According to the present invention, when Z of a compound of Formula (I) as
described herein is a radioisotope (hereinafter referred to as a "radiolabeled
compound of Formula (I)"), it can be any radioisotope known in the art. Preferably, Z
is a radioisotope suitable for imaging (e.g., PET, SPECT). More preferably Z is a
radioisotope suitable for PET imaging. Even more preferably, Z is 18F, 7,6Br, 1'"2I3, 1"24 ,
or 125I . Even more preferably, Z is 18F .
According to the present invention, Q of a compound of Formula (I) as
described herein can be any anionic counterion known in the art suitable for cationic
ammonium compounds. Suitable examples of Q include anionic: bromide (Br ),
chloride (CI ), acetate (CH CH2C(0)0 ), or tosylate (OTos). In a preferred
embodiment of the invention, Q is bromide (Br ) or tosylate (OTos). In a preferred
embodiment of the invention, Q is chloride (CI ) or acetate (CH3CH2C(0)0 ) . In a
preferred embodiment of the invention, Q is chloride (CI ) .
According the invention, a preferred embodiment of a compound of Formula
(I) is the following compo
(la)
wherein:
Ri, R2, R3, and R4 are each independently deuterium (D);
R5, R6, and R 7 are each hydrogen;
X and Y are each independently hydrogen;
Z is F;
Q is c .
According to the invention, a preferred compound of Formula (la) is
[ F]fluoromethyl-[l,2- H4]-choline ([ F]-D4-FCH). [ F]-D4-FCH is a more
18 metabolically stable fluorocholine (FCH) analog. [ FJ-D4-FCH offers numerous
advantages over the corresponding 18F-non-deuterated and/or 18F-di-deuterated
analog. For example, [ F]-D4-FCH exhibits increased chemical and enzymatic
18 18 oxidative stability relative to [ FJfluoromethylcholine. [ FJ-D4-FCH has an
improved in vivo profile (i. e. , exhibits better availability for in vivo imaging) relative
to dideuterofluorocholine, [ F]fluoromethyl-[l- H2]choline, that is over and above
what could be predicted by literature precedence and is, thus, unexpected. [ F]-D4-
FCH exhibits improved stability and consequently will better enable late imaging of
18 tumors after sufficient clearance of the radiotracer from systemic circulation. [ F]-
D4-FCH also enhances the sensitivity of tumor imaging through increased availability
of substrate. These advantages are discussed in further detail below.
The present invention further provides a precursor compound of Formula (II):
(P)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF2)mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
The present invention further provides a method of making a precursor
compound of Formula (II).
The present invention provides a compound of Formula (III):
(HI)
wherein:
Ri, R2, R , and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen
selected from F, CI, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl
group; and
Q is an anionic counterion; with the proviso the compound of Formula (III) is
not C-choline.
According to the invention, C* of the compound of Formula (III) can be any
radioisotope of carbon. Suitable examples of C* include, but are not limited to, C,
C, and 4C. Q is a described for the compound of Formula (I).
In a preferred embodiment of the invention, a compound of Formula (III) is
provided wherein C* is C; X and Y are each hydrogen; and Z is F.
In a preferred embodiment of the invention, a compound of Formula (III) is
provided wherein C* is C; X, Y and Z are each hydrogen H; Ri, R2, R3, and R4 are
each deuterium (D); and R5, R6, and R 7 are each hydrogen ( C-[l,2- H4]choline or
" C-D4-choline".
Pharmaceutical or Radiopharmaceutical Composition
The present invention provides a pharmaceutical or radiopharmaceutical
composition comprising a compound for Formula (I), including a compound of
Formula (la), each as defined herein together with a pharmaceutically acceptable
carrier, excipient, or biocompatible carrier. According to the invention when Z of a
compound of Formula (I) or (la) is a radioisotope, the pharmaceutical composition is
a radiopharmaceutical composition.
The present invention further provides a pharmaceutical or
radiopharmaceutical composition comprising a compound for Formula (I), including a
compound of Formula (la), each as defined herein together with a pharmaceutically
acceptable carrier, excipient, or biocompatible carrier suitable for mammalian
administration.
The present invention provides a pharmaceutical or radiopharmaceutical
composition comprising a compound for Formula (III), as defined herein together
with a pharmaceutically acceptable carrier, excipient, or biocompatible carrier.
The present invention further provides a pharmaceutical or
radiopharmaceutical composition comprising a compound for Formula (III), as
defined 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 or excipient can be any pharmaceutically acceptable carrier or
excipient known in the art.
The "biocompatible carrier" can be any fluid, especially a liquid, in which a
compound of Formula (I), (la), or (III) 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.
The pharmaceutical or radiopharmaceutical composition 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 para-aminobenzoic acid). Where
a compound of Formula (I), (la), or (III) is provided as a radiopharmaceutical
composition, the method for preparation of said compound may further comprise the
steps required to obtain a radiopharmaceutical composition, e.g., removal of organic
solvent, addition of a biocompatible buffer and any optional further ingredients. For
parenteral administration, steps to ensure that the radiopharmaceutical composition is
sterile and apyrogenic also need to be taken. Such steps are well-known to those of
skill in the art.
Preparation of a Compound of the Invention
The present invention provides a method to prepare a compound for Formula
(I), including a compound of Formula (la), wherein said method comprises reaction of
the precursor compound of Formula (II) with a compound of Formula (Ilia) to form a
compound of Formula (I) (Scheme A):
(I)
Scheme A
wherein the compounds of Formulae (I) and (II) are each as described herein and the
compound of Formula (Ilia) is as follows:
ZXYC-Lg (Ilia)
wherein X, Y and Z are each as defined herein for a compound of Formula (I) and
"Lg" is a leaving group. Suitable examples of "Lg" include, but are not limited to,
bromine (Br) and tosylate (OTos). A compound of Formula (Ilia) can be prepared by
any means known in the art including those described herein.
Synthesis of a compound of Formula (Ilia) wherein Z is F; X and Y are both H
and the Lg is OTos (i.e., fluoromethyltosylate) can be achieved as set forth in Scheme
3 below:
CH2l'2 CH2OTos2 FCH2OTos
SCHEME 3
wherein: i : Silver p-toluenesulfonate, MeCN, reflux, 20 h;
ii: KF, MeCN, reflux, 1 h.
According to Scheme 3 above:
(a) Synthesis of methylene ditosylate
Commercially available diiodomethane can be reacted with silver tosylate
using the method of Emmons and Ferris, to give methylene ditosylate (Emmons,
W.D., et al, "Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of
Alkyl Sulfonates", Journal of the American Chemical Society, 1953; 75:225).
(b) Synthesis of cold Fluoromethyltosylate
Fluoromethyltosylate can be prepared by nucleophilic substitution of
Methylene ditosylate from step (a) using potassium fluoride/Kryptofix K222 in
acetonitrile at 80°C under standard conditions.
When Z is a radioisotope, the radioisotope can be introduced by any means
known by one of skill in the art. For example, the radioisotope [ F]-fluoride ion
(18F ) is normally obtained as an aqueous solution from the nuclear reaction
180(p,n) 18F and is made reactive by the addition of a cationic counterion and the
subsequent removal of water. Suitable cationic counterions should possess sufficient
solubility within the anhydrous reaction solvent to maintain the solubility of 18F .
Therefore, counterions that have been used include large but soft metal ions such as
rubidium or caesium, potassium complexed with a cryptand such as Kryptofix™, or
tetraalkylammonium salts. A preferred counterion is potassium complexed with a
cryptand such as Kryptofix™ because of its good solubility in anhydrous solvents and
enhanced 18F- reactivity. 18F can also be introduced by nucleophilic displacement of a
suitable leaving group such as a halogen or tosylate group. A more detailed
discussion of well-known 18F labelling techniques can be found in Chapter 6 of the
"Handbook of Radiopharmaceuticals" (2003; John Wiley and Sons: M.J. Welch and
C.S. Redvanly, Eds.). For example, [18F]Fluoromethyltosylate can be prepared by
nucleophilic substitution of Methylene ditosylate with [ F]-fluoride ion in acetonitrile
containing 2-10%water (see Neal, T.R., et al., Journal of Labelled Compounds and
Radiopharmaceuticals 2005; 48:557-68).
Automated Synthesis
In a preferred embodiment, the method to prepare a compound for Formula (I),
including a compound of Formula (la), is automated. For example, [18F]-radiotracers
may be conveniently prepared in an automated fashion by means of an automated
radiosynthesis apparatus. There are several commercially-available examples of such
platform apparatus, including TRACERlab™ {e.g., TRACERlab™ MX) 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.
Optionally, in a further embodiment of the invention, the automated radiosynthesis
apparatus can be linked to a high performance liquid chromatograph (HPLC).
The present invention therefore provides a cassette for the automated synthesis
of a compound of Formula (I), including a compound of Formula (la), each as defined
herein comprising:
i) a vessel containing the precursor compound of Formula (II) as defined
herein; and
a. means for eluting the contents of the vessel of step (i) with a
compound of Formula (Ilia) as defined herein.
For the cassette of the invention, the suitable and preferred embodiments of the
precursor compound of Formulae (II) and (Ilia) are each as defined herein.
In one embodiment of the invention, a method of making a compound of
Formula (I), including a compound of Formula (la), each as described herein, that is
compatible with FASTlab™ from a protected ethanolamine precursor that requires no
HPLC purification step is provided.
The radiosynthesis of [ F]fluoromethyl-[l,2- H4]choline ( F-D4-FCH) can
be performed according to the methods and examples described herein. The
radiosynthesis of 18F-D4-FCH can also be performed using commercially available
synthesis platforms including, but not limited to, GE FASTlab™ (commercially
available from GE Healthcare Inc.).
An example of a FASTlab™ radiosynthetic process for the preparation of
[18F]fluoromethyl-[l,2- 2H4]choline from a protected precursor is shown in Scheme 5 :
[ F]FCH OTs /Ts-[ F]F
Scheme 5
wherein:
a . Preparation o f [18F]KF/K222/K2CC>3 complex a s described in more detail
below;
b . Preparation o f [18F]FCH 20Ts a s described in more detail below;
c . SPE purification o f [18F F C O T s a s described in more detail below;
d . Radiosynthesis o f O-PMB - [ F ] -D 4 -Choline (0-PMB-[ F]-D4-FCH) a s
described in more detail below; and
ee.. PPuurriiffiiccaattiioonn & ffoorrmmuullaattiioonn oofi [ s F]-D 4 -Choline ( SF-D4-FCH) a s the
hydrochloric salt a s described in more detail below.
18 2 18 The automation o f [ F]fluoro-[l,2- H 4]choline o r [ FJfluorocholine (from the
protected precursor) involves an identical automated process (and are prepared from
tthhee flfluuoorroommeetthhyyllaattiioonn ooff 00--PPMMBB--NN,,NN--ddiimrrethyl-[l,2- ¾]ethanolamine and O-PMB-
N,N-dimethylethanolamine respectively) .
According to one embodiment o f the present invention, FASTlab™ syntheses
o f [ F]fluoromethyl-[l,2- H 4 ]choline o r [ F]fluoromethylcholine comprises the
following sequential steps :
(i) Trapping o f [ F]fluoride onto QMA;
(ii) Elution o f [ F]fluoride from a QMA;
(iii) Radiosynthesis o f [ F]FCH 2OTs;
(iv) SPE clean u p o f [ F]FCH 2OTs;
(v) Reaction vessel clean up;
(vi) Drying reaction vessel and [ F]fluoromethyl tosylate retained o n SPE t-C18
plus simultaneously;
(vii) Alkylation reaction;
(viii) Removal o f unreacted O-PMB-precursor; and
(ix) Deprotection & formulation.
Each o f steps (i)-(ix) are described in more detail below.
In one embodiment o f the present invention, steps (i)-(ix) above are performed
o n a cassette a s described herein. One embodiment o f the present invention i s a
cassette capable o f performing steps (i)-(ix) for use in a n automated synthesis
platform. One embodiment o f the present invention i s a cassette for the
radiosynthesis o f [ F]fluoromethyl-[l,2- H 4]choline ( [ F]-D4-FCH) o r
[18 F]fluoromethylcholine from a protected precursor. A n example o f a cassette o f the
present invention i s shown in Figure 5b.
(i) Trapping of [ F]fluoride onto QMA
[18F]fluoride (typically in 0.5 to 5mL H2
18O) is passed through a pre
conditioned Waters QMA cartridge.
(ii) Elution of [ 8F]fluoride from a QMA
The eluent, as described in Table 1 is withdrawn into a syringe from the eluent
vial and passed over the Waters QMA into the reaction vessel. This procedure elutes
[ F]fluoride into the reaction vessel. Water and acetonitrile are removed using a
well-designed drying cycle of "nitrogen/vacuum/heating/cooling".
(iii) Radiosynthesis of [ 8F]FCH2OTs
Once the K[ F]Fluoride/K222/K 2C0 complex of (ii) is dry, CH2(OTs) 2
methylene ditosylate in a solution containing acetonitrile and water is added to the
reaction vessel containing the K[18F]fluoride/K222/K 2CC>3 complex. The resulting
reaction mixture will be heated (typically to 110°C for 10 min), then cooled down
(typically to 70°C).
(iv) SPE clean up of [ 8F]FCH2OTs
Once radiosynthesis of [18F]FCH 2OTs is completed and the reaction vessel is
cooled, water is added into the reaction vessel to reduce the organic solvent content in
the reaction vessel to approximately 25%. This diluted solution is transferred from the
reaction vessel and through the t-C18-light and t-C18 plus cartridges - these
cartridges are then rinsed with 12 to 15mL of a 25% acetonitrile / 75% water solution.
At the end of this process:
the methylene ditosylate remains trapped on the t-C18-light and
- the [ F]FCH 2OTs, tosyl-[ F]fluoride remains trapped on the t-C18
plus.
(v) Reaction vessel clean up
The reaction vessel was cleaned (using ethanol) prior to the alkylation of
[ FJfluoroethyl tosylate and O-PMB-DMEA precursor.
(vi) Drying reaction vessel and [ 8F]fluoromethyl tosylate retained on SPE t-
C18 plus simultaneously
Once clean up (v) was completed, the reaction vessel and the
[18FJfluoromethyl tosylate retained on SPE t-C18 plus was dried simultaneously.
(vii) Alkylation reaction
Following step (vi), the [ F]FCH2OTs (along with tosyl-[ FJfluoride)
retained on the t-C18 plus was eluted into the reaction vessel using a mixture of OPMB-
N,N-dimethyl-[l,2- H4]ethanolamine (or O-RMB-N,N-dimethylethanolamine)
in acetonitrile.
The alkylation of [18F]FCH2OTs with O-PMB-precursor was achieved by
heating the reaction vessel (typically 110°C for 15min) to afford [ F]fluoro-[l,2-
H4]choline (or 0-PMB-[ F]fluorocholine).
(viii) Removal of unreacted O-PMB-precursor
Water (3 to 4mL) was added to the reaction and this solution was then passed
through a pre-treated CM cartridge, followed by an ethanol wash - typically 2 x 5mL
(this removes unreacted O-PMB-DMEA) leaving "purified" [ F]fluoro-[l,2-
H4]choline (or 0-PMB-[ F]fluorocholine) trapped onto the CM cartridge.
(ix) Deprotection & formulation
Hydrochloric acid was passed through the CM cartridge into a syringe: this
resulted in the deprotection of 0-PMB-[ F]fluorocholine (the syringe contains
[ F]fluorocholine in a HC1 solution). Sodium acetate was then added to this syringe
to buffer to pH 5 to 8 affording [18F]-D4-choline (or [18FJcholine) in an acetate
buffer. This buffered solution is then transferred to a product vial containing a
suitable buffer.
Table 1 provides a listing of reagents and other components required for preparation
of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) (or [ F]fluoromethylcholine)
radiocassette of the present invention:
Table 1
According to one embodiment of the present invention, FASTlabTM
synthesis of [18F]fluoromethyl-[l,2- 2H4]choline via an unprotected precursor
comprises the following sequential steps as depicted in Scheme 6 below:
Scheme 6
18 1. Recovery of [ F]fluoride from QMA;
2 Preparation of K[ F]F/K2 22/K2C0 3 complex;
3 Radiosynthesis of FCH2OTs;
4 SPE cleanup of FCH2OTs;
5 Clean up of reaction vessel cassette and syringe;
6 Drying of reaction vessel and C18 SepPak;
7 Elution off and coupling of FCH2OTs with D4-DMEA;
8 Transfer of reaction mixture onto CM cartridge;
9 Clean up of cassette and syringe;
10 Washing of CM cartridge with dilute aq ammonia solution, Ethanol
and water;
11 Elution of [ F]fluoromethyl-[l,2- H4]choline from CM cartridge with
0.09% sodium chloride (5 ml), followed by water (5ml).
In one embodiment of the present invention, steps (l)-(ll) above are
performed on a cassette as described herein. One embodiment of the present
invention is a cassette capable of performing steps (l)-(l 1) for use in an automated
synthesis platform. One embodiment of the present invention is a cassette for the
radiosynthesis of [ F]fluoromethyl-[l,2- H4]choline ([ F]-D4-FCH) from an
unprotected precursor. An example of a cassette of the present invention is shown in
Figure 5a.
Table 2 provides a listing of reagents and other components required for preparation
of [ F]fluoromethyl-[l,2- H4]choline (D4-FCH) (or [ F]fluoromethylcholine) via an
unprotected precursor radiocassette of the present invention:
Table 2
Imaging Method
The radiolabeled compound of the invention, as described herein, will be
taken up into cells via cellular transporters or by diffusion. In cells where choline
kinase is overexpressed or activated the radiolabeled compound of the invention, as
described herein, will be phosphorylated and trapped within that cell. This will form
the primary mechanism of detecting neoplastic tissue.
The present invention further provides a method of imaging comprising the
step of administering a radiolabeled compound of the invention or a pharmaceutical
composition comprising a radiolabeled compound of the invention, each as described
herein, to a subject and detecting said radiolabeled compound of the invention in said
subject. The present invention further provides a method of detecting neoplastic
tissue in vivo using a radiolabeled compound of the invention or a pharmaceutical
composition comprising a radiolabeled compound of the invention, each as described
herein. Hence the present invention provides better tools for early detection and
diagnosis, as well as improved prognostic strategies and methods to easily identify
patients that will respond or not to available therapeutic treatments. As a result of the
ability of a compound of the invention to detect neoplastic tissue, the present
invention further provides a method of monitoring therapeutic response to treatment
of a disease state associated with the neoplastic tissue.
In a preferred embodiment of the invention, the radiolabeled compound of the
invention for use in a method of imaging of the invention, as described herein, is a
radiolabeled compound of Formula (I).
In a preferred embodiment of the invention, the radiolabeled compound of the
invention for use in a method of imaging of the invention, as described herein, is a
radiolabeled compound of Formula (III).
As would be understood by one of skill in the art the type of imaging (e.g.,
PET, SPECT) will be determined by the nature of the radioisotope. For example, if
the radiolabeled compound of Formula (I) contains 18F it will be suitable for PET
imaging.
Thus the invention provides a method of detecting neoplastic tissue in vivo
comprising the steps of:
i) administering to a subject a radiolabeled compound of the
invention or a pharmaceutical composition comprising a
radiolabeled compound of the invention, each as defined herein;
ii) allowing said a radiolabeled compound of the invention to bind
neoplastic tissue in said subject;
iii) detecting signals emitted by said radioisotope in said bound
radiolabeled compound of the invention;
iv) generating an image representative of the location and/or amount
of said signals; and,
v) determining the distribution and extent of said neoplastic tissue in
said subject.
The step of "administering" a radiolabeled compound of the invention is
preferably carried out parenterally, and most preferably intravenously. The
intravenous route represents the most efficient way to deliver the compound
throughout the body of the subject. Intravenous administration neither represents a
substantial physical intervention nor a substantial health risk to the subject. The
radiolabeled compound 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 imaging method of
the invention. As such, the imaging method of the invention can also be understood
as comprising the above-defined steps (ii)-(v) carried out on a subject to whom a
radiolabeled compound of the invention has been pre-administered.
Following the administering step and preceding the detecting step, the
radiolabeled compound of the invention is allowed to bind to the neoplastic tissue.
For example, when the subject is an intact mammal, the radiolabeled compound of the
invention will dynamically move through the mammal's body, coming into contact
with various tissues therein. Once the radiolabeled compound of the invention comes
into contact with the neoplastic tissue it will bind to the neoplastic tissue.
The "detecting" step of the method of the invention involves detection of
signals emitted by the radioisotope comprised in the radiolabeled compound of the
invention by means of a detector sensitive to said signals, e.g., 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 radioisotope. The signals emitted directly
correlate with the amount of enzyme or neoplastic tissue 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 "disease state associated with the neoplastic tissue" can be any disease
state that results from the presence of neoplastic tissue. Examples of such disease
states include, but are not limited to, tumors, cancer (e.g., prostate, breast, lung,
ovarian, pancreatic, brain and colon). In a preferred embodiment of the invention the
disease state associated with the neoplastic tissue is brain, breast, lung, espophageal,
prostate, or pancreatic cancer.
As would be understood by one of skill in the art, the "treatment" will be
depend on the disease state associated with the neoplastic tissue. For example, when
the disease state associated with the neoplastic tissue is cancer, treatment can include,
but is not limited to, surgery, chemotherapy and radiotherapy. Thus a method of the
invention can be used to monitor the effectiveness of the treatment against the disease
state associated with the neoplastic tissue.
Other than neoplasms, a radiolabeled compound of the invention may also be
useful in liver disease, brain disorders, kidney disease and various diseases associated
with proliferation of normal cells. A radiolabeled compound of the invention may
also be useful for imaging inflammation; imaging of inflammatory processes
including rheumatoid arthritis and knee synovitis, and imaging of cardiovascular
disease including artherosclerotic plaque.
Precursor Compound
The present invention provides a precursor compound of Formula (II):
(P)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
Ri, R2, R , and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
R and R2 are each hydrogen;
R3 and R are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
In a preferred embodiment of the invention, a compound of Formula (II) is
provided wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR ,
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I,
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4.
According to the invention, compound of Formula (II) is a compound of
Formula (Ila):
(Ila)
In one embodiment of the invention, a compound of Formula (lib) is provided:
(lib)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R 7 are each independently hydrogen, R8, -(CH2)mR , -(CD2)mRs, -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; and
Pg is a hydroxyl protecting group.
In a preferred embodiment of the invention, a compound of Formula (lib) is
provided wherein Pg is a p-methoxybenyzl (PMB), trimethylsilyl (TMS), or a
dimethoxytrityl (DMTr) group.
In a preferred embodiment of the invention, a compound of Formula (lib) is
provided wherein Pg is a p-methoxybenyzl (PMB) group.
In one embodiment of the invention, a compound of Formula (lie) is provided:
(lie)
wherein:
Ri, R2, R3, and R4 are each independently hydrogen or deuterium (D);
R5, R6, and R7 are each independently hydrogen, R8, -(CH2)mR , -(CD2)mR , -
(CF )mR , -CH(R ) , or -CD(R ) ;
R is independently hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -
CH2Br, -CH2I, -CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4;
with the proviso that when R , R2, R3, and R4 are each hydrogen, R5, R , and
R7 are each not hydrogen; and with the proviso that when Ri, R2, R3, and R 4 are each
deuterium, R5, R6, and R7are each not hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
Ri, R2, R3, and R4 are each independently hydrogen;
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; with the proviso that R5, R6, and R7 are each not
hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
Ri, R2, R3, and R4 are each deuterium (D);
R5, R6, and R7 are each independently hydrogen, R , -(CH2)mR , -(CD2)mR , -
(CF )mR , or -CD(R ) ;
R is hydrogen, -OH, -CH , -CF , -CH2OH, -CH2F, -CH2C1, -CH2Br, -CH2I, -
CD , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5; and
m is an integer from 1-4; with the proviso that R5, R6, and R 7 are each not
hydrogen.
In a preferred embodiment of the invention, a compound of Formula (lie) is
provided wherein:
R and R2 are each hydrogen; and
R and R 4 are each deuterium (D).
A precursor compound of Formula (II), including a compound of Formula
(Ila), (lib) and (lie), can be prepared by any means known in the art including those
described herein. For example, the compound of Formula (Ila) can be synthesized by
alkylation of dimethylamine in THF with 2-bromoethanol-l,l,2,2-d4 in the presence
of potassium carbonate as shown in Scheme 1 below:
SCHEME 1
wherein i = K2CO3, THF, 50°C, 19 h. The desired tetra-deuterated product can be
purified by distillation. The H NMR spectrum of the compound of Formula (Ila)
(Figure 3) in deuteriochloroform showed only the peaks associated with the N,Ndimethyl
groups and the hydroxyl of the alcohol; no peaks associated with the
hydrogens of the methylene groups of the ethyl alcohol chain were observed.
Consistent with this, the C NMR spectrum (Figure 3) showed the large singlet
associated with the N,N -dimethyl carbons; however, the peaks for the ethyl alcohol
methylene carbons at 60.4 ppm and 62.5 ppm were substantially reduced in
magnitude, suggesting the absence of the signal enhancement associated with the
presence of a covalent carbon-hydrogen bond. In addition, the methylene peaks are
both split into multiplets, indicating spin-spin coupling. Since C NMR is typically
run with H decoupling, the observed multiplicity must be the result of carbondeuterium
bonding. On the basis of the above observations the isotopic purity of the
desired product is considered to be > 98% in favour of the H isotope (relative to the
H isotope).
A di-deuterated analog of a precursor compound of Formula (II) can be
synthesized from N,N-dimethylglycine via lithium aluminium hydride reduction as
shown in Scheme 2 below:
SCHEME 2
wherein i = LiAlD4, THF, 65°C, 24 h. C NMR analysis indicated that isotopic
purity of greater than 95% in favor of the H isomer (relative to the H isotope) can be
achieved.
According to the invention, the hydroxyl group of a compound of Formula
(II), including a compound of Formula (Ila) can be further protected with a protecting
group to give a comp
(lib)
wherein Pg is any hydroxyl protecting group known in the art. Preferably, Pg is any
acid labile hydroxyl protecting group including, for example, those described in
""Protective Groups in Organic Synthesis", 3rd Edition, A Wiley Interscience
Publication, John Wiley & Sons Inc., Theodora W. Greene and Peter G. M. Wuts, pp
17-200. Preferably, Pg is a p-methoxybenzyl (PMB), trimethylsilyl (TMS), or a
dimethoxytrityl (DMTr) group. More preferably, Pg is a p-methoxybenyzl (PMB)
group.
Validation of r Flfluoromethyl-ri,2- 2H4lcholine (D4-FCH)
Stability to oxidation resulting from isotopic substitution was evaluated in in
vitro chemical and enzymatic models using [ F]fluoromethylcholine as standard.
[ F]Fluoromethyl-[l,2- H 4]choline was then evaluated in in vivo models and
11 18 18 compared to [ CJcholine, [ FJfluoromethylcholine and [ F]Fluoromethyl-[l-
H 2]choline:
OH
C-Choline
[ 1 8F]fluo ro methylcholine [ 1 8F] Fluo ro methyl-[1 -2H2]choline
[1 8F]Fluoromethyl-[1 ,2-2H4]choline
Potassium Permanganate oxidation study
The effect of deuterium substitution on bond strength was initially tested by
evaluation of the chemical oxidation pattern of [18FJfluoromethylcholine and
[18F]Fluoromethyl-[l,2- 2H4]choline using potassium permanganate. Scheme 6 below
details the base catalyzed potassium permanganate oxidation of
[18FJfluoromethylcholine and [18F]Fluoromethyl-[l,2- 2H4]choline at room
temperature, with aliquots removed and analyzed by radio-HPLC at pre-selected time
oints:
c) R -| ,R , R3, R4
Scheme 6
Reagents and Conditions: i) KMn0 4, Na2C0 , H20 , rt.
The results are summarized in Figures 6 and 7. The radio-HPLC
chromatogram (Figure 6) showed a greater proportion of the parent compound
remaining at 20 min for [18F]Fluoromethyl-[l,2- 2FLJcholine. The graph in Figure 7
further showed a significant isotope effect for the deuterated analogue,
[ F]Fluoromethyl-[l,2- H4]choline, with nearly 80% of parent compound still present
1 hour post-treatment with potassium permanganate, compared to less than 40% of
parent compound [18FJFluoromethylcholine still present at the same time point.
Choline oxidase model
[18FJfluoromethylcholine and [18F]fluoromethyl-[l,2- 2H4]choline were
evaluated in a choline oxidase model (Roivainen, A., et al. , European Journal of
Nuclear Medicine 2000; 27:25-32). The graphical representation in Figure 8 clearly
shows that, in the enzymatic oxidative model, the deuterated compound is
significantly more stable than the corresponding non-deuterated compound. At the 60
minute time point the radio-HPLC distribution of choline species revealed that for
[18FJfluoromethylcholine the parent radiotracer was present at the level of 11+8%; at
60 minutes the corresponding parent deuterated radiotracer [ F]fluoromethyl-[l,2-
H4]choline was present at 29+4%. Relevant radio-HPLC chromatograms are shown
in Figure 9 and further exemplify the increased oxidative stability of
[18F]fluoromethyl-[l,2- 2H4]-choline relative to [18FJfluoromethylcholine. These radio-
HPLC chromatograms contain a third peak, marked as 'unknown' , that is speculated
to be the intermediate oxidation product, betaine aldehyde.
In vivo stability analysis
[18F]fluoromethyl-[l,2- 2H4]-choline is more resistant to oxidation in vivo. The
relative rates of oxidation of the two isotopically radiolabeled choline species,
[18FJfluoromethylcholine and [18F]fluoromethyl-[l,2- 2H4]-choline to their respective
metabolites, [18FJfluoromethylcholine -betaine ( [18F]-FCH-betaine) and
[ F]fluoromethyl-[l,2- H4]-choline-betaine ([ F]-D4-FCH-betaine) was evaluated by
high performance liquid chromatography (HPLC) in mouse plasma after intravenous
(i.v.) administration of the radiotracers. [18F]fluoromethyl-[l,2- 2H4]-choline was
found to be markedly more stable to oxidation than [ F]fluoromethylcholine. As
shown in Figure 10, [ F]fluoromethyl-[l,2- H4]-choline was markedly more stable
than [18FJfluoromethylcholine with -40% conversion of r1F8]fluoromethyl-[l,2-2H4]-
choline to [18F]-D4-FCH-betaine at 15 min after i.v. injection into mice compared to ~
80% conversion of [ F]fluoromethylcholine to [ F]-FCH-betaine. The time course
for in vivo oxidation is shown in Figure 10 showing overall improved stability of
[18F]fluoromethyl- [1,2- 2Ή 4]-choline over [18FJfluoromethylcholine.
Biodistribution
Time course biodistribution
Time course biodistribution was carried out for [ FJfluoromethylcholine,
18 2 [ F]fluoromethyl-[l- H 18 2 2]choline and [ F]fluoromethyl-[l,2- H4]choline in nude
mice bearing HCT116 human colon xenografts. Tissues were collected at 2, 30 and 60
minutes post-injection and the data summarized in Figure 11A-C. The uptake values
for [18FJfluoromethylcholine were in broad agreement with earlier studies (DeGrado,
T.R., et al. , "Synthesis and Evaluation of 18F-labeled Choline as an Oncologic Tracer
for Positron Emisson Tomography: Initial Findings in Prostate Cancer", Cancer
Research 2000; 61:110-7). Comparison of the uptake profiles revealed a reduced
uptake of radiotracer in the heart, lung and liver for the deuterated compounds
[18F]fluoromethyl-[l- 2H 18 2 2]-choline and [ F]fluoromethyl-[l,2- H4]-choline. The tumor
uptake profile for the three radiotracers is shown in Figure 11D and shows increased
localization of radiotracer for the deuterated compounds relative to
[18FJfluoromethylcholine at all time points. A pronounced increase in tumor uptake of
[18F]fluoromethyl-[l,2- 2H4]choline at the later time points is evident.
Distribution of choline metabolites
Metabolite analysis of tissues including liver, kidney and tumor by HPLC was
also accomplished. Typical HPLC chromatograms of [18F]FCH and [18F]D4-FCH and
their respective metabolites in tissues are shown in Figure 12. Tumor distribution of
metabolites was analyzed in a similar fashion (Figure 13). Choline and its metabolites
lack any UV chromophore to permit presentation of chromatograms of the cold
unlabelled compound simultaneously with the radioactivity chromatograms. Thus, the
presence of metabolites was validated by other chemical and biological means. Of
note the same chromatographic conditions were used for characterization of the
metabolites and retention times were similar. The identity of the phosphocholine peak
was confirmed biochemically by incubation of the putative phosphocholine formed in
untreated HCT116 tumor cells with alkaline phosphatase (Figure 14).
A high proportion of liver radioactivity was present as phosphocholine at 30 min post
injection for both [ F]FCH and [ F]D4-FCH (Figure 12). An unknown metabolite
(possibly the aldehyde intermediate) was observed in both the liver (7.4 + 2.3%) and
kidney (8.8 + 0.2%) samples of [ F]D4-FCH treated mice. In contrast, this unknown
metabolite was not found in liver samples of [18F]FCH treated mice and only to a
smaller extent (3.3 + 0.6%) in kidney samples. Notably 60.6 + 3.7% of [ F]D4-FCH
derived kidney radioactivity was phosphocholine compared to 31.8 + 9.8% from
[ F]FCH (P = 0.03). Conversely, most of the [ F]FCH-derived radioactivity in the
kidney was in the form of [ F]FCH-betaine; 53.5 + 5.3% compared to 20.6 + 6.2%
for [18F]D4-FCH (Figure 12). It could be argued that levels of betaine in plasma
reflected levels in tissues such as liver and kidneys. Tumors showed a different HPLC
profile compared to liver and kidneys; typical radio-HPLC chromatograms obtained
from the analysis of tumor samples (30 min after intravenous injection of [18F]FCH,
[18FJD4-FCH and [11dcholine) are shown in Figure 12. In tumors, radioactivity was
mainly in the form of phosphocholine in the case of [ F]D4-FCH (Figure 13). In
contrast [ F]FCH showed significant levels of [ F]FCH-betaine. In the context of
late imaging, these results indicate that [18FJD4-FCH will be the superior radiotracer
for PET imaging with an uptake profile that is easier to interpret.
The suitable and preferred aspects of any feature present in multiple aspects of the
present invention are as defined for said features in the first aspect in which they are
described herein. The invention is now illustrated by a series of non-limiting
examples.
Isotopic Carbon Choline Analogs
The present invention provides a compound of Formula (III) as described
herein. Such compounds are useful as PET imaging agents for tumor imaging, as
described herein. In particular, a compound of Formula (III), as described herein,
may not be excreted in the urine and hence provide more specific imaging of pelvic
malignancies such as prostate cancer.
The present invention provides a method to prepare a compound for Formula
(III), wherein said method comprises reaction of the precursor compound of Formula
(II) with a compound of Formula (IV) to form a compound of Formula (III) (Scheme
Scheme A
wherein the compounds of Formulae (I) and (III) are each as described herein and the
compound of Formula (IV) is as follows:
ZXYC*-Lg (IV)
wherein C*, X, Y and Z are each as defined herein for a compound of Formula (III)
and "Lg" is a leaving group. Suitable examples of "Lg" include, but are not limited
to, bromine (Br) and tosylate (OTos). A compound of Formula (IV) can be prepared
by any means known in the art including those described herein (e.g. , analogous to
Examples 5 and 7).
Examples
Reagents and solvents were purchased from Sigma-Aldrich (Gillingham, UK) and
used without further purification. Fluoromethylcholine chloride (reference standard)
was purchased from ABCR Gmbh & Co. (Karlsruhe, Germany). Isotonic saline (0.9
% w/v) was purchased from Hameln Pharmaceuticals (Gloucester, UK). NMR
Spectra were obtained using either a Bruker Avance NMR machine operating at 400
MHz ( H NMR) and 100 MHz ( C NMR) or 600 MHz ( H NMR) and 150 MHz ( C
NMR). Accurate mass spectroscopy was carried out on a Waters Micromass LCT
Premier machine in positive electron ionisation (EI) or chemical ionisation (CI) mode.
Distillation was carried out using a Biichi B-585 glass oven (Biichi, Switzerland).
Example 1. Preparation of N,N-dimethyl-[l,2- 2H ]-ethanolamine (3)
1 2
To a suspension of K2CO (10.50 g, 76 mmol) in dry THF (10 mL) was added
dimethylamine (2.0 M in THF) (38 mL, 76 mmol) followed by 2-bromoethanoll
,l,2,2-d 4 (4.90 g, 38 mmol) and the suspension heated to 50°C under argon. After 19
h, thin layer chromatography (TLC) (ethyl acetate/alumina/I 2) indicated complete
conversion of (2) and the reaction mixture was allowed to cool to ambient
temperature and filtered. Bulk solvent was then removed under reduced pressure.
Distillation gave the desired product (3) as a colorless liquid, b.p. 78°C/88 mbar (1.93
g, 55%). H NMR (CDCI3, 400 MHz) d 3.40 (s, 1H, OH), 2.24 (s, 6H , N(CH )2) . C
NMR (CDCI3, 75 MHz) d 62.6 (NCD2 D2OH), 60.4 (N D2CD2OH), 47.7 (N(CH )2) .
HRMS (EI) = 93. 1093 (M+) . C4H7 H4NO requires 93. 1092.
Example 2. Preparation of N,N-dimethyl-[l- 2H2]- (5)
5
To a suspension of N,N-dimethylglycine (0.52 g, 5 mmol) in dry THF(10 mL) was
added lithium aluminium deuteride (0.53 g, 12.5 mmol) and the resulting suspension
refluxed under argon. After 24 h the suspension was allowed to cool to ambient
temperature and poured onto sat. aq. Na2S0 4 (15 mL) and adjusted to pH 8 with 1 M
Na2CC>3, then washed with ether (3 x 10 mL) and dried (Na 2S0 4) . Distillation gave
the desired product (5) as a colorless liquid, b.p. 65°C/26 mbar (0.06 g, 13%). H
NMR (CDC1 , 400 MHz) d 2.43 (s, 2H, NCH2CD2) , 2.25 (s, 6H, N(C H )2), 1.43 (s,
1H, OH) . C NMR (CDCI 3, 150 MHz) d 63.7 (N H2CD2OH), 57.8 (NCH 2 D2OH),
45.7 (N(CH )2) .
Example 3. Preparation of Fluoromethyltosylate (8)
CH2OTos2
FCH2OTos
7 8
Methylene ditosylate (7) was prepared according to an established literature procedure
and analytical data was consistent with reported values (Emmons, W.D., et al. ,
Journal of the American Chemical Society, 1953; 75:2257; and Neal, T.R., et al.,
Journal of Labelled Compounds and Radiopharmaceuticals 2005; 48:557-68).
To a solution of methylene ditosylate (7) (0.67 g, 1.89 mmol) in dry acetonitrile (10
mL) was added Kryptofix K222 [4,7,13,16,21,24-hexaoxa-l,10-
diazabicyclo[8.8.8]hexacosane] (1.00 g, 2.65 mmol) followed by potassium fluoride
(0.16 g, 2.83 mmol). The suspension was then heated to 110°C under nitrogen. After
1 h TLC (7:3 hexane/ethyl acetate/silica/UV 254 ) indicated complete conversion of (7).
The reaction mixture was diluted with ethyl acetate (25 mL), washed with water (2 x
15 mL) and dried over MgS0 4. Chromatography (5 ® 10% ethyl acetate/hexane)
gave the desired product (8) as a colorless oil (40 mg, 11%). H NMR (CDC1 , 400
MHz) d 7.86 (d, 2H, = 8 Hz, aryl CH), 7.39 (d, 2 H, = 8 Hz, aryl CH), 5.77 (d, 1
H, = 52 Hz, CH2F), 2.49 (s, 3H, tolyl CH ) . C NMR (CDC1 ) d 145.6 (aryl), 133.8
(aryl), 129.9 (aryl), 127.9 (aryl), 98.1 (d, = 229 Hz, CH2F), 21.7 (tolyl CH ) . HRMS
(CI) = 222.0604 (M + NH4)+. Calcd. for C H FN0 S 222.0600.
Example 4. Preparation of N,N-Dimethylethanolamine(0-4-methoxybenzyl)
ether (O-PMB-DM
N,N-Dimethylethanolamine(0-4-methoxybenzyl) ether
To a dry flask was added dimethylethanolamine (4.46 g, 50 mmol) and dry DMF (50
mL). The solution was stirred under argon and cooled in an ice bath. Sodium hydride
(2.0 g, 50 mmol) was then added portionwise over 10 min and the reaction mixture
then allowed to warm to room temperature. After 30 min 4-methoxybenzyl chloride
(3.92 g, 25 mmol) was added dropwise over 10 min and the resulting mixture left to
stir under argon. After 60 h GC-MS indicated reaction completion (disappearance of
4-methoxybenzyl chloride) and the reaction mixture was poured onto 1M sodium
hydroxide (100 mL) and extracted with dichloromethane (DCM)(3 x 30 mL) then
dried (Na 2SC>4). Column chromatography (0®10 methanol/DCM; neutral silica)
gave the desired product (O-PMB-DMEA) as a yellow oil (1.46 g, 28 ). H NMR
(CDC1 , 400 MHz) d 7.28 (d, 2H, J = 8.6 Hz, aryl CH), 6.89 (d, 2H, J = 8.6 Hz, aryl
CH), 4.49 (s, 2H, -CH 2-), 3.81 (s, 3H, OCH ) , 3.54 (t, 2H, = 5.8, NCH 2CH20), 2.54
(t, 2H, = 5.8, NCH2CH20), 2.28 (s, 6H, N(CH )2) . HRMS (ES) = 210.1497 (M+H +) .
C 2H20NO2 requires 210.1494.
Example 4a. Preparation of Dueterated Analogues of N,NDimethylethanolamine(
0-4-methoxybenzyl) ether (O-PMB-DMEA)
The di- and tetra-deuterated analogs of N,N-Dimethylethanolamine(0-4-
methoxybenzyl) ether can be prepared according to Example 4 from the appropriate
di- or tetra-deuterated dimethylethanolamine.
Example 5. Preparation of Synthesis of [ 8F]fluoromethyl tosylate (9)
CH2OTos2
1 FCH2OTos
7 9
To a Wheaton vial containing a mixture of K2C0 3 (0.5 mg, 3.6 mihoΐ , dissolved in 100
water), 18-crown-6 (10.3 mg, 39 mihoΐ ) and acetonitrile (500 ) was added
[ FJfluoride (-20 mCi in 100 water). The solvent was then removed at 110°C
under a stream of nitrogen (100 mL/min). Afterwards, acetonitrile (500 ) was
added and distillation to dryness continued. This procedure was repeated twice. A
solution of methylene ditosylate (7) (6.4 mg, 18 mihoΐ ) in acetonitrile (250 )
containing 3 % water was then added at ambient temperature followed by heating at
100°C for 10-15 min., with monitoring by analytical radio-HPLC. The reaction was
quenched by addition of 1:1 acetonitrile/water (1.3 mL) and purified by semipreparative
radio-HPLC. The fraction of eluent containing [ F]fluoromethyl tosylate
(9) was collected and diluted to a final volume of 20 mL with water, then immobilized
on a Sep Pak C18 light cartridge (Waters, Milford, MA, USA) (pre-conditioned with
DMF (5 mL) and water (10 mL)). The cartridge was washed with further water (5
mL) and then the cartridge, with [ F]fluoromethyl tosylate (9) retained, was dried in
a stream of nitrogen for 20 min. A typical HPLC reaction profile for synthesis of
[ F](13) is shown in Figure 4A/4B below.
Example 6. Radiosynthesis of [ 8F]fluoromethylcholine derivatives by reaction
with [ 8F]fluorobromomethane
lla-c
11a: R,, R2, R3, R4 = H
lib: R , R2= H; R3, R4
11c: R R2, R3, R4 = D
[ FJFluorobromomethane (prepared according to Bergman et al (Appl Radiat Isot
2001;54(6):927-33)) was added to a Wheaton vial containing the amine precursor
N,N-dimethylethanolamine (150 uL) or N,N-dimethyl-[l,2- H4]ethanolamine (3) (150
) in dry acetonitrile (1 mL), pre-cooled to 0°C. The vial was sealed and then heated
to 100°C for 10 min. Bulk solvent was then removed under a stream of nitrogen, then
the sample remaining was redissolved in 5% ethanol in water (10 mL) and
immobilized on a Sep-Pak CM light cartridge (Waters, Milford, MA, USA) (pre
conditioned with 2 M HCl (5 mL) and water (10 mL)) to effect the chloride anion
exchange. The cartridge was then washed with ethanol (10 mL) and water (10 mL)
followed by elution of the radiotracer (11a) or (11c) using saline (0.5-2.0 mL) and
passing through a sterile filter (0.2 mih) (Sartorius, Goettingen, Germany).
Example 7. Radiosynthesis of [ 8F]Fluoromethylcholine, [ 8F]fluoromethyl-[l-
2H2]choline and [ 8F]fluoromethyl-[l,2- 2H ]choline by reaction with
[ 8F]fluoromethylmethyl tosylate
[ FJFluoromethyl tosylate (9)(prepared according to Example 5) and eluted from the
Sep-Pak cartridge using dry DMF (300 ), was added in to a Wheaton vial
containing one of the following precursors: N,N-dimethylethanolamine (150 )
N,N-dimethyl-[l,2- F 4]ethanolamine (3) (150 ) (prepared according to Example
1); or N,N-dimethyl-[l- H 2]ethanolamine (5)(150 i ) (prepared according to
Example 2 ), and heated to 100°C with stirring. After 20 min the reaction was
quenched with water (10 mL) and immobilized on a Sep Pak CM light cartridge
(Waters) (pre-conditioned with 2M HCl (5 mL) and water (10 mL)) in order to effect
the chloride anion exchange and then washed with ethanol (5 mL) and water (10 mL)
followed by elution of the radiotracer [ F]Fluoromethylcholine (12a),
[ F]fluoromethyl-[l- H2]choline (12b) or [ F]fluoromethyl-[l,2- H4]choline [ F]
(12c) with isotonic saline (0.5-1.0 mL).
Example 8. Synthesis of cold Fluoromethyltosylate (15)
i ii
CH2I2 CH2OTos2 FCH2OTos
13 14 5
Scheme 3
i : Silver p-toluenesulfonate, MeCN, reflux, 20 h;
ii: KF, MeCN, reflux, 1 h.
According to Scheme 3 above:
(a) Synthesis of methylene ditosylate (14)
Commercially available diiodomethane (13) (2.67 g, 10 mmol) was reacted
with silver tosylate (6. 14 g, 22 mmol), using the method of Emmons and Ferris, to
give methylene ditosylate (10) (0.99g) in 28% yield (Emmons, W.D., et a ,
"Metathetical Reactions of Silver Salts in Solution. II. The Synthesis of Alkyl
Sulfonates", Journal of the American Chemical Society, 1953; 75:225).
(b) Synthesis of cold Fluoromethyltosylate (15)
Fluoromethyltosylate (11) (0.04g) was prepared by nucleophilic substitution of
Methylene ditosylate (10) (0.67 g, 1.89 mmol) of Example 3(a) using potassium
fluoride (0.16 g, 2.83 mmol)/Kryptofix K222 (1-0 g, 2.65 mmol) in acetonitrile (10
mL) at 80°C to give the desired product in 11% yield .
Example 9. Synthesis of [ 8F]fluorobromomethane (17)
[ 8F]KF
CH2Br2
18FCH2Br
16 17
Adapting the method of Bergman et al (Appl Radiat Isot 2001;54(6):927-33),
commercially available dibromomethane (16) is reacted with [ F]potassium
fluoride/Kryptofix K222 in acetonitrile at 110°C to give the desired
[ F]fluorobromomethane (17), which is purified by gas-chromatography and trapped
by elution into a pre-cooled vial containing acetonitrile and the relevant choline
precursor.
Example 10. Analysis of radiochemical purity
18 Radiochemical purity for [ FJFluoromethylcholine, [18F]fluoromethyl-[l- 2tycholine
and [18F]fluoromethyl-[l,2- 2H 18 4]choline [ F] was confirmed by co-elution with a
commercially available fluorocholine chloride standard. An Agilent 1100 series
HPLC system equipped with an Agilent G1362A refractive index detector (RID) and
a Bioscan Flowcount FC-3400 PIN diode detector was used. Chromatographic
separation was performed on a Phenomenex Luna C reverse phase column (150 mm
x 4.6 mm) and a mobile phase comprising of 5 mM heptanesulfonic acid and
acetonitrile (90:10 v/v) delivered at a flow rate of 1.0 mL/min.
Example 11. Enzymatic oxidation study using choline oxidase
This method was adapted from that of Roivannen et al (Roivainen, A., et al,
European Journal of Nuclear Medicine 2000; 27:25-32). An aliquot of either
[ F]Fluoromethylcholine or [ F]fluoromethyl-[l,2- H4]choline [ F] (100 uL, -3.7
MBq) was added to a vial containing water (1.9 mL) to give a stock solution. Sodium
phosphate buffer (0.1 M, pH 7) (10 uL) containing choline oxidase (0.05 units/uL)
was added to an aliquot of stock solution (190 uL) and the vial was then left to stand
at room temperature, with occasional agitation. At selected time-points (5, 20, 40 and
60 minutes) the sample was diluted with HPLC mobile phase (buffer A, 1.1 mL),
filtered (0.22 mih filter) and then ~ 1 mL injected via a 1mL sample loop onto the
HPLC for analysis. Chromatographic separation was performed on a Waters C
Bondapak (7.8 x 300 mm) column (Waters, Milford, Massachusetts, USA) at
3mL/min with a mobile phase of buffer A, which contained acetonitrile, ethanol,
acetic acid, 1.0 mol/L ammonium acetate, water, and 0.1 mol/L sodium phosphate
(800:68:2:3:127:10 [v/v]) and buffer B, which contained the same constituents but in
different proportions (400:68:44:88:400:10 [v/v]). The gradient program comprised
100% buffer A for 6 minutes, 0-100% buffer B for 10 minutes, 100-0% B in 2
minutes then 0% B for 2 minutes.
Example 12. Biodistribution
Human colon (HCT116) tumors were grown in male C3H-Hej mice (Harlan, Bicester,
United Kingdom) as previously reported (Leyton, J., et al., Cancer Research 2005;
65(10):4202-10). Tumor dimensions were measured continuously using a caliper and
tumor volumes were calculated by the equation: volume = (p/6) x a x b x c, where a,
b, and c represent three orthogonal axes of the tumor. Mice were used when their
tumors reached approximately 100 mm3. [18FJFluoromethylcholine,
[ F]fluoromethyl-[l- H2]choline and [ F]fluoromethyl-[l,2- H4]choline (-3.7 MBq)
were each injected via the tail vein into awake untreated tumor bearing mice. The
mice were sacrificed at pre-determined time points (2, 30 and 60 min) after
radiotracer injection under terminal anesthesia to obtain blood, plasma, tumor, heart,
lung, liver, kidney and muscle. Tissue radioactivity was determined on a gamma
counter (Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK)
and decay corrected. Data were expressed as percent injected dose per gram of tissue.
Example 13. Oxidation potential of [ 8F]Fluoromethylcholine ([ 8F]FCH) and
[ 8F]fluoromethyl-[l,2- 2H ]choline ([ 8F]D4-FCH) in vivo
[ F]FCH or [ F](D4-FCH) (80-100 m ) was injected via the tail vein into
anesthetized non-tumor bearing C3H-Hej mice; isofluorane/0 2/N20 anesthesia was
used. Plasma samples obtained at 2, 15, 30 and 60 minutes after injection were snap
frozen in liquid nitrogen and stored at -80°C. For analysis, samples were thawed and
kept at 4°C. To approximately 0.2 mL of plasma was added ice-cold acetonitrile (1.5
mL). The mixture was then centrifuged (3 minutes, 15,493 x g; 4°C). The supernatant
was evaporated to dryness using a rotary evaporator (Heidoloph Instruments GMBH
& CO, Schwabach, Germany) at a bath temperature of 45°C. The residue was
suspended in mobile phase (1.1 mL), clarified (0.2 mih filter) and analyzed by HPLC.
Liver samples were homogenized in ice-cold acetonitrile (1.5 mL) and then
subsequently treated as per plasma samples. All samples were analyzed on an Agilent
1100 series HPLC system equipped with a g -RAM Model 3 radio-detector (IN/US
Systems inc., FL, USA). The analysis was based on the method of Roivannen
(Roivainen, A., et ah, European Journal of Nuclear Medicine 2000; 27:25-32) using a
Phenomenex Luna SCX column (10m, 250 x 4.6 mm) and a mobile phase comprising
of 0.25 M sodium dihydrogen phosphate (pH 4.8) and acetonitrile (90:10 v/v)
delivered at a flow rate of 2 ml/min.
Example 14. Distribution of choline metabolites
Liver, kidney, and tumor samples were obtained at 30 min. All samples were snapfrozen
in liquid nitrogen. For analysis, samples were thawed and kept at 4°C
immediately before use. To -0.2 mL plasma was added ice-cold methanol (1.5 mL).
The mixture was then centrifuged (3 min, 15,493 x g , 4jC). The supernatant was
evaporated to dryness using a rotary evaporator (Heidoloph Instruments) at a bath
temperature of 40°C. The residue was suspended in mobile phase (1.1 mL), clarified
(0.2 Am filter), and analyzed by HPLC. Liver, kidney, and tumor samples were
homogenized in ice-cold methanol (1.5 mL) using an IKA Ultra-Turrax T-25
homogenizer and subsequently treated as per plasma samples (above). All samples
were analyzed by radio-HPLC on an Agilent 1100 series HPLC system (Agilent
Technologies) equipped with a g-RAM Model 3 g -detector (IN/US Systems) and
Laura 3 software (Lablogic). The stationary phase comprised a Waters Bondapak
C18 reverse-phase column (300 x 7.8 mm)(Waters, Milford, MA, USA). Samples
were analyzed using a mobile phase comprising solvent A
(acetonitrile/water/ethanol/acetic acid/1.0 mol/L ammonium acetate/0.1mol/L sodium
phosphate; 800/127/68/2/3/10) and solvent B (acetonitrile/water/ethanol/acetic
acid/1.0 mol/L ammonium acetate/0.1 mol/L sodiumphosphate; 400/400/68/44/88/10)
with a gradient of 0% B for 6 min, then 0 ® 100% B in 10 min, 100% B for 0.5 min,
100®0% B in 1.5 min then 0% B for 2 min, delivered at a flow rate of 3 mL/min.
Example 15. Metabolism of [ 8F]D4-FCH and [ 8F]FCH by HCT116 tumor cells.
HCT116 cells were grown in T150 flasks in triplicate until they were 70% confluent
and then treated with vehicle (1% DMSO in growth medium) or 1 mihoI/L
PD0325901 in vehicle for 24 h. Cells were pulsed for 1 h with 1.1 MBq of either
[ F]D4- FCH or [ F]FCH. The cells were washed three times in ice-cold phosphate
buffered saline (PBS), scraped into 5 mL PBS, and centrifuged at 500 x g for 3 min
and then resuspended in 2 mL ice-cold methanol for HPLC analysis as described
above for tissue samples. To provide biochemical evidence that the 5'-phosphate was
the peak identified on the HPLC chromatogram, cultured cells were treated with
alkaline phosphatase as described previously (Barthel, H., et a , Cancer Res 2003;
63(13):3791-8). Briefly, HCT116 cells were grown in 100 mm dishes in triplicate and
incubated with 5.0 MBq [ F]FCH for 60 min at 37°C to form the putative [ F]FCHphosphate.
The cells were washed with 5 mL ice-cold PBS twice and then scraped
and centrifuged at 750 x g (4°C, 3 min) in 5 mL PBS. Cells were homogenized in 1
mL of 5 mmol/L Tris- H (pH 7.4) containing 50% (v/v) glycerol, 0.5mmol/L
MgCl2, and 0.5mmol/L ZnCl2 and incubated with 10 units bacterial (type III) alkaline
phosphatase (Sigma) at 37°C in a shaking water bath for 30 min to dephosphorylate
the [18F]FCH-phosphate. The reaction was terminated by adding ice-cold methanol.
Samples were processed as per plasma above and analyzed by radio-HPLC.
Control experiments were done without alkaline phosphatase.
Example 16. Small animal PET imaging
PET imaging studies. Dynamic [18F]FCH and [18F]D4-FCH imaging scans were
carried out on a dedicated small animal PET scanner, quad-HIDAC (Oxford Positron
Systems). The features of this instrument have been described previously (Barthel, H.,
et a , Cancer Res 2003; 63(13):3791-8). For scanning the tail veins, vehicle- or drugtreated
mice were cannulated after induction of anesthesia (isofluorane/0 2/N20). The
animals were placed within a thermostatically controlled jig (calibrated to provide a
rectal temperature of ~37°C) and positioned prone in the scanner. [ F]FCH or
[18FJD4-FCH (2.96-3.7 MBq) was injected via the tail vein cannula and scanning
commenced. Dynamic scans were acquired in list mode format over a 60 min period
as reported previously (Leyton, J., et a , Cancer Research 2006; 66(15):7621-9). The
acquired data were sorted into 0.5 mm sinogram bins and 19 time frames (0.5 x 0.5 x
0.5 mm voxels; 4 x 15, 4 x 60, and 11 x 300 s) for image reconstruction, which was
done by filtered back-projection using a two-dimensional Hamming filter (cutoff 0.6).
The image data sets were visualized using the Analyze software (version 6.0;
Biomedical Imaging Resource, Mayo Clinic). Cumulative images of 30 to 60 min
dynamic data were used for visualization of radiotracer uptake and to draw regions of
interest. Regions of interest were defined manually on five adjacent tumor regions
(each 0.5 mm thickness). Dynamic data from these slices were averaged for each
tissue (liver, kidney, muscle, urine, and tumor) and at each of the 19 time points to
obtain time versus radioactivity curves. Corresponding whole body time versus
radioactivity curves representing injected radioactivity were obtained by adding
together radioactivity in all 200 x 160 x 160 reconstructed voxels. Tumor
radioactivity was normalized to whole-body radioactivity and expressed as percent
injected dose per voxel (%ID/vox). The normalized uptake of radiotracer at 60 min
(%ID/vox60) was used for subsequent comparisons. The average of the normalized
maximum voxel intensity across five slices of tumor %IDvox60max was also use for
comparison to account for tumor heterogeneity and existence of necrotic regions in
tumor. The area under the curve was calculated as the integral of %ID/vox from 0 to
60 min.
Example 17. Effect of PD0325901 treatment in mice. Size-matched HCT1 16
tumor bearing mice were randomized to receive daily treatment by oral gavage of
vehicle (0.5% hydroxypropyl methylcellulose + 0.2% Tween 80) or 25 mg/kg (0.005
mL/g mouse) of the mitogenic extracellular kinase inhibitor, PD0325901, prepared in
vehicle. [18FJD4-FCH-PET scanning was done after 10 daily treatments with the last
dose administered 1 h before scanning. After imaging, tumors were snap-frozen in
liquid nitrogen and stored at ~80°C for analysis of choline kinase A expression. The
results are illustrated in Fig. 18 and 19.
This exemplifies use of [ F]D4-FCH-PET as an early biomarker of drug response.
Most of the current drugs in development for cancer target key kinases involved in
cell proliferation or survival. This example shows that in a xenograft model for which
tumor shrinkage is not significant, growth factor receptor-Ras-MAP kinase pathway
inhibition by the MEK inhibitor PD0325901 leads to a significant reduction in tumor
[18FJD4-FCH uptake signifying inhibition of the pathway. The figure also shows that
inhibition of [18F]D4-FCH uptake was due at least in part to the inhibition of choline
kinase activity.
Example 18. Comparison of [ 8F]FCH and [ 8F]D4-FCH for Imaging
As illustrated in Figure 16, [ F]FCH and [ F]D4-FCH were both rapidly
taken up into tissues and retained. Tissue radioactivity increased in the following
order: muscle < urine < kidney < liver. Given the predominance of phosphorylation
over oxidation in the liver (Figure 12), little differences were found in overall liver
radioactivity levels between the two radiotracers. Liver radioactivity at levels 60 min
after [ F]D4-FCH or [ F]FCH injection, %ID/vox60, was 20.92 + 4.24 and 18.75 +
4.28, respectively (Figure 16). This is also in keeping with the lower levels betaine
with [ F]D4-FCH injection than with [ F]FCH injection (Figure 12). Thus,
pharmacokinetics of the two radiotracers in liver determined by PET (which lacks
chemical resolution) were similar. The lower kidney radioactivity levels for [18F]D4-
FCH compared to [ F]FCH (Figure 16), on the other hand, reflect the lower oxidation
potential of [ F]D4-FCH in kidneys. The %ID/vox60 for [ F]FCH and [ F]D4-FCH
were 15.97 + 4.65 and 7.59 + 3.91, respectively in kidneys (Figure 16). Urinary
excretion was similar between the radiotracers. Regions of interest (ROIs) that were
drawn over the bladder showed %ID/vox6ovalues of 5.20 + 1.71 and 6.70 + 0.71 for
[18FJD4-FCH and [18F]FCH, respectively. Urinary metabolites comprised mainly of
the unmetabolized radiotracers. Muscle showed the lowest radiotracer levels of any
tissue.
Despite the relatively high systemic stability of [18FJD4-FCH and high
proportion of phosphocholine metabolites, higher tumor radiotracer uptake by PET in
mice that were injected with [ F]D4-FCH compared to the [ F]FCH group was
observed. Figure 17 shows typical (0.5 mm) transverse PET image slices
demonstrating accumulation of [18F]FCH and [18FJD4-FCH in human melanoma
SKMEL-28 xenografts. In this mouse model, the tumor signal-to-background contrast
was qualitatively superior in the [ F]D4-FCH PET images compared to [ F]FCH
images. Both radiotracers had similar tumor kinetic profiles detected by PET (Figure
17). The kinetics were characterized by rapid tumor influx with peak radioactivity at
~ 1 min (Figure 17). Tumor levels then equilibrated until ~5 min followed by a
plateau. The delivery and retention of [18FJD4-FCH were quantitatively higher than
those for FCH (Figure 17). The %ID/vox60 for [ F]D4-FCH and [ F]FCH were 7.43
+ 0.47 and 5.50 + 0.49, respectively (P=0.04). Because tumors often present with
heterogeneous population of cells, another imaging variable that is probably less
sensitive to experimental noise was exploited - an average of the maximum pixel
%ID/vox6o across 5 slices (%IDvox6om a x)- This variable was also significantly higher
for [ F]D4-FCH (P=0.05; Figure 17). Furthermore, tumor area under the time versus
radioactivity curve (AUC) was higher for D4-FCH mice than FCH (P =0.02).
Although the 30 min time point was selected for a more detailed analysis of tissue
samples, the percentage of parent compound in plasma was consistently higher for
[ F]D4-FCH compared to [ F]FCH at earlier time points. Regarding imaging, tumor
uptake for both radiotracers was similar at the early (15 min) and late (60 min) time
points (Supplementary Tablel). The earlier time points may be appropriate for pelvic
imaging.
Example 19. Imaging response to treatment
Having demonstrated that [18FJD4-FCH was a more stable fluorinated-choline
analog for in vivo studies, the use of this radiotracer to measure response to therapy
was investigated. These studies were performed in a reproducible tumor model
system in which treatment outcomes had been previously characterized, i.e., , the
human colon carcinoma xenograft HCT116 treated with PD0325901 daily for 10 days
(Leyton, J., et al., "Noninvasive imaging of cell proliferation following mitogenic
extracellular kinase inhibition by PD0325901", Mol Cancer Ther 2008; 7(9):3112-
21). Drug treatment led to tumor stasis (reduction in tumor size by only 12.2% at day
10 compared to the pretreatment group); tumors of vehicle-treated mice increased by
375%. Tumor [ F]D4-FCH levels in PD0325901 -treated mice peaked at
approximately the same time as those of vehicle-treated ones, however, there was a
marked reduction in radiotracer retention in the treated tumors (Figure 18). All
imaging variables decreased after 10 days of drug treatment (P=0.05, Figure 18). This
indicates that [ F]D4-FCH can be used to detect treatment response even under
conditions where large changes in tumor size reduction are not seen (Leyton, J., et al.,
"Noninvasive imaging of cell proliferation following mitogenic extracellular kinase
inhibition by PD0325901", Mol Cancer Ther 2008; 7(9):3 112-21). To understand the
biomarker changes, the intrinsic cellular effect of PD0325901 on D4-FCHphosphocholine
formation was examined by treating exponentially growing HCT1 16
cells in culture with PD0325901 for 24 h and measuring the 60-min uptake of
[ F]D4-FCH in vitro. As shown in Figure 18, PD0325901 significantly inhibited
[18F]D4-FCH-phosphocholine formation in drug-treated cells demonstrating that the
effect of the drug in tumors is likely due to cellular effects on choline metabolism
rather than hemodynamic effects.
To understand further the mechanisms regulating [ F]D4-FCH uptake with
drug treatment, changes in CHKA expression in PD0325901 and vehicle-treated
tumors excised after PET scanning were assessed. A significant reduction in CHKA
protein expression was seen in vivo at day 10 (P=0.03) following PD0325901
treatment (Figure 19) indicating that reduced CHKA expression contributed to the
lower D[18FJ4-FCH uptake in drug-treated tumors. The drug-induced reduction of
CHKA expression also occurred in vitro in exponentially growing cells treated with
PD0325901.
Example 20. Statistics.
Statistical analyses were done using the software GraphPad Prism version 4
(GraphPad). Between-group comparisons were made using the nonparametric Mann-
Whitney test. Two-tailed P < 0.05 was considered significant.
Example 21.
Materials and Methods
Cell lines
HCT116 (LGC Standards, Teddington, Middlesex, UK) and PC3-M cells (donation
from Dr Matthew Caley, Prostate Cancer Metastasis Team, Imperial College London,
UK) were grown in RPMI 1640 media, supplemented with 10% fetal calf serum, 2
mM L-glutamine, 100 U.mL- 1 penicillin and 100 ^g.mL - streptomycin (Invitrogen,
Paisley, Refrewshire, UK). A375 cells (donation from Professor Eyal Gottlieb,
Beatson Institute for Cancer Research, Glasgow, UK) and were grown in high glucose
(4.5 g/L) DMEM media, supplemented with 10% fetal calf serum, 2 mML-glutamine,
100 U.mL- 1 penicillin and 100 m . streptomycin (Invitrogen, Paisley,
Refrewshire, UK). All cells were maintained at 37°C in a humidified atmosphere
containing 5% C0 2.
Western blots
Western blotting was performed using standard techniques. Cells were harvested and
lysed in RIPA buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA). Membranes
were probed using a rabbit anti-human choline kinase alpha polyclonal antibody
(Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:500). A rabbit anti-actin antibody
(Sigma-Aldrich Co. Ltd, Poole, Dorset, UK; 1:5000) was used as a loading control
and a peroxidase-conjugated donkey anti-rabbit IgG antibody (Santa Cruz
Biotechnology Inc., Santa Cruz, CA, USA; 1:2500) as the secondary antibody.
Proteins were visualized using the Amersham ECL kit (GE Healthcare, Chalfont St
Giles, Bucks, UK). Blots were scanned (Bio-Rad GS-800 Calibrated Densitometer;
Bio-Rad, Hercules, CA, USA) and signal quantification was performed by
densitometry using scanning analysis software (Quantity One; Bio-Rad).
For analysis of tumor choline kinase expression, tumors at ~ 100 mm3 were excised,
placed in a Precellys 24 lysing kit 2 mL tube (Bertin Technoologies, Montigny-leBretonneux,
France), containing 1.4 mm ceramic beads, and snap-frozen in liquid
nitrogen. For homogenization, 1mL of RIPA buffer was added to the lysing kit tubes
which were homogenized in a Precellys 24 homogenizer (6500 RPM; 2 x 17 s with 20
s interval). Cell debris were removed by centrifugation prior to western blotting as
described above.
In vitro 8F-D4-choline uptake
Cells (5 x 105) were plated into 6-well plates the night prior to analysis. On the day of
the experiment, fresh growth medium, containing 40 18F-D4-choline, was added
to individual wells. Cell uptake was measured following incubation at 37°C in a
humidified atmosphere of 5% C0 2 for 60 min. Plates were subsequently placed on
ice, washed 3 times with ice-cold PBS and lysed in RIPA buffer (Thermo Fisher
Scientific Inc., Rockford, IL, USA; 1 mL, 10 min). Cell lysate was transferred to
counting tubes and decay-corrected radioactivity was determined on a gamma counter
(Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK). Aliquots
were snap-frozen and used for protein determination following radioactive decay
using a BCA 96-well plate assay (Thermo Fisher Scientific Inc., Rockford, IL, USA).
Data were expressed as percent of total radioactivity per mg protein. For
hemicholinium-3 treatment (5 mM; Sigma-Aldrich), cells were incubated with the
compound 30 min prior to addition of radioactivity and for the duration of the uptake
time course.
In vivo tumor models
All animal experiments were performed by licensed investigators in accordance with
the United Kingdom Home Office Guidance on the Operation of the Animal
(Scientific Procedures) Act 1986 and within the newly-published guidelines for the
welfare and use of animals in cancer research (Workman P, Aboagye EO, Balkwill F,
et al. Guidelines for the welfare and use of animals in cancer research. Br J
C n r.2010;102:1555-1577). Male BALB/c nude mice (aged 6 - 8 weeks; Charles
River, Wilmington, MA, USA) were used. Tumor cells (2 x 106) were injected
subcutaneously on the back of mice and animals were used when the xenografts
reached 100 mm3. Tumor dimensions were measured continuously using a caliper
and tumor volumes were calculated by the equation: volume = (p / 6) x a x b x c,
where a, b, and c represent three orthogonal axes of the tumor.
In vivo tracer metabolism
Radiolabeled metabolites from plasma and tissues were quantified using a method
adapted from Smith G, Zhao Y, Leyton J, et al. Radiosynthesis and pre-clinical
evaluation of [(18)F]fluoro-[l,2-(2)H(4)]choline. Nucl Med 5/o/.2011;38:39-51.
Briefly, tumor-bearing mice under terminal anaesthesia were administered a bolus
i.v. injection of one of the following radiotracers: C-choline, C-D4-choline (-18.5
MBq) or F-D4-choline (~ 3.7 MBq), and sacrificed by exsanguination via cardiac
puncture at 2, 15, 30 or 60 min post radiotracer injection. For automated
radiosynthesis methodology, see Example 22. Tumor, kidney and liver samples were
immediately snap-frozen in liquid nitrogen. Aliquots of heparinized blood were
rapidly centrifuged (14000 g, 5 min, 4°C) to obtain plasma. Plasma samples were
subsequently snap-frozen in liquid nitrogen and kept on dry ice prior to analysis.
For analysis, samples were thawed and kept at 4°C immediately before use. To ice
cold plasma (200 mΐ ) was added ice cold methanol (1.5 mL) and the resulting
suspension centrifuged (14000 g 4°C; 3 min). The supernatant was then decanted and
evaporated to dryness on a rotary evaporator (bath temperature, 40°C), then
resuspended in HPLC mobile phase (Solvent A: acetonitrile/water/ethanol/acetic
acid/1.0 M ammonium acetate/0.1 M sodium phosphate [800/127/68/2/3/10]; 1.1
mL). Samples were filtered through a hydrophilic syringe filter (0.2 mih filter; Millex
PTFE filter, Millipore, MA., USA) and the sample (~1 mL) then injected via a 1 mL
sample loop onto the HPLC for analysis. Tissues were homogenized in ice-cold
methanol (1.5 mL) using an Ultra-Turrax T-25 homogenizer (IKA Werke GmbH and
Co. KG, Staufen, Germany) and subsequently treated as per plasma samples.
Samples were analyzed on an Agilent 1100 series HPLC system (Agilent
Technologies, Santa Clara, CA, USA), configured as described above, using the
method of Leyton J, Smith G, Zhao Y, et al. [18F]fluoromethyl-[l,2-2H4]-choline: a
novel radiotracer for imaging choline metabolism in tumors by positron emission
tomography. Cancer /¾s.2009;69:7721-7728. A Bondapak C18 HPLC column
(Waters, Milford, MA, USA; 7.8x3000 mm), stationary phase and a mobile phase
comprising of Solvent A (vide supra) and Solvent B (acetonitrile/water/ethanol/acetic
acid/1.0 M ammonium acetate/0.1 M sodium phosphate (400/400/68/44/88/10)),
delivered at a flow rate of 3 mL/min were used for analyte separation. The gradient
was set as follows: 0% B for 5 min; 0% to 100% B in 10 min; 100% B for 0.5 min;
100% to 0% B in 2 min; 0% B for 2.5 min.
PET imaging studies
Dynamic C-choline, C-D4-choline and F-D4-choline imaging scans were carried
out on a dedicated small animal PET scanner (Siemens Inveon PET module, Siemens
Medical Solutions USA, Inc., Malvern, PA, USA) following a bolus i.v. injection in
18 tumor-bearing mice of either -3.7 MBq for F studies, or -18.5 MBq for 1"1C.
Dynamic scans were acquired in list mode format over 60 min. The acquired data
were then sorted into 0.5 mm sinogram bins and 19 time frames for image
reconstruction (4 x 15 s, 4 x 60 s, and 11 x 300 s), which was done by filtered back
projection. For input function analysis, data were sorted into 25 time frames for image
reconstruction (8 x 5 s, 1 x 20 s, 4 x 40 s, 1 x 80 s, and 11 x 300 s). The Siemens
Inveon Research Workplace software was used for visualization of radiotracer uptake
in the tumor; 30 to 60 min cumulative images of the dynamic data were employed to
define 3-dimensional (3D) regions of interest (ROIs). Arterial input function was
estimated as follows: a single voxel 3D ROI was manually drawn in the center of the
heart cavity using 2 to 5 min cumulative images. Care was taken to minimize ROI
overlap with the myocardium. The count densities were averaged for all ROIs at each
time point to obtain a time versus radioactivity curve (TAC). Tumor TACs were
normalized to injected dose, measured by a VDC-304 dose calibrator (Veenstra
Instruments, Joure, The Netherlands), and expressed as percentage injected dose per
mL tissue. The area under the TAC, calculated as the integral of %ID/mL from 0 -
60 min, and the normalized uptake of radiotracer at 60 min (%ID/mL o) were also
used for comparisons.
Biodistribution studies
C-choline, C-D4-choline (-18.5 MBq) and F-D4-choline (-3.7 MBq) were each
injected via the tail vein of anaesthetized BALB/c nude mice. The mice were
maintained under anesthesia and sacrificed by exsanguination via cardiac puncture at
2, 15, 30 or 60 min post radiotracer injection to obtain blood, plasma, heart, lung,
liver, kidney and muscle. Tissue radioactivity was determined on a gamma counter
(Cobra II Auto-Gamma counter, Packard Biosciences Co, Pangbourne, UK) and
decay corrected. Data were expressed as percent injected dose per gram of tissue.
Statistics
Data were expressed as mean + standard error of the mean (SEM), unless otherwise
shown. The significance of comparison between two data sets was determined using
Student's t test. ANOVA was used for multi-parametric analysis (Prism v5.0 software
for windows, GraphPad Software, San Diego, CA, USA). Differences between groups
were considered significant if P <0.05.
Results
Deuteration leads to enhanced renal radiotracer uptake
Time course biodistribution was performed in non-tumor-bearing male nude mice
with C-choline, C-D4-choline and F-D4-choline tracers. Figure 20 shows tissue
distribution at 2, 15, 30 and 60 min. There were minimal differences in tissue uptake
between the three tracers over 60 min, with uptake values in broad agreement with
data previously published for F-choline and F-D4-choline (DeGrado TR, Baldwin
SW, Wang S, et al. Synthesis and evaluation of (18)F-labeled choline analogs as
oncologic PET tracers. J Nucl .2001 ;42:sl805-1814; Smith G, Zhao Y, Leyton J,
et al. Radiosynthesis and pre-clinical evaluation of [(18)F]fluoro-[l,2-
(2)H(4)]choline. Nucl Med 5/o/.2011;38:39-51). In all tracers there was rapid
extraction from blood, with the majority of radioactivity retained within the kidneys,
evident as early as 2 min post injection. Deuteration of C-choline led to a significant
1.8-fold increase in kidney retention over 60 min (P < 0.05; Figure 20L), with a 3.3-
18 fold increase in kidney retention observed for F-D4-choline when compared to 11Ccholine
at this time point (P < 0.01;). There was a trend towards increased urinary
excretion for 11C-D4-choline and 18F-D4-choline, in comparison to the nature
identical tracer, C-choline, although this increase did not reach statistical
significance.
Deuteration of C-choline results in modest resistance to oxidation in vivo
Tracer metabolism in tissues and plasma was performed by radio-HPLC (Figure 21).
Peaks were assigned as choline, betaine, betaine aldehyde and phosphocholine, using
enzymatic (alkaline phosphatase and choline oxidase) methods to determine their
identity (Figures 27 and 28, respectively) (Leyton J, Smith G, Zhao Y, et al.
[18F]fluoromethyl-[l,2-2H4]-choline: a novel radiotracer for imaging choline
metabolism in tumors by positron emission tomography. Cancer /¾s.2009;69:7721-
7728).
In the liver, both C-choline and C-D4-choline were rapidly oxidized to betaine
(Figure 2 1L), with 49.2 + 7.7 % of C-choline radioactivity already oxidized to
betaine by 2 min. Deuteration of C-choline provided significant protection against
oxidation in the liver at 2 min post injection, with 24.5 + 2.1 % radioactivity as
betaine (51.2 % decrease in betaine levels; P = 0.037), although this protection was
lost by 15 min. Notably, a high proportion of liver radioactivity (-80 ) was present
as phosphocholine by 15 min with 18F-D4-choline. This corresponded to a much
reduced liver- specific oxidation when compared to the two carbon-11 tracers (15.0 +
3.6 % of radioactivity as betaine at 60 min; P =0.002).
In contrast to the liver, deuteration of C-choline resulted in protection against
oxidation in the kidney over the entirety of the 60 min time course (Figure 215).
With C-D4-choline there was a 20 - 40 % decrease in betaine levels over 60 min
when compared to C-choline (P < 0.05), corresponding to a proportional increase in
phosphocholine (P < 0.05). 18F-D4-choline was more resistant to oxidation in the
kidney than both carbon-11 labeled choline tracers. There was a relationship between
levels of radiolabeled phosphocholine and kidney retention when data from all three
tracers were compared (R2 = 0.504; Figure 29). In the plasma, the temporal levels of
betaine for both C-choline and C-D4-choline were almost identical; it should be
noted that total radioactivity levels were low for all radiotracers. At 2 min, 12.1 + 2.6
% and 8.8 + 3.8 % of radioactivity was in the form of betaine for C-choline and CD4-
choline respectively, rising to 78.6 + 4.4 % and 79.5 + 2.9 % at 15 min. Betaine
levels were significantly reduced with F-D4-choline, with 43.7 + 12.4 % of activity
present as betaine at 15 min. A further increase in plasma betaine was not observed
with 18F-D4-choline over the remainder of the time course.
Fluorination protects against choline oxidation in tumor
C-choline, C-D4-choline and F-D4-choline metabolism were measured in
HCT116 tumors (Figure 22). With all tracers, choline oxidation was greatly reduced
in the tumor in comparison to levels in the kidney and liver. At 15 min, both C-D4-
choline and 18F-D4-choline had significantly more radioactivity corresponding to
phosphocholine than C-choline; 43.8 + 1.5 % and 45.1 + 3.2 % for C-D4-choline
and F-D4-choline respectively, in comparison to 30.5 + 4.0 % for C-choline (P =
0.035 and P = 0.046 respectively). By 60 min, the majority of radioactivity was
phosphocholine for all three tracers, with phosphocholine levels increasing in the
order of C-choline < C-D4-choline < F-D4-choline. There was no difference in
the tumor metabolic profile for C-choline and C-D4-choline at 60 min, although
reduced choline oxidation was observed for F-D4-choline; 14.0 + 3.0 % betaine
radioactivity with 18F-D4-choline compared with 28.1 + 2.9 % for 11C-choline (P =
0.026).
Choline tracers have similar sensitivity for imaging tumors by PET
Despite the high systemic stability of 18F-D4-choline, tumor radiotracer uptake in
mice by PET was no higher than with C-choline or C-D4-choline (Figure 23).
Figure 23L shows typical (0.5 mm) transverse PET image slices showing
accumulation of all three tracers in HCT116 tumors. For all three tracers there was
heterogeneous tumor uptake, but tumor signal-to-background levels were identical;
confirmed by normalized uptake values at 60 min and values for the tumor area under
the time verses radioactivity curve (data not shown). It should be noted that the PET
data represent total radioactivity. In the case of C-choline or C-D4-choline, a
significant proportion of this radioactivity is betaine (Figure 22).
Tumor tracer kinetics
Despite there being no difference in overall tracer retention in the tumor, the kinetic
profiles of tracer uptake varied between the three choline tracers, detected by PET
(Figure 235). The kinetics for the three tracers were characterized by rapid tumor
influx over the initial 5 min, followed by stabilization of tumor retention. Initial
delivery of 18F-D4-choline over the first 14 min of imaging was higher than for both
C-choline and C-D4-choline (expanded TAC for initial 14 min shown in Figure
30). Slow wash-out of activity was observed with both 18F-D4-choline and 11C-D4-
choline between 30 and 60 min, in contrast to the gradual accumulation detected with
C-choline. Parameters for the irreversible trapping of radioactivity in the tumor, K
and , were calculated from a two-tissue irreversible model, using metabolitecorrected
TAC from the heart cavity as input function (Figure 24L and B). A double
input (DI) model, accounting for the contribution of metabolites to the tissue TAC,
was used for kinetic analysis, described in supplemental data. There was no
significant difference in flux constant measurements between deuterated and
undeuterated C-choline. Addition of methylfluoride, however, resulted in 49.2 % (n
= 3; P =0.022) and 75.2 % n = 3; P =0.005 decreases in K\ and ¾, respectively; i.e.,
18 when F-D4-choline was compared to 11C-D4-choline. K ' values were similar
between all three tracers: 0.106 + 0.026; 0.114 + 0.019; 0.142 + 0.027 for C-choline,
C-D4-choline and F-D4-choline respectively. It is possible that intracellular
betaine formation (not just presence of betaine in the extracellular space) led to a
higher than expected irreversible uptake; there was a significant 388 and 230%
increase in the ratio of betaine :phophocholine at 15 and 60 min respectively (P =
0.045 and 0.036) with C-choline in comparison to F-D4-choline (Figure 5C).
8F-D4-choline shows good sensitivity for the PET imaging of prostate
adenocarcinoma and malignant melanoma
Having confirmed that 18F-D4-choline is a more stable choline analogue for in vivo
studies, with good sensitivity for the imaging of colon adenocarcinoma, it was desired
to evaluate its suitability for cancer detection in other models of human cancer
including malignant melanoma A375 and prostate adenocarcinoma PC3-M. In vitro
uptake of 18F-D4-choline was similar in the three cell lines over 30 min (Figure 31),
relating to near-identical levels of choline kinase expression (Figure 3 1 insert).
Retention of radioactivity was shown to be choline kinase-dependent as treatment of
cells with the choline transport and choline kinase inhibitor, hemicholinium-3,
resulted in > 90 % decrease in intracellular tracer radioactivity in all three cell lines.
Similar intracellular trapping of F-D4-choline in these cancer models were
translated to their uptake in vivo (Figure 25L)), showing similar values for flux
constant measurements and PET imaging variables (Supplemental Table 1). There
was a trend towards increased tumor retention of 18F-D4-choline in the order of A375
< HCT116 < PC3-M; reflected by the expression of choline kinase in these lines
(Figure 25C). There was no discernable difference in tumor metabolite profiles
between the three cell cancer models at either 15 or 60 min post injection (Figure
255).
Tumor size affects 8F-D4-choline uptake and retention but not tumor
pharmacokinetics
For PET imaging, tumors were grown to 100 mm3 prior to imaging. One small cohort
of animals with implanted PC3-M xenografts were, however, imaged when the tumor
size had reached 200 mm3 (See Figure 32 for typical transverse PET images). These
tumors showed a distinct pattern of 18F-D4-choline uptake around the tumor rim,
corresponding to a substantial decrease in tumor radioactivity when compared to
smaller PC3-M tumors (Figure 26). As with HCT116 tumors, maximal tumorspecific
radioactivity was achieved within 5 min of tracer injection in both PC3-M
cohorts, followed by a plateau. The magnitude of radiotracer retention at 60 min was
substantially higher in the smaller tumors, with a normalized uptake value of 1.97 +
0.07 % ID/mL versus 0.82 + 0.12 % ID/mL in the larger tumors (2.4-fold increase; P
= 0.0002; n = 3-5). Analysis of tumor uptake, taking the maximal voxel radioactivity
value from the tumor ROI, resulted in a smaller difference in tracer uptake at 60 min,
with an %ID/mLmax of 4.75 + 0.38 measured in the -100 mm3 tumor in comparison to
3.34 + 0.08 %ID/mLmax measured in the -200 mm3 tumor (1.4-fold increase; P =
0.019; n = 3-5). Interestingly, there was no significant change in the kinetic
parameters measuring the irreversible trapping of radioactivity, K\ and ¾, between
both tumor cohorts.
Kidney retention increased in the order of C-choline < C-D4-choline < F-D4-
choline over the 60 min time course (Fig. 20), with total kidney radioactivity shown to
be proportional to the % radioactivity retained as phosphocholine (Figure 29; R2 =
0.504). Protection against choline oxidation by deuteration of C-choline was shown
to be tissue specific, with a decrease in betaine radioactivity measured in the liver at
just 2 min post injection when compared to C-choline (Fig. 21).
Despite systemic protection against choline oxidation with 18F-D4-choline, the
reduction in the rate of choline oxidation was much more subtle in implanted HCT1 16
tumors (Fig. 22). At 15 min post injection there were 43.6 % and 47.9 % higher
levels of radiolabeled-phosphocholine when 11C-D4-choline and 18F-D4-choline,
respectively, were injected relative to C-choline. By 60 min there was no difference
in phosphocholine levels between the three tracers, although there was a significant
decrease in betaine-specific radioactivity with 18F-D4-choline. This equilibration of
phosphocholine-specific activity can be explained by a saturation effect, with parent
tracer levels reduced to a minimum by 60 min, severely limiting substrate levels
available for choline kinase activity. Lower betaine levels were observed in the tumor
with all three tracers over the entire time course when compared to liver and kidney,
likely resulting from a lower capacity for choline oxidation or increased washout of
betaine.
Comparison of the three choline radiotracers by PET showed no significant
differences in overall tumor radiotracer uptake and hence sensitivity (Figure 23)
despite large changes observed in other organs. Initial tumor kinetics (at time points
when metabolism was lower), however, varied between tracers, with 18F-D4-choline
characterized by rapid delivery over ~5 min, followed by slow wash-out of activity
from the tumor. This compared to the slower uptake, but continuous tumor retention
of C-choline. At 60 min a 2.7-fold and 4.0-fold higher un-metabolized parent tracer
was seen with F-D4-choline in tumor compared to C-choline and C-D4-choline,
respectively, (Figure 22). Deuteration did not, however, alter total tumor radioactivity
levels and the modeling approach used did not distinguish between different
intracellular species. While all tracers were converted intracellularly to
phosphocholine, the higher rate constants for intracellular retention (K, and Figures
24L and B ) of C-choline and C-D4-choline, compared to F-D4-choline were
explained by the rapid conversion of the non-fluorinated tracers to betaine within
HCT1 16 tumors, indicating greater specificity with F-D4-choline. Compared to FD4-
choline, the tumor betaine-to-phosphocholine metabolite ratio increased by 388%
(P = 0.045) and 259% (P = 0.061 , non-significant) for C-choline and C-D4-
choline, respectively (Figure 24C).
Example 22
General
Materials were used as purchased without further purification. 1,2- H4-
Dimethylethanolamine (DMEA) was a custom synthesis by Target Molecules Ltd
(Southampton, UK). Water for irrigation was from Baxter (Deerfield, IL, USA) and
soda lime was purchased from VWR (Lutterworth, Leicestershire, UK). 0.9 % sodium
chloride for injection was from Hameln pharmaceuticals Ltd (Gloucester, UK) a
0.045% solution of NaCl was prepared from this stock and water for irrigation.
Lithium aluminium hydride (0.1 M in THF) and hydriodic acid (57%) were from
ABX (Radeburg, Germany). Methylene ditosylate was obtained from the Huayi
Isotope Company (Toronto, Canada). All other chemicals were from Sigma-Aldrich
Co. Ltd (Poole, Dorset, UK). For C-methylations on the iPhase 11C-PRO, iPhase
disposable synthesis kits were obtained from iPhase Technologies Pty Ltd
(Melbourne, Australia). For F-fluoromethylations on the GE FASTlab (GE
Healthcare, Chalfont St. Giles, UK) the partly assembled GE FASTlab cassette
contained a FASTlab water bag, N2 filter, pre-conditioned QMA cartridge and
reaction vessel. Waters Sep-Pak Accell CM light, tC18 light and tC18 Plus cartridges
were obtained from Waters Corporation (Milford, Ma., USA).
Synthesis of C-Choline and C-ri ,2- H l -choline
C-Methyl iodide was prepared using a standard wet chemistry method. Briefly, Ccarbon
dioxide was transferred to the iPhase platform via a custom attached cryogenic
trap and reduced to C-me thane using lithium aluminium hydride (0.1 M in THF)
(200 uL) over 1 min at RT. Concentrated hydroiodic acid (200 ) was then added to
the reactor vessel and the mixture heated to 140°C for 1 min. C-methyl iodide was
then distilled through a short column containing soda lime and phosphorus pentoxide
desiccant into a 2 mL stainless steel loop containing the precursor
dimethylethanolamine or 1,2- H 4 -dimethylethanolamine (20 mΐ) . The methylation
reaction was allowed to proceed at room temperature for 2.5 min. The crude product
was then flushed on to a CM cartridge using ethanol (20 mL) at a flow rate of 5 mL
/min. The CM cartridge had previously been pre-conditioned with 0.045 % sodium
chloride (5 mL) then water (5 mL). The CM cartridge was then washed with aqueous
ammonia (0.08 %, 15 mL) then water (10 mL). The choline product was then eluted
from the cartridge using sodium chloride solution (0.045 %, 10 mL).
Synthesis of F-fluoromethyl -ri ,2- H4l-choline
The system was configured with an eluent vial comprising of 1:4 K2CO solution in
water: Kryptofix K222 solution in acetonitrile (1.0 mL), 180 mg K2CC>3 in water (10.0
mL) and 120 mg Kryptofix K222 in acetonitrile (10.0 mL), methylene ditosylate (4.2-
4.4 mg) in acetonitrile (2 % water;1.25 mL), precursor l,2- H 4-dimethylethanolamine
(150 mΐ ) in anhydrous acetonitrile (1.4 mL).
Fluorine- 18 drawn onto system and immobilised on Waters QMA light cartridge then
eluted with 1 mL of a mixture of carbonate and kryptofix into the reaction vessel.
After the KrF18]F/K 2 22 K2C0 drying cycle was complete, methylene ditosylate in
acetonitrile (2 % water; 1.25 mL) was added and reaction vessel heated to 110°C for
10 minutes. The reaction was quenched with water (3 mL) and the resulting mixture
was passed through both t-C18 light and t-C18 plus cartridges (pre-conditioned with
acetonitrile and water; 2 mL each); 15% acetonitrile in water was then passed through
the cartridges. After completion of the clean-up cycle, methylene ditosylate was
trapped on the t-C18 light cartridge and 18F-fluoromethyl tosylate (together with 18Ftosyl
fluoride) was retained on the t-C18 plus, with other reactants going to waste.
The washing cycles ethanol®vacuum®nitrogen were employed to clean the reaction
vessel after this first stage of radiosynthesis. The reaction vessel and the t-C18 plus
cartridge with immobilized 18F-fluoromethyl tosylate were then simultaneously dried
under a stream of nitrogen. 18F-fluoromethyl tosylate was then eluted from the t-C18
plus cartridge with 150 mΐ of l,2- H 4-dimethylethanolamine in 1.4 mL of
acetonitrileinto the reaction vessel. The reactor vessel was then heated to 110°C for 15
minutes then cooled and the reaction vessel contents washed with water on to a CM
cartridge (conditioned with 2 mL water). The cartridge was washed by withdrawing
ethanol from the bulk ethanol vial and passing it through CM cartridge; the washing
cycle was repeated once followed by 0.08 % ammonia solution (4.5 mL). The CM
cartridge then was subjected to final washes with ethanol followed by water. The
product, F-fluoro-[l,2- H2]choline, was washed off the CM cartridge with 0.09%
sodium chloride solution (4.5 mL) to afford F-fluoro-[l,2- H2]choline in sodium
chloride buffer as the final formulated product.
Assessment of Chemical/Radiochemical Purity
C-Choline, C-[1,2- H4]-choline and F-fluoro-[l,2- H2]choline were analyzed for
chemical/radiochemical purity on a Metrohm ion chromatography system (Runcorn,
UK) with a Metrosep C4 cation column (250 x 4.0 mm) attached. The mobile phase
was 3 mM Nitric acid: Acetonitrile (75:25 v/v) running in isocratic mode at 1.5
mL/min. All radiotracers were >95 % radiochemical purity after formulation.
Kinetic analysis in HCT116 tumors
A 2-tissue irreversible compartmental model was employed to fit the TACs, as has
been previously established for C-choline (Kenny LM, Contractor KB, Hinz R, et al.
Reproducibility of [llCJcholine-positron emission tomography and effect of
trastuzumab. Clin Cancer Res. Aug 15 2010;16(16):4236-4245; and Sutinen E, Nurmi
M, Roivainen A, et al. Kinetics of [(ll)C]choline uptake in prostate cancer: a PET
study. Eur J Nucl Med Mol Imaging. Mar 2004;31(3):317-324). An estimate of the
whole blood TAC (wbTAC(t)) was derived from the PET image itself, as described
above. As the wbTAC was obtained from one voxel only it was relatively noisy.
Therefore it was fitted with a sum of 3 exponentials from the peak on and the fitted
function was used as input function in the kinetic modeling (after metabolite
correction, see below). The parent fraction values, pf, were calculated from plasma
metabolite analysis: at 2, 15, 30 and 60 minutes they were [0.96,0.55,0.47,0.26] for
F-D4-choline, [0.92,0.25,0.20,0.12] for C-choline and [0.91,0.18,0.08,0.03] for
C-D4-choline, respectively. The pf values were fitted to a sum of two exponentials
with the constraint pf(t=0)=l to obtain the function pf(t). The parent whole blood
TAC wbTACpA (t) was then computed by multiplying wbTAC(t) and pf(t) and used
as input function to estimate the parameters K (mL/cm /min), (1/min), k (1/min)
and Vb (unitless). The steady state net irreversible uptake rate constant K(mL/cm /min) was calculated from the estimated microparameters as I ( 2 + ).
Because the quality of fits obtained using the wbTACpA (t) as only input function to
the model was poor, and because F-D4-choline, C-choline and C-D4-choline are
quickly metabolized in vivo in the mouse, a double input (DI) model accounting for
the contribution of metabolites to the tissue TAC was also considered (Huang SC, Yu
DC, Barrio JR, et al. Kinetics and modeling of L-6-[18F]fluoro-dopa in human
positron emission tomographic studies. J Cereb Blood Flow Metab. Nov
1991;11(6):898-913). In the DI model the metabolite whole blood TAC
wbTACMET(t) computed as wbTAC(t)x[l-pf(t)] together with wbTACpA (t) was
employed as input function; the parent tracer was modeled with a 2-tissue irreversible
model whereas a simple 1-tissue reversible model was used to describe the metabolite
kinetics, thus computing the metabolite influx and efflux K and å in addition to the
parameters estimated for the parent. The standard Weighted Non-Linear Least
Squares (WNLLS) was used as estimation procedure. WNLLS minimizes the
Weighted Residual Sum of Squares (WRSS) function
WRSS(p) = ¾ , )™ -CO ,.)]2 (A)
=l
with C ti ) and ti indicating respectively the decay-corrected concentration computed
from the PET image and the mid-time of the i-th frame and n denoting number of
frames. In Eq.l weights w were set to
- (B)
with D ; and l representing the duration of the i-th frame and the half-life of F (for
F-D4-choline) or C (for C-choline and C-D4-choline) (Tomasi G, Bertoldo A,
Bishu S, Unterman A, Smith CB, Schmidt KC. Voxel-based estimation of kinetic
model parameters of the L-[l-(ll)C]leucine PET method for determination of
regional rates of cerebral protein synthesis: validation and comparison with region-ofinterest-
based methods. J Cereb Blood Flow Metab. Jul 2009;29(7):1317-1331).
WNLLS estimation was performed with the Matlab function lsqnonlin; parameters
were constrained to be positive but no upper bound was applied.
SUPPLEMENTAL TABLE 1. Kinetic parameters from dynamic F-D4-choline
PET in tumors. Decay-corrected uptake values at 60 min (NUV60) and the area under
the curve (AUC) were taken from tumor TACs. Flux constant measurements, K , Kand were obtained by fitting tumor TAC and derived input function, corrected for
radioactive plasma metabolites of 18F-D4-choline, to a 2-tissue irreversible model of
tracer delivery and retention. Mean values n =3) + SEM are shown.
NUV 0 AUC K i k
0.008 ± 0.039 ±
HCT116 1.81 ± 0.11 114.5 ± 7.0 0.142 ± 0.027 0.001 0.003
0.006 ± 0.030 ±
A375 1.71 ± 0.14 107.3 ± 7.7 0.111 ± 0.021 0.002 0.008
0.009 ± 0.040 ±
PC3-M 1.97 ± 0.07 121.3 ± 3.1 0.090 ± 0.007 0.002 0.006
All patents, journal articles, publications and other documents discussed and/or cited
above are hereby incorporated by reference.

Claims:
1. A compound of
wherein:
Ri, R2 , R 3 , and R4 are each independently hydrogen or deuterium (D);
R5, R6 , and R 7 are each independently hydrogen, R 8 , -(CH2) m R8, -(CD2) mRs, -
(CF2)mR , -CH(R¾, or -CD(R¾;
R is independently hydrogen, -OH, -CH3, -CF3, -CH2OH, -CH2F, -CH2C1,
-CH2Br, -CH2I, -CD 3 , -CD2OH, -CD2F, CD2C1, CD2Br, CD2I, or -C6H5;
m is an integer from 1-4;
C* is a radioisotope of carbon;
X, Y and Z are each independently hydrogen, deuterium (D), a halogen
selected from F, CI, Br, and I, alkyl, alkenyl, alkynl, aryl, heteroaryl, heterocyclyl
group; and
Q is an anionic counterion; with the proviso that said compound of Formula
(III) is not C-choline.
2 . The compound according to Claim 1 wherein C* is C, C, or 4C.
3. The compound according to Claim 1 wherein C* is C; X and Y are each
hydrogen; and Z is F.
4. The compound according to Claim 1 wherein C* is C; X, Y and Z are each
hydrogen H ; Ri, R2, R3 , and R4 are each deuterium (D); and R5, R 6 , and R 7 are each
hydrogen.
5. A pharmaceutical composition comprising a compound of claim 1 and a
pharmaceutically acceptable carrier or excipient.
6. A pharmaceutical composition comprising a compound of claim 2 and a
pharmaceutically acceptable carrier or excipient.
7. A pharmaceutical composition comprising a compound of claim 3 and a
pharmaceutically acceptable carrier or excipient.
8. A pharmaceutical composition comprising a compound of claim 4 and a
pharmaceutically acceptable carrier or excipient.