Note: Descriptions are shown in the official language in which they were submitted.
CA 02266298 1999-03-19
Su et al., page 2
INTRODUCTION
Detection of regions of hypoxia is important in several medical conditions. It
is
important to delineate hypoxic but viable tissue following interruption of
blood flow in
cerebral or myocardial infarction {Nunn 1995 } . Furthermore, it has recently
been
recognised that hypoxia in tumors plays a role not only in response to
radiation, through
the oxygen effect in fixation of DNA damage, but also in metastatic potential
and
response to other forms of therapy {Hockel 1993 } {Graeber 1996} {Brizel
1996}.
However, hypoxia is heterogeneous and tumors which appear identical by all
other
clinical measures can vary greatly in their proportion of hypoxic cells. At
present,
hypoxia is measured clinically with a polarographic oxygen electrode inserted
into
tumors; however, this is invasive, operator-dependent, limited to accessible
tumors, and
not readily repeatable {Hockel 1993}{Brizel 1996}. In recent years several
radiopharmaceuticals have been developed which are targeted to hypoxic tissues
via a 2-
nitroimidazole (2-NI) moiety {Nunn 1995}{Chapman 1998}. 2-NIs are electron-
affinic
compounds which undergo an enzyme-mediated one-electron reduction; in normoxic
tissue the resultant radical anion is immediately oxidized back to the
starting compound,
whereas under hypoxic conditions there is further reduction to products which
are trapped
by binding to macromolecules {Rauth 1984}. A radiolabeled 2-NI would therefore
be
selectively trapped in hypoxic tissues.
Clinical studies have been performed with several radiolabeled 2-NIs,
including
'8F-fluoromisonidazole (FMISO) {Grierson 1989} {Koh 1992} and '23I-
iodoazomycin
arabinoside (IAZA) {Mannan 1991 } {Parliament 1992}. However, the search
continues
for a 99mTc-labeled analog that would be more widely applicable. BMS181321, a
2-NI
2
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Su et al., page 3
linked to a PnAO chelator, has been evaluated in several model systems and
shows
promise, but its high lipophilicity and metabolic instability result in high
background
levels {binder 1994} {DiRocco 1997} {Barron 1996} {Ballinger 1996}. BRU59-21
(previously known as BMS 194796) appears to possess improved characteristics
but only
limited information is available about it {Johnson 1998} {Melo 1998}. In
addition,
butyleneamine oxime (BnAO or HL91; Prognox, Nycomed-Amersham) is a non-nitro
99mTc complex that shows localization in hypoxic myocardium and tumors via an
undetermined mechanism {Archer 1995} {Okada 1997} {Cook 1998} {Zhang 1998}.
Recent work involving the radioiodinated sugar derivatives of 2-NI has
suggested
that an oil/water partition coefficient (PC) of ~ 1 confers optimal
biodistribution
properties for tumor imaging {Chapman 1998}. BMS181321 and BRU59-21 have PCs
which are much higher (40 and 12, respectively) and cannot be readily modified
to
achieve a lower PC. As part of our radiopharmaceutical development program, we
designed two compounds in which peptidic chelators for 99"'Tc were linked to 2-
NI and
evaluated the complexes in an in vitro model of tumor hypoxia. Dimethylglycyl-
L-seryl-
L-cysteinylglycinamide (RP294, Figure 1 ), which contains a sulfur atom
(protected by an
acetoamidomethyl group), one amine nitrogen atom, and two amide nitrogen atoms
is an
N3S class chelator for 99mTc0" and Re0" in a distorted square pyramidal
geometry with
the oxo moiety in the apical position. The metal complexes exist in syn and
anti isomers
with respect to the position of the oxo bond and serine CHZOH side chain.
Interconversion of the two isomers in aqueous solution at room temperature has
been
observed { Wong 1997 } .
3
CA 02266298 1999-03-19
Su et al., page 4
In the present work, dimethylglycyl-1.-seryl-1.-cysteinyl-lysyl{NE-[1-(2-nitro-
1H
imidazolyl)acetamido]}-glycine (RP435, Figure 1) was prepared by attaching 2-
NI-acetic
acid to a lysine linker connected to the RP294 chelator. RP435 chelates 99mTc
at room
temperature and two peaks, assumed to be the syn and anti isomers, were
detected by
HPLC. However, the PC of 99"'Tc0-RP435 is far lower than what is believed to
be the
optimum value. Accordingly, a new N3S class peptide, dimethylglycyl-1.-tert-
butylglycyl-L-cysteinylglycin[2-(2-nitro-1H imidazolyl)ethyl]amide (RP535,
Figure 1)
was designed and synthesized. The chelator (RP455, Figure I) contains a tent-
butyl
group, which should increase the lipophilicity of the corresponding 99"'Tc
complex and
inhibit the interconversion of syn and anti isomers. The labeling, stability,
and in vitro
cellular accumulation of these compounds was studied.
EXPERIMENTAL PROCEDURES
2-Nitroimidazole, 2-bromoethylphthalimide, N,N-dimethylformamide (DMF), 1-
(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (DECD, 2,3,5,6-
tetrafluorophenol (TFP), and trifluoroacetic acid (TFA) were purchased from
Aldrich
Chemical Co. (Milwaukee, WI). Dimethylglycyl-tent-butylglycyl-1.-
cysteinylglycine
(RP455) was custom synthesized by Bachem Bioscience Inc. (Philadelphia, PA).
Dimethylglycyl-1.-Beryl-1.-cysteinylglycinamide (RP294) and Re0-RP455
precursor were
provided by Resolution Pharmaceuticals Inc. (Mississauga, ON). Sodium 99mTCO4
In
saline was obtained from a 99Mo~'9mTc generator (DuPont Pharma, Billerica,
MA). a-
MEM and fetal bovine serum were purchased from Sigma Chemical Co. (St. Louis,
MO).
4
CA 02266298 1999-03-19
Su et al., page 5
NMR data ('H, '3C, COSY, HSQC, HMQC, TOCSY) were recorded on a Varian
UNITYpIus-500 spectrometer (500 Mhz) with TMS as external standard. Mass
spectra
(electrospray) were obtained on a Sciex API#3 mass spectrometer in the
positive ion
mode. Reversed phase high pressure liquid chromatography (HPLC) analyses of
peptide
and 99'"Tc-peptide complexes were carried out on a Beckman model 125 System
Gold
(Fullerton, CA), with Zorbax SB or Beckman ODS 4.6x250 mm 5-~m C,g columns.
Purification of peptides was accomplished by HPLC with a Waters C,8 RCM 8x10
semi-
preparative column. UV and radiometric detectors were connected in series. Two
mobile
phases were used for HPLC analysis of 99'"TC tracers. Mobile phase I was
composed of
Hz0/ACN containing 0.1 % TFA, whereas mobile phase II was 0.02 M pH 4.6 NH40Ac
buffer/MeOH; both were run as gradients. The flow rate was set at 1.0 mL/min
for
analysis and 2.0 mL/min for purification.
Synthesis of dimethylglycyl-L-Beryl-L-cysteinyl-lysyl{N~ [1-(2-vitro-1H
imidazolyl)acetamido]}-glycine (RP435): The peptide backbone was prepared on
an
automated peptide synthesizer (Applied Biosystems Inc., model 433A, Foster
City, CA)
using Sasrin resin and FMOC-protected amino acids. When the backbone was
complete,
the Dde protecting group on the s-amine on lysine was cleaved with 2%
hydrazine and
the free amine was coupled with 2-nitroimidazole acetic acid (synthesized from
2-
nitroimidazole and bromoethyl acetate). The peptide was cleaved from the resin
by
stirring with 95% aqueous TFA at 0 °C for 30 min, then at room
temperature for 60 min.
The resin was filtered and the filtrate was evaporated under vacuum. The
residue was
washed with a minimum amount of tent-butyl methyl ether, which caused the
CA 02266298 1999-03-19
Su et al., page 6
precipitation of the peptide. The ether was removed and the residue was
dissolved in
water/ACN and then lyophilized to yield 190 mg of crude product, of which 110
mg was
further purified by HPLC, yielding 35 mg (31%) pure RP435. 'H NMR (600 Mhz, db-
DMSO): b 1.33 (m, 2H, H(18)), 1.43 (m, 2H, H(19)), 1.59 (m, 1H, H(17a)), 1.72
(m, 1H,
H(17b)), 1.86 (s, 3H, H(30)), 2.58 (broad, 6H, H(1,2)), 2.68 (dd, 1H, H(13a),
JH~3a-HI1-
9.89 Hz, JH~3a-HI3b 14.04 Hz), 2.95 (dd, 1H, H(13b), JH,3b-H»- 3.91 Hz, JH,sb-
Hl3a 13.92
Hz), 3.05 (m, 2H, H(20)), 3.61 (m, 2H, H(7)), 4.13 (m, 1 H, H( 15)), 4.20 (dd,
1 H, H(27a),
JH27a-H28- 5.86 Hz, JHZ7a-H27b 13.67 Hz), 4.32 (dd, 1H, H(27b), JHZ7b-HZS-
5.13 Hz, JH2sb-H28a
13.67 Hz), 4.44 (m, 1 H, H(6)), 4.56 (m, 1 H, H( 11 )), 5.07 (s, 2H, H(23)),
5.09 (broad, 1 H,
H(8)), 7.18 (d, H(25), JHZS-H24- 0.97 Hz), 7.61 (d, 1 H, H(24), JHZa-HZS= 0.98
Hz), 8.0 (d, 1 H,
H(14), JH14-15- 757 Hz), 8.31 (m, 2H, H(10, 21)), 8.56 (t, 1H, H(28), JHZS-H27-
6.47 Hz).
Electrospray mass spectrum: mle= 646.15 ([M+H]+, Cz4H4°N9O,°S,
calculated: 646.69).
2-(2-Nitro-1H imidazolyl)ethylamine Hydrochloride {Hay 1994}. 0.56 g (5.0
mmol) 2-nitroimidazole, 1.33 g (5.2 mmol) N (2-bromoethyl)phthalimide, and
0.72 g (5.2
mmol) potassium carbonate were mixed in 15 mL DMF, and heated at 110 °C
for 2 h.
After removing the solvents, water was added to the residue to dissolve the
salts. The
precipitate was collected, washed by water, and dried under vacuum to yield
0.8 g (2.8
mmol, yield 56%) of raw product. The raw product was refluxed with 0.28 g (5.6
mmol)
hydrazine monohydrate in 20 mL EtOH for 2 h, and then cooled to 4 °C,
filtered, and
evaporated to dryness. The residue was dissolved in 20 mL 1 N HCI, filtered,
and
brought to dryness again. The residue was recrystallized from MeOH/EtOAc to
yield 240
mg (1.25 mmol, yield 44%) of product.
6
CA 02266298 1999-03-19
Su et al. , page 7
Dimethylglycyl-tert-butylglycyl-L-cysteinylglycine-[2-(2-nitro-1H imidazolyl)-
ethyl]amide (RP535): 80 mg (0.18 mmol) RP455, 55.4 mg (0.29 mmol) DECI, 48 mg
(0.29 mmol) TFP were dissolved in 1 mL DMF. The mixture was stirred in an ice-
bath
for 10 min, followed by 30 min at room temperature, and then in a 45°C
oil bath for 60
min. 37.2 mg (0.19 mmol) 2-(2-nitro-1H imidazolyl)ethylamine hydrochloride was
added to the mixture which was set in an ice bath. To the mixture was added
dropwise
74.7 mg (0.58 mmol) DIEA (di-isopropyl ethylamine) in 1 mL DMF. The mixture
was
stirred at 45°C for 1 h. The solvent was removed after the reaction was
completed.
About 2 mL of water was added and then removed. The crude product was purified
by
HPLC, yielding 50 mg (0.085 mmol, 29%) pure RP535. 'H-NMR (500 MHz, db-
DMSO): b 0.93 (s, 9H, H(8,9,10)), 1.86 (s, 3H, H(28)), 2.71 (dd, 1H, H(14a),
JH~4a-H13-
9.3 Hz, JH~4a-Hl4b 13.9 Hz), 2.79 (t, 6H, H(1,2), J=4.0 Hz), 2.90 (dd, 1H,
H(14b), JH,ab-H13-
4.6 Hz, JH~46-Hl4a 13.9 Hz), 4.25 (d, 1H, H(25a), JHZSa-HZ6- 6.59 Hz), 4.27
(d, 1H, H(25b),
JH256-H26- 6.59 Hz), 4.37 (d, 1 H, H(6), JH6-HS- 9.27 Hz), 4.47 (m, 1 H, H(
13)), 7.16 (d, 1 H,
H(23), JH23-H22- 1.2), 7.51 (d, 1H, H(22), JH22-HZS- 0.98 Hz), 7.89 (t, 1H,
H(19), JH~g_H20
5.98 Hz), 8.14 (t, 1H, H(16), JH~6-H17- 5.6 Hz), 8.39 (d, 1H, H(12), JH~2-H13-
7.57 Hz),
8.45 (t, 1H, H(26), JH26-H25- 6.59 Hz), 8.61 (d, 1H, H(5), JHS-H6 9.27 Hz).
Electrospray
mass spectrum: mle = 586.11 ([M+H]+, C23H40N907S, calculated: 586.69).
Synthesis of 99"'Tc0-RP294 and 99"'Tc0-RP455: 100 to 200 ~g of RP294 or
RP455 was added to Na99mTcO4 (2 to 10 mCi) in 200 pL of saline. To the mixture
was
added 100 pL of stannous gluconate which contained 40 ~g of SnClz and 0.25 to
1.0 mg
of sodium gluconate. The mixture was incubated in a 95°C water bath for
15 min.
7
CA 02266298 1999-03-19
Su et al. , page 8
99mTc0-RP294 and 99mTc0-RP455 were analyzed by HPLC (Zorbax SB column,
HZO/ACN containing 0.1 % TFA; gradient 100% to 90% HZO over 45 min for 99mTc0-
RP294, and 100% to 50% HZO over 45 min for 99mTc0-RP455). The labeling
efficiency
for 99'"Tc0-RP294 was >97%, while that for 99"'Tc0-RP455 was >99%.
Synthesis of 99"'Tc0-RP435 and 99"'Tc0-RP535: To a 3-mL tube were added 100
to 200 ~,g of RP435 or RP535, Na99mTcO4 (2 to 10 mCi) in 200 ~.L saline, and
100 ~L of
stannous gluconate solution which contained 10 to 40 ~g of SnClz and 1.0 mg of
sodium
gluconate. The labeling of 99"'Tc0-RP435 was carried out at room temperature,
while that
of 99mTcO-RP535 was incubated in a boiling water bath for 30 min. The products
were
analyzed by HPLC (Zorbax SB column, H20/ACN containing 0.1 % TFA; gradient for
99mTc0-RP435 was 100% to 70% Hz0 over 45 min, while that for 99mTc0-RP535 was
100% to 50% H20 over 45 min). The yield of 99'"Tc0-RP435 was >68%, while that
for
99mTcO-RP535 was >78%. The two compounds were purified for further experiments
by
HPLC in the same conditions stated above.
Synthesis of Re0-RP535: 10 mg (0.017 mmol) of Re0-RP455 and 13.3 mg
(0.070 mmol) of DECI were dissolved in 0.6 mL ACN solution which contained 20%
0.01 M NaOAc/HOAc. To the mixture was added 2.89 mg (0.017 mmol) of TFP
dissolved in 0.3 mL ACN-acetate solution. The mixture was stirred at room
temperature
for 30 min, and then at 45°C for 1 h. To the mixture was added 3.34 mg
(0.017 mmol) of
2-(2-nitro-1H imidazolyl)ethylamine HCl in 0.5 mL ACN-acetate solution,
followed by
2.6 mg (0.20 mmol) of DIEA. After stirring at 45 to 50°C for 2 h, the
solvent was
removed, and the crude product was purified by HPLC (Zorbax SB column, HZO/ACN
8
CA 02266298 1999-03-19
Su et al., page 9
containing 0.1 % TFA, flow rate 1.0 mL/min; gradient 100% to 50% H20 in 45
min). The
fraction with a retention time of 11.62 min was collected and lyophilised. 'H
NMR (500
MHz, d6-DMSO): 8 1.03 (s, 9H, H(8,9,10)), 2.44 (s, 3H, H(1)), 3.55 (s, 3H,
H(2)), 3.83
(d, 1H, H(14a), JH,4a_H,3-12.2 Hz), 4.02 (d, 1H, H(3a), JH3a-H3b 14.9 Hz),
4.40 (t, 1H,
H(21 a), J,jzla-H20 5.62 Hz), 4.41 (t, 1 H, H(21 b), JH216-H20 5.6 Hz), 4.46
(s, 1 H, H(6)], 4.82
[d, 1H, H(3b), J,~3b-H3a 14.9 Hz), 5.08 (d, 1H, H(14b), JH,4b-H13-6~84 Hz),
6.99 (d, 1H,
H(23), JH~3_H22 1.22 Hz), 7.44 (d, 1H, H(22), JHZZ-HZS-1.22 Hz), 7.72 (t, 1H,
H(19)), JH,9_
Hzo 5.86 Hz), 8.19 (t, 1H, H(16)), JH,6_H"=5.62 Hz). ES-MS: mle 712.93
([M+H]+,
Cz°H3zNg0 ,'85ReS, calculated: 712.53) and mle 714.94 ([M+H]+,
CzoH3zNg0,'g'ReS,
calculated: 714.54), confirming a 1:1 ratio of ligand to metal ion.
Partition Coefficient (n-OctanoUPBS) Determination for 99'"Tc0-RP435 and
99m.1.c0-RP535: 10 pL of purified 99'"Tc0-RP435 or 99mTc0-RP535 was added to a
tube
which contained 1.0 mL of n-octanol and 1.0 mL of 0.1 M pH 7.4 PBS. The tube
was
shaken for 1 min. After partial separation of the phases by gravity, 0.7 mL of
each phase
was transferred to a tube and centrifuged at 12,000 g for 5 min. Duplicate 0.2-
mL
aliquots of each phase were taken for y-radioactivity counting.
Cellular Accumulation in an In Vitro Model: Each complex was evaluated in
an in vitro model which has been used in the study of other tracers of hypoxia
{Ballinger
1996} {Melo 1998} {Zhang 1998} {Ballinger 1993}. Suspensions of Chinese
hamster
ovary (CHO) cells were incubated at 37°C with stirring under an
atmosphere of air or
nitrogen (both containing 5% COz) to generate aerobic or hypoxic conditions,
respectively. The tracer was added and aliquots were removed over the course
of 4 h,
9
CA 02266298 1999-03-19
Su et al., page 10
centrifuged, and the radioactivity associated with the cell pellet was
measured in a y well
counter. The results were expressed as the ratio of concentration of
radioactivity in the
cell pellet to that in an equivalent volume of supernatant medium (C;"/Co",)
as a function
of time, as described previously {Ballinger 1996}. In experiments in which 5
mM
misonidazole or 8 mM metronidazole were added, the competitor was added 30 min
before the tracer, as described previously {Melo 1997}.
RESULTS
Synthesis of RP435 and RP535. RP435 was prepared by attaching 2-NI-acetic
acid to a lysine connected to a peptidic N3S chelator (Figure 1). After being
cleaved from
the resin, RP435 was purified by reversed phase HPLC. The purity was shown to
be
greater than 98%. RP535 was prepared by coupling 2-(2-nitro-1H
imidazolyl)ethylamine
to RP455 (Figure 1). The purity of RP535 was analyzed by two HPLC gradients
and
shown to be greater than 97%.
The molecular weight of RP435 was determined by electrospray mass
spectrometry (ES-MS) with mle 646.15 ([M+H]+, C24H39N9010S requires 645.69).
The'H
NMR (500 MHz) spectrum of RP435 revealed two protons of the 2-nitroimidazole
at
7.19 and 7.61 ppm with a weak mutual coupling (J= 0.97 Hz). The proton of the
hydroxyl, H(8), appeared at 5.09 ppm as a broad peak. Four of the five amide
protons of
RP43 5, H( 11 ), H( 14), H(21 ), and H(28), were identified. H( 10) and H(21 )
overlapped
each other at 8.31 ppm. The six protons of H( 1 ) and H(2) formed a broad peak
at 2.57
CA 02266298 1999-03-19
Su et al. , page 11
ppm, while the two protons of H(3) were merged in the solvent peak ranging
from 3.26 to
3.36 ppm.
The molecular weight of RP535 was determined by ES-MS which showed the
molecular ion signal at mle 586.11 ([M+H]+, C23H40N907S requires 585.69). The
'H
NMR spectrum of RP535 exhibited signals of all five amide protons (Figure 1),
with H(5)
at 8.61 ppm, H(26) at 8.45 ppm, H(12) at 8.39 ppm, H(16) at 8.13 ppm, and
H(19) at 7.89
ppm, respectively. Similar to RP435, the two protons of the 2-nitroimidazole
of RP535
showed a weak mutual coupling (JHZZ-HZS- 1.1 Hz). The nine protons of the tent-
butyl
group, H(8,9,10), appeared as a singlet peak at 0.91 ppm. It is interesting to
note that the
six protons, H(1,2), of the two methyl groups of dimethylglycine produced
triplet peaks,
instead of a singlet peak, at 2.80 ppm. The signals of H(17) and H(20) were
partly
merged in the solvent peak ranging from 3.50 to 3.66 ppm, while H(13) and
H(21)
overlapped each other at 4.45 ppm (m, 3H). These data indicated that RP535
contained
the N3S chelator, a 2-nitroimidazole group, and the acetamidomethyl protecting
group for
the sulfur atom of cysteine.
99m~j~C labeling of RP294, RP455, and RP435. 99"'TC labeling of RP294 and
RP435, which have the same N3S chelator, can be carried out at room
temperature via
transchelation from 99mTc-gluconate. However, 99'"Tc labeling of RP455, which
has a
tent-butyl group on the chelator, require heating in a boiling water bath for
10 to 15 min.
99mTc0-RP294 showed two peaks on HPLC in 0.1 % aqueous TFA/ACN in a total
yield
>97%. In contrast, 99"'Tc0-RP455 showed one single peak (yield >99%). When
analyzed
in acetate/MeOH, 99'"Tc-RP294 showed two major peaks, at 33.69 min (39.6%) and
36.81
11
CA 02266298 1999-03-19
Su et al., page 12
min (59.3%), with a gradient of 100% to 10% acetate over 45 min, while 99mTc-
RP4SS
showed a major peak at 35.55 min (84.8%) with the same gradient.
Stannous gluconate solution was used as reducing agent for Na99mTc04 in the
presence of 100 to 300 ~g of RP435. Two main peaks (peak A: 32.42 min, yield
26.4%;
peak B: 35.59 min, yield 42.5%) were detected on HPLC (Figure 2) with aqueous
TFA/ACN (gradient: 100% to 70% water over 45 min). The ratio of peaks A and B
in
the reaction mixture varied with time, while their retention times remained
unchanged.
The difference in retention times of peaks A and B of 99"'Tc0-RP435 was 3.1
min when
measured by HPLC with elution gradient of 100% to 70% water over 45 min, while
that
of the two peaks from 99'"Tc0-RP294 was 0.7 min.
Stability of 99'"Tc0-RP435. Peaks A and B of 99'"Tc0-RP435, after being
separated, were reanalyzed by HPLC after different time intervals. A slow
interconversion of the two isomers in aqueous solution containing 0.1 % TFA
was
observed. For example, 4.2% of B, which came from the conversion of A, was
detected
103 minutes after the isolation of A from the labeling mixture; and 4.0% of A,
which
came from the conversion of B 118 minutes after isolation, was detected. The
interconversion of A and B could be greatly accelerated when they were mixed
with an
equal volume of 0.25 M pH 7.4 phosphate buffer (PB). Figure 3 shows the
interconversion of purified A to B and of purified B to A, and it is evident
that an
approximate 1:1 equilibrium was reached within 1 hour.
99mTC labeling of RP535. RP535 (100 to 300 fig) was labeled with 2 to 20 mCi
99"'Tc with stannous gluconate as reducing agent. Higher temperatures (60 to
100°C)
12
CA 02266298 1999-03-19
Su et al., page 13
were required to label the compound. One major labeled peak (yield over 78%)
has been
obtained when the reaction mixture was incubated in a boiling water bath for
30 min
(Figure 4, lower trace). Varying the pH from 2 to 7 did not significantly
affect the
labeling yield. Although the nitroimidazole group of RP535 was stable under
the labeling
conditions, significant reduction was noted (analyzed by HPLC and monitored by
the UV
detector at 214 and 320 nm) when larger amounts of SnCl2 were added, resulting
in a
dramatic decrease in the yield of 99'"Tc0-RP535. For example, the yield of
99"'Tc0-
RP535 dropped to less than 5% of the total detected radioactivity on HPLC when
100 ~g
of SnClz was used, and no intact RP535 remained when 600 ~g of SnCl2 was
added.
Determination of partition coefficients (PCB of 99"'Tc0-RP435 and 99m.1.cO-
RP535 between n-octanol and phosphate buffered saline (PBS). It is impossible
to
isolate individually the syn and anti isomers of 99'"Tc0-RP435 by HPLC with
mobile
phase I or II due to their rapid interconversion in aqueous solution at room
temperature.
The reported PC (0.0013) of 99"'Tc0-RP435 is in fact the average PCs of the
two isomers
of the 99mTc0-RP435. The PC of 99'"Tc0-RP535 was 2.8~0.1, n=4. Thus, the
difference
in partition coefficients of 99'"Tc0-RP535 and 99'"Tc0-RP435 is over 2,000
fold.
Preparation of Re0-RP535 and co-injection with 99"'Tc0-RP535. It is difficult
to determine directly the structure of a 99"'Tc complex because of the
extremely small
amount of the compound present. 99Tc is a long-lived radioactive isotope
(t"2=2.1x105 y)
which restricts its application. However, the composition and structure of
99'"Tc0-RP535
could be indirectly determined by comparison with Re0-RP535, which can be
prepared
in weighable quantities and characterized. Re belongs to the same group (VIIb)
of the
13
CA 02266298 1999-03-19
Su et al., page 14
periodic table and possesses many similar chemical characteristics with Tc
because of the
lanthanide contraction.
Re0-RP535 was prepared by coupling 2-(2-nitro-1H imidazolyl)ethylamine with
Re0-RP455. The compound was purified by HPLC. 'H NMR data showed that the two
amide protons, H(5) and H(12), disappeared after the chelation of RP535 to
Re03+, while
those of H(16) and H(19) of the uncoordinated RP535 (Figure 1) remained. The
two
methyl groups and the methylene group of dimethylglycine could no longer
freely rotate
around the C(3)-N~",;~e bond in Re0-RP535, which made the protons of these
groups
become chemically non-equivalent. For example, the protons of H( 1 ) shifted
from 2.80
ppm to 2.44 ppm, and H(2) shifted from 2.80 ppm to 3.55 ppm upon the formation
of
Re0-RP535. This indicated that the N atom of the dimethylglycine co-ordinate
to Re03+
core. The two protons of H(3) of Re0-RP535 were in a chemically non-equivalent
environment as well. H(3a) appeared at 4.02 ppm and H(3b) at 4.82 ppm with a
coupling
constant JH3a-H3b at 14.9 Hz. H(14a) coupled H(13) Wlth JHl4a-H13 for 6.8 ppm,
while
H(14b) coupled H(13) Wlth JH~4a-H13 for 12.2 ppm. However, no signals of
H(14a)
coupling with H(14b) were found. All this evidence supported the assumption
that the
Re03+ core was complexed with the N3S chelator. The signals of H(17) and H(20)
overlapped each other at the range of 3.46 to 3.54 ppm, while that of H(13)
was partly
merged in solvent peak (in the range of 3.28 to 3.36 ppm). No evidence of the
co-
existence of the syn and anti isomers of Re0-RP535 was found in the'H NMR
spectrum.
The prepared Re0-RP535 can be either a syn or anti isomer. The electrospray
mass
spectrum exhibited the characteristic two molecular ions at mle 712.93 ([M+1
]+) which
corresponded to a 1:1 'g5Re0 to RP535 compound and mle 714.94 ([M+1]+) which
14
CA 02266298 1999-03-19
Su et al., page 15
corresponded to another 1:1 'g'Re0 to RP535 compound (the required molecular
weights
are 712.53 and 714.54). Moreover, the relative abundances of the two peaks
(57.4% and
100%) are consistent with the natural abundances of'85Re and'8'Re (37.4% and
62.6%).
An aqueous solution of Re0-RP535 was mixed with the 99"'Tc0-RP535 labeling
mixture and co-injected into the HPLC for analysis. Two gradients of aqueous
TFA/ACN were used: 100% to 30% and 100% to 50% water over 45 min. Two
detectors, UV and radiometric, were connected in series with the eluent
entering the UV
detector first and then the radiometric detector. A time delay of 0.3 to 0.4
min for signal
response between the UV and radiometric detectors existed. Figure 4 shows the
chromatogram of the co-injection of Re0-RP535 with 99'"Tc0-RP535 with the 100%
to
30% water gradient. The retention time of Re0-RP535 was 33.04 min, while that
of
99'"Tc0-RP535 was 33.35 min. The UV peak at 23.37 min was free RP535. The
retention times of Re0-RP535 and 99"'Tc0-RP535 became 39.91 min and 40.51 min,
when 100% to 50% water gradient was used. The retention times of Re0-RP535 and
99"'Tc0-RP535 are the same when the time delay in signal response is taken
into account.
This implies that 99"'Tc0-RP535 has the same composition and structure as Re0-
RP535;
i.e. 99mTc is coordinated by the N3S chelator of RP535, there is a 99'"Tc=O
core, and the
ratio of 99'"Tc to RP535 is 1:1.
Stability of 99"'Tc0-RP535. 99mTc0-RP535 was isolated by HPLC and the
collected 99mTcO-RPS3S in aqueous TFA/ACN was re-injected into the HPLC for
analysis after different time intervals at room temperature. It was evident
that 99'"TCO-
RP535 was more stable than 99"'Tc0-RP435 in aqueous solution which contained
0.1%
CA 02266298 1999-03-19
Su et al., page 16
TFA, because neither decomplexation nor change in the radioactive peak was
seen over
26 hours.
Theoretically, the formation of syn and anti isomers of 99"'Tc0-RP535 is
possible,
when the tent-butyl side chain on the N3S chelator is in the syn and anti
conformations
with respect to the 99'"Tc=O core. When the isolated 99mTc0-RP535 was mixed
with an
equal volume of 0.25 M pH 7.4 PB and reinjected after different time
intervals, a
decomplexation of the complex, instead of interconversion of syn and anti
isomers, was
observed (Figure SA). 99"'Tc0-RP535 (retention time 40.27 min) decomplexed in
PB and
formed three products which appeared in the region of 22.1 to 23.7 min on the
HPLC.
These decomplexed compounds cannot be the isomers of 99'"Tc0-RP535 because
they did
not exist in the labeling mixture (Figure 4, lower trace) and they did not
reach any fixed
equilibrium with 99"'Tc0-RP535 over time. The decomplexation might occur when
one
or more coordination bonds of the 99"'Tc0-RP535 complex broke. No free
pertechnetate
was detected from the decomplexation of 99"'Tc0-RP535. The half time for
decomplexation was about 200 min (Figure SB). Interestingly, 99mTc0-RP535
decomposed immediately with release of pertechnetate when mixed with 0.01 N
NaOH.
These results indicate that the tent-butyl group on the N3S chelator acts as
an important
steric hindrant that inhibits the co-existence and interconversion of syn and
anti isomers
of 99"'Tc0-RP535. However, the configuration of 99mTc0-RP535 remains to be
determined.
In Vitro Tests of 99'"Tc0-RP294, 99"'Tc0-RP455, 99"'Tc0-RP435, and 99",TcO-
RP535. The ability of these 99mTc labeled compounds to be taken up by
mammalian cells
16
CA 02266298 1999-03-19
Su et al. , page 17
under aerobic or hypoxic conditions was tested using CHO cells in suspension
culture in
equilibrium with 95%air/5% COZ or 95% Nz/5% COz gas mixtures. Samples were
removed as a function of time and ratios of cell associated radioactivity
(C;n) to that in the
medium (Co~,) were calculated. The control compounds, 99"'Tc0-RP294 and
99"'TCO-
RP455, which do not contain 2-NI groups, did not show selective accumulation
in
hypoxic CHO cells. In contrast, 99'"Tc0-RP435 showed differential accumulation
between hypoxic and aerobic cells. The C;~/CoU, for aerobic cells at 4 hours
was 0.3, while
that of hypoxic cells was 0.8 (Figure 6A), for a hypoxic/aerobic differential
of 2.1 ~ 1.0
(n=8). In comparison, 99"'Tc0-RP535 revealed a greater differential
accumulation
between hypoxic and aerobic cells than 99"'Tc0-RP435. When 99mTc0-RP535 was
added
to a suspension of CHO cells in vitro, there was a modest accumulation of
radioactivity in
the cells under aerobic conditions, reaching a C;n/CoU, value of 0.7 at 4
hours (Figure 6B).
In contrast, under hypoxic conditions there was a further increase to a
C;n/Cout value of
2.1, for a 3.3 ~ 1.2 (n=9) fold hypoxic/aerobic differential.
Previous work {Melo 1997} with the 99"'Tc-labeled 2-NI BMS181321, using the
same cell line and in vitro system, showed that a large molar excess of the 2-
NI
compound misonidazole prevented selective accumulation in hypoxic cells while
the 5-
nitroimidazole metronidazole stimulated it. In the present work, addition of 5
mM
misonidazole abolished the hypoxia-specific accumulation of 99'"Tc0-RP535,
whereas 8
mM metronidazole selectively increased hypoxic accumulation to 130% of the
control
value.
17
CA 02266298 1999-03-19
Su et al., page 18
DISCUSSION
Based on promising results obtained with 'gF-FMISO and 'z3I-IAZA {Grierson
1989} {Koh 1992} {Mannan 1991 } {Parliament 1992}, there has been great
interest in the
development of 99"'Tc-labeled markers of hypoxia which would be convenient to
prepare
and widely applicable. However, the standard 99'"Tc-chelation systems have not
all
proved to be useful. The first attempt using the BATO approach resulted in a
complex
which was not efficiently reduced and trapped, and which showed inadequate
permeability to lipophilic membranes {Linder 1993}. A bis(amine-phenol)
complex
similarly failed to cross cell membranes {Ramalingam 1994}. Greatest success
thus far
has been obtained with amineoxime-type chelators, specifically BMS181321
{Linder
1994}, BRU59-21 {Wedeking 1995}, and BnAO {Archer 1995}; however, each has its
limitations. BMS 181321 has a PC of 40, which results in slow clearance from
the blood
and background tissues, and extensive elimination via the gastrointestinal
tract {Ballinger
1996}. BRU59-21 has a lower PC of 12 with resultant improved clearance from
the
blood, but the extent of hepatobiliary excretion remains high {Johnson 1998}
{Melo
1998}. In contrast, BnAO undergoes extensive renal elimination but, lacking a
nitro
group, its mechanism of hypoxia-specific localization is unclear {Zhang 1998}.
BnAO
has a low PC of 0.1, although this does not appear to limit its penetration of
tissue
{Zhang 1998}. Work in the radioiodinated sugar 2-NI series has suggested an
optimum
PC of ~ 1 { Chapman 1998 } .
The RP294 chelation system for 99"'Tc was developed by Resolution
Pharmaceuticals Inc. {along 1997} and is used in RP128, a peptide for imaging
18
CA 02266298 1999-03-19
Su et al., page 19
inflammation {Caveliers 1996}. The free carboxylic acid group of the molecule
contributes to the hydrophilicity of the complex and RP435, the 2-NI-
containing
derivative of RP294, has an extremely low PC of 0.0013. Therefore, an analog
containing tent-butyl glycine in place of serine was developed. In the present
work, the
utility of this chelator on a 2-NI compound targeted to hypoxic tumor cells
was evaluated.
The partition coefficient of 99"'Tc0-RP535 was 2.8, which is similar to that
reported for
IAZA {Mannan 1991 }.
Although the presence of the tert-butyl group was desirable to increase the PC
of
the complex, it made the chelator more difficult to label. While 99"'Tc
labeling of RP294
and RP435 by transchelation from gluconate proceeded efficiently at room
temperature,
the tent-butyl analogs RP455 and RP535 required heating. Maximal yields
obtained were
26% and 43% for the two peaks of 99mTc0-RP435 (Figure 2) and 78% for 99"'Tc0-
RP535
(Figure 4, lower trace). It was shown that the nitro group was not reduced by
the typical
quantities of stannous chloride used in 99"'Tc labeling, but that it could be
reduced by
excessive amounts.
The structure of RP294 was determined by '3C and 'H NMR, except the
assignments of the amide protons { Wong 1997 } . In addition, Re0-RP294 and
99TcO-
RP294 have been structurally confirmed by 'H NMR and X-ray crystallography,
which
indicated that the metal ions were co-ordinated by the N3S chelator. RP435,
containing
the same N3S chelator as RP294, can be presumed to chelate the 99'"TCO3+ core
in the
same manner. As has been shown previously for 99mTc0-RP294 { Wong 1997 }, two
complexes of 99mTc0-RP435 were formed, presumably syn and anti isomers (Figure
2).
Following separation by HPLC these were quite stable in acidic aqueous
solution (4%
19
CA 02266298 1999-03-19
Su et al., page 20
interconversion in 2 hours) but equilibrated in 0.25 M pH 7.4 phosphate buffer
with a
half time of ~l hour (Figure 3). In contrast, 99'"Tc0-RP535 existed as a
single isomer
(Figure 4, lower trace), confirmed by co-injection with its Re analog (Figure
4, upper
trace), which was extremely stable in acidic aqueous medium. When diluted in
PB,
99"'Tc0-RP535 decomplexed with a half time of ~2 hours, resulting in the
formation of
several hydrophilic species but no release of free pertechnetate (Figure 5).
However,
when 99'"Tc0-RP535 was diluted in 0.01 N NaOH it decomposed rapidly to
pertechnetate.
Following HPLC purification, 99"'Tc0-RP435 and 99"'Tc0-RP535 were evaluated
in an in vitro model of cellular hypoxia that has been used extensively in
radiation
biology and investigation of bioreductive drugs, including other 99mTc- and
'gF-labeled
hypoxia tracers {Ballinger 1996} {Melo 1998} {Zhang 1998} {Ballinger 1993}.
Both
tracers showed a modest level of uptake in aerobic CHO cells, which increased
slightly
over the course of four hours. In contrast, hypoxic cells accumulated 2 to 3
times as
much radioactivity over that time period, a significant degree of hypoxia-
specific
accumulation (Figure 6). Moreover, the selective accumulation of 99mTc0-RP535
could
be modulated by co-incubation with millimolar concentrations of unlabeled
nitro
compounds (data not shown). The 2-NI misonidazole abolished the hypoxia-
specific
accumulation of 99"'Tc0-RP535 while metronidazole, a 5-NI of lower electron
affinity,
enhanced this accumulation. The same effects have been reported previously
with the 2-
NI 99mTc-BMS 181321 and suggest a bioreductive mechanism of localization of
the tracer
{Melo 1997}.
CA 02266298 1999-03-19
Su et al. , page 21
CONCLUSIONS
RP435 and RP535 have a similar N3S chelator in structure, but their ability to
form chelates with 99mTC 1S different. The 99"'Tc0-RP435 chelate could be
prepared at
room temperature, and showed interconversion of syn and anti isomers in
aqueous
solution. The interconversion of the two isomers was much faster in neutral
than in
acidic medium. In contrast, the tent-butyl group on the backbone of the N3S
chelator of
RP535 hindered the chelation with 99mTc, presumably by steric effects, and
blocked the
formation and interconversion of the theoretically existing syn and anti
isomers of
99mTc0-RP535 in acidic and neutral aqueous solution at room temperature.
However, the
tent-butyl group of RP535 did have the desired effect of significantly
increasing the
lipophilicity of the corresponding 99mTc chelate. Both 99"'Tc0-RP435 and
99mTc0-RP535
showed selective uptake in hypoxic cells, suggesting that 99'"Tc0-peptidic
complexes
containing the 2-nitroimidazole group can be a new class of hypoxia imaging
agents.
21
CA 02266298 1999-03-19
Su et al., page 22
REFERENCES
1. Nunn, A.; Linden K.; Strauss, H. W. Nitroimidazoles and hypoxia imaging.
Eur. J.
Nucl. Med. 1995, 22, 265-280.
2. Hockey M.; Knoop, C.; Schlenger, K.; Vorndran, B.; Baussmann, E.; Mitze,
M.;
Knapstein, P. G.; Vaupel, P. Intratumoral p0z predicts survival in advanced
cancer
of the uterine cervix. Radiother. Oncol. 1993, 26, 45-50.
3. Graeber, T. G.; Osmanian, C.; Jacks, T.; Housman, D. E.; Kock, C. J.; Lowe,
S. W.;
Giaccia, A. J. Hypoxia mediated selection of cells with diminished apoptotic
potential in solid tumors. Nature, 1996, 379, 88-91.
4. Brizel, D. M.; Scully, S. P.; Harrelson, J. M.; Layfield, L. J.; Bean, J.
M.; Prosnitz,
L. R.; Dewhirst, M. W. Tumor oxygenation predicts for likelihood of distant
metastases in human soft tissue sarcoma. Cancer Res. 1996, 56, 941-943.
5. Chapman, J. D.; Engelhardt, E. L.; Stobbe, C. C.; Schneider, R. F.; Hanks,
G. E.
Measuring hypoxia and predicting tumor radioresistance with nuclear medicine
assays. Radiother. Oncol. 1998, 46, 229-237.
6. Rauth, A. M. Pharmacology and toxicology studies of sensitizers. Int. J.
Radiat.
Oncol. Biol. Phys. 1984,10, 1293-1300.
7. Grierson, J. R.; Link, J. M.; Mathis, C. A.; Rasey, J. A.; Krohn, K. A. A
radiosynthesis of fluorine-18 fluoromisonidazole. J. Nucl. Med. 1989, 30, 343-
350.
8. Koh, W.-J.; Rasey, J. S.; Evans, M. L.; Grierson, J. R.; Lewellen, T. K.;
Graham, M.
M.; Krohn, K. A.; Griffin, T. W. Imaging of hypoxia in human tumors with ['gF]-
fluoromisonidazole. Int. J. Radiat. Oncol. Biol. Phys. 1992, 22, 199-212.
22
CA 02266298 1999-03-19
Su et al., page 23
9. Mannan, R. H.; Somayaji, V. V.; Lee, J.; Mercer, J. R.; Chapman, J. D.;
Wiebe, L. I.
Radioiodinated 1-(5-iodo-5-deoxy-beta-D-arabinofuranosyl)-2-nitroimidazole
(Iodoazomycin arabinoside: IAZA): a novel marker of tissue hypoxia. J. Nucl.
Med.
1991, 32, 1764-1770.
10. Parliament, M. B.; Chapman, J. D.; Urtasun, R. C.; McEwan, A. J.; Golberg,
L.;
Mercer, J. R.; Mannan, R. H.; Wiebe, L. I. Non-invasive assessment of human
tumor hypoxia with 'z3I-iodoazomycin arabinoside: preliminary report of a
clinical
study. Br. J. Cancer 1992, 65, 90-95.
11. Linder, K. E.; Chan, Y.-W.; Cyr, J. E.; Malley, M. F.; Nowotnik, D. P.;
Nunn, A. D.
Tc0(PnAO-1-(2-nitroimidazole)) [BMS-181321], a new technetium-containing
nitroimidazole complex for imaging hypoxia: synthesis, characterization, and
xanthine oxidase-catalyzed reduction. J. Med. Chem. 1994, 37, 9-17.
12. DiRocco, R. J.; Bauer, A. A.; Pirro, J. P.; Kuczynski, B. L.; Belnavis,
L.; Chan, Y.-
W.; Linden K. E.; Narra, R. K.; Nowotnik, D. P.; Nunn, A. D. Delineation of
the
border zone of ischemic rabbit myocardium by a technetium-labeled
nitroimidazole.
Nucl. Med. Biol. 1997, 24, 201-207.
13. Baryon, B.; Grotta, J.; Lamki, L.; Villar, C.; Ephron, V.; Patel, D.;
Linder, K. E.;
Nunn, A. D. Preliminary experience with technetium-99m BMS-181321, a
nitroimidazole, in the detection of cerebral ischemia associated with acute
stroke. J.
Nucl. Med. 1996, 37, 272P-273P.
14. Ballinger, J. R.; Wan Min Kee, J.; Rauth, A.M. In vitro and in vivo
evaluation of a
technetium-99m-labeled 2-nitroimidazole (BMS 181321 ) as a marker of tumor
hypoxia. J. Nucl. Med. 1996, 37, 1023-1031.
23
CA 02266298 1999-03-19
Su et al., page 24
15. Johnson, L. L.; Schofield, L.; Mastrofrancesco, P.; Donahay, T.; Nott, L.
Technetium-99m-nitroimidazole uptake in a swine model of demand ischemia. J.
Nucl. Med 1998, 39, 1468-1475.
16. Melo, T.; Duncan, J.; Ballinger, J. R.; Rauth, A. M. BMS 194796, a second-
generation Tc-99m-labelled 2-nitroimidazole for imaging hypoxia in tumours. J.
Nucl. Med. 1998, 39, 219P.
17. Archer, C. M.; Edwards, B.; Kelly, J. D.; King, A. C.; Burke, J. F.;
Riley, A. L. M.
Technetium labelled agents for imaging tissue hypoxia in vivo. In Technetium
and
rhenium in chemistry and nuclear medicine, Nicolini M, Bandoli G, Mazzi U,
Eds.; Padova: S G Editoriali; 1995; pp 535-539.
18. Okada, R. D.; Johnson, G.; Nguyen, K. N.; Edwards, B.; Archer, C. M.;
Kelly, J. D.
99mTc-HL91: Effects of low flow and hypoxia on a new ischemia-avid myocardial
imaging agent. Circulation, 1997, 95, 1892-1899.
19. Cook, G. J. R.; Houston, S.; Barrington, S. F.; Fogelman, I. Technetium-
99m-labeled
HL91 to identify tumor hypoxia: Correlation with fluorine-18-FDG. J. Nucl. Med
1998, 39, 99-103.
20. along, E.; Fauconnier, T.; Bennett, S.; Valiant, John.; Nguyen, T.; Lau,
F.; Lu, L. F.
L.; Pollak, A.; Bell, R. A.; Thornback, J. R. Rhenium(V) and Technetium(V) oxo
complexes of an NZN'S peptidic chelator: Evidence of interconversion between
the
syn and anti conformations. Inorg. Chem. 1997, 36, 5799-5808.
24
CA 02266298 1999-03-19
Su et al., page 25
21. Melo, T.; Hua, H. A.; Ballinger, J. R.; Rauth, A. M. Modifying the in
vitro
accumulation of BMS181321, a technetium-99m-nitroimidazole, with unlabelled
nitroaromatics. Biochem. Pharmacol. 1997, 54, 685-693.
22. Linder, K.; Chan, Y.-W.; Cyr, J. E.; Nowotnik, D. P.; Eckelman, W. C.;
Nunn, A. D.
Synthesis, characterization, and in vitro evaluation of nitroimidazole-BATO
complexes: new technetium compounds designed for imaging hypoxic tissue.
Bioconj. Chem. 1993, 4, 326-333.
23. Ramalingam, K.; Raju, N.; Nanjappan, P.; Linder, K. E.; Pirro, J.; Zeng,
W.;
Rumsey, W.; Nowotnik, D. P.; Nunn, A. D. The synthesis and in vitro evaluation
of
a 99mTechnetium-nitroimidazole complex based on a bis(amine-phenol) ligand:
Comparison to BMS-181321. J. Med. Chem. 1994, 37, 4155-4163.
24. Wedeking, P.; Yost, F.; Wen, M.; Patel, B.; Eaton, S.; Romero, V.; Linden
K. E.;
Rumsey, W.; Nunn, A. D. Comparison of the biologic activity of the isomers of
Tc-
99m-nitroimidazole complex BMS-194796. J. Nucl. Med. 1995, 36, 17P.
25. Zhang, X.; Melo, T.; Ballinger, J. R.; Rauth, A. M. Evaluation of Tc-99m
butyleneamine oxime (BnAO), a non-nitroaromatic agent for imaging hypoxia in
tumours. Int. J. Radiat. Oncol.Biol. Phys. 1998, 42, 737-740.
26. Caveliers, V.; Goodbody, A.; Tran, L.; Bossuyt, A.; Thornback, J. Human
dosimetry of TC99m-RP 128, a potential inflammation imaging agent. Eur. J.
Nucl.
Med. 1996, 23, 1131.
CA 02266298 1999-03-19
Su et al., page 26
27. Ballinger, J. R.; Cowan, D. S. M.; Boxen, L; Zhang, Z. M.; Rauth, A. M.
Effect of
hypoxia on the accumulation of technetium-99m-glucarate and technetium-99m
gluconate by Chinese hamster ovary cells in vitro. J. Nucl. Med. 1993, 34, 242-
245.
28. Hay, M. P.; Wilson, W. R.; Moselen, J. W.; Palmer, B. D.; Denny W. A.
Hypoxia-Selective Antitumor Agents. 8.
Bis(nitroimidazolyl)alkanecarboxamides: A new class of hypoxia-selective
cytotoxins and hypoxia cell radiosensitisers. J. Med. Chem.,1994, 37, 381-391
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