Supplementary Material
Direct in vitro and in vivo comparison of terbium-161 and
lutetium-177 using a tumour-targeting folate conjugate
Cristina Müller1*, Josefine Reber1, Stephanie Haller1, Holger Dorrer2,3, Peter
Bernhardt4, Konstantin Zhernosekov2, Andreas Türler2,3, Roger Schibli1,5*
1
Center for Radiopharmaceutical Sciences ETH-PSI-USZ, Paul Scherrer Institute,
Villigen-PSI, Switzerland
2
Laboratory of Radiochemistry and Environmental Chemistry, Paul Scherrer
Institute, Villigen-PSI, Switzerland
3
Laboratory of Radiochemistry and Environmental Chemistry, Department of
Chemistry and Biochemistry, University of Bern, Bern, Switzerland
4
Department of Radiation Physics, The Sahlgrenska Academy, University of
Gothenburg, Sweden
5
Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich,
Switzerland
* Corresponding authors:
Prof. Roger Schibli and Dr. Cristina Müller
Center for Radiopharmaceutical Sciences ETH-PSI-USZ
Paul Scherrer Insitute
5232 Villigen-PSI
Switzerland
e-mail: roger.schibli@psi.ch; cristina.mueller@psi.ch
phone: +41-56-310 28 37; +41-56-310 44 54
fax:
+41-56-310 28 49
1
1. Comparative SPECT imaging studies with 161Tb- and 177Lu-phantoms
Purpose
161
Phantom studies were performed with
Tb and
177
Lu using a small-animal SPECT
scanner in order to compare imaging quality and resolution of these radionuclides.
Experimental procedure
SPECT phantom studies were performed with a 4-head multiplexing multipinhole
camera (NanoSPECT/CT, Bioscan Inc. U.S.) using collimators of 4 x 9 holes of a
diameter of 1.0 mm. The scans were acquired using Nucline software (version 1.02,
Bioscan). Derenzo phantoms with hole-diameters of 0.8-1.3 mm were filled with
~13 MBq of
161
Tb and ~11 MBq of
177
Lu, respectively. For
161
Tb the time per view
was 86 s resulting in a scan time of 40 min to achieve approximately the same total
177
number of counts per view (60.000) which was obtained for
Lu with a time per
view of 200 s and a scan time of 93 min.
Results and conclusion of the phantom studies
Acquisition of the same total number of counts per view for each of the two isotopes,
revealed an imaging resolution of
imaging resolution obtained with
161
Tb (~1 mm) which was comparable to the
177
Lu (Fig. S1). These findings demonstrate the
excellent characteristics of 161Tb for (pre)clinical SPECT.
Fig. S1 SPECT images of Derenzo phantoms filled with
161
Tb (a) and
177
Lu (b).
Images were obtained by acquiring the same total number of counts per view for
each radionuclide. (Numbers on the images indicate the diameter of the
corresponding holes in mm).
2
2. Cell uptake and internalization studies of 161Tb-cm09 and 177Lu-cm09
using KB and IGROV-1 tumour cells
Purpose
In order to compare the in vitro behaviour of
161
Tb-cm09 and
177
Lu-cm09 we
performed cell uptake and internalization studies with KB and IGROV-1 tumour
cells.
Experimental procedure
KB and IGROV-1 cells were seeded in 12-well plates (~ 700,000 cells in 2 ml
FFRPMI medium/well) allowing cell adhesion and growth overnight at 37 °C. After
removal of the supernatant, the cells were washed once with PBS prior to the
addition of FFRPMI medium (975 L/well) without supplements.
161
Tb-cm09 and
Lu-cm09, respectively, (25 L, ~ 38 kBq, ~ 1.5 pmol) were added to each well. In
177
some cases cells were incubated with excess folic acid (100 M) to block FRs on the
surface of the tumour cells. After incubation of the well-plates for different time
periods (0 min, 5 min, 15 min, 30 min, 60 min, 120 min, and 240 min) at 37 °C, the
cells were washed twice with ice-cold PBS to determine total uptake of
and
177
Lu-cm09. To assess the fraction of internalized
161
Tb-cm09 and
161
Tb-cm09
177
Lu-cm09,
KB and IGROV-1 cells were additionally washed with a stripping buffer (aqueous
solution of 0.1 M acetic acid and 0.15 M NaCl, pH 3) to release FR-bound
radiofolates from the cell surface. Cell samples were lysed by addition of NaOH
(1 M, 1 mL per well) and counted in a -counter after transfer of the suspension to
4 mL-tubes. The concentration of proteins was determined for each sample by a
Micro BCA Protein Assay kit (Pierce, Thermo Scientific) in order to standardize
measured radioactivity to the average content of protein in each well (0.5 mg protein
for KB cells and 0.1 mg protein for IGROV-1 cells).
Results and conclusion of the cell uptake and internalization studies
Direct comparison of
161
Tb-cm09 and
177
Lu-cm09 revealed largely the same results
for both radioconjugates (Fig. S2). Cell uptake was increasing over time reaching a
plateau after incubation of about 2 h. The internalized fraction of the total amount of
FR-bound radiofolates (161Tb-cm09 and
177
Lu-cm09) was slightly increasing over
time reaching a maximum value of 60-80 % for KB cells and 50-60 % for IGROV-1
3
cells after 4 h. In spite of an only small difference among KB and IGROV-1 cells in
terms of the internalized fraction, there was a tendency of a faster internalization in
the case of KB cells. Analysis of cell samples, which were co-incubated with excess
folic acid to block FRs on the cellular surfaces reduced the radiofolates’ uptake to
less than 1 % (data not shown).
Fig. S2. Time-dependent cell uptake and internalization of
161
Tb-cm09 (green) and
177
Lu-cm09 (red) in KB (a) and IGROV-1 cells (b).
3. Unspecific effects of 161Tb and 177Lu on tumour cell viability
Purpose
Effects on the viability of KB and IGROV-1 cells upon exposure to external and
internal radiation from 161Tb and 177Lu were compared. Previously, it was found that
these radiolanthanides are not internalized into cancer cells if they are chelated by
DTPA, but able to penetrate the cellular membrane by simple diffusion in their ionic
form added as chloride salts (results not shown). In order to investigate potentially
different effects on cancer cells caused by the different energies of these
radioisotopes cells were incubated with both
161
TbCl3 and 177LuCl3 for several days.
4
161
Tb-DTPA and
177
Lu-DTPA or
Experimental procedure
Cell viability experiments were performed by 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assays as described in the main manuscript.
Cells were incubated in 200 μL FFRPMI medium (with supplements) containing
variable radioactivity concentrations (1, 5, and 10 MBq/mL) of
161
Tb-DTPA and
Lu-DTPA, respectively. In a different setting, cells were incubated in 200 μL
177
FFRPMI
medium
(with
supplements)
concentrations (0.05 – 5.00 MBq/mL) of
containing
variable
radioactivity
161
TbCl3 and 177LuCl3. After incubation for
4 days at 37 °C without changing the medium, the cell samples were treated with
MTT reagent as described in the main manuscript. Analysis was performed by
determination of the absorbance at 560 nm using microplate reader (Victor X3,
Perkin Elmer). To quantify cell viability, the absorbance of the test samples was
expressed as percentage of the absorbance of untreated control cell samples set to
100 %.
Results and conclusion of unspecific effects of
161
Tb and
177
Lu on tumour cell
viability
The results of cell viability obtained upon exposure of KB (a) and IGROV-1 cells (b)
161
to variable radioactivity concentrations of
Tb-DTPA and
177
Lu-DTPA are shown
in Figure S3.
Fig. S3. Percent cell viability upon exposure of KB (a) and IGROV-1 cells (b) to
variable radioactivity concentrations of
difference between the effects of
161
Tb-DTPA and
161
Tb-DTPA and
5
177
177
Lu-DTPA. A significant
Lu-DTPA was not determined.
No significant differences were determined between the effects of
161
Tb-DTPA and
177
Lu-DTPA on a particular cell line. These findings indicate a similar effect of 161Tb
and 177Lu if these nuclides are not specifically associated to the cellular membrane of
cancer cells nor internalized into the tumour cell interior.
The results of cell viability obtained upon exposure of KB and IGROV-1 cells to
variable radioactivity concentrations of
161
TbCl3 and
Figure S4. A significantly increased effect of
177
LuCl3 are shown in
161
TbCl3 compared to
177
LuCl3 was
determined at several radioactivity concentrations (Fig. S4, * p <0.05) in both KB
and IGROV-1 cells. At very low radioactivity concentrations (≤0.05 MBq/mL)
which did not affect cell viability and at very high radioactivity concentrations (≥5.0
MBq/mL) which inhibited cell growth almost completely a significant difference
among the effect of 161TbCl3 and 177LuCl3 was not determined.
Fig. S4 Percent cell viability upon exposure of KB (a) and IGROV-1 cells (b) to
variable radioactivity concentrations of 161TbCl3 and 177LuCl3. (* p <0.05)
It is important to recognize that the inhibitory effect on cell viability caused by
161
TbCl3 and
161
177
LuCl3 was much more pronounced than if cells were exposed to
Tb-DTPA and
177
Lu-DTPA of the same radioactivity concentration. These
findings indicate the dependency on internalization of the radioisotopes to achieve a
distinct inhibition of the cell viability.
6
4. FR-specific effects of
161Tb-cm09
177Lu-cm09
and
on tumour cell
viability
Purpose
MTT cell viability assays were performed with variable radioactivity concentrations
of 161Tb-cm09 and 177Lu-cm09 with and without co-incubation of excess folic acid to
block FRs. The aim of these studies was to investigate FR-specific effects of the
folate radioconjugates on FR-positive KB and IGROV-1 tumour cells.
Experimental procedure
Investigation of the viability upon exposure of KB and IGROV-1 cells to 161Tb-cm09
and
177
Lu-cm09
were
performed
by
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assays as described in the main manuscript.
Cells were incubated in 200 μL FFRPMI medium (without supplements) containing
variable radioactivity concentrations (1, 5, and 10 MBq/mL) of
161
Tb-cm09 and
177
Lu-cm09 at specific activities of 20 MBq/nmol. For each of these concentrations
additional cell samples were pre-incubated with excess folic acid (100 M) to block
FRs. After 4 h incubation at 37 °C, cells were washed once with 200 μL PBS
followed by addition of 200 μL of FFRPMI medium (with supplements) to each
well. After 4 days of incubation the MTT reagent was added to each cell sample as
described in the main manuscript. Analysis was performed by determination of the
absorbance at 560 nm using a microplate reader (Victor X3, Perkin Elmer). To
quantify cell viability, the absorbance of the test samples was expressed as
percentage of the absorbance of untreated control cell samples (not shown) which
was set to 100 %.
Results and conclusion of effects of 161Tb-cm09 and 177Lu-cm09 on cell viability
Cell viability was reduced in KB and IGROV-1 cells upon exposure to increasing
radioactivity concentrations of
161
Tb-cm09 and
177
Lu-cm09 (Fig. S5). At the same
radioactivity concentration reduction of cell viability was more pronounced in KB
cells compared to IGROV-1 cells. This was most probably a result of the higher FRexpression level in KB cells compared to IGROV-1 cells [1] and, hence, an
increased uptake of radiolabelled cm09 in KB cells. The inhibition of cell viability
achieved by incubation of cells with
161
Tb-cm09 at a concentration of 1 MBq/mL or
5 MBq/mL was significantly more effective (p <0.05) compared to the inhibition
7
obtained by incubation of the cells with the same concentrations of 177Lu-cm09. At a
concentration of 10 MBq/mL inhibition of cell viability was reduced to about 10 %
and in the same range for both 161Tb-cm09 and 177Lu-cm09.
Fig. S5 Effects of
161
Tb-cm09 on cell viability of KB (a) and IGROV-1 cells (b)
compared to the effects of 177Lu-cm09 on cell viability of KB (c) and IGROV-1 cells
(d). A FR-specific effect of
161
Tb-cm09 and
177
Lu-cm09 was proven by abolishment
of the inhibitory effect on cell viability upon co-incubation of cells with excess folic
acid to block FRs.
8
161Tb-cm09
5. Biodistribution study of
and
177Lu-cm09
in IGROV-1
tumour-bearing mice
Purpose
IGROV-1 cells (ovarian cancer cell line) are known to express the FR [1]. However,
compared to KB cells which are used as a standard tumour model for FR-targeting
research IGROV-1 cells express the FR at lower levels and hence, they represent the
situation which would be expected in human patients better. Therefore, we chose to
use mice with IGROV-1 tumour xenografts as an additional in vivo model to test
161
Tb-cm09 and to compare it with 177Lu-cm09.
Experimental procedure
Upon arrival of six to eight-week-old female, athymic nude mice (CD-1 Foxn-1/nu)
they were fed with a folate-deficient rodent diet (Harlan Laboratories) starting 5 d
prior to tumour cell inoculation. Mice were then inoculated with IGROV-1 cells
(6.5 x 106 cells in 100 L PBS) into the subcutis of each shoulder. Biodistribution
studies were performed in triplicate approximately 16 d after tumour cell
inoculation.
161
Tb-cm09 and
177
Lu-cm09 (3 MBq, 0.5 nmol per mouse) were
administered via a lateral tail vein. The animals were euthanized at pre-determined
time points after administration of the radiofolates. Selected tissues and organs were
collected, weighed, and counted for radioactivity in a -counter. The results were
listed as the percentage of the injected dose per gram of tissue weight [%ID/g], using
reference counts from a defined volume of the original injection solution that was
counted at the same time.
Results and conclusion of the biodistribution study in IGROV-1 tumour-bearing
mice
The results of the tissue distribution of
161
Tb-cm09 in IGROV-1 tumour-bearing
mice (Table S1) was largely the same as what was previously obtained in KB
tumour-bearing mice (Table S2) [2]. Interestingly we found the same high tumour
uptake in IGROV-1 xenografts (22.25 ± 3.88 %ID/g, 4 h p.i.) as was previously
determined in KB tumour xenografts (23.81 ± 2.53 %ID/g, 4 h p.i.) although it is
known that the FR-expression level in IGROV-1 cells is significantly lower
compared to KB cells [1]. The reason for this observation might be that application
of 0.5 nmol of radiolabelled cm09 was below the concentration which would have
9
saturated FRs in the tumour tissue and/or that FR-expression levels of these
xenografts similar in vivo although they are different in cultured cells. Also, the fact
that the albumin-binding conjugate cm09 is circulating longer in blood might allow
high tumour uptake even if the FR-level was not as high as it is the case for KB
tumour cells.
Based on the theory of equal coordination chemistry of lanthanides and our previous
findings with the
161
Tb- [2] and 177Lu-labelled cm09 [3] in KB tumour-bearing mice
the tissue distribution results obtained with
be representative also for
177
161
Tb-cm09 in IGROV-1 tumours would
Lu-cm09. Hence, equal tumour uptake of
161
Tb-cm09
and 177Lu-cm09 could be assumed in both, KB and IGROV-1 tumour xenografts.
Table S1 Biodistribution of
161
Tb-cm09 in IGROV-1 tumour-bearing female nude
mice
161Tb-cm09
%injected dose per gram tissue*
1 h p.i.
4 h p.i.
24 h p.i.
48 h p.i.
96 h p.i.
168 h p.i.
Blood
8.50 ± 0.49
3.77 ± 0.86
0.86 ± 0.11
0.45 ± 0.15
0.18 ± 0.03
0.03 ± 0.01
Lung
4.37 ± 0.45
2.69 ± 0.23
0.97 ± 0.03
0.72 ± 0.16
0.51 ± 0.13
0.21 ± 0.07
Spleen
2.03 ± 0.34
1.20 ± 0.23
0.79 ± 0.14
0.69 ± 0.21
0.64 ± 0.13
0.38 ± 0.05
Kidneys
25.60 ± 1.51
33.93 ± 2.51
28.42 ± 0.77
27.39 ± 2.07
18.46 ± 3.82
8.94 ± 0.32
Stomach
1.90 ± 0.40
1.43 ± 0.27
0.59 ± 0.18
0.37 ± 0.14
0.25 ± 0.10
0.11 ± 0.03
Intestines
1.53 ± 0.30
0.79 ± 0.30
0.24 ± 0.13
0.25 ± 0.07
0.17 ± 0.06
0.04 ± 0.00
Liver
4.27 ± 0.29
3.70 ± 0.22
2.55 ± 1.18
1.43 ± 0.28
1.62 ± 0.51
0.83 ± 0.25
Salivary glands
8.19 ± 1.03
6.60 ± 1.21
3.73 ± 0.39
3.91 ± 0.59
2.25 ± 0.18
1.08 ± 0.38
Muscle
1.50 ± 0.01
1.85 ± 0.52
0.79 ± 0.18
0.62 ± 0.14
0.42 ± 0.23
0.19 ± 0.07
Bone
1.64 ± 0.05
1.63 ± 0.22
0.70 ± 0.09
0.48 ± 0.11
0.38 ± 0.13
0.17 ± 0.03
Tumour
12.29 ± 1.08
22.25 ± 3.88
23.17 ± 3.04
18.36 ± 4.75
12.39 ± 3.14
6.35 ± 1.61
Tumour-to-blood
1.78 ± 0.58
6.00 ± 1.06
27.29 ± 4.83
41.72 ± 6.77
67.57 ± 13.09
189.85 ± 27.71
Tumour-to-liver
2.88 ± 0.24
6.04 ± 1.21
7.00 ± 1.33
13.41 ± 4.64
8.12 ± 2.98
7.80 ± 1.57
Tumour-to-kidney
0.48 ± 0.04
0.66 ± 0.14
0.82 ± 0.10
0.66 ± 0.13
0.67 ± 0.16
0.61 ± 0.11
* values shown represent the mean ± S.D. of data from three animals (n=3) per cohort
10
Table S2 Biodistribution of
161
Tb-cm09 in KB tumour-bearing female nude mice
[[2]]
161Tb-cm09
%injected dose per gram tissue*
1 h p.i.
4 h p.i.
24 h p.i.
48 h p.i.
96 h p.i.
168 h p.i.
Blood
11.23 ± 1.12
6.11 ± 0.25
1.53 ± 0.11
0.72 ± 0.18
0.16 ± 0.01
0.04 ± 0.01
Lung
6.30 ± 0.51
4.13 ± 0.28
1.64 ± 0.08
1.03 ± 0.33
0.54 ± 0.08
0.21 ± 0.06
Spleen
2.52 ± 0.52
1.78 ± 0.23
0.99 ± 0.19
0.86 ± 0.09
0.71 ± 0.03
0.44 ± 0.11
Kidneys
20.52 ± 2.67
27.54 ± 1.06
27.84 ± 5.03
24.45 ± 1.59
15.48 ± 3.15
7.36 ± 1.74
Stomach
2.56 ± 0.51
2.09 ± 0.33
0.99 ± 0.37
0.65 ± 0.19
0.39 ± 0.08
0.17 ± 0.05
Intestines
1.94 ± 0.30
1.16 ± 0.22
0.31 ± 0.05
0.34 ± 0.06
0.13 ± 0.02
0.06 ± 0.03
Liver
5.48 ± 0.27
5.13 ± 0.59
3.75 ± 0.66
2.33 ± 0.39
1.56 ± 0.19
0.86 ± 0.16
Salivary glands
9.90 ± 1.14
9.29 ± 0.81
5.78 ± 1.26
3.91 ± 0.59
2.62 ± 0.12
1.28 ± 0.50
Muscle
1.78 ± 0.05
1.87 ± 0.19
1.26 ± 0.14
0.70 ± 0.03
0.45 ± 0.06
0.16 ± 0.08
Bone
2.03 ± 0.20
1.76 ± 0.05
1.30 ± 0.25
0.56 ± 0.23
0.46 ± 0.04
0.22 ± 0.01
Tumour
14.06 ± 0.63
23.81 ± 2.53
22.01 ± 4.39
18.45 ± 1.80
10.34 ± 1.79
5.68 ± 1.85
Tumour-to-blood
1.23 ± 0.13
3.89 ± 0.32
14.33 ± 2.00
26.83 ± 6.54
64.72 ± 8.43
129.40 ± 17.95
Tumour-to-liver
2.56 ± 0.21
4.66 ± 0.51
5.86 ± 0.64
8.12 ± 1.70
6.54 ± 1.72
6.46 ± 1.23
Tumour-to-kidney
0.68 ± 0.07
0.87 ± 0.12
0.79 ± 0.05
0.75 ± 0.04
0.70 ± 0.09
0.76 ± 0.12
* values shown represent the mean ± S.D. of data from three animals (n=3) per cohort
6. Dosimetric considerations of the biological effectiveness of
161Tb-cm09
and 177Lu-cm09
Purpose
In order to get an idea about the radioactive dose burden of
161
Tb-cm09 and
177
Lu-
cm09 to KB and IGROV-1 tumour xenografts and kidneys, respectively, we made a
dose estimation while taking only the self-radiation dose into account for these
161
tissues. Moreover to compare the biological effectiveness of
Tb and
177
Lu the
relative absorbed dose for these two radioisotopes was determined for a sphere shape
which should represent the tumour xenograft.
Experimental procedure
To estimate the equivalent absorbed radiation dose for
161
Tb-cm09 and
177
Lu-cm09
in tumour xenografts and in the kidneys, the following assumptions were made.
First, the biodistribution data were considered as equal for
11
161
Tb-cm09 and
177
Lu-
cm09 and second, the uptake in KB and IGROV-1 tumour xenografts was
considered as the same (Supplemental Data, chapter 5).
Based on these assumptions the following calculations were made: (i) the cumulative
radioactivity was calculated by fitting an exponential curve to the non-decay
corrected biodistribution data (%ID/g) by using the time points 4 h to 168 h p.i. The
AUCs (MBq∙s) were determined by integrating the mono-exponential function to
infinity. The initial accumulation phase of the radiopharmaceuticals was estimated
by linear integration of the AUCs between the time points 0 and 4 h p.i. (ii) The
mean absorbed dose to the tumour xenografts was assessed for the tumour size at the
time of injection. This means about 35-58 mg for the tumours, and 125 mg for one
kidney. The absorbed fraction to these tumours and kidney volumes, with the
activity uniformly distributed was simulated by PENELOPE [4]. (iii) The absorbed
dose (mGy/MBq) was calculated by multiplying the AUC (s, normalized to 1 MBq
ID) with the absorbed fraction and the emitted energy per decay for
177
Lu and
161
Tb
(ENSDF decay data in the MIRD format, www.nndc.bnl.gov) and multiplied with a
conversion factor. (iv) The dose (mGy) was calculated by multiplying the absorbed
dose (mGy/MBq) with the amount of injected radioactivity.
Results and conclusion of the dosimetric considerations of the biological
effectiveness of 161Tb-cm09 and 177Lu-cm09
The absorbed fractions for the assumed spherical size ranged between 0.90-0.95. In
the case of
161
Tb-cm09 an absorbed dose of 3.3 Gy/MBq was estimated for tumour
xenografts whereas for
177
Lu-cm09, the estimated absorbed dose was 2.4 Gy/MBq.
This results in an absorbed dose of ~33 Gy (161Tb-cm09) and ~24 Gy (177Lu-cm09)
in tumours upon a single injection of 10 MBq of radiolabelled cm09. For kidneys, an
absorbed dose of 4.5 Gy/MBq was estimated for
161
Tb-cm09 and 3.4 Gy/MBq for
177
Lu-cm09 resulting in a dose of ~45 Gy and ~34 Gy for injection of 10 MBq 161Tb-
cm09 and
177
Lu-cm09, respectively. For the additional study where we wanted to
investigate kidney damage, 20 MBq of
161
Tb-cm09 or
177
Lu-cm09 was injected
which resulted in a kidney dose of ~90 Gy (161Tb-cm09) and ~68 Gy (177Lu-cm09).
12
7. In vivo plasma stability of 161Tb-cm09 and 177Lu-cm09
Purpose
In order to obtain a high and FR-specific tumour uptake it is crucial that the
radioconjugates are not metabolized in the blood circulation. Radioactive
metabolites could eventually accumulate in non-targeted tissues and organs and
potentially result in undesired side effects. Therefore, the in vivo stability of
cm09 and
177
161
Tb-
Lu-cm09 was assessed in the blood samples of non-tumour-bearing
nude mice.
Experimental procedure
Two groups of 6 animals (CD-1 Foxn-1/nu) each were injected with either
cm09 or
177
161
Tb-
Lu-cm09. At 4 h, 24 h and 48 h after injection of the radioconjugates
blood was taken from the sublingual vein of 2 animals of each group immediately
before euthanasia. The blood samples (~500 L) were centrifuged for 20 min
allowing separation of the plasma (~150 L) from the blood cells. Methanol
(~200 L) was added to precipitate plasma proteins. After centrifugation (2 x
10 min) the supernatants were analyzed by HPLC.
Results and conclusion of the in vivo plasma stability of 161Tb-cm09 and 177Lu-cm09
The analysis of blood plasma showed intact radioconjugates 4 h after injection
(Fig. S6). Also, at 24 h and 48 h after injection metabolites were not detected and
only a very small quantity (≤2 % and ≤6 %, respectively) of free
177
161
Tb(III) and
Lu(III) was found at 24 h and 48 h after injection (Fig. S6). These results
confirmed an excellent in vivo stability of 161Tb-cm09 and 177Lu-cm09.
13
Fig. S6 HPLC chromatograms of
161
Tb-cm09 (a, c, e) and
177
Lu-cm09 (b, d, f)
obtained from plasma samples taken at 4 h p.i. (a, b), 24 h p.i. (c, d) and 48 h (e, f)
after injection of the radioconjugates.
8. Comparison of long-term effects of
161Tb-cm09
and
177Lu-cm09
on
body weights and survival rates of mice
Purpose
In a separate experiment, non-tumour-bearing nude mice were investigated with
regard to body weight and survival rate upon administration of
161
Tb-cm09 and
177
Lu-cm09 at high amounts of radioactivity (20 MBq, 1 nmol).
Experimental procedure
Three groups (A-C) of non-tumour-bearing nude mice (CD-1 Foxn-1/nu) were
intravenously injected with either only PBS (group A, n = 3), with 20 MBq
cm09 (group B, n = 6) or
177
161
Tb-
Lu-cm09 (group C, n = 3) at day 0 of the study. Mice
were weighed 3-4 times a week over a time period of 6 months. Body weight loss of
>15 % of the initial body weight (at day 0) or signs of unease were defined as
endpoint criteria which required euthanasia.
14
Results and conclusion of comparison of the long-term effects of
161
Tb-cm09 and
177
Lu-cm09 in non-tumour-bearing mice
The relative body weights were comparable for all three groups of animals (Fig. S7).
Towards the end of the study one of the control mice lost weight which required
euthanasia at day 166 after start of the study. The reason for body weight loss was
most probably an infection as a consequence of frequent manipulation of these mice.
Fig. S7 Relative body weight of control mice (group A, blue), mice which received
20 MBq 161Tb-cm09 (group B, green) and mice which received 20 MBq 177Lu-cm09
(group C, red).
In addition, one of the
161
Tb-cm09 treated mice lost weight which required
euthanasia at day 140 after injection. Whether or not the observed body weight loss
of the
161
Tb-cm09 treated mouse was related to the
161
Tb-based therapy remained
unclear. The average survival time remained undefined since most of the mice were
still alive (group A: 2/3, group B: 5/6, group C: 3/3) at the end of the study at day
174 after start of the therapy (Fig. S8). These findings indicate that treatment with 20
MBq of
161
Tb-cm09 or 20 MBq of
177
Lu-cm09 was well tolerated although it has to
be critically acknowledged that the number of test animals was too low to allow
drawing final conclusions about the tolerability of high-dosed 161Tb-cm09 and 177Lucm09.
15
Fig. S8 Survival curve of control mice (group A, blue), mice which received
20 MBq 161Tb-cm09 (group B, green) and mice which received 20 MBq 177Lu-cm09
(group C, red). The average survival time remained undefined since most of the mice
(10/12) were still alive at the end of the study at day 174.
9. Investigation of long-term effects of
161Tb-cm09
and
177Lu-cm09
according to plasma and blood parameters
Purpose
As additional parameters to investigate test animals upon injection of high quantities
of radioactivity several blood plasma parameters were measured at different time
points after start of the therapy study. The goal of this analysis was to determine
potential impairment of kidney and liver functions as a consequence of radionuclide
therapy. Moreover, at the end of the study immediately before euthanasia fractions
of viable, apoptotic and dead white blood cells were determined by flow cytometry.
Experimental procedure
Several plasma parameters were measured from mice at day 29, 69, 127 and 147
after start of the therapy. Determination of these values was performed according to
the procedure reported in the main manuscript.
Before euthanasia at day 174, blood samples were collected from the retrobulbar
vein of each mouse. In order to assess viable, apoptotic and dead white blood cells, a
viability assay kit (Guava® ViaCount® Assay, Millipore) was used for determination
of each fraction by flow cytometry. The kit consists of a membrane permeant DNAbinding dye allowing determination of nucleated cells and a membrane impermeant
DNA-binding dye which discriminates among viable, apoptotic and dead white
16
blood cells based on the cellular membrane’s permeability. After dilution of blood
samples (10 L) of each mouse with PBS (90 L) they were mixed with a 10-fold
excess of Guava® ViaCount® assay reagent and incubated at room temperature in the
dark for 10 min. From each sample duplicates were added in a 96-well plate (200
L/well) allowing measurement by using a flow cytometer (Guava® EasyCyteTM
Plus Flow Cytometry System, Millipore). The different cell populations were
quantified from a number of 1.000 cells. Data processing was carried out using the
GuavaSoft software (version 2.2).
Results and conclusion of examination of blood cells and plasma parameters
Determination of the two most important plasma parameters, blood urea nitrogen
(BUN) and alkaline phosphatase (ALK PHOS), are reported in the main manuscript
(Table 3). All of the other plasma parameters which were determined are shown in
Table S3. Aspartate-aminotransferase (AST) previously designated as glutamateoxalacetate transaminase (GOT) is an enzyme found in liver and muscle cells.
Alanine-aminotransferase (ALT) previously designated as glutamate-pyruvate
transaminase (GPT) is found primarily in liver cells. Increased plasma levels of these
transaminases may be an indication for injury of liver cells (Table S3). Albumin
(ALB) is a plasma protein which is synthesized in the liver and hence, reduced
plasma albumin levels may indicate liver disease (hepatitis, cirrhosis, ascites etc).
However, a reduced plasma level of albumin might also be a consequence of a renal
disease. Bilirubin (TBIL) is a leftover after removal of the older red blood cells of
which a certain amount is renewed every day. An increased level of TBIL may
indicate hemolytic anemia, or impaired liver health. The absolute values which were
determined for the transaminases correlated well with published data obtained from
C57BL/6J mice whereas the values for albumin were somewhat lower in our study
compared to those obtained from C57BL/6J mice [5]. It has to be recognized that
different strains of mice may reveal different values for blood plasma parameters. At
no time during therapy plasma parameters (AST, ALT, ALB, TBIL) of treated mice
(group B and C) differed significantly from those of control mice (group A). These
findings indicate that impaired function of liver cells or hemolytic anemia as a
consequence of radionuclide therapy with
161
Tb-cm09 and
177
Lu-cm09, respectively,
did not occur within the scope of this preliminary long-term study.
17
Table S3 List of plasma values of mice of group A (n=3), group B (n=6) and group
C (n=3) (AST = aspartate aminotransferase, ALT = alanine aminotransferase,
TBIL = total bilirubin and ALB = albumin)
AST (IU/L)
Group
Day 29
Day 69
Day 127
Day 174
A (control)
58 ± 6
50 ± 6
76 ± 10
54 ± 6
B (161Tb-cm09)
65 ± 17
95 ± 21
66 ± 17
47 ± 14
123 ± 53
73 ± 12
84 ± 17
59 ± 12
C
(177Lu-cm09)
ALT (IU/l)
Group
Day 29
Day 69
Day 127
Day 174
A (control)
23 ± 5
20 ± 6
26 ± 18
31 ± 12
B (161Tb-cm09)
23 ± 4
21 ± 5
20 ± 5
18 ± 5
(177Lu-cm09)
26 ± 8
19 ± 6
18 ± 5
22 ± 6
C
ALB (g/L)
Group
Day 29
Day 69
Day 127
Day 174
A (control)
25 ± 1
24 ± 2
24 ± 3
26 ± 2
B (161Tb-cm09)
24 ± 0
24 ± 1
23 ± 1
23 ± 0
C (177Lu-cm09)
23 ± 1
23 ± 2
22 ± 1
23 ± 1
TBIL (mol/L)
Group
Day 29
Day 69
Day 127
Day 174
A (control)
8±0
9±0
10 ± 2
11 ± 1
B
(161Tb-cm09)
10 ± 2
11 ± 3
15 ± 11
9±3
C
(177Lu-cm09)
12 ± 2
9±1
8±2
11 ± 1
No significance determined
In all three groups of mice (A-C) blood analysis revealed over 95 % of viable white
blood cells. The two exceptions were a control mouse in group A (mouse A2:
87.1 %) and a mouse treated with 161Tb-cm09 in group B (mouse B4: 86.9 %) which
reduced the average values shown in Table S4. Less than 3 % of the cells were
apoptotic in mice of all groups except in mouse 2 of group A (mouse A2: 9.3 %).
The number of dead cells was below 4 % of the total cell number. Only in one case
of group B (mouse B4) the fraction of dead cells was 11.1 %.
Table S4 FACS measurement of viable, apoptotic and dead white blood cells
Group A
Group B
Group C
Treatment
saline
161Tb-cm09
177Lu-cm09
Viable cells
91.3 ± 5.9
95.0 ± 4.9
97.7 ± 1.9
Apoptotic cells
5.3 ± 5.7
1.4 ± 0.9
1.3 ± 0.7
Dead cells
3.5 ± 0.1
3.7 ± 4.6
1.0 ± 1.2
No significance determined
18
In average there was no significant difference determined in treated mice of groups
B and C compared to untreated controls of group A (Table S4). However, the
number of mice which were used for this experiment was too small to draw final
conclusions about potential long-term toxicity of 161Tb-cm09 and 177Lu-cm09.
10. Determination of kidney function by quantification of
99mTc-DMSA
uptake using small-animal SPECT
Purpose
99m
SPECT experiments using
Tc-DMSA were performed to determine kidney
function of mice as previously reported [6]. Forrer et al. demonstrated the possibility
for quantification of the renal uptake of
99m
Tc-DMSA by small-animal SPECT and
to correlate it with renal function which may be impaired as a consequence of
radionuclide therapy [6]. Very recently, it was shown that
99m
Tc-DMSA uptake in
the kidneys is dependent on the presence of megalin/cubilin and hence it correlates
with endocytotic function of the proximal tubule cells in the kidneys [7].
Experimental procedure
Kidney function of mice was investigated by quantitative SPECT after injection of
99m
Tc-DMSA every month (day 40, 71, 105, 141 and 167) starting 6 weeks after
injection of the
161
Tb-cm09 and
injected with 25-35 MBq
177
Lu-cm09, respectively. Mice were intravenously
99m
Tc-DMSA. SPECT scans of the kidney region (30 mm)
were performed 2 h p.i. (time per projection 20-25 sec, total scan time <7 min).
Uptake of radioactivity was analyzed in each kidney by determination of
accumulated radioactivity in a cylindric volume around each kidney (%ID/kidney)
using the InVivoScope post-processing software (version 2.0, Bioscan Inc.). The
data were expressed in percent uptake of the average uptake (= 100 %) in control
mice (group A).
Results and conclusion of the determination of kidney function
SPECT experiments performed to determine kidney function of mice did not
revealed any significant differences among renal uptake of
99m
Tc-DMSA in control
mice of group A (set to 100 %) and renal uptake in mice of group B and C,
19
respectively (Fig S9). Nevertheless, there was a trend of reduced uptake in treated
animals compared to controls. The only significant reduction of accumulated
DMSA was determined for the uptake in kidneys of mice treated with
99m
Tc-
177
Lu-cm09
(group C) after 4 months at day 141 (Fig. S9, asterisk). Clearly, a critical issue of
this study was the fact that only a small number of mice were included in each
group. Hence statistical analysis was not significant and gave only a vague idea of
the tolerability of these therapies. More extended studies will be required in order to
allow final conclusions about potential damage to the kidneys upon therapy with
high-dosed 161Tb-cm09 in comparison to 177Lu-cm09.
Fig. S9 Uptake of 99mTc-DMSA in kidneys of control mice (group A, blue, values set
to 100 %) in comparison to the renal uptake of
99m
Tc-DMSA found in mice treated
with either 161Tc-cm09 (group B, green) or 177Lu-cm09 (group C, red). (* p <0.05)
20
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