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Department of Research-ZLF (S.F., A.N.E., A.-M.M., M.H.), Department of Radiology and Radiopharmacy Unit (A.H., M.B., P.P., H.R.M.), and Department of Internal Medicine (C.B.); University Hospital and University Children’s Hospital, CH-4031 Basel, Switzerland
Address all correspondence and requests for reprints to: Dr. S. Froidevaux, Department of Research, Kantonsspital Basel, Hebelstrasse 20, CH-4031 Basel, Switzerland. E-mail: sylvie.froidevaux{at}unibas.ch
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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scintigraphy after radiolabeling either with 123I
after substitution of Phe3 by Tyr, or with
111In after conjugation of the chelator
diethylenetriaminepentaacetic acid (DTPA) to octreotide, yielding
DTPA-octreotide (OctreoScan) (6, 7, 8, 9, 10).
This success prompted us and others to envisage the use of radiolabeled
somatostatin analogs for selective internal radiotherapy (9, 11, 12, 13, 14).
For this purpose, the choice of the appropriate radionuclide is
critical. Among potential candidates, 90Y is of
special interest because of its well-developed coordination chemistry,
its ready availability from a
90Sr/90Y generator, and its
physical characteristics (including a 64.1-h half-life and a pure
ß-emission of high energy. 111In can be another
suitable radionuclide because, besides its -emitting properties, it
is also an Auger emitter that makes it interesting for treatment of
micrometastases if one can direct it into the cell nucleus.
Whatever the selected radiometal, the success of internal therapy depends on the accumulation of high amounts of radioactivity within the tumor and metastases, along with a rapid clearance of radioactivity from the other tissues. This may be possible to achieve by the development of radiolabeled somatostatin analogs with very high affinity for SSTRs, favorable biodistribution profile regarding hepatic and kidney clearance, and sufficient stability to prevent nonspecific uptake in nontarget organs attributable to radiometal loss in vivo. Initial attempts to label antibodies or small molecules with 90Y for radiotherapeutic applications, using DTPA or DTPA derivatives as the chelating agent, have been hampered by the dose-limiting myelotoxicity attributable to the in vivo instability of the 90Y-chelator complex and subsequent uptake of the released 90Y into the bone (15, 16, 17, 18).
To circumvent this problem, we used the macrocyclic DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) as radiometal chelator. DOTA, as opposed to DTPA or DTPA-derivatives, has proved to form stable metal complexes with various metals when conjugated to monoclonal antibodies (16, 17, 19, 20). As somatostatin analog, we assessed the three clinically relevant somatostatin agonists, namely octreotide, vapreotide, and lanreotide. Furthermore, we designed a fourth somatostatin analog consisting of octreotide with Tyr substituted for Phe3, with the aim of: 1) increasing peptide hydrophilicity, thus presumably kidney excretion; and 2) allowing concomitant labeling, i.e. iodination of Tyr3, which can be of additional benefit in therapeutic applications if the Auger-emitter 125I or the ß-emitter 131I is used. These somatostatin analogs show little difference in their binding properties, i.e. a high or intermediate affinity for SSTR2, SSTR3, and SSTR5 and no or very-low affinity for SSTR1 and SSTR4 (21). Therefore, they constitute attractive targeting peptides for cancer cells expressing SSTR2 that have been detected at high levels in a wide range of human tumors (22).
In this study, we compare the aforementioned somatostatin analog constructs, DOTA0-(D)Phe1-octreotide (DOTAOc), DOTA0-(D)Phe1-Tyr3-octreotide (DOTATOC), DOTA0-(D)ßNal1-lanreotide (DOTALan), and DOTA0-(D)Phe1-vapreotide (DOTAVap). They were evaluated with regard to their in vivo behavior in AR4–2J tumor-bearing mice after radiolabeling with either 90Y or 111In. This included biodistribution in the presence or absence of an excess of cold octreotide and tumor penetration, as indicated by the ratio tumor uptake to tumor size. Furthermore, because we observed intriguing differences in the uptakes of the four radiolabeled constructs in the SSTR-positive tissues (i.e. tumor; adrenals; and, to a lesser extent, pancreas), we investigated the pattern of SSTR subtype expression in these tissues, by reverse transcriptase PCR (RTPCR), to see whether there is a correlation between the retention of a somatostatin analog and the expression of a given SSTR subtype. Finally, we report the in vitro binding affinity of the four Y-DOTA-somatostatin analogs for AR4–2J tumor membranes.
Overall, our results show the superiority of radiolabeled DOTATOC over the other DOTA-somatostatin analogs for targeting SSTR2-expressing malignancies, as clearly indicated by the highest binding affinity for AR4–2J membranes correlated in vivo with the highest tumor-to-kidney uptake ratio. This makes this radioligand very promising for both internal radiotherapy and tumor diagnosis.
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Materials and Methods |
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Vapreotide and Lys5(BOC)-octreotide were received
from Debiopharma (Lausanne, Switzerland) and Sandoz Pharmaceuticals Corp. Pharma (Basel, Switzerland), respectively.
The
DOTA0-(D)Phe1-octreotide
(DOTAOc),
DOTA0-(D)ßNal1-lanreotide
(DOTALan),
DOTA0-(D)Phe1-Tyr3-octreotide
(DOTATOC), and
DOTA0-(D)Phe1-vapreotide
conjugates were synthesized as previously reported (23). Incorporation
of the radiometals 90Y or
111In was performed as indicated in a previous
publication (23). Briefly, the DOTA-somatostatin analogs were dissolved
in sodium acetate buffer, pH 4.8–5.5, and heated, at 95 C for 25 min,
after addition of either
111InCl3 (in 0.1
M HCl; Mallinckrodt, Inc., Petten, The
Netherlands) or 90YCl3 (in
0.05 M HCl; Pacific Northwest National Laboratory, Hanford,
WA). DTPA-(D)Phe1-octreotide was
labeled at room temperature. The resulting radioligands were of high
purity, as indicated by HPLC quality controls, and the specific
activity was always greater than 40 GBq/µmol.
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HPLC analysis
HPLC was performed on a Hewlett Packard 1050 chromatography
system (Hewlett Packard Co., Palo Alto, CA) connected to a
flow-through Berthold LB506C1 -detector (Berthold Ltd., Bundoora,
Australia) and a C18-RP (Hewlett Packard Co.) column
(Vydac 218TP54, Western Analytical Products Inc., Murrieta, CA). The
conditions were as followed: eluent A = 0.1% trifluoroacetic acid
in water; eluent B = acetonitrile; gradient = 0–5 min
at 100% A, 5–25 min at 100%–70% A, 25–35 min at 70% A, 35–40
min at 70%–0% A, 40–45 min at 0%–100% A; flow 1.5
ml/min.
In vitro binding assay
Octreotide was purchased from Novartis Pharmaceuticals (Basel, Switzerland), and
Tyr3-octreotide was obtained from
Mallinckrodt, Inc. (St Louis, MO). DOTAOc, DOTATOC,
DOTALan, and DOTAVap were labeled with cold yttrium as described above.
Their precise concentration was determined by OD measurement (
= 280 nm) after determination of the respective molar extinction
coefficient by two methods: 1) assessment of the optical density at
= 280 nm of a mixture of all amino acids plus chelator; and 2)
labeling experiment using a defined excess of radiometal (M1), in the
presence of DTPA, followed by a comparison of the two peaks (M1-DTPA
vs. M1-DOTA-peptide) obtained after injection in a HPLC
system connected to a -detector. Stock solutions were prepared in
0.01 M acetic acid at 0.5–10
mM and further diluted in binding medium
consisting of modified Eagle’s medium supplemented with 25
mM HEPES, 0.4% BSA and 1
mM 1,10-phenanthroline. The stock solutions, as
well as the dilutions, were made in prelubricated microcentrifuge
tubes (Costar, Cambridge, MA) to minimize loss by
absorption. Competitive binding experiments were performed in
BSA-precoated 96-well plates, as previously described (24), by
using 125I-Tyr3-octreotide
as radioligand and AR4–2J tumor membranes as a source of SSTR2.
IC50 (inhibitory concentration 50%) was
calculated using the Prism software (GraphPad Software, Inc., San Diego, CA).
SSTR messenger RNA (mRNA) expression in tissues
Total RNA was extracted from adrenals, pancreas, or AR4–2J
tumors implanted into female Swiss nude mice, in 10 vol TriZOL reagent
(Life Technologies, Basel, Switzerland), according to the
manufacturer’s protocol. Fifty micrograms of total RNA were subjected
to a DNase RQ1 (Promega Corp., Madison, WI) digest,
followed by a classical phenol/chloroform extraction. First-strand
complementary DNA was produced by Moloney murine leukemia virus reverse
transcriptase (Promega Corp.), using 1 µg total RNA and
200 ng oligo(dT)15, in a final vol of 20 µl. For each RNA
preparation, genomic DNA removal was controlled by omitting reverse
transcriptase. The first-strand complementary DNA (2 µl) was then
amplified by PCR in 20-µl reaction volumes containing 0.05% Tween
20, 5% dimethylsulfoxide, 250 µM of each deoxynucleotide
triphosphate, 1 U Taq DNA Polymerase (Boehringer,
Mannheim, Germany), 1X Taq PCR reaction buffer (Boehringer),
and 1 µM of each primer. Primer pair sequences
were as followed: 5'-tcagctgggatgttccccaatg-3' and
5'-gtcgtcttgctcggcgaacacg-3' for mouse/rat SSTR1,
5'-caccgccccctctcccacct-3' and 5'-gtcgtcttgctcggcgaacacg-3' for rat
SSTR1, 5'-tgggtgtcctctccatttg-3' and 5'-gattgatgccatctacagtc-3' for
mouse SSTR2, 5'-gaggacacgatggcctgg-3' and 5'-cacgcgcggaactttga-3' for
rat SSTR2, 5'-ctgggaacacatcctcgacc-3' and 5'-taggacagggcgttctgagc-3'
for mouse SSTR3, 5'-tctcggcgagtacggagcca-3' and
5'-acagatggctcagcgtgctg-3' for rat SSTR3,
5'-gatgccactgtcaaccatgtgtccct-3' and 5'-gtcctacccccatggaggtg-3' for
mouse SSTR4, 5'-gatgccactgtcaaccatgtgtccct-3' and
5'-acggagttgtccttggagccagtcag-3' for rat SSTR4,
5'-agatacatgtgctctggcat-3' and 5'-aggatgtacacattagtaac-3' for mouse
SSTR5, 5'-gacacgcgtggtctggcacc-3' and 5'-aataatacgtcagccacggc-3' for
rat SSTR5. After the initial denaturation step, at 95 C for 3 min,
amplifications were carried out for 60 cycles, with a final 10-min
elongation step at 72 C. Each cycle consisted of denaturation at 95 C
for 45 sec; annealing at 62 C for SSTR1/SSTR3/rat SSTR5, 58 C for
SSTR2/SSTR4, 55 C for mouse SSTR5 for 1 for 45 sec, and elongation at
72 C for either 90 sec (SSTR1) or 45 sec (SSTR2–5). As positive and
species-specificity controls, 100 ng of either rat or mouse genomic DNA
(CLONTECH Laboratories, Inc., Palto Alto, CA) were used,
because SSTRs lack introns. Integrity of RNA was controlled with actin
primers. PCR products were then visualized with ethidium bromide after
electrophoresis on 1–2% agarose gel.
Biodistribution in AR4–2J tumor-bearing mice
All animal experiments were performed in compliance with the
Swiss regulation for animal treatment.
Standard protocol. Female Swiss nude mice (6–10 weeks old,
IFFA-CREDO, L’ Arbresle, France) were implanted sc with 5
million AR4–2J cells (ATCC, Manassas,), previously
expanded in DMEM medium containing 10% FCS, 2 mM
L-glutamine, 50 U/ml penicillin, and 50 µg/ml
streptomycin. One week later, 5 µCi radioligand, diluted in NaCl,
0.1% BSA (pH 7.5) was injected iv into the lateral tail vein, in 200
µl. For determination of nonspecific uptake, 50 µg cold octreotide
was coinjected with the radioligand. The animals were killed at the
indicated time points. Organs and tissues of interest were dissected
and rinsed of excess blood, weighed, and assayed for radioactivity in a
-counter. In the case of 90Y, the organs were
dissolved in 2 ml 1N HCl, and the Bremsstrahlung was measured in a
-counter. Percent of injected dose per gram (I.D./g) was calculated
for each tissue. The total counts injected per animal were calculated
by extrapolation from counts of a standard taken from the injected
solution for each animal.
Comparison of preblockage vs. coblockage. CB17 scid mice (6–7 weeks old, breeding pairs obtained from IFFA-CREDO) were implanted with AR4–2J cells as described above. One week later, the mice were divided into three groups. The first group was given 5 µCi radioligand iv; the second one, a mixture of 50 µg cold octreotide and 5 µCi radioligand; and the third one, 50 µg cold octreotide ip 30 min before 5 µCi radioligand iv. The animals were killed 4 h after radioligand administration and processed as indicated above.
Analysis of data
Unless otherwise stated, results are expressed as mean ±
SEM. Statistical evaluation was performed using the one-way
ANOVA test. When significant overall effects were obtained by ANOVA,
multiple comparisons were made with the Bonferroni correction. A
P value less than 0.05 was considered a statistically
significant difference.
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Results |
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In vitro receptor-binding studies
To characterize the receptor-binding properties of the
somatostatin analogs, competitive binding experiments were performed
using 125I-Tyr3-octreotide
as radioligand and AR4–2J membranes. Because some peptides were only
available in tiny amounts, a limited number of experiments could be
performed; therefore these results have to be considered as preliminary
studies. Table 1 lists the
IC50 for the four DOTA-somatostatin analogs
labeled with cold yttrium. As controls, OC, TyrOc, and unlabeled
DOTATOC were included. We used Y-complexed somatostatin analogs because
the metal by itself can influence the binding affinity of the complex.
The IC50 values were all in the nanomolar range,
but Y-DOTATOC showed the highest affinity to SSTR2. The comparison of
the IC50 obtained for TyrOc, DOTATOC, and
Y-DOTATOC (0.33, 2.44, and 2.37 nM; respectively) showed
that DOTA influenced the binding affinity of the somatostatin analog
but that incorporation of yttrium did not.
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presents the tissue
distribution of the four DOTA-somatostatin analogs labeled with
90Y or 111In in AR4–2J
tumor-bearing mice, 4 h post injection. As internal standard, we
included the widely used OctreoScan. For the DOTA-somatostatin analogs,
the uptake in the tumor was at least similar to that obtained with
OctreoScan and clearly much higher for DOTATOC (4.6 x) and DOTALan
(2.6 x). The accumulation in the other SSTR-positive tissues,
i.e. pancreas and adrenals, was variable and, interestingly,
did not correlate with the tumor uptake. For example, the ratio
tumor/adrenals was 1.7 for OctreoScan, 14.5 for DOTATOC, 4.2 for
DOTAOc, 4.9 for DOTA Lan, and only 0.9 for DOTAVap. The blood clearance
was, overall, more rapid for the four DOTA-somatostatin analogs than
for OctreoScan. This was associated with a similar faster elimination
of radioactivity from the SSTR-negative tissues not involved in the
excretion process. Bone uptake was low for the four DOTA-somatostatin
analogs tested, irrespective of the incorporated radiometal,
i.e. 90Y or
111In, indicating that DOTA is a stable chelator
for 90Y. As observed with OctreoScan, the
DOTA-somatostatin analogs were mainly, or even exclusively (DOTATOC and
DOTAOc), excreted through the kidney, leading to very low (DOTATOC and
DOTAOc) or moderate liver uptake (OctreoScan, DOTALan, and DOTAVap). In
return, the kidney uptake was high for all the radioligands tested,
including OctreoScan; however, it was substantially lower for
DOTAOc, DOTATOC, and (to a lesser extent) DOTAVap.
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Tumor uptake, as compared with kidney, liver, muscle, and blood
uptakes
To better evaluate the potential suitability of the four
radiolabeled somatostatin analogs for tumor targeting, the ratios
tumor-to-liver, tumor-to-muscle, tumor-to-kidney, and tumor-to-blood
were calculated 4 h post injection (Fig. 2). The values obtained for the two
radiometals were pooled, because they were statistically similar. These
ratio values clearly demonstrated the superiority of DOTATOC, not only
over OctreoScan but also over the other DOTA-somatostatin analogs.
Indeed, the tumor-to-liver, tumor-to-muscle, tumor-to-kidney, and
tumor-to-blood ratios calculated for DOTATOC were 23 x, 44 x, 9.2 x,
and 86 x higher than those found with OctreoScan and 8 x, 13 x,
4 x, and 12 x higher than those obtained for the second-best
somatostatin analog, i.e. DOTAOc, respectively. It is to be
noted, however, that DOTATOC was the only DOTA-somatostatin analog
giving a tumor-to-kidney ratio superior to 1.
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Specific uptake in tissues
Tumor-bearing mice were either coinjected or preinjected with 50
µg octreotide to determine the specific uptake of DOTATOC in tissues
4 h post injection. As shown in Fig. 3, the uptake of DOTATOC in SSTR-rich
tissues, such as tumor or pancreas, represented mainly specific
receptor-mediated uptake, because radioligand accumulation was
decreased by 93% in the tumor and 81% in the pancreas in SSTR-blocked
(compared with unblocked) mice. This injection of octreotide had no
statistically significant effects on radioligand uptake in tissues such
as liver or small intestine. Stomach and lung uptake was only partially
displaceable, indicating that only a fraction of the observed uptake
was presumably receptor-mediated. Both blockage protocols,
i.e. preblockage or coblockage, were essentially equivalent
for all tissues, including SSTR-rich tissues (exemplified by tumor and
pancreas), with the exception of the kidney. Surprisingly, whereas
preinjection of octreotide did not change kidney uptake of DOTATOC
significantly, its coinjection resulted in a 1.7-fold increase in
kidney uptake. Similar kidney uptake increases were also observed with
the three other DOTA-somatostatin analogs when octreotide was
coinjected with the radioligand (not shown).
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Kinetics of tumor and kidney uptake
Tumor and kidney clearance in tumor-bearing mice of the four
DOTA-somatostatin analogs and OctreoScan, from 4–48 h post injection,
are presented in Fig. 4. Maximum tumor
uptake was observed 4 h post injection for all the radioligands
tested. At that time, only DOTATOC and DOTALan showed a
statistically higher tumor uptake than that of OctreoScan. DOTALan was
then cleared rapidly from the tumor (25% of maximum remaining at
24 h), resulting in a tumor uptake that was essentially equivalent
to that of OctreoScan by 24 and 48 h. In contrast, DOTATOC showed
slower tumor clearance (62% of maximum remaining at 24 h); and
therefore, its retention in the tumor remained the highest during the
observation period of 48 h.
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DOTATOC < DOTAVap < OctreoScan < DOTALan. By
24 h, excretion clearly predominated, leading to much lower kidney
uptakes. It is to be noted that the clearance rate from the kidney was
not similar for all radioligands, as indicated by the fact that 24
h post injection, all kidney uptakes were virtually equal. A good indicator for the suitability of the DOTA-somatostatin analogs for internal radiotherapy is the ratio of the area under the curve of tumor-to-kidney. These ratios were calculated for the period ranging from 4–48 h and were as follows: 1.15 for DOTATOC, 0.33 for DOTAOc, 0.29 for DOTALan, 0.22 for DOTAVap, and 0.16 for OctreoScan, indicating again the most favorable behavior for DOTATOC.
Thus, the kinetics of tumor and kidney uptakes strongly argued in favor of DOTATOC as the best candidate for tumor targeting, even though kidney uptake remained high.
Tumor penetration
Tumor penetration was evaluated for each DOTA-somatostatin analog
by plotting tumor uptake as a function of tumor size (ranging from
8–2300 mg) (Fig. 5). Tumor uptake was
normalized to 100% for each radioligand to compare the four
DOTA-somatostatin analogs. For all radioligands, there was no
statistically significant linear relationship between tumor size and
tumor uptake. This indicates that tumor penetration was excellent for
the four DOTA-somatostatin analogs.
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Thus, mouse adrenals expressed mRNA for SSTR2, SSTR3, and SSTR5; and mouse pancreas for SSTR2 and SSTR3. The rat AR4–2J tumor contained only SSTR2 mRNA but was infiltrated by host cells expressing SSTR1and SSTR2 mRNA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The success of both the diagnostic and therapeutic strategies relies on the quantity of radioactivity that can be accumulated in the tumor and on its rapid clearance from the other organs. For radiotherapeutic applications, additional criteria have to be taken into account, such as biological half-life of the radioligand in the tumor and tumor penetration, because homogenous radioligand distribution is necessary for complete elimination of the tumor, particularly with low- or medium-energy radionuclides. All DOTA-somatostatin analogs tested showed a comparably favorable behavior in vitro and in vivo: 1) They retained their capacity to bind SSTR2, with IC50 in the nanomolar range, as indicated by displacement of 125I-Tyr3-octreotide from AR4–2J tumor membranes; 2) They accumulated preferentially in SSTR-expressing tissues including the tumor; 3) Their main route of excretion was via the kidneys; 4) In contrast to radiolabeled antibodies that were shown to localize only in the periphery of the tumors (29), they diffused within the tumor, as evidenced by the absence of correlation between tumor size and tumor uptake. In addition, our study clearly demonstrated that the new chelator DOTA enables successful labeling of somatostatin analogs with both diagnostic and therapeutic radionuclides. Indeed, for all DOTA-somatostatin analogs tested, the biodistributions of 111In- vs. 90Y-labeled somatostatin analogs were comparable. The low amounts of radioactivity found in the bones, for the 48-h period of observation, suggested excellent 90Y-DOTA complex stability in vivo, because dissociated 90Y rapidly accumulates in bone (15). This strongly indicates that DOTA can hold 90Y with high stability in vivo and, in that respect, is superior to the DTPA chelator that was reported to form unstable complexes with 90Y and thereby caused some bone-marrow toxicity (15). DOTA seems, therefore, to be the chelator of choice, particularly if internal radiotherapy is envisaged.
Despite these overall similarities, one DOTA-somatostatin analog, i.e. DOTATOC, was clearly found to be far superior to the other DOTA-somatostatin analogs, for both diagnostic and therapeutic use. DOTATOC showed the highest tumor uptake at all time points tested. Its rapid clearance from nontarget tissues led to excellent tumor-to-background ratios, mostly superior to the ones obtained with OctreoScan or the other DOTA-somatostatin analogs. Moreover, its accumulation tended to be higher in the tumor than in the normal SSTR-positive organs, which is of importance for radiotherapeutic applications when high doses of radioactivity are injected. Retention of radioactivity in the kidneys was most favorable with DOTATOC. It was indeed the only radioligand tested, which resulted in higher radioactivity exposure of tumor cells than kidney cells, as indicated by a ratio of tumor-to-kidney area under the curve that was greater than 1. Taken together, the remarkable performances of DOTATOC in our mouse tumor model makes it very promising for treatment or imaging of SSTR2-expressing neuroendocrine tumors. It is very likely that renal accumulation, which is still problematic in the clinic, can be lowered by infusion of lysine, as reported previously for several other radiolabeled proteins and peptides (30, 31). Preliminary data confirm the usefulness of this radiolabeled peptide in internal radiotherapy (13, 14).
The highest tumor uptake of DOTATOC was correlated with the highest binding affinity to tumor membrane in vitro. For the other DOTA-somatostatin analogs, there was no direct correlation between binding activity in vitro and accumulation in the tumor. This is very well exemplified by DOTALan, which showed the lowest binding affinity in vitro despite being the second-best radiolabeled DOTAsomatostatin analog, with regard to tumor uptake in vivo. Obviously, other parameters influence tumor uptake of radiopeptides, such as in vivo stability, internalization, and tumor penetration. We demonstrated, in this study, that tumor diffusion of the four DOTA-somatostatin analogs was essentially identical. Radioligand stability has not yet been carefully evaluated, but it is not expected to differ markedly because the four peptides used for coupling with DOTA are stable in vivo. Internalization of DOTATOC, after binding to SSTRs, was described recently (32); but in the absence of data concerning the other DOTA-somatostatin analogs, it is hard to speculate further on the role of internalization in the observed tumor uptake.
An interesting observation arising from this study was that kidney uptake of DOTA-somatostatin analogs was increased when an excess of cold octreotide was coinjected, suggesting the presence of saturable carriers for DOTA-somatostatin analogs in kidney. At this stage of the investigation, we can only speculate on the type of saturable carriers involved in DOTA-somatostatin transport in this organ. A possible candidate is the P-glycoprotein that was shown to be expressed in the proximal tubules (33, 34), where it mediates transport of various compounds, including peptides (35, 36). Ongoing studies are focusing on the identification of DOTATOC carriers in kidney to set up strategies to lower its renal accumulation.
Our investigation of the pattern of SSTR subtype expression in adrenals, pancreas, and tumor (by RTPCR) revealed the presence of different SSTR subtypes, which can presumably explain the differential uptakes of the radioligands tested in SSTR-positive tissues. Adrenals were found to express mRNA for SSTR2, SSTR3, and SSTR5; whereas in the pancreas, only SSTR2 and SSTR3 mRNA were detected. The tumor cells per se contained exclusively SSTR2, but the tumor was infiltrated by cells of host origin expressing mRNA for SSTR1 and SSTR2. To our knowledge, no data are available regarding the distribution of mRNA encoding the five cloned SSTRs in peripheral tissues in mouse. Human adrenals were reported to contain mRNA encoding either all five SSTRs (37) or only SSTR1 and SSTR2 (38); whereas in human pancreas, SSTR1, SSTR2, and SSTR4 mRNA were identified (38). In rat pancreas, SSTR2 mRNA was found to be the most abundant (39), but the presence of SSTR3 mRNA was also noted (39). Taken together, our results obtained in mouse indicate an overall closer similarity with the rat, at least for the pancreas.
The finding that our rat tumor was infiltrated by host cells expressing SSTR1 and SSTR2 mRNA is interesting per se and can have some implications in the observed tumor uptake if these transcripts give rise to significant amounts of corresponding proteins. Additionally, the presence of SSTR mRNA of nontumor origin in tumors can strongly influence the interpretation of the results concerning the identification of SSTR subtype expression in human malignancies, by Northern blotting or RTPCR protocols. Similar host infiltration of SSTR2-mRNA-expressing cells was also observed in scid mice implanted with AR4–2J cells (not shown). The origin of these SSTR1- and SSTR2-mRNA-expressing cells is unclear. These two SSTR subtypes were identified in activated lymphocytes/thymocytes (40, 41, 42); but given the immunodeficient status of the nude/scid mice used as hosts, it is very unlikely that the implanted tumor was infiltrated by lymphocytes. Expression of SSTRs in blood vessels in the immediate vicinity of tumors has been described (43). Nevertheless, further investigations, such as in situ hybridization, would be necessary for further speculation.
Interestingly, based on the recently reported affinity profiles of the DOTA-somatostatin analogs for human SSTR subtypes (25), a good correlation was found between the uptake of DOTA-somatostatin analogs in adrenals, pancreas, and tumor and the observed pattern of SSTR subtype expression in these tissues. For instance, we found that adrenals differed from pancreas by the presence of SSTR5; and, interestingly, the two radioligands exhibiting the highest adrenal-to-pancreas uptake ratio, namely DOTALan and DOTAVap, were reported to display the highest affinity for SSTR5. Similarly, the difference between tumor and pancreas lay in the presence of SSTR3 in the latter and, again, the highest tumor-to-pancreas uptake ratio was obtained with the radioligands exhibiting the lowest SSTR3 affinity, DOTATOC and DOTALan. Clearly this interpretation is based on the assumption that DOTA-somatostatin analogs display a similar affinity profile for mouse and human SSTR subtypes; this needs further clarification, although mouse and human SSTRs share many similarities (between 85% and 98%, depending on the subtype).
In conclusion, our study clearly showed that the use of DOTA enables successful radiolabeling of various somatostatin analogs with both diagnostic and therapeutic radionuclides. DOTATOC is most promising and clearly superior to the other DOTA-somatostatin analogs tested and to the widely used OctreoScan, with regard to both potential applications in nuclear oncology, i.e. tumor localization and internal radiotherapy of SSTR2-expressing malignancies. Even though these results were obtained in an animal model and thus raise the question of whether we can extrapolate them to human, the high homology between the human and the rat SSTR2 (95%) together with the predominance of the expression of this subtype in human malignancies foresee a high clinical relevance.
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Acknowledgments |
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Footnotes |
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Received December 30, 1999.
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I. Velikyan, A. L. Sundberg, O. Lindhe, A. U. Hoglund, O. Eriksson, E. Werner, J. Carlsson, M. Bergstrom, B. Langstrom, and V. Tolmachev Preparation and Evaluation of 68Ga-DOTA-hEGF for Visualization of EGFR Expression in Malignant Tumors J. Nucl. Med., November 1, 2005; 46(11): 1881 - 1888. [Abstract] [Full Text] [PDF]
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D. Storch, M. Behe, M. A. Walter, J. Chen, P. Powell, R. Mikolajczak, and H. R. Macke Evaluation of [99mTc/EDDA/HYNIC0]Octreotide Derivatives Compared with [111In-DOTA0,Tyr3, Thr8]Octreotide and [111In-DTPA0]Octreotide: Does Tumor or Pancreas Uptake Correlate with the Rate of Internalization? J. Nucl. Med., September 1, 2005; 46(9): 1561 - 1569. [Abstract] [Full Text] [PDF]
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S. Froidevaux, M. Calame-Christe, H. Tanner, and A. N. Eberle Melanoma Targeting with DOTA-{alpha}-Melanocyte-Stimulating Hormone Analogs: Structural Parameters Affecting Tumor Uptake and Kidney Uptake J. Nucl. Med., May 1, 2005; 46(5): 887 - 895. [Abstract] [Full Text] [PDF]
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L. J. Hofland, A. Capello, E. P. Krenning, M. de Jong, and M. P. van Hagen Induction of Apoptosis with Hybrids of Arg-Gly-Asp Molecules and Peptides and Antimitotic Effects of Hybrids of Cytostatic Drugs and Peptides J. Nucl. Med., January 1, 2005; 46(1_suppl): 191S - 198S. [Abstract] [Full Text] [PDF]
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 M. Schottelius, T. Poethko, M. Herz, J.-C. Reubi, H. Kessler, M. Schwaiger, and H.-J. Wester First 18F-Labeled Tracer Suitable for Routine Clinical Imaging of sst Receptor-Expressing Tumors Using Positron Emission Tomography Clin. Cancer Res., June 1, 2004; 10(11): 3593 - 3606. [Abstract] [Full Text] [PDF]
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 M. Wikman, A.-C. Steffen, E. Gunneriusson, V. Tolmachev, G.P. Adams, J. Carlsson, and S. Stahl Selection and characterization of HER2/neu-binding affibody ligands Protein Eng. Des. Sel., May 1, 2004; 17(5): 455 - 462. [Abstract] [Full Text] [PDF]
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G. Vaidyanathan, H. S. Friedman, D. J. Affleck, M. Schottelius, H.-J. Wester, and M. R. Zalutsky Specific and High-Level Targeting of Radiolabeled Octreotide Analogues to Human Medulloblastoma Xenografts Clin. Cancer Res., May 1, 2003; 9(5): 1868 - 1876. [Abstract] [Full Text] [PDF]
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S. Froidevaux, M. Calame-Christe, H. Tanner, L. Sumanovski, and A. N. Eberle A Novel DOTA-{alpha}-Melanocyte-Stimulating Hormone Analog for Metastatic Melanoma Diagnosis J. Nucl. Med., December 1, 2002; 43(12): 1699 - 1706. [Abstract] [Full Text] [PDF]
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