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Departments of Nuclear Medicine (P.M.S.-J., C.B., M.L., D.G., T.P., T.T., I.V.), Gastroenterology (M.P.-R.), Surgery (G.H.), Pathology (K.K.), Urology (A.K.), Dermatology (H.S.-W.), University of Vienna,A-1090 Vienna; and the Department of Radiochemistry, Research Center (P.A.), A-2444 Seibersdorf, Austria
Address all correspondence and requests for reprints: Irene Virgolini, M.D., Department of Nuclear Medicine, University of Vienna, Währinger Gürtel 18–20, Ebene 3L, A-1090 Vienna, Austria. E-mail: irene.virgolini{at}akh-wien.ac.at
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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200 nM). The rank order of displacement
of [125I]Tyr11-SST binding to
hsst1 was: SST (IC50, 0.5 nM) >>
DOTALAN (IC50, 154 nM) > lanreotide
(LAN) Tyr3-octreotide (TOCT)
DOTA-Tyr3-octreotide (DOTATOCT) DOTA-vapreotide
(DOTAVAP; IC50, >1000 nM); that to
hsst2 was: DOTATOCT TOCT DOTALAN
SST LAN DOTAVAP (IC50, 1.4
nM); that to hsst3 was: SST (IC50,
1.2 nM) > DOTALAN = LAN (IC50, 15
nM) TOCT (IC50, 20
nM) DOTAVAP (IC50, 28
nM) > DOTATOCT (IC50, 73 nM);
that to hsst4 was: SST (IC50, 1.8
nM) DOTALAN (IC50, 2.5
nM) > LAN (IC50, 22 nM) >>
DOTATOCT DOTAVAP TOCT (IC50, >500
nM); and that to hsst5 was: DOTALAN
(IC50, 0.45 nM) > SST (IC50,
0.9 nM) > TOCT (IC50, 1.5
nM) > DOTAVAP (IC50, 5.4 nM)
>> LAN (IC50, 21 nM) > DOTATOCT
(IC50, 260 nM). In Sprague Dawley rats (n
= 10), 90Y-DOTALAN was rapidly cleared from the circulation
and concentrated in hsst-positive tissues such as pancreas or
pituitary. Taken together, our results indicate that
111In-/90Y-DOTALAN binds to a broad range of
primary human tumors and tumor cell lines, probably via binding to
hsst2-5. We conclude that this radiolabeled
peptide can be used for hsst-mediated diagnosis
(111In-DOTALAN) as well as systemic radiotherapy
(90Y-DOTALAN) of human tumors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Native SST exists in two forms (14 or 28 amino acids), but it is readily attacked by aminopeptidases and endopeptidases and has a short in vivo half-life. Consequently, synthetic SST analogs, which incorporate a Phe-(D)Trp-Lys-Thr (or similar sequence) and which are metabolically stabilized at both the N- and C-terminals, were developed for clinical applications. To date, three commercially available SST analogs (i.e. octreotide [(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr(ol); OCT (19)], lanreotide [(D)ßNal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH2; LAN (20)], and vapreotide [(D)Phe-Cys-Tyr-(D)Trp-Lys-Val-Cys-Trp-NH2; VAP (21)]) have been shown to be effective in controlling the growth of some human tumors. These SST analogs all have similar binding profiles for four of the five hsst subtypes (i.e. a high affinity for hsst2 and hsst5, moderate affinity for hsst3, and very low affinity for hsst1), but LAN and VAP have a moderate affinity for hsst4, whereas OCT has little or no affinity for this hsst (for review, see Ref. 22).
A number of radiolabeled SST analog conjugates have been synthesized
for diagnostic (23, 24, 25, 26, 27) or therapeutic applications (28). In general,
these small peptides not only have a rapid blood clearance after iv
injection, but also, for some tumor types, a high accumulation of
radioactivity at the tumor (23). This in combination with a large dose
of a diagnostic radionuclide led to the successful application of
receptor-mediated radiotherapy (28). Although the proposed mechanism
includes the internalization of the radioactivity and a localized auger
electron shower irradiation of the cell DNA (29), 111In is
not an ideal radiotherapeutic nuclide, because a majority of the decay
energy is in the form of 
-rays, which irradiate both healthy tissue
and the surrounding personnel. Similarly, attempts to use
diethylene-triamine-penteacetic
acid-D-Phe1-OCT with other
radiotherapeutic nuclides have not been successful, probably because
the metal chelate was unstable under physiological conditions (30).
In this work we present the in vitro binding characteristics and hsst subtype specificity of a novel SST analog that is a conjugate of DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid) coupled directly to the N-terminus of lanreotide [(D)ßNal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH2]. This conjugate has already been successfully used for diagnostic (31, 32) and therapeutic (33) applications.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) was synthesized
in a three-step reaction using the commercially available LAN
[Somatuline,
(D)ßNal-Cys-Tyr-(D)Trp-Lys-Val-Cys-Thr-NH2]
and
1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic
acid (DOTA) as starting materials. First, the 
-amino group of LAN
was blocked by using ditert-butyl dicarbonate to produce -Boc LAN.
Eighty milligrams of DOTA, 74 mg N-hydroxysuccinimide, and
80 mg [-boc-Lys5]LAN were dissolved in 15 ml water and
30 ml N,N-dimethylformamide. One hundred
milligrams of N,N'-dicyclohexylcarbodiimide were
added, and the solution was stirred at room temperature for 16 h.
The product,
[
-DOTA-(D)ßNal1,ß-Boc-Lys5]LAN,
was recovered and purified on a Silica Gel 60 column using a methylene
chloride-methanol-50% acetic acid (9:1:0.125
7:3:1) solvent system.
The Boc-protecting group was cleaved by dissolving the residue in 4 ml
methylene chloride and 2 ml 95% trifluoroacetic acid and allowing the
reaction to proceed for 30 min at room temperature. The product,
[DOTA-(D)ßNal1]LAN was precipitated with
ether and purified on a C18 reverse phase HPLC column using
a water-acetonitrile-0.1% trifluoroacetic acid (TFA) solvent system.
HPLC was performed with a Jasco (Maarssen, The Netherlands) HPLC
system (dual 880PU pumps and 875 UV detector) and an on-line Bertold
-detector (LB 506, Bad Wildbad, Germany) coupled to a 386 PC
running Bertold software. A second HPLC purification using
water-acetonitrile-1% acetic acid yielded the DOTALAN compound as an
acetate salt [purity, 97% (C18 reverse phase HPLC);
MS:MH+:1482 (californium-252 time of flight, Biolon
AB, Stockholm, Sweden)]. DOTATOCT and DOTAVAP were synthesized
in an analogous manner by the direct coupling of either DOTA and
[Tyr3, -Boc-Lys5]octreotide
[i.e.
(D)Phe-Cys-Phe-(D)Trp-(-Boc)Lys-Thr-Cys-Thr(ol)]
or tri-tBu-DOTA and
[Tyr3,-Boc-Lys5]vapreotide
[i.e.
(D)Phe-Cys-Tyr-(D)Trp-(-Boc)Lys-Val-Cys-Trp-NH2)
(Powel, P., and H. R. Mäcke, unpublished data). The
respective products, when analyzed by mass spectroscopy, gave
MH+ peaks at 1423.2 and 1518.0 (Powel, P., and H. R.
Mäcke, unpublished data).
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The 90Y complex was prepared in a similar manner as the 111In complex, but 90YCl3 was used at a reaction ratio of about 35 MBq 90Y:1 nmol DOTALAN. 90YCl3 (no carrier added; >18,500 MBq/ml in 0.05 M HCl) was obtained from Pacific Northwest National Laboratory (Richland, WA). The one-step radiolabeling procedure typically produced specific activities of about 20 and 35 MBq/nmol for 111In and 90Y, respectively, with the fraction of uncomplexed radionuclide being below 2%. The labeling results obtained for 111In and 90Y were critically dependent on the amount of other metal cations in the radiochemical grade isotopes, as increasing the reaction time did not appreciably increase the radiolabeling yield.
The stability of 90Y-DOTALAN was examined by studying the transchelation of 90Y to DTPA. Briefly, a sample of either 90Y-DOTALAN (50 µl, 50 pmol) or 111In-DOTALAN (25 µl, 50 pmol) was diluted to 2 ml with 4 mM DTPA (pH 4.0) and incubated at 37 C. Fifty-microliter portions of this DTPA solution were then periodically withdrawn and analyzed by HPLC to determine the transchelation rate of 90Y and 111In from the respective DOTALAN chelate to DTPA over a period of 1 week. Both the 111In- and 90Y-labeled DOTALAN were stable in 4 mM DTPA (pH 4.0) up to a period of 7 days postmixing, with less than 0.1% of the radioactivity being associated with DTPA at any one time point.
Preparation of primary tumor tissue
Written informed consent was obtained from all patients
undergoing surgery. Tumor tissue specimens (0.5–1 ml) were collected
at surgery and immediately placed into liquid nitrogen. The respective
diagnoses were established by histological examination and
immunohistochemistry according to WHO criteria. Tissue was stored at
-70 C until used for in vitro studies. Tumor cell membrane
fractions were prepared according to established techniques (16).
Briefly, tissue was thawed, cut into pieces, put into 20 mM
HEPES-KOH buffer (pH 7.4), and homogenized by means of a glass
homogenizer. The cell homogenate was centrifuged at 5000 x
g for 10 min at 4 C, washed and resuspended in assay buffer
containing 20 mM HEPES-KOH (pH 7.4), and measured by the
method of Bradford et al. (34).
Tumor cell lines
The colonic adenocarcinoma cell line HT29, the pancreatic
adenocarcinoma cell line PANC1, the prostate cancer cell lines PC3 and
DU145, and the breast cancer cell lines T47D, ZR75–1, and MCF7 were
purchased from American Type Culture Collection (Manassas,
VA). These cell lines were cultured in RPMI 1640 supplemented with 10%
FCS, L-glutamine, and antibiotics in 5%
CO2-95% air at 37 C. The melanoma cell line 518A2 was
provided by Dr. Schrier (University Hospital, Leiden, The Netherlands)
and cultured in DMEM supplemented with 8% FCS. The cells were fed two
or four times per week. Adherent cells were passaged with trypsin
(Worthington Biochemical Corp., Freehold, NJ) after
confluence was reached. Before being used in binding experiments, cells
were washed and then resuspended in the assay buffer (4 C). Peripheral
mononuclear cells were also obtained from healthy volunteers and used
for binding studies.
Transfection
Plasmids containing the receptor complementary DNAs for the
human SST receptors (hsst1–4) were provided by Prof. Dr.
G. Bell (Howard Hughes Medical Institute, Chicago, IL). The plasmid
carrying hsst5 was provided by Dr. O’Carroll (NIH,
Bethesda, MD). The precision of each receptor clone was confirmed by
both agarose gel analysis of restriction fragments and complete
sequence analysis. Isolation of plasmid DNA was carried out with the
QIAGEN plasmid purification kit (QIAGEN,
Hilden, Germany).
COS-7 cells were grown in RPMI 1640 containing 10% FBS and antibiotics (Life Technologies, Inc./BRL, Vienna, Austria). Transient transfection of COS-7 cells with different receptor complementary DNAs was performed using the DOTAP transfection reagent (Roche Molecular Biochemicals, Germany) according to the manufacturer’s description. Twenty-four hours after transfection, the medium was exchanged, and after an additional 48 h in culture, the cells were harvested. An aliquot of the cells was recovered for RNA extraction and Northern analysis immediately before these cells were used for binding studies.
RNA extraction and Northern blot analysis
Tumor specimens, previously snap-frozen in liquid
nitrogen, were homogenized in Trizol reagent (Life Technologies, Inc./BRL), using a glass homogenizer. Total RNA from the
homogenate was extracted according to the manufacture’s protocol, a
modification of the RNA extraction method described by Chomczynski and
Sacchi (35). The RNA of transfected and control COS-7 cells was
extracted using the same technique. The integrity of the RNA was
confirmed by agarose gel electrophoresis and staining with ethidium
bromide. Northern blot analysis of receptor subtype expression was
preformed as previously described (35, 36). Northern transfer of about
20 µg total RNA was performed using a Nytran membrane
(Schleicher & Schuell, Inc., Vienna, Austria) by capillary
blotting overnight. RNA was fixed to the membranes by UV cross-linking.
Specific probes for Northern hybridization were generated by
restriction cutting of the plasmids carrying the probes with the
appropriate restriction enzymes. After separation with an agarose gel,
the probes were purified with the Qiaex gel purification kit
(QIAGEN) and labeled using the Redivue random prime
labeling kit (Amersham Pharmacia Biotech, Aylesbury, UK)
and [32P]deoxy-CTP (Amersham Pharmacia Biotech). Hybridization was carried out as described previously
(35, 36). Briefly, the membranes were prehybridized at 42 C in a
hybridization solution containing 50% formamide, 5 x Denhardt’s
solution, 5 x SSC (saline sodium citrate), 0.2% SDS, and 100
µg/ml salmon sperm DNA. After 4 h of prehybridization, the
labeled probe was added in fresh hybridization buffer, and
hybridization was carried out overnight. Thereafter, blots were rinsed
twice at room temperature with 2 x SSC buffer containing 0.1%
SDS and then twice at 42 C with 0.2 x SSC buffer containing 0.1%
SDS, and finally exposed to an x-ray film (Hyperfilm, Amersham).
Radioligand binding of
111In-/90Y-DOTALAN in
vitro
The radiolabeled or unlabeled DOTALAN was evaluated in vitro by
performing saturation and displacement binding studies with membranes
derived from primary human tumors, human tumor cell lines, or COS-7
cells transfected with one of the five hssts.
Saturation studies employed 50 µg of membranes in 20 mM HEPES-KOH buffer (5 mM MgCl2, 40 µg/ml bactracin, 0.3% BSA) and increasing amounts of either 111In- or 90Y-DOTALAN. The mixture was incubated at room temperature for 60 min before being rapidly filtered through a glass-fiber filter (GF/C, Whatman, Haverhill, MA) presoaked in 1% BSA. The filters were washed four times with 1 ml ice-cold 10 mM Tris-HCl (0.85% NaCl, pH 7.4), before being counted with an automatic Na(Tl)I detector. Each assay was made in triplicate, and nonspecific binding was defined as binding in the presence of 1 µM SST.
The displacement studies were performed in parallel to determine the IC50 of LAN, DOTALAN, and SST for the binding of 111In-/90Y-DOTALAN or of [125I]Tyr11-SST (Amersham Pharmacia Biotech; SA, 74 MBq/nmol), to primary tumor cells, tumor cell lines, or the five hsst subtypes expressed by COS7 cells. The assay conditions were similar to those employed above, but used a constant amount of radioligand (50–100 pM) and concentrations of the unlabeled peptide ranging from 0.001–1000 nM. In selected experiments, also unlabeled Tyr3-OCT (TOCT), DOTA-Tyr3-OCT (DOTATOCT), and DOTA-vapreotide (DOTAVAP) were used as competitors. The saturation binding data were evaluated using the least squares fitting routine of the computer program Origin (version 3.54, Microcal Software, MA) as well as by Scatchard transformation of the data (37). All experiments were performed in triplicate, and for cell binding studies they were repeated at least three times.
Animal biodistribution of 90Y-DOTALAN
The animal studies were performed in accordance with Austrian
regulations. The biodistribution of 90Y-DOTALAN was
evaluated in normal female Sprague Dawley rats. The animals (180–220
g) were injected with the 90Y-labeled peptide (4 MBq, 160
pmol) via the tail vein. After periods of 1, 24, or 48 h, groups
of animals (n = 3–4) were killed, and the organs of interest were
removed and assayed for radioactivity. Statistical analysis was
performed using a two-sample t test to compare the means and
the respective SDs at the three time points evaluated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All primary tumors bound either 111In- and/or
90Y-labeled DOTALAN in a saturable manner with a high
binding affinity (Table 1). The
respective dissociation constants (Kd) ranged between 1 and
15 nM (median, 5 nM), and the estimated number
of specific binding sites (Bmax) ranged between 6.6 x
1010 and 5.4 x 1012 sites/mg protein
(i.e. 110 and 9000 fmol/mg protein; median, 1.8 x
1012 sites/mg protein, i.e. 3000 fmol/mg
protein). The calculated numbers of binding sites and the binding
affinities for the different tumor types are depicted in Table 1. The
highest binding densities were found for intestinal adenocarcinomas
(median, 2100; range 150-4000 fmol/mg protein), ductal breast cancers
(mean, 2000; range, 250-9000 fmol/mg protein), and carcinoid tumors
(mean, 3000; range, 300-6000 fmol/mg protein).
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, A–C, show an
example for the specific binding of 111In-DOTALAN,
90Y-DOTALAN, and [125I]Tyr11-SST
to membranes prepared from a carcinoid tumor. The messenger RNA (mRNA)
analysis (Fig. 2D) showed the strong expression of hsst3
and hsst4 as well as the presence of hsst2 and
hsst5. For the three tumor types no differences were
observed in either the binding affinity or the binding capacity of
111In-DOTALAN or 90Y-DOTALAN. Furthermore, for
all three primary tumor specimens, the maximal binding capacity was
comparable to that observed with
[125I]Tyr11-SST.
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, the binding of
111In-/90Y-DOTALAN was inhibited by (unlabeled)
DOTALAN and SST in a dose-dependent manner, with resultant
IC50 values in the lower nanomolar range (0.2–10
nM). TOCT was less potent than either DOTALAN or SST in
inhibiting the binding of 111In-/90Y-DOTALAN to
intestinal adenocarcinomas or ductal breast cancers. In contrast, the
four ligands had a similar affinity for the binding sites expressed by
the carcinoid tumor.
An extended set of peptides also included LAN as a competing ligand for
the binding of 111In-DOTALAN, 90Y-DOTALAN,
or 125I-Tyr11-SST to a carcinoid tumor. In
these studies, the resultant IC50 values were in the same
high affinity ligand range of 0.1–5 nM, with the same rank
order of potency (Fig. 3, A and B).
However, for a colorectal tumor, DOTATOCT and TOCT were unable to
totally displace 111In-DOTALAN, whereas both LAN and
unlabeled DOTALAN were potent in displacing all of the radioligand
(Fig. 4A). This tumor was also found to
express only the mRNAs for hsst3, hsst4, and
hsst5 (Fig. 4B).
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), the
prostate adenocarcinoma cancer cell lines PC3 (Fig. 5B) and DU145, the
colonic adenocarcinoma cell line HT29, and the melanoma cell line 518A2
(Fig. 5C). The respective binding data are listed in Table 2, which indicates that approximately
10,000–100,000 sites/cell were expressed by the various tumor cell
lines. In contrast, the MCF7 breast cancer cell line did not bind
111In-/90Y-DOTALAN in a saturable manner.
Furthermore, on the cell surface of peripheral mononuclear cells, only
a small number of binding sites (6,000/cell) for DOTALAN could be
detected. No substantial difference could be observed for the binding
behavior of 111In- and 90Y-DOTALAN (Fig. 5).
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).
111In-/90Y-DOTALAN bound to either T47D or
ZR75–1 breast cancer cells, PC3 or DU145 prostate cancer cells, 518A2
melanoma cells, and HT29 and PANC1 adenocarcinoma cells was displaced
by SST and DOTALAN in a similar manner (IC50, 1–9
nM). TOCT and LAN were slightly less effective competitors
(IC50, 1–30 nM) and were noticeably weaker
competitors with the T47D breast cancer cell line. DOTATOCT and DOTAVAP
were even more selective in inhibiting the binding of radiolabeled
DOTALAN, and they were noticeably weaker for the ZR75–1 breast cancer,
518A2 melanoma, and HT29 colonic adenocarcinoma cell lines
(IC50, 66–320 nM).
Identification of the molecular interaction site
The specific binding of radiolabeled DOTALAN to
hsst2–5 was saturable and typical of a high affinity
interaction between a ligand and a single class of receptors (Fig. 6, A–E).
111In-/90Y-DOTALAN bound with high affinity to
hsst2 (Kd, 4.3 nM),
hsst3 (Kd, 5.1 nM),
hsst4 (Kd, 3.8 nM), and
hsst5 (Kd, 10 nM) and with low
affinity to hsst1 (Kd, 200 nM).
There was no appreciable difference in the Kd or
Bmax values when the ligand was labeled with either
111In or 90Y.
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and 4.
The rank order of displacement of
[125I]Tyr11-SST-14 binding to transfected
COS7 cells was: for hsst1, SST >> DOTALAN > LAN
TOCT DOTATOCT DOTAVAP; for hsst2,
DOTATOCT TOCT DOTALAN SST LAN
DOTAVAP; for hsst3, SST > DOTALAN =
LAN TOCT DOTAVAP > DOTATOCT; for
hsst4, SST DOTALAN > LAN >> DOTATOCT
DOTAVAP TOCT; and for hsst5, DOTALAN >
SST > DOTAVAP > TOCT > LAN > DOTATOCT.
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) showed that there is a high
accumulation of radioactivity in organs that are known to contain high
densities of somatostatin receptors, such as pancreas, adrenals, and
pituitary. In all other organs the uptake of activity was low, except
for the kidneys, which reflects the predominant renal clearance of the
ligand. There was a high and significant (P < 0.05)
washout of radioactivity from all normal organs over the first 24
h, with the exception of the hepatobiliary organs and the kidneys. Over
the second 24-h period, only the liver and lungs had a significant
(P < 0.05), but slight, reduction in activity. Most
importantly, there was no increase in the bone-associated radioactivity
over the second 24-h period, indicating that there was no significant
release of 90Y from the 90Y-DOTALAN
chelate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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200 nM). DOTALAN also binds to a broad
range of primary human tumors and human tumor cell lines. All of these
tumor types have been shown to express SSTR subtypes (18). Subsequent
human studies have already indicated that when labeled with
111In or 90Y, DOTALAN can be used to localize
(31, 32) and treat (33) human tumors, respectively. The binding of 111In-/90Y-DOTALAN to primary human tumors demonstrates the suitability of this peptide for in vivo applications in humans. The high affinity binding exhibited for this wide range of tumors [intestinal adenocarcinomas (n = 17), ductal breast cancers (n = 4), carcinoids (n = 4), melanoma (n = 1), non-Hodgkin lymphoma (n = 1), papillary thyroid cancer (n = 3), and hepatocellular cancer (n = 1)] is further enhanced by the high receptor densities expressed by these tumors (for an overview, see Ref. 18). Furthermore, 111In-/90Y-DOTALAN bound to the tumor types that, by mRNA analysis, expressed no hsst2 mRNA. All of these in vitro binding studies demonstrate that radiolabeled DOTALAN maintains a wider sst recognition profile compared with other commercially available and experimental radiolabeled SST analogs (23, 24, 25, 26, 27).
The binding of DOTALAN to primary tumor cell lines further demonstrates that both 111In-DOTALAN and 90Y-DOTALAN have similar high affinities (Kd) for the naturally expressed hssts, and the similar Bmax values indicate that these two radioligands have similar recognition profiles for these expressed hssts. In a majority of these cell lines, only one binding site was detected despite the fact that more than one sst mRNA was expressed. Although this could be due to the expression of the mRNA and not the receptor, it is probably due to the close similarity in the binding affinity of DOTALAN for the hsst2–5.
The data for the binding of radiolabeled DOTALAN to human tumors and cell lines suggests that the radioligand is suitable for in vivo detection or therapy of tumors, but it does not explain the apparent differences in the ability of the unlabeled ligands to displace it. Consequently, the final set of binding studies, performed with COS-7 cells, gives a greater understanding of the binding behavior of the ligands. The binding profile of DOTALAN for the five hssts is different from that of TOCT, LAN, DOTAVAP, and DOTATOCT. DOTALAN has a high affinity (4–10 nM) to the hsst2–5 and a moderate (200 nM) affinity to hsst1. In this respect it is similar to the parent peptide LAN. hsst2 (and hsst3) seem to be the main receptor subtypes targeted in vivo by DTPA-M-Phe1-OCT (28) and DOTATOCT (38) radiotherapy. Although hsst2 is frequently expressed by human tumors, it is often expressed in combination with other subtypes. Furthermore, as current research (12, 13, 14) seems to suggest that any response observed in the various octreotide/lanreotide/vapreotide therapies is mediated by hsst2 and hsst5, the selective targeting of hsst2 by a radiotherapeutic ligand is unnecessary. However, in some cases hsst2 is not expressed although other hssts are present (18). These findings suggest that DOTALAN should recognize and bind to a larger variety of malignant tissues.
The reason for the different binding behaviors of DOTA modified and unmodified peptides is difficult to explain, and little has appeared in the literature to demonstrate these effects. Stolz et al. (39) have shown that when OCT is modified to form DOTATOCT, the affinities (Kd) for hsst1, hsst2, and hsst4 remain similar at 200/more than 100, 0.6/0.9, and 1000/more than 1000 nM, respectively, but there is a 5-fold reduction in the affinity of DOTATOCT for hsst3 (down from 10–50 nM) and a 100-fold reduction in the affinity for hsst5. Apart from these data, other studies, using rat cortex membranes, have shown the effects of modifying the N-terminus of OCT. The same group (40) reported that DTPA-M-Phe1-OCT was weaker than OCT in displacing [125I]Tyr3-OCT from rat cortex membranes (pKi 9.1 vs. 9.8) and that another DTPA-modified analog, DTPA-benzly-acetamido-M-Phe1-Tyr3-OCT and its yttrium complex were even weaker (pKi, 8.4 and 8.5). Using the same test system, another group (41) showed that when a lipophilic propylenediamine dioxime chelate (PnAO) was conjugated to OCT, this new analog was more effective in displacing [125I]Tyr3-OCT from rat cortex membranes (pKi 9.89 vs. 9.45). These data seem to suggest that the conjugation of hydrophilic groups to the N-terminus of OCT can diminish the binding affinity of OCT, whereas the conjugation of a lipophilic group can increase the binding affinity. Why no such dramatic reduction is seen in the binding affinity of LAN when it is modified with DOTA is unclear, but the very lipophilic side-chain of the N-terminus amino acid of LAN might contribute to this effect.
Although the cellular expression of specific hsst mRNA does not specifically predict the expression of the same hsst on the cell surface, it is a reliable indicator of which receptor subtypes are present. Consequently, in the particular case of the MCF7 breast cancer cell line, the lack of hsst1–5 mRNA expression precluded the expression of hssts on the cell surface and made it a suitable control cell line. It is important to note that a previous study (42) with the MCF7 cell line detected no sst1–4 and only a slight expression of sst5. Similarly, another group (43) has reported that MCF7 cells specifically bound [125I]TOCT, but they estimated a very low number of binding sites (<0.5 fmol/mg). The reason why we found no expression of sst5 or other ssts could be explained by either the growth medium on this estrogen-dependent cell line or the low densities of receptors and the sensitivity of our assay.
If one combines mRNA data with the binding data performed with the COS-7 cells transfected with the individual hssts, then binding behavior of both the radioligands and the competing ligands with the tumor cells and primary tumor membranes is comprehensible. For example, in the case of the colorectal tumor, the expression of only sst3–5 mRNA helped to explain why the hsst2-selective ligand (DOTATOCT) was unable to compete with 111In-DOTALAN (hsst2–5 selective) for all of the specific binding sites. The similar behaviors of DOTATOCT and TOCT (hsst2 and hsst5 selective) also suggest that despite the presence of hsst5 mRNA, hsst5 either represents only a small population of the overall hssts expressed, or it is not expressed on the cell surface. Conversely, in the case of the carcinoid tumor expression of hsst2–5 mRNA, the hsst2-selective ligand was able to compete with 111In-DOTALAN for all specific binding sites. A similar argument could be used to explain the inferior IC50 values observed with DOTATOCT for the ZR75–1 breast cancer, 518A2 melanoma, and HT29 colonic adenocarcinoma cell lines.
Despite the broader hsst subtype recognition profile of DOTALAN, it exhibited a similar rapid blood clearance and general biodistribution in normal rats compared with those in other OCT-based ligands. This would seem to indicate that the expression of sst2–5 is low in normal tissue. Most importantly, the accumulation of radioactivity in the bone was low and did not increase over the study period, which confirms the high in vitro stability of the complex.
In comparison with 111In-DTPA-D-Phe1-OCT (25), the uptake by the adrenals was significantly higher, i.e. 4.3 ± 0.61% vs. 2.5 ± 0.3% injected dose (at 1 h postinjection). Interestingly, the renal retention was reduced by about 20%, and there was a corresponding 20% increase in the liver radioactivity compared with 111In-DTPA-D-Phe1-OCT (25). This slightly higher liver and lower kidney accumulation might reflect the more lipophilic nature of the DOTALAN peptide and suggests that in humans a lower radiation dose to the kidneys (a critical organ) might be achieved. In fact, recent clinical data (31, 32, 33) have proven this concept.
The long residence time at hsst-positive tissues in experimental animals would seem not to arise from a simple ligand-receptor interaction, but rather from an internalization of the ligand and subsequent trapping within the cell (44, 45). Previous studies have shown that primary metabolites of chelates conjugated to both monoclonal antibodies and OCT (45, 46) consist of the chelate conjugated to the single N-terminal amino acid. This suggests that the main cellular metabolite is the lipophilic of 90Y-DOTA-(M)ßNal.
DOTA is one of the more effective chelators for yttrium, but, like most
polyaminocarboxilic acid chelators, is not a selective complexing agent
(47, 48). It is known to form thermodynamically stable chelates with
most transition metals, lanthanides, and even some of the alkaline
earth metals. However, this lack of selectivity is not a serious
drawback to labeling of DOTALAN with 90Y, because the major
commercial suppliers of 90Y can produce it with a
sufficiently high chemical purity. In addition, the incorporation of
DOTA into the conjugate allows the ligand to also be used with
67/68Ga and 111In for positron emission
tomography and planar -scintigraphy. This is an important aspect
because it is difficult to perform dosimetry with the pure ß-emitter
90Y, and few centers have the necessary infrastructure to
produce and perform positron emission tomography studies with
86Y.
Receptor-mediated radiotherapy relies on the use of a receptor-specific ligand to transport a radionuclide to tumor cells that overexpress the target receptor. In this concept, the radioligand should be rapidly cleared from the circulation to prevent high whole body irradiation, the chelate containing the radionuclide should be stable under physiological conditions to reduce the nontarget radiation dose, the tumor cells should have a sufficiently high density of hssts (ideally the radioligand should bind with a high affinity to all of the hsst subtypes expressed), the biological residence time of radioactivity should be as long as possible (ideally the radioligand should be internalized), and it or its radioactive metabolites should be trapped in the cell. The radiation dose to the tumor should be homogeneous to prevent cell-selective irradiation of cells expressing high concentrations of receptors and, hence, an overall down-regulation of tumor receptor status. In short, all of these points have been demonstrated for DOTALAN.
In summary, we present a novel hsst radiopharmaceutical that is able to
target a large variety of human tumor entities. This novel ligand,
named MAURITIUS (Multicenter Analysis of a
Universal Receptor and Imaging and
Treatment Initiative: a European
Study), is distinct from all other reported SST analogs,
which are based on OCT. DOTALAN binds to all SST receptors: to
hsst2–5 with high affinity (Kd, 4–10
nM) and to hsst1 with low affinity
(Kd, 200 nM). DOTALAN also binds to a broad
range of human tumor cell lines and primary human tumors. Subsequent
human studies have already shown that when labeled with
111In or 90Y, DOTALAN can be used to localize
(31, 32) or treat human tumors (33).
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Received February 16, 1999.
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, SST; 
, DOTALAN; •, TOCT; 
, LAN;
0.001–1000 nM), and primary tumor membrane fractions.
, DOTATOCT; 0.001–1000 nM), and primary
tumor membrane fractions. The mRNA analysis clearly shows that only
hsst3, hsst4, and hsst5 mRNA were
expressed by this tumor.
