Original article
`
`The somatostatin receptor-targeted radiotherapeutic
`[90Y-DOTA-DPhe1,Tyr3]octreotide (90Y-SMT 487) eradicates
`experimental rat pancreatic CA 20948 tumours
`
`Barbara Stolz, Gisbert Weckbecker, Peter M. Smith-Jones*, Rainer Albert, Friedrich Raulf, Christian Bruns
`
`Novartis Pharma AG, Basel, Switzerland
`
`&misc:Received 14 January and in revised form 16 March 1998
`
`&p.1:Abstract. Somatostatin receptor-expressing tumours are
`potential targets for therapy with radiolabelled somato-
`statin analogues. We have synthesized a number of such
`analogues in the past and identified [DOTA-DPhe1,
`Tyr3]octreotide (SMT 487) as the most promising candi-
`date molecule because of its advantageous properties in
`cellular and in vivo tumour models. In the current paper
`we describe the radiotherapeutic effect of yttrium-90 la-
`belled SMT 487 in Lewis rats bearing the somatostatin
`receptor-positive rat pancreatic tumour CA 20948. SMT
`487 binds with nanomolar affinity to both the human and
`the rat somatostatin receptor subtype 2 (sst2) (human sst2
`IC50=0.9 nM, rat sst2 IC50=0.5 nM). In vivo, 90Y-SMT
`487 distributed rapidly to the sst2 expressing CA 20948
`rat pancreatic tumour, with a tumour-to-blood ratio of
`49.15 at 24 h post injection. A single intravenous admin-
`istration of 10 mCi/kg 90Y-SMT 487 resulted in a com-
`plete remission of the tumours in five out of seven CA
`20948 tumour-bearing Lewis rats. No regrowth of the tu-
`mours occurred 8 months post injection. Control animals
`that were treated with 30 m g/kg of unlabelled SMT 487
`had to be sacrificed 10 days post injection due to exces-
`sive growth or necrotic areas on the tumour surface. Up-
`on re-inoculation of tumour cells into those rats that had
`shown complete remission, the tumours disappeared af-
`ter 3–4 weeks of moderate growth without any further
`treatment. The present study shows for the first time the
`curative potential of 90Y-SMT 487-based radiotherapy
`for somatostatin receptor-expressing tumours. Clinical
`phase I studies with yttrium-labelled SMT 487 have
`started in September 1997.
`
`&kwd:Key words: Somatostatin – Octreotide – Receptor target-
`ed radionuclide therapy – Yttrium-90
`
`Eur J Nucl Med (1998) 25:668–674
`
`* Present address: University Hospital AHK, Department of Nu-
`clear Medicine, Währinger Gürtel 18–20, A-1090 Vienna, Austria
`Correspondence to: B. Stolz, Novartis Pharma AG, K-125.15.14,
`CH-4002 Basel, Switzerland&/fn-block:
`
`Many human tumours express somatostatin receptors
`with a high incidence [1]. These include neuroendocrine
`tumours, such as pituitary adenomas, carcinoids, islet
`cell carcinomas, paragangliomas and phaeochromocyto-
`mas, lymphomas [2], small cell lung cancer [1, 3], tu-
`mours of the nervous system and breast cancer [4]. The
`majority of these tumours express the somatostatin re-
`ceptor subtype 2 (sst2) [5]. For diagnostic purposes these
`tumours can be visualized with [111In-DTPA-DPhe1]oct-
`reotide (OctreoScan 111) [6]. In order to make radio-
`therapeutic treatment of somatostatin receptor-express-
`ing tumours possible, we synthesized a series of octreo-
`tide analogues that can tightly chelate beta-emitting rare
`earths (e.g. yttrium-90). Among these compounds were
`[DTPA-benzyl-acetamido-DPhe1,Tyr3]octreotide
`[7],
`[DTPA-benzyl-acetamido-DPhe1]octreotide, [DOTA-ben-
`zyl-acetamido-DPhe1]octreotide and [DOTA-benzyl-acet-
`amido-DPhe1,Tyr3]octreotide (manuscript
`in prepara-
`tion).
`Since octreotide, as observed with most hydrophilic
`low molecular weight proteins [8], is accumulated exten-
`sively at the proximal tubules of the kidneys, a series of
`octreotide analogues were synthesized and evaluated that
`contain cleavable linkers [9]. The basic idea in this study
`was that those conjugates containing kidney-specific
`cleavable linkers would be rapidly and selectively
`cleaved in the kidneys and that the hydrophilic cleavage
`product would be rapidly excreted without further degra-
`dation. Thus the accumulation of the radiotherapeutic
`drug in the somatostatin receptor-expressing tumours
`would be enhanced in relation to the amount accumulat-
`ed in the kidneys. Finally [DOTA-DPhe1,Tyr3]octreotide
`(SMT 487) [10] was selected from among various
`DOTA-coupled octreotide analogues because of its ad-
`vantageous properties in tumour models. In the present
`study we investigated the extension of the concept of re-
`ceptor-targeted radionuclides to its radiotherapeutic ap-
`plication by using 90Y-SMT 487.
`90Y-SMT 487 is a radiotherapeutic drug that has been
`developed for somatostatin receptor-targeted radiothera-
`py of somatostatin receptor-expressing tumours. In pre-
`liminary studies SMT 487 was shown to specifically
`
`European Journal of Nuclear Medicine
`Vol. 25, No. 7, July 1998 – © Springer-Verlag 1998
`
`NOVARTIS EXHIBIT 2086
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`bind to somatostatin receptors with nanomolar affinity
`[11], and this was confirmed by others using [111In-
`DOTA-DPhe1,Tyr3]octreotide [12]. The chelate stability
`of 90Y-SMT 487 is of high importance since any free
`90Y3+ is known to mimic Ca2+ and will be accumulated
`by the bones. Therefore, and prior to in vivo experi-
`ments, the stability of 90Y-SMT 487 was evaluated. The
`chelate was stable against isotopic exchange with natural
`yttrium and transchelation by either DTPA or serum pro-
`teins (manuscript in preparation).
`The aim of the present work was to investigate the ra-
`diotherapeutic effect of 90Y-SMT 487 in rats bearing the
`CA 20948 rat pancreatic tumour, which has previously
`been shown to be somatostatin receptor-positive [13].
`
`Materials and methods
`
`Materials. &p.2:Unless otherwise stated, all reagents and solvents were
`obtained from commercial sources and were used without further
`purification. Somatostatin-14, octreotide (SMS 201-995), Tyr3-
`octreotide (SDZ 204-090) and [DOTA-DPhe1-Tyr3]octreotide
`(SMT 487) were synthesized at Novartis Pharma AG Basel. 90Y
`was obtained from the Pacific Northwest National Laboratory
`(United States) in the form of YCl3 in a 0.05 M HCl solution con-
`taining a Sr/Y ratio of 9.0×10–9 at calibration. The specific activi-
`ty of the 90Y was 50 mCi/ml at a reference time of 10 h post deliv-
`ery. SMT 487 was dissolved in 10 m
`l of AcOH and 10 m
`l of
`MeOH before being diluted with water to create a 1 mM solution.
`Aliquots of this solution were stored at –20°C until required. All
`buffer solutions used were passed over a Chelex filter to remove
`any divalent or trivalent metals and stored in polypropylene tubes.
`All cold solutions were degassed and saturated with either He
`(HPLC solvents) or N2 (labelling/binding assay reagents) before
`use. Analytic high-performance liquid chromatography (HPLC)
`was performed using an RP18 column (Waters Novapak, 4 m m,
`3.9×150 mm) with a MeCN/NH4OAc solvent system initially
`composed of 100% A, followed by a linear gradient of 100% A
`(25 mM NH4OAc, pH 4.0) to 100% B (25 mM MeCN, 60%
`MeCN, pH 4.0) over 15 min, and then 100% B for 5 min (flow
`rates 1.2 ml/min). The eluate was monitored by a UV detector
`(l =240 nm) and by a radioactivity detector (B-Ram, Inus, United
`States) in series. Large amounts of radioactivity were assayed
`with an ionization chamber (CDC-202, Veenstra Instrumenten,
`The Netherlands). 90Y activity was determined with an automatic
`NaI(Tl) counter (No. 1282, Compugamma, LKB, Finland) adjust-
`ed to an appropriate counting window. Iodine-125 activity was de-
`termined with an automatic NaI(Tl) counter (No. 1274, Riagam-
`ma, LKB, Finland).
`
`Radioligand preparation. &p.2:To prepare 90Y-SMT 487, 40 m
`l of 90Y
`was added to 40 m
`l of 0.15 mM NH4OAc (pH 4.5) and 2 m
`l of
`1 mM SMT 487. The reaction mixture was placed in a water bath
`at 100°C for 15 min. A 1-m
`l portion was then removed and diluted
`to 2 ml with 4 mM DTPA (pH 4.0) to challenge transchelation of
`any free 90Y present in the radioligand solution. Fifty microlitres
`of this solution was then analysed by HPLC to determine the
`amount of unchelated 90Y. Typically >99.5% of the 90Y was bound
`to SMT 487. The
`specific activity was adjusted
`to
`1.0–1.2 Ci/m mol. The radioligand was diluted in HEPES buffer,
`pH 7.5, containing 1% BSA, and a final volume of 100 m
`l was in-
`jected intravenously. The 125I-labelled Tyr3-octreotide (SDZ 204-
`
`European Journal of Nuclear Medicine Vol. 25, No. 7, July 1998
`
`669
`
`090) was prepared using the chloramine T method as described
`previously [14]. The 125I-Tyr11-somatostatin-14 was obtained from
`Amersham Switzerland (product no. IM 161).
`
`Receptor binding. &p.2:For in vitro somatostatin receptor binding stud-
`ies 125I-Tyr3-octreotide or 125I-Tyr11-somatostatin-14 was used as
`the radioligand. Somatostatin-14, octreotide and SMT 487 were
`used as competitors. Radioligand binding assays with the five hu-
`man somatostatin receptor subtypes (hsst1–5) were performed us-
`ing cell membrane preparations of CHO and COS cells express-
`ing the respective receptor subtypes. Somatostatin receptor bind-
`ing studies were also performed using membrane preparations of
`CA 20948 tumours. The binding of 125I-Tyr3-octreotide and 125I-
`Tyr11-somatostatin-14 was assayed as described previously [15,
`16]. The data from the displacement experiments were analysed
`according to a one-site model to obtain values for the half-maxi-
`mal-inhibitory concentration (IC50) of the respective somatostatin
`analogues. The dissociation constant (Kd) and the number
`of binding sites (Bmax) were calculated according to Scatchard
`[17].
`
`RT-PCR. &p.2:The CA 20948 tumour was assessed for the presence of
`somatostatin receptor subtype (sst1–5) mRNA to confirm the pres-
`ence of the target receptor subtype for 90Y-SMT 487, which is
`mainly the sst2. Preparation of poly(A)+-RNA, Dnase I digestion
`prior to reverse transcription, and RT-PCR analyses were per-
`formed as described in [18]. The following primer pairs were ap-
`plied: for rat sst1, RS190 5’-TCAGCTGGGATGTTCCCCAATG-
`3’ and RS190 5-GTCGTCTT GCTCGGCGAACACG-3’; for sst2,
`2A and 2B as described [19]; for sst3, RS35 5’-CT GGCG
`AACAGCCTTCATCATCTA-3’ and RS39 5’-TAGGAGAGGAA
`GCCGTAGAGGATGG-3’; for sst4 RS46 5’-GATGCCACTGT
`CAACCATGTGTCCCT-3’ and RS47 5’-ACGGAGTTGTCCTT
`GGAGCCAGTCAG-3’; and for sst5, RS51 5’-GTATTAGTG
`CCTGTGCTCTACCTGTTGG-3’ and RS55 5’-GGCAGATGT
`GGGTTCCTCGGGCAGTGT-3’.
`
`Tumour cell inoculation and tumour growth assessment. &p.2:In all ani-
`mal experiments the “Principles of laboratory animal care” (NIH
`publication No. 86-23, revised 1985) were followed, and the spe-
`cific Swiss laws “Art. 13a Eidg. Tierschutzgesetz, TschG; Art. 60
`bis 62 Eidg. Tierschutzverordnung, TschV” were also applicable.
`The CA 20948 pancreatic tumours were grown in Lewis rats as
`previously described [13]. Briefly, male Lewis rats (Iffa Credo,
`France) were injected subcutaneously into both flanks, each with
`1 ml of a 2 mg/ml cell suspension of the CA 20948 tumour. The
`tumour cells were suspended in F-12 Nutrient Mixture HAM, sup-
`plemented with 0.1 g/100 ml NaHCO3 and 10% fetal calf serum.
`When the experiments started at about 14 days post injection
`(p.i.), multiple tumours had grown per inoculation site and the to-
`tal tumour load per rat was approximately 11 000 mm3. The body
`weight of the rats at this time was approximately 300 g. To those
`rats that exhibited complete remission, CA 20948 tumour cells
`were re-inoculated as described above. Control rats of the same
`age were also inoculated with tumor cells.
`
`Tissue distribution. &p.2:Seven tumour-bearing rats received each
`50 m Ci 90Y-SMT 487 in 100 m
`l HEPES (10 mM, pH 7.6, contain-
`ing 1% BSA) into the v. jugularis under isoflurane anaesthesia.
`After 24 h p.i. the animals were sacrificed by CO2 asphyxiation;
`the organs of interest were removed, weighed and counted in a
`gamma counter, along with a standard of the injectate. The radio-
`activity in each tissue was expressed as percent of the injected
`dose per gram tissue (%ID/g).
`
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`670
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`Measurement of radioactivity. &p.2:All tissue samples were made up to
`a 1 ml volume with water to establish a linear relationship be-
`tween counting rate and tissue size; the samples where counted
`for 2 min in a gamma counter measuring the bremsstrahlung from
`the beta decay. The validity of this measuring method was estab-
`lished by Hnatowich et al. [20]. Activity standards (10%, 1%,
`0.1% and 0.01% of the injected dose) were prepared from the 90Y-
`SMT 487 treatment solution for each experiment and measured
`with each batch of samples to provide an automatic decay correc-
`tion. A linear relationship between the observed counting rate and
`the time activity was found.
`
`Experimental radiotherapy. &p.2:Ten to 12 days following tumour cell
`inoculation, animals were randomized to control and treatment
`groups. One treatment group received 5 mCi/kg (n=5) and the oth-
`er 10 mCi/kg (n=7) as a single dose intravenously (i.v.). The con-
`trol group received unlabelled SMT 487 (8.3 nmol/kg), the same
`amount of peptide as present in the radioligand preparation (n=7).
`The tumours were measured on the day of radioligand administra-
`tion and every 3–4 days thereafter. Tumour volumes were deter-
`mined with a caliper in a blinded fashion and were calculated by
`using the formula for the ellipsoid V=p /6(d1×d2×d3), where d1, d2
`and d3 represent the three largest diameters. Individual and mean
`growth curves were calculated using Excel (Microsoft). Tumour
`volume refers to the sum of volumes of individual tumours on
`each animal. The tumour volumes were normalized and expressed
`as times of their initial volume. This procedure was chosen be-
`cause of the differences in the tumour size at the start of the study
`and in the multiplicity of the tumours in a single animal. Excel or
`Origin statistical procedures were used for data evaluation (Stu-
`dent’s t test).
`At progressed stages the CA 20948 tumour becomes soft and
`haemorrhagic; cystic spaces and areas of necrosis are common
`[13]. In our experiments tumour necrosis was a strict indication to
`sacrifice the rats.
`
`Fig. 1. RT-PCR analyses of sst1–5 receptor expression in the CA
`20948 rat exocrine pancreatic tumour. Lane 1, CA 20948 tumour
`cDNA. Negative controls: lane 2, CA 20948 tumour poly(A)+-
`RNA without reverse transcription; lane 3, water control. Positive
`control: lane 4, rat genomic DNA (since sst receptor genes are in-
`tron-less). Further controls: lane 5, mouse sst1; lane 6, rat sst2;
`lane 7, mouse sst3; lane 8, rat sst4; and lane 9, rat sst5 cDNA&/fig.c:
`
`Results
`
`In vitro pharmacology
`
`Both octreotide and SMT 487 bound with high, sub-
`nanomolar affinity to the human somatostatin receptor
`subtype 2 (hsst2) expressed on COS cells (Table 1).
`While the affinity of SMT 487 and octreotide was simi-
`lar for hsst2, there was a 100-fold drop in the affinity of
`SMT 487 for hsst5 relative to octreotide, indicating in-
`creased sst2 specificity of SMT 487. RT-PCR revealed
`that the CA 20948 rat tumour strongly expressed rat sst2.
`Furthermore, low expression of sst5 receptor mRNA
`could be detected (Fig. 1). Receptor binding experiments
`
`Fig. 2. Binding of 125I-Tyr3-octreotide to membranes prepared
`from a CA 20948 rat pancreatic tumour. The membranes (0.33
`mg/ml assay mixture) were incubated with the radioligand and in-
`creasing concentrations (each in triplicate) of octreotide or SMT
`487 (x-axis). l, Single measurements of SMT 487, (cid:209)
`, single mea-
`surements of octreotide. Inset: SMT 487 binding data plotted by
`the method of Scatchard. The maximum binding capacity (Bmax)
`was calculated from the intercept on the x-axis&/fig.c:
`
`demonstrated that both octreotide (n=10) and SMT 487
`(n=3) inhibited the binding of 125I-Tyr3-octreotide to CA
`20948 tumour membranes in a monophasic manner
`(Fig. 2), with IC50 values for octreotide of 0.22±0.02 nM
`and for SMT 487 of 0.39±0.02 nM. The Scatchard trans-
`formation of the binding data was linear, demonstrating
`
`Table 1. Binding affinity of octreotide and
`SMT 487 to hsst1–5 using 125I-SRIF-14 as a
`specific ligand (n=3, mean; SEM within
`10% of mean). Membranes were prepared
`from transfected CHO or COS cells&/tbl.c:&tbl.b:
`
`Compound
`
`Binding affinity, IC50-value (nM)
`
`SRIF-14
`(Somatostatin)
`Octreotide
`SMT 487
`
`hsst1
`
`hsst2
`
`> 0.4
`
`>200
`>100
`
`0.1
`
`0.6
`0.9
`
`hsst3
`
`0.3
`
`10
`50
`
`hsst4
`
`hsst5
`
`>
`
`0.8
`
`0.6
`
`>1000
`>1000
`
`7
`700
`
`European Journal of Nuclear Medicine Vol. 25, No. 7, July 1998
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`Table 2. Tissue distribution of 90Y-SMT 487 in CA 20948 tu-
`mour-bearing rats 24 h post i.v. injection (n=7, average±SEM)&/tbl.c:&tbl.b:
`
`Tissue
`
`90Y-SMT
`
`487
`
`(%ID/g)
`
`Adrenals
`Blood
`Duodenum
`Femur
`Heart
`Kidneys
`Large Intestine
`Liver
`Muscle
`Ovaries
`Pancreas
`Pituitary
`Small Intestine
`Spleen
`Stomach
`Tumour
`
`Average
`
`5.462
`0.013
`0.104
`0.105
`0.014
`1.511
`0.305
`0.156
`0.004
`0.013
`0.656
`2.873
`0.290
`0.379
`0.227
`0.639
`
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`±
`
`SEM
`
`0.269
`0.003
`0.020
`0.040
`0.003
`0.126
`0.045
`0.024
`0.001
`0.002
`0.152
`0.798
`0.122
`0.161
`0.059
`0.118
`
`the binding of SMT 487 to a single class of high-affinity
`binding sites in CA 20948 cell membranes (inset in
`Fig. 2). For SMT 487 the apparent equilibrium dissocia-
`tion constant Kd was 0.30 nM and the maximum binding
`Bmax was 489 fmol/mg. The results from both the RT-
`PCR and the ex vivo binding experiments indicated that
`SMT 487 targets selectively sst2 binding sites with high
`affinity in the CA 20948 tumour cell inoculates.
`
`Tissue distribution
`
`The tissue distribution of 90Y-SMT 487 was studied in
`CA 20948 tumour-bearing rats. Twenty-four h after i.v.
`administration of the radioligand, the amount of activity
`that accumulated in the tumour was 0.64%±0.12% of the
`injected dose per gram (%ID/g) (Table 2). Somatostatin
`receptor-expressing normal tissues like pancreas, stom-
`ach and small and large intestine exhibited a low accu-
`mulation of the radioligand per gram tissue, whereas the
`distribution of 90Y-SMT 487 to the adrenals and the pitu-
`itary was high, at 5.5%±0.3%ID/g and 2.9%±0.8%ID/g,
`respectively. The rapid distribution of 90Y-SMT 487 in
`vivo results in high tumour to tissue ratios at 24 h post
`injection. For selected organs this ratio was 49.15
`(blood), 159.75 (muscle), 6.08 (femur), 4.09 (liver) and
`2.20 for the intestines. Except for the kidneys, the rela-
`tive exposure to 90Y-SMT 487 in somatostatin receptor-
`negative tissues was low after 24 h.
`
`Tumour growth
`
`The tumours of the animals in the control group grew ei-
`ther excessively (Fig. 3a), with a doubling time of 11
`days, and became necrotic; consequently, those rats had
`
`European Journal of Nuclear Medicine Vol. 25, No. 7, July 1998
`
`671
`
`Fig. 3. Effect of 90Y-SMT 487 (single i.v. administration of 5 or
`10 mCi/kg at day 0) on tumour growth in Lewis rats bearing CA
`20948
`tumours. Mean
`start volume
`at day 0 was
`12 805±1140 mm3. The tumour volume change in single animals
`is plotted against time post injection (n=4–7/group). a Tumor
`growth in animals of the control group (SMT 487, 8.3 nmol/kg); b
`tumour growth in rats treated with 90Y-SMT 487, 5 mCi/kg; c tu-
`mour growth in rats treated with 90Y-SMT 487, 10 mCi/kg. The
`asterisks indicate that these rats had to be sacrificed because of tu-
`mour necrosis. large figure: 0–50 days post injection; inset: 0–200
`days post injection&/fig.c:
`
`to be sacrificed 10 days post injection (p.i). The tumour-
`bearing rats were treated with either 5 or 10 mCi/kg i.v.
`since similar radiotherapeutic doses had been successful-
`ly used in tumour-bearing nude mice [7]. This treatment
`induced a partial to complete tumour remission. After
`administration of a single i.v. dose of 5 mCi/kg 90Y-SMT
`487 in one out of five rats the somatostatin receptor-ex-
`pressing tumours disappeared 3 weeks after injection
`(Fig. 3b). In three rats of this group a tumour growth de-
`lay of about 1 week was observed. Interestingly, in the
`10 mCi/kg group all tumours started to shrink at 5 days
`p.i. In five out of seven animals the tumour disappeared
`at seven weeks p.i. In these rats no regrowth of the tu-
`mours occurred over the entire observation period of 8
`months. Two rats out of this group (Fig. 3c, asterisks)
`had to be sacrificed 3 weeks p.i., because the tumours
`became necrotic, which is a common observation of ad-
`vanced stages of this exocrine pancreatic tumour. Necro-
`sis was not observed in any of the tumours that exhibited
`complete remission.
`Both the 5 mCi/kg dose and the 10 mCi/kg dose were
`well tolerated, with only transient loss in body weight.
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`Fig. 4. Effect of re-inoculation of CA 20948 tumour cells into rats
`that previously exhibited complete remission (Fig. 3) (n=5/group).
`For comparison the same amount of CA 20948 tumour cells was
`inoculated into previously untreated rats (control). *, Tumour
`growth in control rats; l, tumour growth in rats that previously
`exhibited complete remission
`
`The nadir in body weight loss was on day 7 p.i.
`(10%–15% of initial weight). Those rats, that showed
`complete remission gained up to 200% of their initial
`body weight.
`After those 8 months during which the animals were
`free of tumour, CA 20948 cells were re-inoculated into
`the same rats. Untreated control rats received the same
`amount of tumour cells. Upon re-inoculation of CA
`20948 tumour material into the cured rats, in five out of
`seven animals no tumour growth occurred, whereas in
`those rats exhibiting moderate tumour growth the tu-
`mour disappeared after 3–4 weeks without any further
`treatment (Fig. 4).
`
`Discussion
`
`The present study demonstrates that the new radiothera-
`peutic 90Y-SMT 487 has the potential to induce complete
`tumour remission in somatostatin receptor-expressing tu-
`mours. This tumour expresses rat sst2, as demonstrated
`by RT-PCR. The human sst2 is 95% homologous to the
`rat sst2 [21]. The latter subtype is most abundant in hu-
`man somatostatin receptor-expressing malignancies [5].
`SMT 487 targets sst2 binding sites with nanomolar affin-
`ity in the CA 20948 tumour cell inoculates, as demon-
`strated by in vitro binding studies on ex vivo tumours
`(Fig. 2). In vivo 90Y-SMT 487 rapidly accumulated in
`the tumour tissue, with a high tumour-to-blood ratio of
`49.15 at 24 h post injection. The amount of activity ac-
`cumulated in the tumour (0.64%ID/g, 24 h p.i.) was
`123I-Tyr3-octreotide
`higher
`than
`reported
`for
`(0.007%ID/g [22]) in the same tumour model. This
`might be due to a deiodination process in the case of
`123I-Tyr3-octreotide. After 24 h p.i. 180%±50%ID/g of
`123I-Tyr3-octreotide was found in the thyroid [23], an or-
`gan which is known to take up free iodine avidly. Nor-
`mal rat tissues such as adrenals, pituitary and kidneys
`
`are exposed to comparably high radiation doses after ad-
`ministration of 90Y-SMT 487. In order to estimate the
`potential side-effects that may arise from the irradiation
`of healthy somatostatin receptor-expressing organs (an-
`terior pituitary, adrenal cortex) or organs through which
`the radiotherapeutic is excreted (kidneys), a preliminary
`side-effect profile of 90Y-SMT 487 has been generated in
`normal rats.
`In this study various parameters were measured, in-
`cluding blood cell counts, endocrine profile, urine bio-
`chemistry and the histology of various organs (data not
`shown). No marked impairment of the health of the ani-
`mals upon a single (30 mCi/kg) or fractionated treatment
`(3×10 mCi/kg) with 90Y-SMT 487 was observed in rats
`(manuscript in preparation). The comparatively high ex-
`posure to activity in the kidneys presumably may be
`lowered by a prior injection with L-lysine. In a mouse tu-
`mour model the kidney load with 90Y-SMT 487 was re-
`duced by 60% by prior administration of 2 g/kg L-lysine,
`and this finding was also confirmed by others using
`[111In-DOTA-DPhe1,Tyr3]octreotide in the CA 20948 rat
`tumour model [12]. The latter effect is in accordance
`with the reduction of kidney exposure to OctreoScan
`111 after an amino acid infusion that is high in L-lysine
`in patients [24].
`In the radiotherapeutic experiment a single intrave-
`nous administration of 10 mCi/kg 90Y-SMT 487 resulted
`in complete remission of the tumours in five out of seven
`CA 20948 tumour-bearing Lewis rats. No regrowth of
`the tumours occurred 8 months post injection. The radio-
`therapeutic effect was dose dependent, since the admin-
`istration of 5 mCi/kg 90Y-SMT 487 resulted in complete
`tumour remission in only one out of 5 tumour-bearing
`rats. The time to tumour progression was delayed for
`about 1 week in the other animals of the same group. In
`the same tumour model Anderson et al. reported on a so-
`matostatin receptor-targeted radiotherapeutic approach
`using a copper-64 labelled TETA-octreotide analogue
`b –
`b +
`[25]. Copper-64 emits
`(0.571 MeV) and
`(0.657 MeV) particles which are accompanied by a
`g -line of 1.34 MeV. The physical half-life time of 64Cu is
`12.7 h. With a single dose of 40 mCi/kg 64Cu-TETA-oct-
`reotide a tumour growth delay of about 1 week was ob-
`served. The repeated administration of 64Cu-TETA-oct-
`reotide (cumulative dose of 60 mCi/kg, Anderson et al.’s
`study) gave a maximum tumour growth delay of about 2
`weeks and thereafter tumour regrowth was observed. For
`comparison, in our own studies with a single dose of
`10 mCi/kg 90Y-SMT 487 complete tumour remission
`was induced. This demonstrates that both the choice of
`the chelating moiety (DOTA) and the radionuclide (90Y,
`b –
`max=2 MeV) in 90Y-SMT 487 make it the preferable
`compound for radiotherapeutic treatment of somatostatin
`receptor-positive tumours.
`The number of binding sites for somatostatin ex-
`pressed by the CA 20948 rat tumour (489 fmol/mg) is
`similar to previously reported data for this tumour type
`[13], and comparable to the number of binding sites that
`
`European Journal of Nuclear Medicine Vol. 25, No. 7, July 1998
`
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`have been determined for human tumours [26]. For other
`experimental tumours, e.g. the AR42J rat pancreatic tu-
`mour, a 10 times higher number of somatostatin recep-
`tors has been reported [27]. Interestingly, the favourable
`therapeutic outcome using 90Y-SMT 487 as reported
`here, was achieved despite a heterogeneous and moder-
`ate expression of somatostatin binding sites by the CA
`20948 tumour. The high beta energy of 90Y, with a mean
`energy of 0.9 MeV and a maximum energy emission of
`2 MeV, results in a comparatively long mean travel
`length of the beta particles in the tissue of about 5 mm.
`Therefore also those tumour cells that express somato-
`statin receptors at low density may be treated effectively
`with 90Y-SMT 487.
`After re-inoculation of CA 20948 tumour cells into
`the cured rats, in the majority of rats no tumour regrowth
`occurred, and in those rats in which the tumour grew
`moderately, it disappeared again without any further
`treatment. To our knowledge, and as observed over a pe-
`riod of 8 years in our laboratory, spontaneous disappear-
`ance of the CA 20948 tumour never occurs. The fact that
`the tumour could not be re-inoculated in the survivors of
`the 90Y-SMT 487 treatment leads to some interesting im-
`munological questions. During the first 10 days during
`which rapid tumour shrinkage is observed, the irradia-
`tion of the tumour cells may induce apoptosis. Later, at
`10–14 days p.i. a cellular or a humoral immune response
`may start. The nature of this response is not yet known.
`As an effect of the high radiation dose in the tumour,
`macrophages may extensively infiltrate there; the num-
`ber of cytotoxic T cells and natural killer cells also may
`be increased. The finding of a dose dependency of the
`complete remission may point to the hypothesis of a
`massive infiltration of macrophages in the irradiated ar-
`ea, which may then give rise to an immune response of
`the host to certain tumour antigens. However, at this
`stage of investigation we can only speculate as to the
`mechanisms underlying the observed effect, and this
`question certainly needs further clarification. A possible
`induction of an immune response through the idiotypic
`network as described by Madiyalakan et al. [28] is also
`an attractive hypothesis.
`The present study ist the first to provide evidence that
`a somatostatin receptor-targeted 90Y-labelled somatosta-
`tin analogue can induce complete tumour remission.
`These results, together with a case report on the thera-
`peutic use of [90Y-DOTA-DPhe1,Tyr3]octreotide in a pa-
`tient [29], strengthen the concept of receptor-targeted ra-
`diotherapy with 90Y-SMT 487. Given that the somatosta-
`tin receptor subtype 2 is expressed in a large variety of
`human tumours [5] and that this subtype is targeted by
`both OctreoScan 111 and 90Y-SMT 487, a high clinical
`relevance can be foreseen for the use of the two radioli-
`gands together in the diagnosis and treatment of somato-
`statin receptor-expressing tumours. Phase I trials with
`yttrium-labelled SMT-487 have started to evaluate our
`concept in patients with somatostatin receptor-positive
`malignancies.
`
`European Journal of Nuclear Medicine Vol. 25, No. 7, July 1998
`
`&p.2:Acknowledgements. The excellent technical assistance of Ms. G.
`Hofmann, Ms. K. Pollehn, Mr. R. Haller and Mr. L. Tolcsvai is
`greatly acknowledged.
`
`673
`
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