Short communication
`
`The addition of DTPA to [177Lu-DOTA0,Tyr3]octreotate prior
`to administration reduces rat skeleton uptake of radioactivity
`
`Wouter A. P. Breeman1, Katy van der Wansem1, Bert F. Bernard1, Arthur van Gameren1, Jack L. Erion3,
`Theo J. Visser2, Eric P. Krenning1, 2, M. de Jong1
`
`1 Department of Nuclear Medicine, Erasmus University Medical Center, Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands
`2 Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
`3 BioSynthema, St Louis, Mo., USA
`
`Received: 17 July 2002 / Accepted: 21 October 2002 / Published online: 29 November 2002
`© Springer-Verlag 2002
`
`Abstract. Peptide receptor-targeted radionuclide therapy
`is nowadays also being performed with DOTA-conjugat-
`ed peptides, such as [DOTA0,Tyr3]octreotate, labelled
`with radionuclides like 177Lu. The incorporation of 177Lu
`is typically ≥99.5%; however, since a total patient dose
`can be as high as 800 mCi, the amount of free 177Lu3+ (=
`non-DOTA-incorporated) can be
`substantial. Free
`177Lu3+ accumulates in bone with unwanted irradiation
`of bone marrow as a consequence. 177Lu-DTPA is report-
`ed to be stable in serum in vitro, and in vivo it has rapid
`renal excretion. Transforming free Lu3+ to Lu-DTPA
`might reroute this fraction from accumulation in bone to
`renal clearance. We therefore investigated: (a) the biodis-
`tribution in rats of 177LuCl3, [177Lu-DOTA0,Tyr3]octreo-
`tate and 177Lu-DTPA; (b) the possibilities of complexing
`the free 177Lu3+ in [177Lu-DOTA0,Tyr3]octreotate to
`177Lu-DTPA prior to intravenous injection; and (c) the
`effects of free 177Lu3+ in [177Lu-DOTA0,Tyr3]octreotate,
`in the presence and absence of DTPA, on the biodistribu-
`tion in rats. 177LuCl3 had high skeletal uptake (i.e. 5% ID
`per gram femur, with localization mainly in the epiphy-
`seal plates) and a 24-h total body retention of 80% in-
`jected dose (ID). [177Lu-DOTA0,Tyr3]octreotate had high
`and specific uptake in somatostatin receptor-positive tis-
`sues, and 24-h total body retention of 19% ID. 177Lu-
`DTPA had rapid renal clearance, and 24-h total body re-
`tention of 4% ID. Free 177Lu3+ in [177Lu-DOTA0,Tyr3]oct-
`reotate could be complexed to 177Lu-DTPA. Accumula-
`tion of 177Lu in femur, blood, liver and spleen showed a
`dose relation to the amount of free 177Lu3+, while these
`accumulations could be normalized by the addition of
`DTPA. After labelling [DOTA0,Tyr3]octreotate with
`177Lu the addition of DTPA prior to intravenous adminis-
`Wouter A. P. Breeman (✉)
`Department of Nuclear Medicine,
`Erasmus University Medical Center, Dr. Molewaterplein 40,
`3015 GD Rotterdam, The Netherlands
`e-mail: breeman@nuge.azr.nl
`Tel.: +31-10-4635317, Fax: +31-10-4635997
`
`tration of [177Lu-DOTA0,Tyr3]octreotate is strongly rec-
`ommended.
`
`Keywords: DOTA – DTPA – Radiolabelling – Biodistri-
`bution
`
`Eur J Nucl Med (2003) 30:312–315
`DOI 10.1007/s00259-002-1054-4
`
`Introduction
`
`Peptide receptor-targeted radionuclide therapy of so-
`matostatin receptor (SSR)-positive tumours is currently
`also being performed using DOTA-chelated SS ana-
`logues such as [DOTA0,Tyr3]octreotate (DOTA-tate), ra-
`diolabelled with 177Lu [1, 2, 3]. Although incorporation
`of the radiolabel is typically ≥99.5%, there is always a
`free fraction. Since therapeutic doses (up to 800 mCi per
`patient) are involved, the absolute amount of free 177Lu3+
`can be significant, and since free 177Lu3+ acts as a Ca2+
`mimic, it accumulates in bone [4]. Muller et al. reported
`in a study with mice that 60% of the injected dose (ID)
`of 177LuCl3 is incorporated in the skeleton at 48 h p.i. [5,
`6]. Li et al. reported the stability of 177Lu-DTPA in vitro
`in serum [4]. In order to investigate the in vivo charac-
`teristics of the three 177Lu-labelled compounds separate-
`ly – i.e. 177LuCl3, 177Lu-DTPA and 177Lu-DOTA-tate –,
`biodistribution studies were performed in rats.
`Based on these results (reported here) with the three
`forms of 177Lu, we hypothesized that after labelling
`DOTA-tate with 177Lu, the addition of excess DTPA
`might be able to complex the fraction of free 177Lu3+ by
`transformation to 177Lu-DTPA, with rerouting from ac-
`cumulation in bone to renal clearance. Therefore, we in-
`vestigated the biodistribution of 177Lu-DOTA-tate with
`0% and free 177Lu3+ (up to 10%), and in the presence and
`absence of DTPA.
`
`European Journal of Nuclear Medicine and Molecular Imaging Vol. 30, No. 2, February 2003
`Evergeen Ex. 1035
`1 of 4
`
`

`

`Materials and methods
`
`177Lu-labelled compounds. 177LuCl3 (IDB Holland, Baarle Nassau,
`The Netherlands) was provided in 0.05 N HCl. The labelling
`(20 min at 80°C), high-performance liquid chromatography and
`instant thin-layer chromatography were performed as described
`previously [2]. The amount of injected of DOTA-tate was 0.5 μg
`and the amount of DTPA (added to 177LuCl3 or 177Lu-DOTA-tate
`prior to the i.v. administration) was 4 μg.
`
`Animals and tissue distribution. Male Wistar rats (10–14 weeks
`old, 225–250 g, Harlan-CPB, Austerlitz, The Netherlands, n≥3
`rats per group) were kept under standard laboratory conditions
`(12 h light/12 h dark) and were given standard laboratory diet
`(Hope Farms, Woerden, The Netherlands) and water ad libitum.
`The experimental protocol adhered to the rules laid down by the
`Dutch Animal Experimentation Act and was approved by the
`Committee on Animal Research of the Erasmus Medical Centre,
`Rotterdam. Radioactive material, 10 MBq 177LuCl3, 177Lu-DTPA
`or 177Lu-DOTA-tate, was injected intravenously in the penis vein.
`The rats were housed in metabolic cages for urine and faeces col-
`lection. Blood, spleen, pancreas, adrenals, pituitary, kidney, liver,
`lungs, thymus, sternum, muscle and femurs were isolated 24 h p.i.
`and concentration of radioactivity measured in the well-type gam-
`ma counter LKB-282 compugamma, as described previously [7].
`From one femur the inner part was rinsed with saline in order to
`collect the bone marrow. Bone and bone marrow were analysed
`for radioactive concentration.
`
`Ex vivo autoradiography. Localization of radioactivity in tissues
`after injection of 177LuCl3, 177Lu-DTPA or 177Lu-DOTA-tate in
`rats was investigated by ex vivo autoradiography, as described
`previously [3]. The sections and spliced femur were exposed to
`phosphor imaging screens (Packard Instruments Co., Meriden,
`USA) for 1 day in X-ray cassettes and analysed using a Cyclone
`phosphor imager and a computer-assisted OptiQuant 03.00 image
`processing system (Packard Instruments Co, Groningen, The
`Netherlands).
`
`Data acquisition. The injections of radioactivity were monitored
`with a gamma camera (Siemens, ROTA-II Erlangen). During the
`first 25 min and at 4 and at 24 h, radioactivity accumulation was
`measured using regions of interest, as described previously [7].
`Statistical analysis was performed using Student’s t test. Statistical
`significance was defined at P<0.05.
`
`Results
`
`The dynamic studies in rats injected with 177LuCl3,
`177Lu-DOTA-tate and 177Lu-DTPA showed rapid distri-
`bution of radioactivity (data not shown). The 177Lu-
`DTPA-injected rats revealed instant uptake of radioactiv-
`ity in kidney and rapid appearance of radioactivity in the
`urinary bladder. Twenty-five minutes following the in-
`jection of 177Lu-DTPA, 50% ID was accumulated in the
`urinary bladder (calculated using regions of interest),
`while at this time point the rats injected with 177LuCl3 or
`177Lu-DOTA-tate had accumulated less than 5% ID there
`(data not shown). The images from static gamma camera
`studies at 4 and 24 h of rats injected with 177LuCl3
`
`313
`
`Fig. 1A, B. The results of a static gamma camera study at 4 h p.i.
`(A) and 24 h p.i. (B) in rats injected with 177LuCl3, 177Lu-DTPA or
`177Lu-DOTA-tate. The arrows indicate: K, kidney; F, head of the
`femur
`
`(Fig. 1) showed mainly uptake in skeleton, with total
`body retention of 83% and 80% ID, respectively. At
`these time points, the 177Lu-DTPA-injected rats had total
`body retention of 20% and 4% ID, respectively. The rats
`injected with 177Lu-DOTA-tate showed clear kidney re-
`tention at both time points, with total body retention of
`28% and 19% ID, respectively. The % excreted radioac-
`tivity, as measured from rats in metabolic cages, corre-
`sponded to these data (i.e. retained and excreted radioac-
`tivity totalled 100%), confirming the total body data ob-
`tained using gamma camera measurements.
`Ex vivo autoradiography of kidney showed uptake of
`radioactivity in the proximal renal tubuli (located in the
`outer part of the renal cortex) of rats injected with
`177Lu-DOTA-tate or 177Lu-DTPA (data not
`177LuCl3,
`shown). The 177LuCl3-injected rats also showed accumu-
`lation of radioactivity on the border of the medulla and
`cortex, and since this is where ions are reabsorbed, the
`accumulation of radioactivity is most probably due to the
`uptake of free 177Lu (data not shown). The distribution of
`radioactivity in liver and spleen was homogeneous (data
`not shown).
`Photographs of femur and the corresponding ex vivo
`autoradiography are presented in Fig. 2. Figure 2A
`shows the femur of a rat injected with 177Lu-DOTA-tate
`(0% free 177Lu). In this rat, 0.28±0.02% ID/g was found
`in the total femur, of which 95% was SSR mediated (as
`
`European Journal of Nuclear Medicine and Molecular Imaging Vol. 30, No. 2, February 2003
`
`Evergeen Ex. 1035
`2 of 4
`
`

`

`314
`
`Fig. 2A, B. Ex vivo autoradiography of femurs at 24 h p.i. of rats
`injected with 177Lu-DOTA-tate (A) or 177LuCl3 (B). The uptake in
`A is 0.28% ID per gram femur. This value could be lowered to
`0.02% ID per gram femur by co-injection with an SSR-blocking
`amount of octreotide. Femoral uptake in the rat injected with
`177LuCl3 (B) was 5.1% ID per gram. This value could be lowered
`to 0.02% ID by the addition of DTPA (data not shown). The arrow
`indicates the epiphyseal plate
`
`found when rats were co-injected with 0.1 mg octreotide,
`data not shown), and localized in the epiphyseal plate.
`Of the total amount of radioactivity in femur, 0.7±0.1%
`was localized in bone marrow: 140 times lower than the
`amount localized in bone. Figure 2B shows the femur of
`a rat injected with 177LuCl3. In this rat, 5.1±0.3% ID/g
`was found in the total femur, of which 0.015±0.002%
`was recovered in the bone marrow: 6,000 times lower
`than the amount localized in bone. The addition of
`DTPA resulted in a >95% reduction in total femoral up-
`take and strongly reduced uptake in the epiphyseal plate
`(data not shown). In 177Lu-DOTA-tate-injected rats with
`10% free 177Lu in DOTA-tate, the femoral uptake was
`1.2±0.1% ID/g, and this dropped to 0.02±0.01% ID/g in
`the presence of DTPA. In 177Lu-DTPA-injected rats the
`femoral uptake was 0.017±0.002% ID/g, of which 8±2%
`was in the bone marrow: 12 times lower than the amount
`localized in bone.
`The concentration of radioactivity in blood, liver and
`spleen in rats injected with 10% free 177Lu in DOTA-tate
`in the absence of DTPA was significantly higher (ex-
`pressed as % ID/g) than that in rats injected with 0% free
`177Lu (data not shown). No significant differences were
`
`found in other organs between rats injected with 0% free
`177Lu in the absence or presence of DTPA and the rats
`injected with 10% free 177Lu in the presence of DTPA.
`
`Discussion
`
`Peptide receptor-targeted radionuclide therapy of SSR-
`positive tumours with 177Lu-DOTA-tate is currently be-
`ing performed [1, 2, 3]. Although the amount of free
`177Lu is ≤0.5%, there are reports on its incorporation in
`the skeleton [5, 6, 8]. Therefore, preclinical experiments
`were performed to obtain detailed morphological infor-
`mation. Autoradiographic studies of femurs of rats in-
`jected with 177LuCl3 or 177Lu-DOTA-tate showed local-
`ization in the epiphyseal plates (Fig. 2). (In humans these
`plates disappear after early adulthood.) In rats injected
`with 177Lu-DOTA-tate, the uptake in the epiphyseal
`plates could be strongly reduced by the co-injection of
`DTPA (if free 177LuCl3 was present) or octreotide. The
`uptake in bone of 177LuCl3 and 177Lu-DOTA-tate can be
`explained by the following facts: (a) Lu3+ mimics Ca2+
`and participates in hydroxyapatite formation [4], and (b)
`the epiphyseal plates are reported to be SSR positive [9].
`The addition of DTPA reduced uptake in epiphyseal
`plates, most probably due to the rapid renal clearance of
`the formed 177Lu-DTPA, as has also been reported for
`177Lu-DTPA in mice [4]. Radioactivity was recovered in
`the bone and not in the bone marrow: the ratio of bone vs
`bone marrow radioactivity was 6,000:1 and 140:1 for
`177LuCl3 and 177Lu-DOTA-tate, respectively. A retro-
`spective study of nine young patients (1.5–14 years old)
`with non-growth-related diseases who were undergoing
`OctreoScan scintigraphy was performed, and the epiphy-
`seal plates were not visualized. The concentration of ra-
`dioactivity in blood, liver and spleen increases when
`10% free 177Lu is present in 177Lu-DOTA-tate, and this
`increase can simply be abolished by the addition of
`DTPA. The explanation for this is most likely the forma-
`tion of 177Lu-DTPA in vitro, which will reroute the free
`177Lu3+ to rapid renal clearance in vivo. Therefore, it
`may be concluded that 177Lu-DOTA-tate with 10% free
`177Lu3+ in the presence of DTPA is comparable with 0%
`free 177Lu3+.
`In conclusion, after labelling DOTA-tate with 177Lu,
`the addition of DTPA prior to intravenous administration
`of 177Lu-DOTA-tate is strongly recommended.
`
`Acknowledgements. The expert technical assistance provided by
`Erik de Blois is gratefully acknowledged.
`
`References
`
`1. Breeman WA, de Jong M, Kwekkeboom DJ, et al. Somatostatin
`receptor-mediated imaging and therapy: basic science, current
`knowledge, limitations and future perspectives. Eur J Nucl Med
`2001; 28:1421–1429.
`
`European Journal of Nuclear Medicine and Molecular Imaging Vol. 30, No. 2, February 2003
`Evergeen Ex. 1035
`3 of 4
`
`

`

`2. Kwekkeboom DJ, Bakker WH, Kooij PP, et al. [177Lu-
`DOTA0Tyr3]octreotate: comparison with [111In-DTPA0]octreo-
`tide in patients. Eur J Nucl Med 2001; 28:1319–1325.
`3. de Jong M, Breeman WA, Bernard BF, et al. [177Lu-
`DOTA(0),Tyr3] octreotate for somatostatin receptor-targeted ra-
`dionuclide therapy. Int J Cancer 2001; 92:628–633.
`4. Li WP, Ma DS, Higginbotham C, et al. Development of an in
`vitro model for assessing the in vivo stability of lanthanide
`chelates. Nucl Med Biol 2001; 28:145–154.
`5. Muller WA, Schaffer EH, Linzner U. Studies on incorporated
`short-lived beta-emitters with regard to the induction of late ef-
`fects. Radiat Environ Biophys 1980; 18:1–11.
`
`6. Muller WA, Linzner U, Schaffer EH. Organ distribution studies
`of lutetium-177 in mouse. Int J Nucl Med Biol 1978; 5:29–31.
`7. Bakker WH, Krenning EP, Reubi JC, et al. In vivo application
`of [111In-DTPA-D-Phe1]-octreotide for detection of somatostatin
`receptor-positive tumors in rats. Life Sci 1991; 49:1593–
`1601.
`8. O’Mara RE, McAfee JG, Subramanian G. Rare earth nuclides
`as potential agents for skeletal imaging. J Nucl Med 1969;
`10:49–51.
`9. Mackie EJ, Trechsel U, Bruns C. Somatostatin receptors are re-
`stricted to a subpopulation of osteoblast-like cells during endo-
`chondral bone formation. Development 1990; 110:1233–1239.
`
`315
`
`European Journal of Nuclear Medicine and Molecular Imaging Vol. 30, No. 2, February 2003
`
`Evergeen Ex. 1035
`4 of 4
`
`