Year 7, Number 28, April 2005

Labeling of DOTA-Tyr3-octreotate with 177Lu - Stability and Biodistribution Study

Caldeira Filho, José de Souza; Muramoto, Emiko; Silva, Constancia Pagano; Araújo, Elaine Bortoleti
Instituto de Pesquisas Energéticas e Nucleares - IPEN
Av. Prof. Lineu Prestes 2242
Cidade Universitária. CEP: 05508-900

Correspondence:

José de S. Caldeira Filho
Instituto de Pesquisas Energéticas e Nucleares - IPEN
Centro de Radiofarmácia Divisao de Pesquisa
Av. Prof. Lineu Prestes 2242 CEP: 05508-900
Cidade Universitária.
Sao Paulo - SP Brasil
Fone: 11 3816 9256
Fax: 11 3816 9257
E-mail: jscaldei@ipen.br

Cita/Reference:
Caldeira Filho, José de Souza; Muramoto, Emiko; Silva, Constancia Pagano. et al. Labeling of DOTA-Tyr3-octreotate with 177Lu - Stability and Biodistribution Study. Alasbimn Journal 7(28): April 2005. Article N° AJ28-3.

 

 

 

Abstract


The labeling with 177Lu and quality control procedures to produced DOTA (1,4,7,10-N,N´,N´´,N´´´, tetraazaciclododecane tetraacetic acid) coupled octreotate labeled peptide was evaluated.

The labeling was performed using 30µg of DOTA-Tyr3-octreotate and 500 and 1110 MBq of 177Lu, buffered with sodium acetate/acetic acid 0.4M pH 4.5 for 20 minutes at 100ºC. The radiochemical purity was determined by ITLC na HPLC. Biological distribution studies were performed on Nude mices with tumours (AR42J rat pancreatic tumour cells) by invasive method.

The stability of the 177Lu-DOTA-Tyr3-octreotate was followed by 7 days, and in both labeling procedures, the radiochemical purity were superior than 98%. Biodistribution studies showed fast blood clearance and the kidneys were the critical organs. The uptake in tumour were significant after 24 hours and the labeled peptide showed high in vivo stability.

Key words: DOTA, octreotate, 177Lu, biodistribuition.

 

 

 

Introduction


The advances in molecular biology opened the way for peptides in nuclear medicine. These biomolecules can be radiolabeled with an appropriated radioisotope to produce radiopharmaceuticals for diagnostic and therapeutic applications [1].

Among the peptides, somatostatin (sst) analogues have been intensively studied with positive clinical results[2-5]. The radioisotope with great perspectives to label these molecules for therapy application is 177Lu. It has some properties such as physical half life of 6.7 days[6], ß- emission of 497 keV (78%), that can be absorbed in a small mass of tissue and the emission of gamma radiation (208,3 keV, 11%) wich allows simultaneous external detection, used for scintigraphy and dosimetric studies.

The aim of this work is to evaluate in vitro stability of DOTA-Tyr3-octreotate labeled with 177Lu and the biodistribution of the complex in Nude tumor bearing mice.

 

 

 

Material and methods


Labeling procedure

Labeling was performed using 30µg of DOTA-Tyr3-octreotate (Pichem-Austria) and 500 and 1110 MBq of 177LuCl3 in 0.05 N HCl, (MDS Nordion, Canada) for in vitro stability studies and 30µg of DOTA-Tyr3-octreotate with 104 MBq of 177LuCl3 to biological distribution studies. The reaction mixtures were buffered with sodium acetate/acetic acid 0.4 M pH 4.5. All reagents were prepared with Chelex 100 treated metal free water. The labeling reactions were allowed to proceed for 20 minutes at 100 şC. The reaction mixtures were mantained at  7 - 8 şC for stability studies. The conditions for radiolabeling were based in the procedures of Breeman et al[7].

Quality Control

The radiochemical purity was determined by instant thin layer chromatography (ITLC-SG), with citrate/citric acid buffer 0.1 M, pH 5.0 as the mobile phase. The stability was evaluated 48 and 168 hours after labeling reaction. The HPLC (WatersTM 600 Controller with an UV/visible detector, 280nm and Radiomatic Flo-ONE\Beta radiodetector, Packard) was performed using C18 column (4.6 x 250 mm, 5µm, Waters), eluted with metanol:sodium acetate 0.06M, pH 5.5 gradient [5].

Biological Distribution Studies

Nude mice weighting 20.5 ±3.5g (n=3/group) were implanted subcutaneously in the back with 106 cells of the AR42J rat pancreatic tumor. Tumors are allowed to grow for about 4 weeks. These animals were injected in a tail vein with 177Lu-DOTA-Tyr3-octreotate diluted in NaCl 0.9% (0.74MBq/0.1mL). Biological distribution studies were performed by invasive method. At 1, 4, 24 and 48 hours after injection the animals were sacrificed. Blood and organs were colected and the radioactivity in these samples and in the diluted standard of the administered dose were determined using a gama counter (Packard).

 

 

 

Results


The complex was found to be stable with radiochemical purity superior than 98% (Table 1) for both activities (500 and 1110 MBq) when stored at low temperature. The biodistribution studies are showed in Table 2, results were expressed as percentage of the injected dose (ID) per tissue (%ID/tissue) and per gram of tissue (%ID/g).

The figure 1 and 2 shows the HPLC outline with a well defined peak in both radiolabeling conditions.


Table 1
Radiochemical purity of 177Lu-DOTA-Tyr3-octreotate (n=4).

Time (hours)
500MBq
1110MBq
0
99.4 ± 0.02%
99.46 ± 0.06%
48
98.84 ± 0.1%
98.77 ± 0.08%

168

98.8 ± 0.07%
98.3 ± 0.24%

Table 2
Biodistribution of 177Lu-DOTA-Tyr3-octreotate in Nude mice
(% ID/g ± SD) (n = 3).

Organs

1 hr
4 hr
24hr
48hr
Brain
0.10 ± 0.05
0.05 ±  0.03
0.04 ±  0.042
0.02 ±  0.007
Lung
2.33 ±  0.30
1.10 ±  0.81
0.61 ±  0.29
0.36 ±  0.13
Heart
0.68 ±  0.19
0.18 ±  0.04
0.09 ±  0.02
0.08 ±  0.01
Spleen
0.82 ±  0.15
0.43 ±  0.09
0.27 ±  0.06
0.24 ±  0.04
Liver
0.92 ±  0.26
0.69 ±  0.05
0.40 ±  0.05
0.36 ±  0.04
Stomach
5.93 ±  1.45
3.80 ± 0.55
2.06 ±  0.40
1.38 ±  0.14
Muscle
0.32 ±  0.11
0.09 ±  0.04
0.03 ±  0.004
0.03 ±  0.01
Kidneys
10.84 ±  0.82
9.99 ±  1.96
4.35 ±  1.79
2.54 ±  0.65
Small Intestine
1.77 ±  0.53
1.43 ± 0.08
0.26 ±  0.01
0.19 ±  0.01
Large Intestine
1.02 ±  0.43
5.16 ± 0.44
0.62 ±  0.08
0.50 ±  0.11
Adrenal
1.14 ±  1.18
1.65 ±  1.51
2.07 ±  0.44
1.06 ±  0.22
Pancreas
8.42 ±  2.94
3.95 ±  1.26
1.64 ±  0.21
0.90 ± 0.28
Bone
1.34 ±  0.79
1.59 ±  0.46
1.34 ±  0.28
1.67 ±  0.32
Tumor
2.45 ±  0.77
1.18 ±  0.32
0.83 ±  0.26
0.57 ±  0.09

Blood/mL

1.57 ±  0.10
0.16 ±  0.07
0.04 ±  0.006
0.03 ±  0.005

 

       
Fig.1 - HPLC outline of 30 µg -500MBq

Fig. 2 - HPLC outline of 30 µg - 1110MBq

 

 

 

Discussion and conclusion


The interest in using somatostatin analogues labeled with radioisotopes to receptor target therapy is increasing[8,9].

In this study, the complex 177Lu-DOTA-Tyr3-octreotate was obtained with high stability. Additional studies will be developed to evaluate radiochemical stability using labeling activities compatibles with therapeutical aplication.

Biological distribution studies showed the high uptake of the compound in tissues with high density of somatostatine receptors like adrenals and pancreas.

Blood clearance was fast and the kidneys are the critical organ, because the compound is mainly excreted in the urine. Bone uptake was very low, showing the great in vivo stability of the compound, considering that free luthetium presents high affinity to the bone tissue.

Tumour uptake of 177Lu-DOTA-Tyr3-octreotate was significant and evidences the applicability of the complex in tumour target therapy.

 

 

 

References


1

Buscombe JR. Radiopeptides from diagnosis to therapy. Brazilian Archives of Biology and Technology 2002;45:91-96.
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2

Krenning EP, Kooij PP, Bakker Wh, et al. Radiotherapy with a radiolabeled somatostatin analogue, [111In-DTPA-D-Phe1]-octreotide. A case history. Ann NY Acad Sci 1994; 733:496-506.
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3

Otte A, Herrmann R, Heppeler A, et al. Yttrium-90 DOTATOC: first clinical results. Eur J Nucl Med 1992;26:1430-47.
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4

Otte A, Mueller-Brand J, Goetze M, et al. Yttrium-90-DOTA-octreotide treatment of somatostatin receptor positive tumors. J Nucl Med 1998;39:70P.
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5

Kwekkeboom DJ, Kam BL, Bakker WH, et al. Treatment with [177Lu-DOTA,Tyr3] octreotate in patients with somatostatin receptor positive tumors: Preliminary results. J Nucl Med 2001;452:37P.
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6

Zimmerman BE, Unterweger MP, Brodack JW. The standardization of 177Lu by 4πß liquid scintillation spectrometry with 3H-standard efficiency tracing. Appl Radiat Isotopes 2001;54:623-31.
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7

Breeman WAP., de Jong M., Visser TJ., Erion JL., Krenning EP. Optimising conditions for radiolabelling of DOTA-peptides with 90Y, 111In and 177Lu at high specific activities. Eur. J. Nucl. Med. Mol. Imaging 2003; vol. 30, nº 6: 917-920.
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8

Krenning EP, Kooji PP, Bakker WH, et al. Radiotherapy with a radiolabeled somatostatin analogue, [111In-DTPA-D-Phe]-Octreotide. A case history. Ann NY Sci 1994; 733: 496-506.
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9

Paganelli G, Zoboli S, Cremosi M, et al. Receptor mediated radiotherapy with 90Y-DOTA-D-Phe-Tyr3- Octreotide. Eur. J. Nucl. Med 2000; 28: 426-34.
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