- Email: [email protected]
Technetium-99m direct radiolabeling of Lanreotide: a Somatostatin analog
Applied Radiation and Isotopes 55 (2001) 647–651
Technetium-99m direct radiolabeling of Lanreotide: a Somatostatin analog Shahid Perveza, A. Mushtaqa,*, Muhammad Arifb a
Nuclear Chemistry Division, Pakistan Institute of Nuclear Science and Technology, P.O. Nilore, Islamabad, Pakistan b Bahauddin Zakariya University, Multan, Pakistan Received 2 January 2001; received in revised form 26 April 2001; accepted 5 June 2001
Abstract Lanreotide, a synthetic octapeptide analog of a native hormone somatostatin, was labeled with a commonly available, inexpensive radionuclide 99mTc. Labeling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelation with 99mTc. Stannous chloride was used as reducing agent, while tartrate acted as transchelating agent. Lanreotide (100 mg), stannous chloride dihydrate (100 mg) and tartaric acid (64 mg) were dissolved in acetate/acetic acid buffer (pH 2.8). After overnight (B18 h) incubation, B444 MBq (12 mCi) 99mTc was added and kept in boiling water for 30 min. More than 97% labeling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 cartridge analysis. Radiolabeling results in one major peak when analyzed by reverse-phase HPLC. The stability of the 99mTc-peptide bond was evaluated by cysteine challenge studies. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Peptide; Direct radiolabeling; Technetium-99m; HPLC
1. Introduction Like monoclonal antibodies, peptides are also receptor-specific. Somatostatin has been demonstrated to exhibit a wide spectrum of biological and oncological actions, and the clinical potential of the peptide has been appreciated for several years. Various studies showed the inhibitory effects of somatostatin on a wide range of tumors (Schally, 1988). However, the therapeutic application of somatostatin was limited by its multiple action and the very short half-life in the circulation. Therefore, analogs were synthesized, which were more potent and long-acting than somatostatin itself (Riever et al., 1975; Bauer et al., 1982; Maina et al., 1994). Radiolabeled receptor-specific biomolecules can detect primary sites, identify occult metastatic lesions, guide surgical intervention, stage tumors, predict efficacy of certain therapeutic agents or, when labeled with suitable *Corresponding author. Fax: +92-51-929-0275. E-mail address: [email protected] (A. Mushtaq).
radionuclides, be useful radiotherapeutic agents. During the past few years, much attention has been paid to the diagnostic applications of radiolabeled peptides. 111InDTPA–Octreotide has been shown to detect a variety of neuroendocrine tumors with high specificity and sensitivity (Krenning et al., 1993; Hoffland et al., 1994; Tenenbaum et al., 1995). Although this agent has became a valuable tool in diagnostic imaging, it suffers from at least one major drawbackFthe cost. The impetus generated by these results have prompted investigators to label peptides with such radionuclides as 99mTc, 123I, 18F, 64Cu, 186,188Re and 90Y. Technetium-99m is more widely accessible because the radionuclide may be produced from the decay of 99 Mo (T1=2 ¼ 67 h) using commercially available 99 Mo-99mTc generator systems. It is therefore inexpensive and is available in every nuclear medical center. Due to its favorable physical characteristics (g decay 140 keV) and a convenient half-life (6 h), it does not deliver an unnecessary radiation burden to a patient, long after examinations are carried out and 140 keV gamma ray of
0969-8043/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 1 ) 0 0 1 1 8 - X
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S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
99m
Tc is highly efficient for external imaging. The purpose of this investigation was to label lanreotide with 99mTc and evaluate its stability and radiochemical purity.
2. Methods and materials
Lanreotide. Each cartridge was washed with 100% ethanol followed by 0.001 M HCl. The cartridge was then serially eluted with a graded series of acidified ethanol solutions. The radioactivity in each eluent fraction (0.001 M HCl, ethanol solutions of 20%, 40%, 60%, 80%, and 100% ethanol, cartridge) was determined by counting in a dose calibrator (CRC-5RH) Capintec.
2.1. Materials 2.4. HPLC analysis Lanreotide (8-mer, Cys 2–7, cyclo) was a product of pi CHEM Austria and was supplied by H. Vera Ruiz International Atomic Energy Agency (IAEA), Vienna. The 99mTc-pertechnetate was obtained from a 99 Mo-99mTc radionuclide generator Pakgen, PINSTECH, Pakistan. [The generator contains fission produced molybdenum-99 adsorbed on alumina. 99mTc may be eluted asceptically using saline as an eluent.] Tartaric acid and stannous chloride were purchased from E. Merek, Germany. Other chemicals and reagents were obtained from commercial suppliers.
The Hitachi L-6200 Intelligent pump and L-4200 UVVis detector systems were used for HPLC analysis. Analytical reverse-phase HPLC analysis was performed with an analytical C18 column (RP-18.5 mm, Lichrosorb 25 0.45 cm) using a continuous gradient of acetonitrile (10–90%) and 0.1% aqueous trifluoroacetic acid. Elutions were performed at a flow rate of 1 ml/min after an injection volume of 20 ml labeled peptide. The eluted radioactivity was monitored on line using a NaI probe (Raytest–Steffi); collected fractions were also measured by well type gamma counter.
2.2. Labeling of Lanreotide 2.5. Cysteine challenge Lanreotide in acetate buffer was reduced by addition of SnCl2 2H2O+tartaric acid with incubation at room temperature for a certain period and a required radioactivity of 99mTc was added. Finally the mixture was kept for 30 min in a boiling water bath. Various parameters, such as the mass ratio of peptide and stannous chloride, concentration of 99mTc and incubation period for the reduction of the peptide were studied. After incubation in a boiling water bath, the reaction mixture was cooled and aliquots were taken for radiochemical analysis. 2.3. Radiochemical analysis To determine the amount of free 99mTc, the sample was chromatographed on ITLC-SG (Gelman Sciences Inc., USA) using 0.9% saline as mobile phase. Free 99m Tc migrated with the solvent front, whereas radiocolloid and labeled Lanreotide remained at the origin. The amount of radiocolloid was determined using acidified ethanol 85% (pH 3.5) to develop the ITLCSG strip. In this system, colloidal material was retained at the origin, while free and labeled Lanreotide migrated with the solvent front. The radioactivity was quantified by cutting the strips (1.5 10 cm) into 1 cm pieces and counting in a well type gamma counter. (LUDLUM Model 261 spectrometer, Texas, USA.) Sometimes a 2p scanner (Berthold, Germany) was used for scanning the radioactivity on ITLC-SG strips. For reverse-phase analysis, C18 cartridges (SepPak, Millipore, Inc., USA) were used as reverse-phase adsorbents to evaluate the binding of 99mTc to the
Cysteine solutions were prepared in phosphate buffered saline (PBS) and the pH was adjusted to 7.4 with 1.0 M NaOH. Aliquots of 100 ml were diluted into separate vials to provide six dilutions. One vial contained only saline. To each vial was added 100 ml of 99mTc-Lanreotide. Samples were incubated for 45 min at 371C. At the end of the incubation period each sample was spotted on to an ITLC-SG strip and chromatographed in PBS, pH 7.4. The chromatogram was developed, each strip was cut in half, and the radioactivity in each half determined by gamma counting. The amount of displacement was expressed as the percentage of total radioactivity associated with the solvent front. The 99mTc displaced by Cysteine migrated near the solvent front.
3. Results Based on the results of initial experiments, 50 mg peptide was used to optimize the labeling of Lanreotide with 99mTc. Lanreotide (50 mg) was dissolved in 1 ml acetate/acetic acid (pH 2.8) buffer. Stannous chloride/ tartaric acid solution (100 ml) containing 50 mg SnCl2. 2H2O and 32 mg tartaric acid were added and incubated for various time intervals at room temperature. After a certain period of incubation, freshly eluted pertechnetate solution (1–2 mCi/ml) was added and vortexed. Finally the reaction vial was kept in a boiling water bath for 30 min. After a 1-h incubation period the labeling yield was 15% which gradually increased with increase in
S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
incubation period. A 5-h incubation period resulted in 80% labeling of Lanreotide with 99mTc. To get a labeling yield of >97% the reaction vial containing peptide and stannous tartrate in acetate buffer was kept overnight (B18 h) at room temperature. In each experiment the vial containing the reaction mixture was sealed under vacuum. The mass ratio of peptide and stannous chloride had a significant effect on the labeling of the peptide with 99m Tc. The best results obtained were near the 1 : 1 mass ratio of peptide and stannous chloride. When the amount of stannous chloride was doubled, the labeling yield was decreased to 30%. Similarly, the labeling yield was also decreased when the mass ratio of peptide and stannous chloride were 1 : 0.5 or 1 : 0.75. The radioactive concentration of 99mTc also had a marked effect on the labeling yield. Quantitative labeling was achieved when 74–92.5 MBq (2–2.5 mCi) of 99mTc was employed for 50 mg of Lanreotide. When the amount of Lanreotide was 100 mg, up to 444 MBq (12 mCi) 99mTc was labeled with >97% labeling efficiency. It is expected that large amounts of Lanreotide will be required with increase in the radioactive concentration of 99mTc. The final preparation contained 100 mg Lanreotide, in 1 ml acetate/acetic acid buffer (pH 2.8), stannous chloride (100 mg), tartaric acid (64 mg). The vial containing the reaction mixture was sealed under vacuum. After overnight incubation at room temperature, 1 ml freshly eluted 99mTc (12 mCi/444 MBq) was added to the reaction vial and finally, the vial was heated in a boiling water bath for 30 min. A typical elution profile of 99mTcLanreotide from an analytical HPLC at 30 min post labeling is shown in Fig. 1. No change in the elution profile of 99mTc-Lanreotide was observed at 6 h post labeling when analytical RP-HPLC profiles revealed one primary peak which accounted for approximately 95% of the radiolabeled material. Step gradient elutions of 99m Tc-Lanreotide on C18 cartridges were used to confirm the results obtained by analytical HPLC. The results from ITLC with saline or acidified ethanol as the mobile phases also indicated a high radiolabeling yield. The amount of radiocolloid was determined by ITLC-SG using acidified ethanol (85%, pH=3.5) as the mobile phase and were frequently less than 1%; less than 2% of the 99mTc was found to migrate in saline with the solvent front. The stability of the 99mTc peptide bond was evaluated by cysteine challenge tests. The amount of cysteine required to displace 50% of the 99mTc from the labeled peptide was approximately 50 mM.
4. Discussion Lanreotide labeled successfully with inexpensive and commonly available 99mTc can be used in all nuclear
649
Fig. 1. Elution profile of 99mTc-Lanreotide at 30 min post labeling from a C18 RP-HPLC column.
medical centres for planar or single photon emission tomography (SPECT) studies. We have developed a method to label Lanreotide with 99mTc that is simple, does not require synthetic modification or blocking or deblocking of functional groups, and yet provides a 99m Tc-labeled imaging agent in high yield. Lanreotide is a cyclic peptide ðD2b2 Nal2Cys2Tyr2D2Trp2Lys2Val2Cys2Thr2NH2 Þ; hence direct labeling with 99mTc is applicable. In this method, the dicysteine bond is reduced with stannous chloride. The sulfahydryls resulted by the reduction serve as strong chelating groups for reduced 99mTc. Tartrate serves as a transchelating agent. The involvement of tin in protein labeling as a tin–protein intermediate was suggested by Rhodes (1991) for ‘‘pretinning’’ procedure when the reduction of native disulfide bridges was carried out by incubating the protein with stannous ions. Direct labeling of peptides with 99mTc involves, in brief: (1) application of a reducing agent to cleave disulfide bonds in order to expose cysteine residues on the peptide; (2) reduction of 99m Tc to the required reduced states with the reducing agent, mainly stannous chloride, in the presence of an
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S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
appropriate complexing agent with simultaneous complexation of reduced 99mTc; and (3) transchelation of reduced 99mTc, from the intermediate complex to the cysteine residues on the peptide. 1. R–S–S–R+SnX2-2R–S–Sn–X 2. TcO 4 +SnX2+Tartrate-Tc(Tartrate) 3. Tc(Tartrate)+R–S–Sn–X-R–S–TcX2 Paik et al. (1985) proved the existence of both highand- low affinity bonding sites by measuring the amount of protein labeling in the presence of varying concentrations of DTPA, a strong chelator for 99mTc metal ions. They titrated the free sulfide groups, and as a result suggested that the high affinity binding was related to the presence of these groups. They also suggested that the pretinning procedure increased the number of high affinity binding sites, i.e., the number of reactive sulphides. In our experiments, overnight (B18 h) incubation period for the peptide+SnCl2 was found sufficient to generate binding sites for quantitative labeling of Lanreotide with 99mTc. In peptide chemistry linear peptides are cyclized via a cysteine bridge to minimize the peptides susceptibility for in-vivo protolysis. Contrary to this, we have reduced the bridge to provide sulfahydryl groups for 99mTc binding. In order to shed light on the structural conformation of this complex another octapeptide, Vapreotide (RC-160), cyclized via cysteine bridge was labeled with stable rhenium (Varnum et al., 1994). With two dimensional H-NMR studies and computer model analysis, they concluded that Re-RC-160 maintained its backbone structure and spatial topography essential for the binding RC-160 to SSTR receptor subtypes. Similarly, directly labeled 99mTc-RC-160 retained its receptor specificity and had reasonable thermodynamic stability in vitro and in-vivo (Thakur et al., 1996). Rhodes (1991) also discussed in detail, the antibody reduction and its immunoreactivity and finally concluded that the immunoreactive fraction of the antibody is not adversely affected by the pretinning process (reduction process). The results obtained by three different techniques, HPLC, ITLC and step gradient elutions of 99mTc Lanreotide are in very good agreement. More than 97% labeling yield was achieved during this study. While hydrolyzed and free, 99mTc was less than 3%. A typical elution profile of 99mTc Lanreotide from an analytical HPLC shows a major peak and few minor peaks. The exact chemical nature of minor peaks is not clearly understood but they may be due to either isomeric forms or to the different oxidation states reduced 99mTc. The radiometal ions may also have been coordinated with other ligands such as Trp available in Lanreotide. The bond strength of the 99mTc to the peptide is high and is resistant to large molar excess of cysteine (Fig. 2).
Fig. 2. Displacement of cysteine as a challenge.
99m
Tc from
99m
Tc-Lanreotide using
The present study demonstrates that 99mTc-Lanreotide can be readily prepared by direct reduction techniques. The labeling method is simple, did not require protecting and deprotecting functional groups and yields are quite high (>97%). The method can also be applied for the formulation of a freeze-dried kit.
Acknowledgements This work was sponsored by the International Atomic Energy Agency (IAEA), Vienna, under research contract No. 10107/RB.
References Bauer, W., Briner, U., Doepfner, W., Haller, R., Huguenin, R., Marbach, P., Petcher, T.J., Pless, J., 1982. SMS 201–995: a very potent and selective octapeptide analog of somatostatin with prolonged action. Life Sci. 31, 1133–1140. Hoffland, L.J., van der Pluijm, M., van Koetsveld, P.M., de Jong, M., Setyono-Han, B., Bakker, W.H., Kwekkeboom, D.K., Visser, T.J., Lamberts, A.W., 1994. A new radiolabeled somatostatin analog [111In-DTPA-D-Phe1]RC-160 preparation, biological activity, receptor scintigraphy in rats and comparison with [111In-DTPA-D-Phe1] Octreotide. Eur. J. Nucl. Med. 21, 328–335. Krenning, E.P., Kwekkeboom, D.J., Bakker, W.H., Breeman, W.A., Kooij, P.P., Oei, H.Y., van Hagen, M., Postema, P.T., de Jong, M., Reubi, J.C., 1993. Somatostatin receptor scintigraphy with [In-111-DTPA-D-Phe] and [I-123-tyr-3] octreotide: the Rotterdam experience with more than 1000 patient. Eur. J. Nucl. Med. 20, 716–731. Maina, T., Stolz, B., Albert, R., Bruns, C., Koch, P., Macke, H., 1994. Synthesis, radiochemistry and biological evaluation of a new somatostatin analogue (SDZ 219-387) labeled with technetium-99m. Eur. J. Nucl. Med. 21, 437–444. Paik, C.H., Phan, L., Hong, J.J., Sahami, M.W., Heald, S.C., Reba, R.C., Steigman, J., Eckelman, W.C., 1985. The
S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651 labeling of high affinity sites of antibodies with 99mTc. Int. J. Nucl. Med. Biol. 12, 3–8. Rhodes, B.A., 1991. Direct labeling of proteins with Tc-99m. Nucl. Med. Biol. 18, 667–676. Riever, J., Brown, M., Vale, W., 1975. D-Trp-8 somatostatin: an analog of somatostatin more potent than the native molecule. Biochem. Bipohys. Res. Commun. 65, 746–751. Schally, A.V., 1988. Oncological applications of somatostatin analogues. Cancer Res. 48, 6977. Tenenbaum, F., Lumbroso, J., Schlumberger, M., Mure, A., Plouin, P.F., Caillou, B., Parmentier, C., 1995. Comparison
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of radiolabeled octreotide and meta-iodobenzylguanidine (MIBG) scintigraphy in malignant pheochromocytoma. J. Nucl. Med. 36, 1. Thakur, M.L., Kolan, H.R., John, E., Rifat, S., Li, J., Rux, A., Halmos, G., Shally, A.V., 1996. Int. J. Oncol. 9, 455. Varnum, J.M., Thakur, M.L., Schally, A.V., Jansen, M., Mayo, K.H., 1994. Rhenium labeled somatostatin analog RC-160 1 H-NMR and computer modeling conformational analysis. J. Biol. Chem. 269, 12583–12588.
Technetium-99m direct radiolabeling of Lanreotide: a Somatostatin analog Shahid Perveza, A. Mushtaqa,*, Muhammad Arifb a
Nuclear Chemistry Division, Pakistan Institute of Nuclear Science and Technology, P.O. Nilore, Islamabad, Pakistan b Bahauddin Zakariya University, Multan, Pakistan Received 2 January 2001; received in revised form 26 April 2001; accepted 5 June 2001
Abstract Lanreotide, a synthetic octapeptide analog of a native hormone somatostatin, was labeled with a commonly available, inexpensive radionuclide 99mTc. Labeling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelation with 99mTc. Stannous chloride was used as reducing agent, while tartrate acted as transchelating agent. Lanreotide (100 mg), stannous chloride dihydrate (100 mg) and tartaric acid (64 mg) were dissolved in acetate/acetic acid buffer (pH 2.8). After overnight (B18 h) incubation, B444 MBq (12 mCi) 99mTc was added and kept in boiling water for 30 min. More than 97% labeling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 cartridge analysis. Radiolabeling results in one major peak when analyzed by reverse-phase HPLC. The stability of the 99mTc-peptide bond was evaluated by cysteine challenge studies. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Peptide; Direct radiolabeling; Technetium-99m; HPLC
1. Introduction Like monoclonal antibodies, peptides are also receptor-specific. Somatostatin has been demonstrated to exhibit a wide spectrum of biological and oncological actions, and the clinical potential of the peptide has been appreciated for several years. Various studies showed the inhibitory effects of somatostatin on a wide range of tumors (Schally, 1988). However, the therapeutic application of somatostatin was limited by its multiple action and the very short half-life in the circulation. Therefore, analogs were synthesized, which were more potent and long-acting than somatostatin itself (Riever et al., 1975; Bauer et al., 1982; Maina et al., 1994). Radiolabeled receptor-specific biomolecules can detect primary sites, identify occult metastatic lesions, guide surgical intervention, stage tumors, predict efficacy of certain therapeutic agents or, when labeled with suitable *Corresponding author. Fax: +92-51-929-0275. E-mail address: [email protected] (A. Mushtaq).
radionuclides, be useful radiotherapeutic agents. During the past few years, much attention has been paid to the diagnostic applications of radiolabeled peptides. 111InDTPA–Octreotide has been shown to detect a variety of neuroendocrine tumors with high specificity and sensitivity (Krenning et al., 1993; Hoffland et al., 1994; Tenenbaum et al., 1995). Although this agent has became a valuable tool in diagnostic imaging, it suffers from at least one major drawbackFthe cost. The impetus generated by these results have prompted investigators to label peptides with such radionuclides as 99mTc, 123I, 18F, 64Cu, 186,188Re and 90Y. Technetium-99m is more widely accessible because the radionuclide may be produced from the decay of 99 Mo (T1=2 ¼ 67 h) using commercially available 99 Mo-99mTc generator systems. It is therefore inexpensive and is available in every nuclear medical center. Due to its favorable physical characteristics (g decay 140 keV) and a convenient half-life (6 h), it does not deliver an unnecessary radiation burden to a patient, long after examinations are carried out and 140 keV gamma ray of
0969-8043/01/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 9 - 8 0 4 3 ( 0 1 ) 0 0 1 1 8 - X
648
S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
99m
Tc is highly efficient for external imaging. The purpose of this investigation was to label lanreotide with 99mTc and evaluate its stability and radiochemical purity.
2. Methods and materials
Lanreotide. Each cartridge was washed with 100% ethanol followed by 0.001 M HCl. The cartridge was then serially eluted with a graded series of acidified ethanol solutions. The radioactivity in each eluent fraction (0.001 M HCl, ethanol solutions of 20%, 40%, 60%, 80%, and 100% ethanol, cartridge) was determined by counting in a dose calibrator (CRC-5RH) Capintec.
2.1. Materials 2.4. HPLC analysis Lanreotide (8-mer, Cys 2–7, cyclo) was a product of pi CHEM Austria and was supplied by H. Vera Ruiz International Atomic Energy Agency (IAEA), Vienna. The 99mTc-pertechnetate was obtained from a 99 Mo-99mTc radionuclide generator Pakgen, PINSTECH, Pakistan. [The generator contains fission produced molybdenum-99 adsorbed on alumina. 99mTc may be eluted asceptically using saline as an eluent.] Tartaric acid and stannous chloride were purchased from E. Merek, Germany. Other chemicals and reagents were obtained from commercial suppliers.
The Hitachi L-6200 Intelligent pump and L-4200 UVVis detector systems were used for HPLC analysis. Analytical reverse-phase HPLC analysis was performed with an analytical C18 column (RP-18.5 mm, Lichrosorb 25 0.45 cm) using a continuous gradient of acetonitrile (10–90%) and 0.1% aqueous trifluoroacetic acid. Elutions were performed at a flow rate of 1 ml/min after an injection volume of 20 ml labeled peptide. The eluted radioactivity was monitored on line using a NaI probe (Raytest–Steffi); collected fractions were also measured by well type gamma counter.
2.2. Labeling of Lanreotide 2.5. Cysteine challenge Lanreotide in acetate buffer was reduced by addition of SnCl2 2H2O+tartaric acid with incubation at room temperature for a certain period and a required radioactivity of 99mTc was added. Finally the mixture was kept for 30 min in a boiling water bath. Various parameters, such as the mass ratio of peptide and stannous chloride, concentration of 99mTc and incubation period for the reduction of the peptide were studied. After incubation in a boiling water bath, the reaction mixture was cooled and aliquots were taken for radiochemical analysis. 2.3. Radiochemical analysis To determine the amount of free 99mTc, the sample was chromatographed on ITLC-SG (Gelman Sciences Inc., USA) using 0.9% saline as mobile phase. Free 99m Tc migrated with the solvent front, whereas radiocolloid and labeled Lanreotide remained at the origin. The amount of radiocolloid was determined using acidified ethanol 85% (pH 3.5) to develop the ITLCSG strip. In this system, colloidal material was retained at the origin, while free and labeled Lanreotide migrated with the solvent front. The radioactivity was quantified by cutting the strips (1.5 10 cm) into 1 cm pieces and counting in a well type gamma counter. (LUDLUM Model 261 spectrometer, Texas, USA.) Sometimes a 2p scanner (Berthold, Germany) was used for scanning the radioactivity on ITLC-SG strips. For reverse-phase analysis, C18 cartridges (SepPak, Millipore, Inc., USA) were used as reverse-phase adsorbents to evaluate the binding of 99mTc to the
Cysteine solutions were prepared in phosphate buffered saline (PBS) and the pH was adjusted to 7.4 with 1.0 M NaOH. Aliquots of 100 ml were diluted into separate vials to provide six dilutions. One vial contained only saline. To each vial was added 100 ml of 99mTc-Lanreotide. Samples were incubated for 45 min at 371C. At the end of the incubation period each sample was spotted on to an ITLC-SG strip and chromatographed in PBS, pH 7.4. The chromatogram was developed, each strip was cut in half, and the radioactivity in each half determined by gamma counting. The amount of displacement was expressed as the percentage of total radioactivity associated with the solvent front. The 99mTc displaced by Cysteine migrated near the solvent front.
3. Results Based on the results of initial experiments, 50 mg peptide was used to optimize the labeling of Lanreotide with 99mTc. Lanreotide (50 mg) was dissolved in 1 ml acetate/acetic acid (pH 2.8) buffer. Stannous chloride/ tartaric acid solution (100 ml) containing 50 mg SnCl2. 2H2O and 32 mg tartaric acid were added and incubated for various time intervals at room temperature. After a certain period of incubation, freshly eluted pertechnetate solution (1–2 mCi/ml) was added and vortexed. Finally the reaction vial was kept in a boiling water bath for 30 min. After a 1-h incubation period the labeling yield was 15% which gradually increased with increase in
S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
incubation period. A 5-h incubation period resulted in 80% labeling of Lanreotide with 99mTc. To get a labeling yield of >97% the reaction vial containing peptide and stannous tartrate in acetate buffer was kept overnight (B18 h) at room temperature. In each experiment the vial containing the reaction mixture was sealed under vacuum. The mass ratio of peptide and stannous chloride had a significant effect on the labeling of the peptide with 99m Tc. The best results obtained were near the 1 : 1 mass ratio of peptide and stannous chloride. When the amount of stannous chloride was doubled, the labeling yield was decreased to 30%. Similarly, the labeling yield was also decreased when the mass ratio of peptide and stannous chloride were 1 : 0.5 or 1 : 0.75. The radioactive concentration of 99mTc also had a marked effect on the labeling yield. Quantitative labeling was achieved when 74–92.5 MBq (2–2.5 mCi) of 99mTc was employed for 50 mg of Lanreotide. When the amount of Lanreotide was 100 mg, up to 444 MBq (12 mCi) 99mTc was labeled with >97% labeling efficiency. It is expected that large amounts of Lanreotide will be required with increase in the radioactive concentration of 99mTc. The final preparation contained 100 mg Lanreotide, in 1 ml acetate/acetic acid buffer (pH 2.8), stannous chloride (100 mg), tartaric acid (64 mg). The vial containing the reaction mixture was sealed under vacuum. After overnight incubation at room temperature, 1 ml freshly eluted 99mTc (12 mCi/444 MBq) was added to the reaction vial and finally, the vial was heated in a boiling water bath for 30 min. A typical elution profile of 99mTcLanreotide from an analytical HPLC at 30 min post labeling is shown in Fig. 1. No change in the elution profile of 99mTc-Lanreotide was observed at 6 h post labeling when analytical RP-HPLC profiles revealed one primary peak which accounted for approximately 95% of the radiolabeled material. Step gradient elutions of 99m Tc-Lanreotide on C18 cartridges were used to confirm the results obtained by analytical HPLC. The results from ITLC with saline or acidified ethanol as the mobile phases also indicated a high radiolabeling yield. The amount of radiocolloid was determined by ITLC-SG using acidified ethanol (85%, pH=3.5) as the mobile phase and were frequently less than 1%; less than 2% of the 99mTc was found to migrate in saline with the solvent front. The stability of the 99mTc peptide bond was evaluated by cysteine challenge tests. The amount of cysteine required to displace 50% of the 99mTc from the labeled peptide was approximately 50 mM.
4. Discussion Lanreotide labeled successfully with inexpensive and commonly available 99mTc can be used in all nuclear
649
Fig. 1. Elution profile of 99mTc-Lanreotide at 30 min post labeling from a C18 RP-HPLC column.
medical centres for planar or single photon emission tomography (SPECT) studies. We have developed a method to label Lanreotide with 99mTc that is simple, does not require synthetic modification or blocking or deblocking of functional groups, and yet provides a 99m Tc-labeled imaging agent in high yield. Lanreotide is a cyclic peptide ðD2b2 Nal2Cys2Tyr2D2Trp2Lys2Val2Cys2Thr2NH2 Þ; hence direct labeling with 99mTc is applicable. In this method, the dicysteine bond is reduced with stannous chloride. The sulfahydryls resulted by the reduction serve as strong chelating groups for reduced 99mTc. Tartrate serves as a transchelating agent. The involvement of tin in protein labeling as a tin–protein intermediate was suggested by Rhodes (1991) for ‘‘pretinning’’ procedure when the reduction of native disulfide bridges was carried out by incubating the protein with stannous ions. Direct labeling of peptides with 99mTc involves, in brief: (1) application of a reducing agent to cleave disulfide bonds in order to expose cysteine residues on the peptide; (2) reduction of 99m Tc to the required reduced states with the reducing agent, mainly stannous chloride, in the presence of an
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S. Pervez et al. / Applied Radiation and Isotopes 55 (2001) 647–651
appropriate complexing agent with simultaneous complexation of reduced 99mTc; and (3) transchelation of reduced 99mTc, from the intermediate complex to the cysteine residues on the peptide. 1. R–S–S–R+SnX2-2R–S–Sn–X 2. TcO 4 +SnX2+Tartrate-Tc(Tartrate) 3. Tc(Tartrate)+R–S–Sn–X-R–S–TcX2 Paik et al. (1985) proved the existence of both highand- low affinity bonding sites by measuring the amount of protein labeling in the presence of varying concentrations of DTPA, a strong chelator for 99mTc metal ions. They titrated the free sulfide groups, and as a result suggested that the high affinity binding was related to the presence of these groups. They also suggested that the pretinning procedure increased the number of high affinity binding sites, i.e., the number of reactive sulphides. In our experiments, overnight (B18 h) incubation period for the peptide+SnCl2 was found sufficient to generate binding sites for quantitative labeling of Lanreotide with 99mTc. In peptide chemistry linear peptides are cyclized via a cysteine bridge to minimize the peptides susceptibility for in-vivo protolysis. Contrary to this, we have reduced the bridge to provide sulfahydryl groups for 99mTc binding. In order to shed light on the structural conformation of this complex another octapeptide, Vapreotide (RC-160), cyclized via cysteine bridge was labeled with stable rhenium (Varnum et al., 1994). With two dimensional H-NMR studies and computer model analysis, they concluded that Re-RC-160 maintained its backbone structure and spatial topography essential for the binding RC-160 to SSTR receptor subtypes. Similarly, directly labeled 99mTc-RC-160 retained its receptor specificity and had reasonable thermodynamic stability in vitro and in-vivo (Thakur et al., 1996). Rhodes (1991) also discussed in detail, the antibody reduction and its immunoreactivity and finally concluded that the immunoreactive fraction of the antibody is not adversely affected by the pretinning process (reduction process). The results obtained by three different techniques, HPLC, ITLC and step gradient elutions of 99mTc Lanreotide are in very good agreement. More than 97% labeling yield was achieved during this study. While hydrolyzed and free, 99mTc was less than 3%. A typical elution profile of 99mTc Lanreotide from an analytical HPLC shows a major peak and few minor peaks. The exact chemical nature of minor peaks is not clearly understood but they may be due to either isomeric forms or to the different oxidation states reduced 99mTc. The radiometal ions may also have been coordinated with other ligands such as Trp available in Lanreotide. The bond strength of the 99mTc to the peptide is high and is resistant to large molar excess of cysteine (Fig. 2).
Fig. 2. Displacement of cysteine as a challenge.
99m
Tc from
99m
Tc-Lanreotide using
The present study demonstrates that 99mTc-Lanreotide can be readily prepared by direct reduction techniques. The labeling method is simple, did not require protecting and deprotecting functional groups and yields are quite high (>97%). The method can also be applied for the formulation of a freeze-dried kit.
Acknowledgements This work was sponsored by the International Atomic Energy Agency (IAEA), Vienna, under research contract No. 10107/RB.
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