Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 123–127
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The fast method of Cu-porphyrin complex synthesis for potential use in positron emission tomography imaging Krzysztof Kilian a,⁎, Maria Pęgier b, Krystyna Pyrzyńska b a b
Heavy Ion Laboratory, University of Warsaw, 5ath Pasteur Str., 02-093 Warsaw, Poland Faculty of Chemistry, University of Warsaw, 1st Pasteur Str., 02-093 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 8 September 2015 Received in revised form 12 January 2016 Accepted 23 January 2016 Available online 25 January 2016 Keywords: Copper-64 Porphyrin Positron emission tomography
a b s t r a c t Porphyrin based photosensitizers are useful agents for photodynamic therapy and fluorescence imaging of cancer. Additionally, porphyrins are excellent metal chelators, forming stable metalo-complexes and 64Cu isotope can serve as a positron emitter (t1/2 = 12.7 h). The other advantage of 64Cu is its decay characteristics that facilitates the use of 64Cu-porphyrin complex as a therapeutic agent. Thus, 64Cu chelation with porphyrin photosensitizer may become a simple and versatile labeling strategy for clinical positron emission tomography. The present study reports a convenient method for the synthesis of Cu complex with tetrakis(4carboxyphenyl)porphyrin (TCPP). The experimental conditions for labeling, such as the metal-to-ligand molar ratio, pH and time of reaction were optimized to achieve a high complexation efficiency in a short period of time as possible. In order to accelerate the metallation, the use of substitution reactions of cadmium or lead porphyrin and the presence of reducing agent, such as ascorbic acid, hydroxylamine and flavonoid – morin, were evaluated. The optimum conditions for the synthesis of the copper complex were borate buffer at pH 9 with the addition of 10-fold molar excess, with respect to Cu2+ ions and TCPP and ascorbic acid which resulted in reduction of the reaction time from 30 min to below 1 min. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The nuclear medicine imaging, in particular positron emission tomography (PET), allows the detection and monitoring of a variety of biological and pathophysiological processes, at tracer quantities of the radiolabeled target agents and at doses free from pharmacological effects. The use of radio metals for labeling has now become one of the essential tools in identifying, screening and development of new target agents. Copper plays significant role in human biochemistry and nutrition [1] and copper-labeled biologically active compounds could be used for imaging molecular background of diseases. 64Cu is described in the literature as a potentially effective diagnostic radioisotope, used in PET. The quality of imaging is in some applications, comparable to the quality of imaging with 18F isotope [2], which is one of the most frequently used in PET. 64Cu is produced in medical cyclotrons via 64 Ni(p,n)64Cu reaction, forming isotopically pure 64Cu. Copper – 64, because of its decay scheme (t1/2 = 12.7 h, β+: 17.4%, Eβ+max = 656 keV; β−: 39%, Eβ-max = 573 keV) is also used as a therapeutic agent [2]. Relatively long half-life of this isotope (in relation to other PET radioisotopes) offers an opportunity to label larger biomolecules. On the other
⁎ Corresponding author. E-mail address: [email protected] (K. Kilian).
http://dx.doi.org/10.1016/j.saa.2016.01.045 1386-1425/© 2016 Elsevier B.V. All rights reserved.
hand, a wide half-live range of other copper isotopes (τ1/2 60Cu = 23.7 min, τ1/2 61Cu = 3.4 h, τ1/2 62Cu = 9.7 min) allows imaging processes with different dynamics, while the identical chemistry of the labeling process is maintained. Various types of chelating ligands were evaluated as the chelators for copper radioisotopes, such as polyaminocarbonoxylates, cyclic polyamines, tetraaza-macrocycles and bis-thiosemicarbazones [3–8]. The selection of reagents for use in binding copper isotopes is largely dependent on the ligands forming very stable complexes. Acyclic chelates lack kinetic stability [6], thus, macrocyclic ligands are preferred [7]. The thermodynamic stability of the respective classes of ligands for Cu(II) complexes decreases in the order: hexaaza cages N tetrazamacrocycles N polyaminophosphonates N polyaminocarboxylate macrocycles N open chain aminocarboxylates [3]. DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid) is the most often used macrocyclic metals chelate, including 64Cu, in radiopharmaceutical research [8]. However, DOTA is not an ideal ligand for 64Cu because of slow reaction kinetics [9]. 1,4,7-triazacyclononane-1,4,7-triacetic acid derivatives [9] as well as cross-bridged 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid [10] have superior stability in comparison to DOTA, but require harsher radiolabeling conditions. The most common in the literature 64 Cu labeled compound is diacetyl-bis(N-4-methyl thiosemicarbazone (ATSM) [11]. 64Cu-ATSM molecules are lipophilic, small and selectively absorbed by the hypoxic tissues and can be used to study the cardiovascular system, with focus on hypoxic areas.
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K. Kilian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 123–127
From the other side, the rapid complexation of metal ion with a ligand is an important criterion in developing radiopharmaceuticals since the radioisotope is decaying throughout its preparation and application. This is a serious limitation of many radiolabeling protocols which usually employ a long incubation times (up to 1 h) at room or elevated temperature and an excess of ligand is needed to achieve sufficient complexation yield. However, the use of ligand excess in formulations should be avoided as many targeting agents are biologically active in themselves. Porphyrins and their derivatives show significant affinity to cancer cells and are used for diagnosis and photodynamic therapy (PDT). In clinical practice, Photofrin® and Visudyne® are used for PDT treatment of lung, esophagus, bladder cancer and skin inflammations [12,13]. Thus, porphyrins are potentially suitable as bifunctional chelates and vehicles for delivery of copper radioisotopes to selected tissues. Porphyrins also have their own useful tissue-targeting properties, such as selective accumulation in inflammatory and lymphatic tissues. Hence, porphyrin labeled with suitable therapeutic or diagnostic radionuclide could be envisaged as potential agents for tumor therapy and diagnostics [14]. They are used in tumor phototherapy in large doses, and they are relatively nontoxic and well tolerated [15]. The disadvantage is the high kinetic barrier to metallation, but copper complexes with porphyrins are exceedingly robust to demetallation [16,17]. Porphyrins have the ability to chelate metal ions, due to the system of four pyrrole nitrogen atoms, highly selective for ions of ionic radius of about 70 pm (ionic radius of Cu2+ is 72 pm). Complexes, although characterized by high values of stability constants, are formed with comparatively poor kinetics, which has led to criticism in earlier work [18]. The above restriction was removed by using the reaction mechanism of sitting-a-top (SAT) [19]. Chelating properties are not significantly affected by the type and number of substituents in the ring, allowing application of the appropriate porphyrin for the intended purpose of imaging (such as hydrophilicity/hydrophobicity and partition coefficient octanol/water) The goal was to develop improved methods for synthesis and physico-chemical characteristics of copper complexes with TCPP, as potential diagnostic and therapeutic radiopharmaceuticals for nuclear medicine. 2. Experimental 2.1. Reagents 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) was obtained from Fluka (Germany). Stock solutions (10−3 mol L−1) of TCPP were prepared by dissolution of 0.1976 g in 200 mL of 10 mmol L −1 sodium hydroxide and further dilution with deionized water up to 250 mL. The structure of this porphyrin is shown in Fig. 1. Under basic conditions the carboxylic groups are in –COONa form. 1000 mg L−1 stock solutions of Cu, Pb and Cd as nitrates in 2–5% nitric acid were obtained from Merck (Germany). Further dilutions were done with deionized water. Fresh stock solution of ascorbic acid (10−1 mol L−1) in acetate buffer at pH = 5 was prepared every day. The components of borate buffer as well as other reagents were of analytical grade and used without further purification. 2.2. Apparatus Double-beam UV–Vis spectrophotometer Lambda 20 (Perkin Elmer, USA) with diode-array detector was used for spectra recording and absorbance measurements. Spectra were recorded in the range from 350 to 700 nm with 0.2 nm resolution in 10 mm quartz cells. Data were processed with WinLab software. A Radelkis (Hungary) pH-meter with Orion (USA) electrode was used for pH measurement and adjustment.
Fig. 1. Chemical structure of TCPP.
2.3. Procedures All experiments were conducted using molar ratio of Cu(II) to TCPP being 1:1. Concentration of the reagents was 10−5 mol L−1. For simple complexation reaction Cu(II) ions were added to TCPP solution in borate buffer at pH = 9. After mixing the spectra were recorded. The experiments using large-radius metal ions (Me = Pb(II) or Cd(II)) were conducted as follows: first the complex with 10−5 mol L−1 TCPP was prepared ([Me]:[TCPP] = 1.5:1) by addition of respective metal ion to a solution of TCPP in borate buffer at pH = 9. The reaction was observed by recording the spectra until the PbTCPP and Cd-TCPP complexes were formed. Then Cu(II) ions were added and the spectra was recorded. For the reaction with the addition of reducing agents, respective compounds – ascorbic acid (in acetate buffer at pH = 5, freshly prepared), hydroxylamine and morin (water solutions, freshly prepared, molar ratio in relation to TCPP 1:1) were added to TCPP solution in borate buffer at pH = 9. Then Cu(II) ions were added and spectra was recorded.
3. Results and discussion TCPP solution at pH 9 in borate buffer exhibits five absorption maxima: one in Soret band at 414 nm (ε = 3.5 × 105 M−1) and four maxima in the Q band at 517, 554, 579 and 634 nm with the molar absorption coefficients of 1.2 × 104, 6.5 × 103, 5.2 × 103 and 3.3. × 103, respectively [19]. Soret band and Q bands are the effect of the π−π∗ transitions. The Soret band is an effect of strong transition from ground to S2 state, the Q bands are weaker transitions to first excited state. For TCPP relation between Q bands responds to “etio” type of spectra, where intensities of band are reduced with increase of wavelength (peak height at 517 nm N 554 nm N 579 nm N 634 nm). Addition of Cu(II) caused significant changes in ligand spectra (Fig. 2). Metallation of the macrocycle gives more symmetrical ring and the Q bands are reduced to two (α and β), representing for Cu the “hypso” type, blue shifted comparing to regular (d-filled) metalloporphyrins. The absorbance of ligand at 517 nm (as well as at 414 nm) was gradually decreasing and a new
K. Kilian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 123–127
Fig. 2. Changes in TCPP spectra upon addition of Cu(II) at pH 9 within insert the enlargement of Q region. [TCPP] = [Cu2+] = 10−5 M. Reaction time 0.5–240 min.
peak of specific absorbance developed slowly at 542 nm, which increased over time. The reaction comes to equilibrium in 120 min. The absorbance plot at 542 nm against the molar fraction of Cu(II) has maximum at XCu = 0.5, confirming that the stoichiometric ratio for the complexation is 1:1 (Fig. 1S). The rate of Cu-TCPP complex formation is much lower than those of acyclic ligands. The formation of metalloporphyrins can be accelerated substantially by the use of an auxiliary complexing agent (L-cysteine or 8-quinolinol) [16,17] or organic ligands with the extended πelectron structures (imidazole or bipyridine) [17,20]. The complexation reaction is also highly accelerated by the presence of metal ions having a large ionic radius such as Pb(II), Hg(II) or Cd(II) [19,21]. Large metal ions do not incorporate well into porphyrin core and just sit on the ligand plane forming so-called a “sitting-atop” (SAT) complex and their favorable complexation kinetic leads to highly expanded or distorted porphyrin skeleton [22]. It makes two diagonal pyrrolic nitrogens more accessible to a smaller metal ion on the other side of the ligand. Our study showed that metal-substitution reaction in Pb-TCPP complex by Zn(II) could be applied for spectrophotometric determination of trace amounts of zinc [19]. There is no large difference in the rate constants for Cd(II) and Pb(II) incorporation into carboxy-phenylporphyrin molecule, however, Cd-TCPP complex is less stable [19].
Fig. 3. Changes in absorption spectra during the addition of Cu(II) to Pb-TCPP complex. [Pb-TCPP] = 5 × 10−5 M, [Cu2+] = 10−5 M, pH 9. Reaction time 0.5–120 min.
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Fig. 3 shows the changes in absorption spectra of Cd-TCPP complex after addition of Cu(II) solution in borate buffer at pH 9. The characteristic spectra at 571 and 613 nm quickly disappeared and the absorption peak at 542 nm for Cu-TCPP complex was increasing over time. Similar situation was observed when Cu(II) ions were added to the solution containing Pb-TCPP complex at pH 9 (Fig. 4). Thus, as expected the presence of these metal ions having a large ionic radius greatly accelerates the formation of Cu-TCCP complex according to the SAT complex formation mechanism. As can be seen from Fig. 5, the reaction rate between copper ions and TCPP is much faster in the presence of Pb(II) or Cd(II). The large deformation of the porphyrin core in the Cu(II)-SAT complex can be interpreted in terms of both steric requirement and by electronic interaction with the Jahn–Teller distorted Cu2+ ion having d9 electronic configuration [16]. The reaction proceeds with the formation of an intermediate heterodinuclear metalloporphyrin in which two different metal ions are simultaneously bounded to TCPP on the opposite sides [22,23]. The rate of metalloporphyrin formation is also sensitive to the oxidation state of a given metal ion. Faster complexation reaction was observed by lower oxidation state metals such as iron(II) and cobalt(II) compared to iron(III) and cobalt(III) [23,24]. Thus, faster metalloporphyrin formation involving Cu(II) and TCPP should be obtained in the presence of some reducing agents. The ionic radius of Cu+ (96 pm) is significantly larger than that of Cu2 + (72 pm) and could deform the porphyrin nucleus allowing to attack Cu2+ from the lower side of the molecule. The generation of a SAT complex as intermediate is similar to the effect of the presence of Cd(II) or Pb(II) described earlier. Fig. 6 shows the catalytic effect of ascorbic acid on the reaction of Cu(II) with TCPP. The disappearance of absorbance at 517 nm (characteristic for ligand) and the increase at 542 nm represent the generation of metalloporphyrin. The reaction rate increased significantly with the increase of ascorbic acid concentration (Fig. 7). The steadystate conditions were reached in 120 min in the absence of ascorbic acid, but in its presence, at the 10-fold molar excess in relation to Cu(II), dropped below 1 min. In order to confirm that the catalytic effect of ascorbic acid on the formation of Cu-TCPP complex results from its reducing properties, we investigated also the formation of metalloporphyrin in the presence of other reducing agents such as hydroxylamine and morin. The last one belongs to flavonols, which are one of the most important groups of compounds occurring in plants. They have attracted considerable interest because of their strong antioxidant activity and associated health properties [25]. The copper(II) solution equilibrated with each of the reducing agents was mixed with the 10−5 M solution of TCPP. The recorded absorption spectra after addition
Fig. 4. Changes in absorption spectra during the addition of Cu(II) to Cd-TCPP complex. [Cd-TCPP] = 10−5 M, [Cu2+] = 10−5 M, pH 9. Reaction time 0.5–120 min.
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Fig. 7. The changes in absorbance at λmax over time for the reaction of Cu(II) and TCPP in the presence of ascorbic acid (AA) at different concentration. [Cu2+] = [TCCP] = 10−5 M. Fig. 5. The changes in absorbance at 524 nm over time for the reaction of Cu(II) and TCPP in the absence and the presence of Cd(II) and Pb(II). [Cu2+] = [TCCP] = 10−5 M, [Cd2+] = [Pb2+] = 1.5 10−5 M.
of morin solution (as an example) in borate buffer at pH 9 are presented in Supplementary Material (Fig. 2S). Fig. 8 presents the influence of reaction time for Cu-TCPP complex formation in the presence of hydroxylamine and morin as the reducing agents. The presence of hydroxylamine significantly improved the rate of Cu-TCPP complex formation. The reaction came to equilibrium in 7 min, while in the presence of morin in 15 min. The attempts to obtain evidence for an intermediate binuclear metalloporphyrin complex, in which two metal ions are simultaneously bound to a porphyrine molecule on the opposite sites, have failed. Funahashi et al. [22] using extended X-ray absorption fine structure method studied reaction between Cu(II) and 5,10,15,20-tetrakis(4sulfonatophenyl)porphyrin in the presence of Hg(II) and concluded that the intermediate was formed within 100 ms during 10 s after mixing the mercury-porphyrin solution and Cu(II) ions. Among different studied compounds that could accelerate the formation of Cu-TCPP complex, the addition of cadmium ions or ascorbic acid seems the most useful. However, as far as the radiopharmaceutical
Fig. 6. Changes in absorption spectra during the reaction of Cu(II) with TCPP in the presence of ascorbic acid. [Cu2+] = [TCCP] = 10−5 M, [AA] = 2 × 10−5 M. Reaction time 0.5–120 min.
application is concerned, the application of heavy metals during synthesis is limited. Thus, the best way to overcome the slow reaction rate is the in situ reduction of Cu2 + to Cu+ with ascorbic acid as reducing agent. The Cu+ ions having larger ionic radius than Cu2 + form SAT complex with porphyrin and thereby accelerate the formation of the final product Cu(II)-TCPP complex. Adopted reducing agent is safe and can be present in pharmaceutical formulations. The conditions for complex formation were optimized. The best reaction rate was achieved at pH 9 (in borate buffer) with the ratio of the reactants Cu:AA:TCPP as 1:10:1. Under these conditions the reaction was almost immediate (below 1 min). There were some published attempts, introducing the 64Cu to the porphyrin core but generally suffered from poor kinetics. Intensive heating was required for the labeling of TCPP (100°, 1 h) or TPPS (50°, 2 h) with ambient yield (58.6% for TCPP and 19.7% for TPPS), which increased after addition of ethanol to 80.4% and 68.6% respectively [26]. Other approach was used for labeling of porphyrin–peptide–folate [4], where the reaction mixture was heated in a waterbath at 60° for 20 min. Room temperature reduced the yield to 50–80%. The 64Cu radiolabeling of the
Fig. 8. The changes in absorbance at 542 nm over time for the reaction of Cu(II) and TCPP in the presence of hydroxylamine and morin. [Cu2+] = [TCCP] = [morin] = [hydroxylamine] = 10−5 M.
K. Kilian et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 159 (2016) 123–127
phtalocyanine, very similar to porphyrins, was conducted with microwave heating in 1 min at 150 W [27] but reached 40–50% yield. 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (H2TFPP) required 60 min heating at 100 °C under reflux condition to complex copper with satisfactory output. Ascorbic acid is used as stabilizer in many intravenously injected 99m Tc preparations, routinely applied in nuclear medicine [28]. The amounts reach several mg/per injection (for 99mTc-EC (ethylcysteine) 24 mg, 7 mg for 99mTcNanocolloids (Lymphoscint, GE Healthcare)) and the formulations are registered by respective regulators (FDA, EMEA). For formulation proposed in this work, total amount of added ascorbic acid is about 0.5 mg per injection. pH of the final formulation is very important for its biological properties and for injectable radiopharmaceuticals is set in range 4.5–8.5 (European Pharmacopoeia). The concentration of NaOH in the solution is relatively small (b10−5 mol L−1) additionally compensed by ascorbic acid to physiological pH. For clinical application addition of injectable buffer (e.g. PBS, acetate) could be recommended. 4. Conclusion The proposed method offers stable and fast chelation in room temperature and allows either labeling of thermally unstable compounds or further investigations on metalloporphyrins labeled with copper isotopes. The reaction is carried out at ambient temperature what is convenient for radiopharmaceutical application and “kit-concept”, known from 99mTc preparations could be concerned as a way of final formulation. The presented strategy of Cu(II)-TCPP synthesis can be potentially used in development of new PET radiopharmaceuticals labeled with 64 Cu or other copper radioisotopes, especially short-lived 60Cu and 62Cu. Acknowledgments Heavy Ion Laboratory gratefully acknowledges funding for the equipment from the Ministry of Health and from European Regional Development Fund under the Innovative Economy Program (POIG.02.02.0014-024/08-00) project: Center of Preclinical Studies and Technology (CePT). References [1] X. Ding, H. Xie, J. Kang, J. Nutr. Biochem. 22 (2011) 301–310, http://dx.doi.org/10. 1016/j.jnutbio.2010.06.010.
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[2] P. Rowshanfarzad, M. Sabetb, A.R. Jaliliana, M. Kamalidehghana, Appl. Radiat. Isot. 64 (2006) 1563–1573. [3] S.V. Smith, J. Inorg. Biochem. 98 (2004) 1874–1901, http://dx.doi.org/10.1016/j. jinorgbio.2004.06.009. [4] J. Shi, Tracy W.B. Liu, J. Chen, D. Green, D. Jaffray, B.C. Wilson, F. Wang, G. Zheng, Theranostics, 1 (2011) 363–370. doi:http://dx.doi.org/10.7150/thno/v01p0363 [5] W. Aung, Z.H. Jin, T. Furukawa, M. Claron, D. Boturyn, C. Sogawa, A.B. Tsuji, H. Wakizaka, T. Fukumura, Y. Fujibayashi, P. Dumy, T. Saga, Mol. Imaging 12 (6) (2013) 1–12, http://dx.doi.org/10.2310/7290.2013.00054. [6] W.C. Cole, S.J. DeNardo, C.F. Meares, M.J. McCall, G.L. DeNardo, A.L. Epstein, J. Nucl. Med. 28 (1987) 83–90. [7] T.M. Jones-Wilson, K.A. Deal, C.J. Anderson, D.W. McCarthy, Z. Kovacs, R. Motekaitis, Nucl. Med. Biol. 25 (1998) 523–530. [8] L.M. De Leon-Rodriguez, Z. Kovacs, Bioconjug. Chem. 19 (2008) 391–402, http://dx. doi.org/10.1021/bc700328s. [9] H.S. Chong, S. Mhaske, M. Lin, S. Bhuniya, H.A. Song, M.W. Brechbiel, X. Sun, Bioinorg. Med. Chem. Lett. 17 (2007) 6107–6110, http://dx.doi.org/10.1016/j.bmcl. 2007.09.052. [10] Sprague J.E., Peng Y., Fiamengo A.L, Woodin K.S., Southwick E.A., Weisman G.R., J. Med. Chem. 50 (2007) 2527–35. doi: http://dx.doi.org/10.1021/jm070204r [11] J.R. Dilworth, S.I. Pascu, P.A. Waghorn, D. Vullo, S.R. Bayly, M. Christlieb, X. Sun, C.T. Supuranc, Dalton Trans. 44 (2015) 4859–4873, http://dx.doi.org/10.1039/ C4DT03206C. [12] M. Ethirajan, Y. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011) 340–362, http://dx.doi.org/10.1039/B915149B. [13] A.E. O'Connor, W.M. Gallagher, A.T. Byrne, Photochem. Photobiol. 85 (2009) 1053–1074, http://dx.doi.org/10.1111/j.1751-1097.2009.00585.x. [14] T. Das, S. Chakraborty, H.D. Sarma, S. Banerjee, M. Venakatesh, Nucl. Med. Biol. 37 (2010) 655–663, http://dx.doi.org/10.1016/j.nucmedbio.2010.02.007. [15] L.B. Josefsen, R.W. Boyl, Theranostics 2 (2012) 916–966, http://dx.doi.org/10.7150/ thno.4571. [16] T. Makino, J. Itoh, Clin. Chim. Acta 111 (1) (1981) 1–8. [17] M. Tabata, M. Tanaka, Trend. Anal. Chem. 10 (1991) 128–133. [18] P.J. Blower, J.S. Lewis, J. Zweit, Nucl. Med. Biol. 23 (1996) 957–980. [19] K. Kilian, K. Pyrzynska, Talanta 60 (2003) 669–678, http://dx.doi.org/10.1016/ S0039-9140(03)00149-8. [20] R. Giovannetti, V. Bartocci, S. Ferraro, M. Gusteri, P. Passamonti, Talanta 42 (1995) 1913–1918. [21] Y. Qi, J.G. Pan, Bull. Kor. Chem. Soc. 35 (2014) 3313–3318, http://dx.doi.org/10.5012/ bkcs.2014.35.11.3313. [22] S. Funahashi, Y. Inada, M. Inamo, Anal. Sci. 17 (2001) 917–927. [23] Y. Inada, Y. Nakano, M. Inamo, M. Nomura, S. Funahashi, Inorg. Chem. 39 (2000) 4793–4801, http://dx.doi.org/10.1021/ic000479w. [24] M. Tabata, M. Babasaki, Inorg. Chem. 31 (1992) 5268–5271, http://dx.doi.org/10. 1021/ic00051a019. [25] N. Khan, H. Mukhar, Life Sci. 81 (2007) 519–533, http://dx.doi.org/10.1016/j.lfs. 2007.06.011. [26] H. Mukai, Y. Wada, Y. Watanabe, Ann.Nucl.Med. 27 (2013) 625–639, http://dx.doi. org/10.1007/s12149-013-0728-2. [27] E.R.Ranyuk, N. Cauchon, H.Ali, R.Lecomte, B.Guerrin, J.E.van Lier, Biorg.Med.Chem.Lett, 2011, 21, 7470–7473. doi: http://dx.doi.org/10.1016/j.bmcl.2011.09.121. [28] I. Zolle (Ed.), Technetium 99 m-Pharmaceuticals: Preparation and Quality Control in Nuclear Medicine, Berlin-Heidelberg 2007.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
The fast method of Cu-porphyrin complex synthesis for potential use in positron emission tomography imaging Krzysztof Kilian a,⁎, Maria Pęgier b, Krystyna Pyrzyńska b a b
Heavy Ion Laboratory, University of Warsaw, 5ath Pasteur Str., 02-093 Warsaw, Poland Faculty of Chemistry, University of Warsaw, 1st Pasteur Str., 02-093 Warsaw, Poland
a r t i c l e
i n f o
Article history: Received 8 September 2015 Received in revised form 12 January 2016 Accepted 23 January 2016 Available online 25 January 2016 Keywords: Copper-64 Porphyrin Positron emission tomography
a b s t r a c t Porphyrin based photosensitizers are useful agents for photodynamic therapy and fluorescence imaging of cancer. Additionally, porphyrins are excellent metal chelators, forming stable metalo-complexes and 64Cu isotope can serve as a positron emitter (t1/2 = 12.7 h). The other advantage of 64Cu is its decay characteristics that facilitates the use of 64Cu-porphyrin complex as a therapeutic agent. Thus, 64Cu chelation with porphyrin photosensitizer may become a simple and versatile labeling strategy for clinical positron emission tomography. The present study reports a convenient method for the synthesis of Cu complex with tetrakis(4carboxyphenyl)porphyrin (TCPP). The experimental conditions for labeling, such as the metal-to-ligand molar ratio, pH and time of reaction were optimized to achieve a high complexation efficiency in a short period of time as possible. In order to accelerate the metallation, the use of substitution reactions of cadmium or lead porphyrin and the presence of reducing agent, such as ascorbic acid, hydroxylamine and flavonoid – morin, were evaluated. The optimum conditions for the synthesis of the copper complex were borate buffer at pH 9 with the addition of 10-fold molar excess, with respect to Cu2+ ions and TCPP and ascorbic acid which resulted in reduction of the reaction time from 30 min to below 1 min. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The nuclear medicine imaging, in particular positron emission tomography (PET), allows the detection and monitoring of a variety of biological and pathophysiological processes, at tracer quantities of the radiolabeled target agents and at doses free from pharmacological effects. The use of radio metals for labeling has now become one of the essential tools in identifying, screening and development of new target agents. Copper plays significant role in human biochemistry and nutrition [1] and copper-labeled biologically active compounds could be used for imaging molecular background of diseases. 64Cu is described in the literature as a potentially effective diagnostic radioisotope, used in PET. The quality of imaging is in some applications, comparable to the quality of imaging with 18F isotope [2], which is one of the most frequently used in PET. 64Cu is produced in medical cyclotrons via 64 Ni(p,n)64Cu reaction, forming isotopically pure 64Cu. Copper – 64, because of its decay scheme (t1/2 = 12.7 h, β+: 17.4%, Eβ+max = 656 keV; β−: 39%, Eβ-max = 573 keV) is also used as a therapeutic agent [2]. Relatively long half-life of this isotope (in relation to other PET radioisotopes) offers an opportunity to label larger biomolecules. On the other
⁎ Corresponding author. E-mail address: [email protected] (K. Kilian).
http://dx.doi.org/10.1016/j.saa.2016.01.045 1386-1425/© 2016 Elsevier B.V. All rights reserved.
hand, a wide half-live range of other copper isotopes (τ1/2 60Cu = 23.7 min, τ1/2 61Cu = 3.4 h, τ1/2 62Cu = 9.7 min) allows imaging processes with different dynamics, while the identical chemistry of the labeling process is maintained. Various types of chelating ligands were evaluated as the chelators for copper radioisotopes, such as polyaminocarbonoxylates, cyclic polyamines, tetraaza-macrocycles and bis-thiosemicarbazones [3–8]. The selection of reagents for use in binding copper isotopes is largely dependent on the ligands forming very stable complexes. Acyclic chelates lack kinetic stability [6], thus, macrocyclic ligands are preferred [7]. The thermodynamic stability of the respective classes of ligands for Cu(II) complexes decreases in the order: hexaaza cages N tetrazamacrocycles N polyaminophosphonates N polyaminocarboxylate macrocycles N open chain aminocarboxylates [3]. DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid) is the most often used macrocyclic metals chelate, including 64Cu, in radiopharmaceutical research [8]. However, DOTA is not an ideal ligand for 64Cu because of slow reaction kinetics [9]. 1,4,7-triazacyclononane-1,4,7-triacetic acid derivatives [9] as well as cross-bridged 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid [10] have superior stability in comparison to DOTA, but require harsher radiolabeling conditions. The most common in the literature 64 Cu labeled compound is diacetyl-bis(N-4-methyl thiosemicarbazone (ATSM) [11]. 64Cu-ATSM molecules are lipophilic, small and selectively absorbed by the hypoxic tissues and can be used to study the cardiovascular system, with focus on hypoxic areas.
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From the other side, the rapid complexation of metal ion with a ligand is an important criterion in developing radiopharmaceuticals since the radioisotope is decaying throughout its preparation and application. This is a serious limitation of many radiolabeling protocols which usually employ a long incubation times (up to 1 h) at room or elevated temperature and an excess of ligand is needed to achieve sufficient complexation yield. However, the use of ligand excess in formulations should be avoided as many targeting agents are biologically active in themselves. Porphyrins and their derivatives show significant affinity to cancer cells and are used for diagnosis and photodynamic therapy (PDT). In clinical practice, Photofrin® and Visudyne® are used for PDT treatment of lung, esophagus, bladder cancer and skin inflammations [12,13]. Thus, porphyrins are potentially suitable as bifunctional chelates and vehicles for delivery of copper radioisotopes to selected tissues. Porphyrins also have their own useful tissue-targeting properties, such as selective accumulation in inflammatory and lymphatic tissues. Hence, porphyrin labeled with suitable therapeutic or diagnostic radionuclide could be envisaged as potential agents for tumor therapy and diagnostics [14]. They are used in tumor phototherapy in large doses, and they are relatively nontoxic and well tolerated [15]. The disadvantage is the high kinetic barrier to metallation, but copper complexes with porphyrins are exceedingly robust to demetallation [16,17]. Porphyrins have the ability to chelate metal ions, due to the system of four pyrrole nitrogen atoms, highly selective for ions of ionic radius of about 70 pm (ionic radius of Cu2+ is 72 pm). Complexes, although characterized by high values of stability constants, are formed with comparatively poor kinetics, which has led to criticism in earlier work [18]. The above restriction was removed by using the reaction mechanism of sitting-a-top (SAT) [19]. Chelating properties are not significantly affected by the type and number of substituents in the ring, allowing application of the appropriate porphyrin for the intended purpose of imaging (such as hydrophilicity/hydrophobicity and partition coefficient octanol/water) The goal was to develop improved methods for synthesis and physico-chemical characteristics of copper complexes with TCPP, as potential diagnostic and therapeutic radiopharmaceuticals for nuclear medicine. 2. Experimental 2.1. Reagents 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (TCPP) was obtained from Fluka (Germany). Stock solutions (10−3 mol L−1) of TCPP were prepared by dissolution of 0.1976 g in 200 mL of 10 mmol L −1 sodium hydroxide and further dilution with deionized water up to 250 mL. The structure of this porphyrin is shown in Fig. 1. Under basic conditions the carboxylic groups are in –COONa form. 1000 mg L−1 stock solutions of Cu, Pb and Cd as nitrates in 2–5% nitric acid were obtained from Merck (Germany). Further dilutions were done with deionized water. Fresh stock solution of ascorbic acid (10−1 mol L−1) in acetate buffer at pH = 5 was prepared every day. The components of borate buffer as well as other reagents were of analytical grade and used without further purification. 2.2. Apparatus Double-beam UV–Vis spectrophotometer Lambda 20 (Perkin Elmer, USA) with diode-array detector was used for spectra recording and absorbance measurements. Spectra were recorded in the range from 350 to 700 nm with 0.2 nm resolution in 10 mm quartz cells. Data were processed with WinLab software. A Radelkis (Hungary) pH-meter with Orion (USA) electrode was used for pH measurement and adjustment.
Fig. 1. Chemical structure of TCPP.
2.3. Procedures All experiments were conducted using molar ratio of Cu(II) to TCPP being 1:1. Concentration of the reagents was 10−5 mol L−1. For simple complexation reaction Cu(II) ions were added to TCPP solution in borate buffer at pH = 9. After mixing the spectra were recorded. The experiments using large-radius metal ions (Me = Pb(II) or Cd(II)) were conducted as follows: first the complex with 10−5 mol L−1 TCPP was prepared ([Me]:[TCPP] = 1.5:1) by addition of respective metal ion to a solution of TCPP in borate buffer at pH = 9. The reaction was observed by recording the spectra until the PbTCPP and Cd-TCPP complexes were formed. Then Cu(II) ions were added and the spectra was recorded. For the reaction with the addition of reducing agents, respective compounds – ascorbic acid (in acetate buffer at pH = 5, freshly prepared), hydroxylamine and morin (water solutions, freshly prepared, molar ratio in relation to TCPP 1:1) were added to TCPP solution in borate buffer at pH = 9. Then Cu(II) ions were added and spectra was recorded.
3. Results and discussion TCPP solution at pH 9 in borate buffer exhibits five absorption maxima: one in Soret band at 414 nm (ε = 3.5 × 105 M−1) and four maxima in the Q band at 517, 554, 579 and 634 nm with the molar absorption coefficients of 1.2 × 104, 6.5 × 103, 5.2 × 103 and 3.3. × 103, respectively [19]. Soret band and Q bands are the effect of the π−π∗ transitions. The Soret band is an effect of strong transition from ground to S2 state, the Q bands are weaker transitions to first excited state. For TCPP relation between Q bands responds to “etio” type of spectra, where intensities of band are reduced with increase of wavelength (peak height at 517 nm N 554 nm N 579 nm N 634 nm). Addition of Cu(II) caused significant changes in ligand spectra (Fig. 2). Metallation of the macrocycle gives more symmetrical ring and the Q bands are reduced to two (α and β), representing for Cu the “hypso” type, blue shifted comparing to regular (d-filled) metalloporphyrins. The absorbance of ligand at 517 nm (as well as at 414 nm) was gradually decreasing and a new
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Fig. 2. Changes in TCPP spectra upon addition of Cu(II) at pH 9 within insert the enlargement of Q region. [TCPP] = [Cu2+] = 10−5 M. Reaction time 0.5–240 min.
peak of specific absorbance developed slowly at 542 nm, which increased over time. The reaction comes to equilibrium in 120 min. The absorbance plot at 542 nm against the molar fraction of Cu(II) has maximum at XCu = 0.5, confirming that the stoichiometric ratio for the complexation is 1:1 (Fig. 1S). The rate of Cu-TCPP complex formation is much lower than those of acyclic ligands. The formation of metalloporphyrins can be accelerated substantially by the use of an auxiliary complexing agent (L-cysteine or 8-quinolinol) [16,17] or organic ligands with the extended πelectron structures (imidazole or bipyridine) [17,20]. The complexation reaction is also highly accelerated by the presence of metal ions having a large ionic radius such as Pb(II), Hg(II) or Cd(II) [19,21]. Large metal ions do not incorporate well into porphyrin core and just sit on the ligand plane forming so-called a “sitting-atop” (SAT) complex and their favorable complexation kinetic leads to highly expanded or distorted porphyrin skeleton [22]. It makes two diagonal pyrrolic nitrogens more accessible to a smaller metal ion on the other side of the ligand. Our study showed that metal-substitution reaction in Pb-TCPP complex by Zn(II) could be applied for spectrophotometric determination of trace amounts of zinc [19]. There is no large difference in the rate constants for Cd(II) and Pb(II) incorporation into carboxy-phenylporphyrin molecule, however, Cd-TCPP complex is less stable [19].
Fig. 3. Changes in absorption spectra during the addition of Cu(II) to Pb-TCPP complex. [Pb-TCPP] = 5 × 10−5 M, [Cu2+] = 10−5 M, pH 9. Reaction time 0.5–120 min.
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Fig. 3 shows the changes in absorption spectra of Cd-TCPP complex after addition of Cu(II) solution in borate buffer at pH 9. The characteristic spectra at 571 and 613 nm quickly disappeared and the absorption peak at 542 nm for Cu-TCPP complex was increasing over time. Similar situation was observed when Cu(II) ions were added to the solution containing Pb-TCPP complex at pH 9 (Fig. 4). Thus, as expected the presence of these metal ions having a large ionic radius greatly accelerates the formation of Cu-TCCP complex according to the SAT complex formation mechanism. As can be seen from Fig. 5, the reaction rate between copper ions and TCPP is much faster in the presence of Pb(II) or Cd(II). The large deformation of the porphyrin core in the Cu(II)-SAT complex can be interpreted in terms of both steric requirement and by electronic interaction with the Jahn–Teller distorted Cu2+ ion having d9 electronic configuration [16]. The reaction proceeds with the formation of an intermediate heterodinuclear metalloporphyrin in which two different metal ions are simultaneously bounded to TCPP on the opposite sides [22,23]. The rate of metalloporphyrin formation is also sensitive to the oxidation state of a given metal ion. Faster complexation reaction was observed by lower oxidation state metals such as iron(II) and cobalt(II) compared to iron(III) and cobalt(III) [23,24]. Thus, faster metalloporphyrin formation involving Cu(II) and TCPP should be obtained in the presence of some reducing agents. The ionic radius of Cu+ (96 pm) is significantly larger than that of Cu2 + (72 pm) and could deform the porphyrin nucleus allowing to attack Cu2+ from the lower side of the molecule. The generation of a SAT complex as intermediate is similar to the effect of the presence of Cd(II) or Pb(II) described earlier. Fig. 6 shows the catalytic effect of ascorbic acid on the reaction of Cu(II) with TCPP. The disappearance of absorbance at 517 nm (characteristic for ligand) and the increase at 542 nm represent the generation of metalloporphyrin. The reaction rate increased significantly with the increase of ascorbic acid concentration (Fig. 7). The steadystate conditions were reached in 120 min in the absence of ascorbic acid, but in its presence, at the 10-fold molar excess in relation to Cu(II), dropped below 1 min. In order to confirm that the catalytic effect of ascorbic acid on the formation of Cu-TCPP complex results from its reducing properties, we investigated also the formation of metalloporphyrin in the presence of other reducing agents such as hydroxylamine and morin. The last one belongs to flavonols, which are one of the most important groups of compounds occurring in plants. They have attracted considerable interest because of their strong antioxidant activity and associated health properties [25]. The copper(II) solution equilibrated with each of the reducing agents was mixed with the 10−5 M solution of TCPP. The recorded absorption spectra after addition
Fig. 4. Changes in absorption spectra during the addition of Cu(II) to Cd-TCPP complex. [Cd-TCPP] = 10−5 M, [Cu2+] = 10−5 M, pH 9. Reaction time 0.5–120 min.
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Fig. 7. The changes in absorbance at λmax over time for the reaction of Cu(II) and TCPP in the presence of ascorbic acid (AA) at different concentration. [Cu2+] = [TCCP] = 10−5 M. Fig. 5. The changes in absorbance at 524 nm over time for the reaction of Cu(II) and TCPP in the absence and the presence of Cd(II) and Pb(II). [Cu2+] = [TCCP] = 10−5 M, [Cd2+] = [Pb2+] = 1.5 10−5 M.
of morin solution (as an example) in borate buffer at pH 9 are presented in Supplementary Material (Fig. 2S). Fig. 8 presents the influence of reaction time for Cu-TCPP complex formation in the presence of hydroxylamine and morin as the reducing agents. The presence of hydroxylamine significantly improved the rate of Cu-TCPP complex formation. The reaction came to equilibrium in 7 min, while in the presence of morin in 15 min. The attempts to obtain evidence for an intermediate binuclear metalloporphyrin complex, in which two metal ions are simultaneously bound to a porphyrine molecule on the opposite sites, have failed. Funahashi et al. [22] using extended X-ray absorption fine structure method studied reaction between Cu(II) and 5,10,15,20-tetrakis(4sulfonatophenyl)porphyrin in the presence of Hg(II) and concluded that the intermediate was formed within 100 ms during 10 s after mixing the mercury-porphyrin solution and Cu(II) ions. Among different studied compounds that could accelerate the formation of Cu-TCPP complex, the addition of cadmium ions or ascorbic acid seems the most useful. However, as far as the radiopharmaceutical
Fig. 6. Changes in absorption spectra during the reaction of Cu(II) with TCPP in the presence of ascorbic acid. [Cu2+] = [TCCP] = 10−5 M, [AA] = 2 × 10−5 M. Reaction time 0.5–120 min.
application is concerned, the application of heavy metals during synthesis is limited. Thus, the best way to overcome the slow reaction rate is the in situ reduction of Cu2 + to Cu+ with ascorbic acid as reducing agent. The Cu+ ions having larger ionic radius than Cu2 + form SAT complex with porphyrin and thereby accelerate the formation of the final product Cu(II)-TCPP complex. Adopted reducing agent is safe and can be present in pharmaceutical formulations. The conditions for complex formation were optimized. The best reaction rate was achieved at pH 9 (in borate buffer) with the ratio of the reactants Cu:AA:TCPP as 1:10:1. Under these conditions the reaction was almost immediate (below 1 min). There were some published attempts, introducing the 64Cu to the porphyrin core but generally suffered from poor kinetics. Intensive heating was required for the labeling of TCPP (100°, 1 h) or TPPS (50°, 2 h) with ambient yield (58.6% for TCPP and 19.7% for TPPS), which increased after addition of ethanol to 80.4% and 68.6% respectively [26]. Other approach was used for labeling of porphyrin–peptide–folate [4], where the reaction mixture was heated in a waterbath at 60° for 20 min. Room temperature reduced the yield to 50–80%. The 64Cu radiolabeling of the
Fig. 8. The changes in absorbance at 542 nm over time for the reaction of Cu(II) and TCPP in the presence of hydroxylamine and morin. [Cu2+] = [TCCP] = [morin] = [hydroxylamine] = 10−5 M.
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phtalocyanine, very similar to porphyrins, was conducted with microwave heating in 1 min at 150 W [27] but reached 40–50% yield. 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (H2TFPP) required 60 min heating at 100 °C under reflux condition to complex copper with satisfactory output. Ascorbic acid is used as stabilizer in many intravenously injected 99m Tc preparations, routinely applied in nuclear medicine [28]. The amounts reach several mg/per injection (for 99mTc-EC (ethylcysteine) 24 mg, 7 mg for 99mTcNanocolloids (Lymphoscint, GE Healthcare)) and the formulations are registered by respective regulators (FDA, EMEA). For formulation proposed in this work, total amount of added ascorbic acid is about 0.5 mg per injection. pH of the final formulation is very important for its biological properties and for injectable radiopharmaceuticals is set in range 4.5–8.5 (European Pharmacopoeia). The concentration of NaOH in the solution is relatively small (b10−5 mol L−1) additionally compensed by ascorbic acid to physiological pH. For clinical application addition of injectable buffer (e.g. PBS, acetate) could be recommended. 4. Conclusion The proposed method offers stable and fast chelation in room temperature and allows either labeling of thermally unstable compounds or further investigations on metalloporphyrins labeled with copper isotopes. The reaction is carried out at ambient temperature what is convenient for radiopharmaceutical application and “kit-concept”, known from 99mTc preparations could be concerned as a way of final formulation. The presented strategy of Cu(II)-TCPP synthesis can be potentially used in development of new PET radiopharmaceuticals labeled with 64 Cu or other copper radioisotopes, especially short-lived 60Cu and 62Cu. Acknowledgments Heavy Ion Laboratory gratefully acknowledges funding for the equipment from the Ministry of Health and from European Regional Development Fund under the Innovative Economy Program (POIG.02.02.0014-024/08-00) project: Center of Preclinical Studies and Technology (CePT). References [1] X. Ding, H. Xie, J. Kang, J. Nutr. Biochem. 22 (2011) 301–310, http://dx.doi.org/10. 1016/j.jnutbio.2010.06.010.
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[2] P. Rowshanfarzad, M. Sabetb, A.R. Jaliliana, M. Kamalidehghana, Appl. Radiat. Isot. 64 (2006) 1563–1573. [3] S.V. Smith, J. Inorg. Biochem. 98 (2004) 1874–1901, http://dx.doi.org/10.1016/j. jinorgbio.2004.06.009. [4] J. Shi, Tracy W.B. Liu, J. Chen, D. Green, D. Jaffray, B.C. Wilson, F. Wang, G. Zheng, Theranostics, 1 (2011) 363–370. doi:http://dx.doi.org/10.7150/thno/v01p0363 [5] W. Aung, Z.H. Jin, T. Furukawa, M. Claron, D. Boturyn, C. Sogawa, A.B. Tsuji, H. Wakizaka, T. Fukumura, Y. Fujibayashi, P. Dumy, T. Saga, Mol. Imaging 12 (6) (2013) 1–12, http://dx.doi.org/10.2310/7290.2013.00054. [6] W.C. Cole, S.J. DeNardo, C.F. Meares, M.J. McCall, G.L. DeNardo, A.L. Epstein, J. Nucl. Med. 28 (1987) 83–90. [7] T.M. Jones-Wilson, K.A. Deal, C.J. Anderson, D.W. McCarthy, Z. Kovacs, R. Motekaitis, Nucl. Med. Biol. 25 (1998) 523–530. [8] L.M. De Leon-Rodriguez, Z. Kovacs, Bioconjug. Chem. 19 (2008) 391–402, http://dx. doi.org/10.1021/bc700328s. [9] H.S. Chong, S. Mhaske, M. Lin, S. Bhuniya, H.A. Song, M.W. Brechbiel, X. Sun, Bioinorg. Med. Chem. Lett. 17 (2007) 6107–6110, http://dx.doi.org/10.1016/j.bmcl. 2007.09.052. [10] Sprague J.E., Peng Y., Fiamengo A.L, Woodin K.S., Southwick E.A., Weisman G.R., J. Med. Chem. 50 (2007) 2527–35. doi: http://dx.doi.org/10.1021/jm070204r [11] J.R. Dilworth, S.I. Pascu, P.A. Waghorn, D. Vullo, S.R. Bayly, M. Christlieb, X. Sun, C.T. Supuranc, Dalton Trans. 44 (2015) 4859–4873, http://dx.doi.org/10.1039/ C4DT03206C. [12] M. Ethirajan, Y. Chen, P. Joshi, R.K. Pandey, Chem. Soc. Rev. 40 (2011) 340–362, http://dx.doi.org/10.1039/B915149B. [13] A.E. O'Connor, W.M. Gallagher, A.T. Byrne, Photochem. Photobiol. 85 (2009) 1053–1074, http://dx.doi.org/10.1111/j.1751-1097.2009.00585.x. [14] T. Das, S. Chakraborty, H.D. Sarma, S. Banerjee, M. Venakatesh, Nucl. Med. Biol. 37 (2010) 655–663, http://dx.doi.org/10.1016/j.nucmedbio.2010.02.007. [15] L.B. Josefsen, R.W. Boyl, Theranostics 2 (2012) 916–966, http://dx.doi.org/10.7150/ thno.4571. [16] T. Makino, J. Itoh, Clin. Chim. Acta 111 (1) (1981) 1–8. [17] M. Tabata, M. Tanaka, Trend. Anal. Chem. 10 (1991) 128–133. [18] P.J. Blower, J.S. Lewis, J. Zweit, Nucl. Med. Biol. 23 (1996) 957–980. [19] K. Kilian, K. Pyrzynska, Talanta 60 (2003) 669–678, http://dx.doi.org/10.1016/ S0039-9140(03)00149-8. [20] R. Giovannetti, V. Bartocci, S. Ferraro, M. Gusteri, P. Passamonti, Talanta 42 (1995) 1913–1918. [21] Y. Qi, J.G. Pan, Bull. Kor. Chem. Soc. 35 (2014) 3313–3318, http://dx.doi.org/10.5012/ bkcs.2014.35.11.3313. [22] S. Funahashi, Y. Inada, M. Inamo, Anal. Sci. 17 (2001) 917–927. [23] Y. Inada, Y. Nakano, M. Inamo, M. Nomura, S. Funahashi, Inorg. Chem. 39 (2000) 4793–4801, http://dx.doi.org/10.1021/ic000479w. [24] M. Tabata, M. Babasaki, Inorg. Chem. 31 (1992) 5268–5271, http://dx.doi.org/10. 1021/ic00051a019. [25] N. Khan, H. Mukhar, Life Sci. 81 (2007) 519–533, http://dx.doi.org/10.1016/j.lfs. 2007.06.011. [26] H. Mukai, Y. Wada, Y. Watanabe, Ann.Nucl.Med. 27 (2013) 625–639, http://dx.doi. org/10.1007/s12149-013-0728-2. [27] E.R.Ranyuk, N. Cauchon, H.Ali, R.Lecomte, B.Guerrin, J.E.van Lier, Biorg.Med.Chem.Lett, 2011, 21, 7470–7473. doi: http://dx.doi.org/10.1016/j.bmcl.2011.09.121. [28] I. Zolle (Ed.), Technetium 99 m-Pharmaceuticals: Preparation and Quality Control in Nuclear Medicine, Berlin-Heidelberg 2007.
