Fonslet, Jesper

Severin, Gregory

Zhuravlev, Fedor

IntroductionTitanium-45, a candidate PET isotope, is under-employed largely because of the challenging aqueous chemistry of Ti(IV). The propensity for hydrolysis of Ti(IV) compounds makes radio-labeling difficult and excludes 45Ti from use in bio-conjugate chemistry. This is unfortunate because the physical characteristics are extremely desirable: 45Ti has a 3 hour half-life, a positron branching ratio of 85 %, a low Eβmax of 1.04 MeV, and negligible secondary gamma emission. In terms of isotope production, 45Ti is transmuted from naturally mono-isotopic 45Sc by low energy proton irradiation. The high cross-section and production rates on an unenriched metal foil target contribute to make 45Ti an ideal PET radionuclide.In order to bring 45Ti to even a preclinical plat-form, the hydrolytic instability of aqueous Ti(IV) needs to be addressed. Recently, the groups of Edit Tshuva (Hebrew University of Jerusalem) and Thomas Huhn (University of Konstanz) have synthesized several stable Ti(IV) compounds based upon the salan ligand [1,2]. Additionally, these compounds have shown heightened cyto-toxicity against HT-29 (human colorectal cancer) cells, amongst others, as compared to traditional metal-based chemotherapeutics such as cisplatin.The aim of our work has been to produce the radioactive analogue of one of these Ti(IV)-salan compounds, Ti-salan-dipic [2], which has hydro-lytic stability on the order of weeks. Not only will this allow us to shed some light on the still un-known mechanism of antiproliferative action of titanium-based chemotherapeutics, but it will also make progress toward bioconjugate 45Ti PET tracers.In the current abstract, we present some of the methods we are using to separate 45Ti from irradiated Sc, and subsequent labeling conditions.Material and Methods45Ti was produced by proton irradiation of 250μm scandium foils at currents ranging from 10-20μA on a GE PETTrace. In order to increase production rate in the thin foil, an 800μm aluminum degrader was used to take the proton energy down from the nominal 16 MeV. The scandium was cooled by contact to a water-cooled silver plate.The activated foil was dissolved in 4M HCl, dried under argon at 120 oC, and taken back up in 12M HCl. Here, four (i-iv below) different approaches to removing the Ti from the Sc and labeling were taken with varying success.Briefly: i. 45Ti was separated on hydroxamate resin, as presented by K. Gagnon [3], only at 12M acid concentration followed by on-column radiolabeling. ii. 45Ti was extracted into 1-octanol [4], stripped with 12M HCl, and used directly for labeling from the organic phase. iii. 45Ti was trapped on a C-18 cartridge that had been pre-loaded with 1-octanol, similar to ion-pairing, and eluted with isopropanol. iv. 45Ti was extracted onto a polystyrene based 1,3 diol resin (RAPP polymers) and labeling commenced on the column.Radiolabeling was slightly different in each condition, but in general the salan and dipic ligands were added to the 45Ti in pyridine and reacted at elevated temperature (60–100 oC) for several (10–30) minutes. Reaction progression and radiochemical purity were assessed with silica TLC in chloroform : ethyl acetate (1 : 1).Results and ConclusionThe trap, release, and yields for the four methods listed above are shown in TABLE 1. The best result was with the 1,3 diol resin which had the added advantage of reacting on-column.Further optimization is underway including a test of a solid supported 1,2 diol, and preclinical imaging with HT-29 xenografts.We conclude that hydrolytically stable 45Ti com-pounds can be synthesized in high yield, and hope that this advances the radiochemistry and use of 45Ti toward more widespread applications

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Downloaded from orbit.dtu.dk on: Dec 21, 2017Hydrolytically stable titanium-45Severin, Gregory; Fonslet, Jesper; Zhuravlev, FedorPublished in:Proceedings of the 15th International Workshop on Targetry and Target ChemistryPublication date:2014Document VersionPublisher's PDF, also known as Version of recordLink back to DTU OrbitCitation (APA):Severin, G., Fonslet, J., & Zhuravlev, F. (2014). Hydrolytically stable titanium-45. In Proceedings of the 15thInternational Workshop on Targetry and Target Chemistry Prague, Czech Republic.*Corresponding author, E-mail: fezh@dtu.dk Hydrolytically Stable Titanium-45 G.W. Severin, J. Fonslet, F. Zhuravlev*  Hevesy Laboratory, Center for Nuclear Technologies, Technical University of Denmark   Introduction  Titanium-45, a candidate PET isotope, is under-employed largely because of the challenging aqueous chemistry of Ti(IV).  The propensity for hydrolysis of Ti(IV) compounds makes radio-labeling difficult and currently excludes 45Ti from use in bio-conjugate chemistry.  This is unfortu-nate because the physical characteristics are extremely desirable (FIG. 1): 45Ti has a 3 hour half-life, a positron branching ratio of 85%, a low Emax of 1.04 MeV, and negligible secondary gamma emission1.  In terms of isotope produc-tion, 45Ti is transmuted from naturally mono-isotopic 45Sc by low energy proton irradiation.  The high cross-section (300-400 mb from 7.8-14.4 MeV2) and resulting production rates on an unenriched metal foil target contribute to make 45Ti an ideal PET radionuclide.       FIGURE 1:  The simplified decay scheme of 45Ti In order to even bring 45Ti to a preclinical setting, the hydrolytic instability of aqueous Ti(IV) needs to be addressed.   Titanium citrate is a good example of a Ti(IV) complex that is hydrolytically stable at physio-logical pH3, and is the basis for two of the very few preclinical radio-tracing publications em-ploying 45Ti4,5. The results from Vavere and Welch4 showed that 45Ti administered as citrate rapidly transchelated to transferrin (Tf) in se-rum.  While this property proved useful for tracking the distribution of Tf, it precludes the use of Ti-citrate as a platform for bioconjugate chemistry.  Recently, the groups of Edit Tshuva (Hebrew University of Jerusalem) and Thomas Huhn (Uni-versity of Konstanz) have synthesized several hydrolytically stable Ti(IV) compounds based upon the salan ligand (a diamine bisphenolato complex)6–9. Interestingly, many of these com-pounds have shown heightened cytotoxicity against HT-29 and other cell lines compared to traditional metal-based chemotherapeutics such as cis-platin8.   The salan-based titanium compounds prompt two interesting topics for radiochemical investi-gation.  First, the biodistribution of these anti-proliferative agents may be traced with 45Ti.  Second, the ligand system may be exploited as a scaffold for bioconjugate chemistry with 45Ti.  The aim of our work is to investigate different methods of producing the radioactive analogue of one of these Ti(IV)-salan compounds, Ti-salan-dipic7 (depicted in FIG. 2), which has hydrolytic stability on the order of weeks, and is TLC-traceable.  In the current report we present some of the methods we surveyed to separate 45Ti from irradiated scandium. Reactions with salan and dipic (pyridine-2,6-dicarboxylic acid) are presented as assessments of the chemical viability of the extracted 45Ti by each method.  FIGURE 2: A structural depiction of Ti-salan-dipic Material and Methods  45Ti was produced by proton irradiation of 250 μm scandium foils (Alfa Aesar) at currents rang-ing from 10-20 μA on a GE PETTrace. A 500 μm aluminum degrader was used to take the proton energy down from the nominal 16.5 MeV.  The scandium was cooled by contact to a water-cooled silver plate.   The activated foil was dissolved in 4 M HCl, dried under argon at 120 oC, and taken back up in 12  M HCl.  The intention of using 12 M HCl was to restrict hydrolysis in the aqueous environment, and revert any titanyl ions to Ti(IV) chlorides 10.  Next, four (i-iv below) different approaches to 45Ti 45Sc 3.08 hstable85% β+ 15 % ECEave β+= 439 keVQEC = 2.06 MeV*Corresponding author, E-mail: fezh@dtu.dk recovering the 45Ti from the aqueous environ-ment were taken with varying success. Radio-chemical purity and reaction yield was assessed with silica TLC in 1:1 chloroform : ethyl acetate.  When necessary, a co-spot of non-radioactive Ti-salan-dipic (clearly visible, distinctive orange color) was added in order to confirm the Rf val-ue, which varied from 0.53-0.60 depending on residual solvents.    i. Hydroxamate Resin:  45Ti was separated on hydroxamate resin, as presented by K. Gagnon11 with minor modifica-tions.  Briefly, hydroxamate resin was prepared as described by Holland et al.12, and washed with 12 M HCl.  The dissolved and reconstituted Sc (16 mg) plus 45Ti in 12 M HCl (1 mL) was passed slowly over approximately 100 mg resin in an 8mm ID column with polyethylene frits. Flow was controlled by gravity. Salan in pyridine (20 mg, 1 mL) was passed through, followed by addition of dipic in pyridine (10 mg, 1 mL) while heating.  The lability of 45Ti remaining on the column was determined by passing 1 M citric acid over the column and trapping the eluted activity on a QMA Sep-Pak cartridge (Waters).  ii. Solvent Extraction:  45Ti was extracted into 1-octanol following the method reported by Siikanen et al13.  First, the activity and Sc laden HCl (2.8 mg in 1.1 mL) was vortexed with 1-octanol (3 mL) and allowed to settle.  To strip the Sc, the aqueous phase was removed, and replaced with 1 mL of fresh 12 M HCl.  The mixture was vortexed again, settled, and the aqueous phase removed. Then the or-ganic phase was stripped again and settled by centrifugation. Finally, 1 mL of the organic phase was used directly for radiolabeling by addition of 1 mL salan in pyridine (50 mg/mL) followed by mixing with 1 mL dipic in pyridine (23 mg/mL) at 60 oC for 15 minutes.   iii. Hydrophobicity Pairing: As a next approach, the solvent extraction method was transferred to solid phase. Here, 45Ti was trapped on a C-18 cartridge that had been pre-loaded with 1-hexanol, similar to ion-pairing.  The C-18 was prepped with 1 mL 1-hexanol, and then washed with 0.5 mL 12 M HCl.  The Sc plus 45Ti in HCl (7 mg Sc, 200 µL) was passed over the cartridge, followed by 1 mL HCl wash. The cartridge was eluted with 1 mL iso-propanol. Salan in pyridine (63 mg/mL) and dipic in pyridine (32 mg/mL) were added to the iso-propanol, and mixed for 15 min at 60 oC.  iv. Extraction onto solid supported 1,3 diol: 158 mg Hypogel 200 diol (RAPP polymers) was equilibrated for 20 minutes in 12 M HCl, and packed into an 8 mm ID column with polyeth-ylene frits. 500 µL of reconstituted Sc/45Ti was added to the top, and dripped through with light pressure (approximately 1 drop per minute).  The resin was then washed with 500 µL more HCl and rinsed with dry acetonitrile. Next, 1 mL salan in pyridine (60 mg/mL) was pushed through slowly, followed by dipic in pyridine (1 mL, 30 mg).    Results and Discussion  The results and observations from each method described above are given in the next sections: i.  Hydroxamate Resin: Upon addition of the dissolved Sc to the column, the top of the resin showed a bright yellow band, and the activity was almost quantitatively trapped (99%).  Addition of salan and dipic had no apparent effect upon the 45Ti, which was not moved from the resin in either case.  Washes with pyridine and methanol also did not remove activity from the column as would be expected if the [45Ti] Ti-salan-dipic had been successfully synthesized.   As a test, the resin was washed with 1M citric acid with the eluent passing over a QMA Sep-Pak.  In our experience citric acid removes large quantities of unreacted 45Ti from the resin in a form that can be trapped on the QMA resin.  This characteristic behavior was observed in the current case, indicating that the hydroxamate resin restricted reaction with salan and dipic, but did not irreversibly bind the titanium.  ii. Solvent extraction: The solvent extraction method did not deviate from the expected behavior. Overall, after three emulsions and settlings, the organic phase con-tained 48% of the 45Ti activity.  Because the organic phase was used directly for synthesis, back extraction methods were not tested. Upon addition of salan in pyridine, white fuming was observed. This is likely due to the quenching of residual HCl in the organic phase by the basic pyridine.  The reaction yield was 53%, giving an overall yield of 25%.  The incomplete reaction can possibly be due to hydrolysis of Ti from residual water in the octanol after the extraction process. Additionally, 1-octanol presented a problem on the TLC by splitting the product peak.  This splitting was also observed with the non-radioactive, non-trace, compound in the presence of octanol, and was therefore attribut-ed to an octanol artifact. *Corresponding author, E-mail: fezh@dtu.dk  iii. Hydrophobicity pairing: After some success with the solvent extraction method, a new approach was taken based on the same chemistry.  The aim was to perform the solvent extraction on solid phase, thereby limiting radiation exposure during phase mixing, and restricting the volume of long-chain alcohol that would be present in the labeling solution. In this method, 1-hexanol was passed over a C-18 plus Sep-Pak in order to functionalize it.  The concept was similar to ion-pairing used in HPLC chromatography, where the functionality of a reversed-phase column is altered by hydropho-bic interactions with a molecule bearing a lipo-philic side-chain and the analyte-interacting moiety. When the activity and Sc in HCl was passed over the resin, 33% of the titanium was trapped (a control experiment without 1-hexanol gave negligible extraction of 45Ti).  This was easily eluted with 100% efficiency by inter-rupting the hydrophobic interaction between 1-hexanol and the solid phase with 1mL of isopro-panol. Reaction with the ligands converted 86% of the 45Ti to [45Ti]Ti-salan-dipic.  iv. Extraction onto solid supported 1,3 diol: The 1,3-diol resin did not swell, nor did it appear to wet very efficiently in HCl. Also, passage of the Sc/HCl solution was slow under gravity, and had to be aided with occasional light pressure. Nevertheless, the resin trapped 96% of the 45Ti that was applied to it.  Pyridine caused signifi-cant swelling of the resin. The reaction with salan removed 15% of the activity, and caused the solid phase to turn a bright yellow color.  The eluent was a deep blue-violet color, which was later found to be due to Fe (by ICPOES measurement), presumably as a soluble salan complex.  The reaction with dipic caused release of 40% of the 45Ti, with >70% being present as the final product [45Ti]Ti-salan-dipic.  In follow-up experiments using this meth-od, we found that heating the recovered eluent leads to full conversion. Summary The trap, release, and yields for the four meth-ods listed above are shown in TAB. 1.  Although the overall yields are very similar for methods ii, iii, and iv, the most promising result was with the 1,3-diol resin because it had the added ad-vantage of reacting on-column, and releasing on addition of the dipic ligand.  This is an important point for moving forward with a pure production of [45Ti]Ti-salan-dipic, because it allows removal of excess salan ligand –a challenging purification due to the similarities between Ti-salan-dipic and salan itself. The other methods appear to be reasonable approaches for isolating 45Ti, but in the specific situation of reactions with salan and dipic, they are not the best choice.     Method % Trap Release Yield i SPE HX. 99 0 0 ii Solv.extr. 48 - 25* iii SPE / hex. 33 100 26 iv SPE diol 96 40 28 TABLE 1. Results for trap and release of 45Ti and subsequent labeling yield (as percentage of activity before trapping, not decay corrected) of [45Ti]Ti-salan-dipic for conditions described above. *The presence of 1-octanol caused ab-normal chromatography (peak splitting).   Conclusion We conclude that the hydrolytically stable 45Ti compound [45Ti]Ti-salan-dipic can be synthesized in high yield after trapping of 45Ti on a 1,3-diol resin, and reacting on-column. Additionally, it is possible that the other methods presented for isolating 45Ti out of HCl mixtures may find use in other applications requiring different solvents or employing different ligands. We hope that this work adds to the base-knowledge of 45Ti radio-chemistry, and helps push 45Ti toward more widespread applications.   References 1 T.W. Burrows, Nucl. Data Sheets 109, 171 (2008). 2 R.G. Thomas and W. Bartolini, Nucl. Phys. A 106, 323 (1967). 3 J.M. Collins, R. Uppal, C.D. Incarvito, and A.M. Valentine, Inorg. Chem. 44, 3431 (2005). 4 A.L. Vavere and M.J. Welch, J. Nucl. Med. 46, 683 (2005). 5 K. Ishiwata, T. Ido, M. Monma, M. Murakami, H. Fukuda, M. Kameyama, K. Yamada, S. Endo, S. Yoshioka, T. Sato, and T. Matsuzawa, Int. J. Radiat. Appl. Instrumentation. Part A. Appl. Radiat. Isot. 42, 707 (1991). 6 J. Schur, C.M. Manna, A. Deally, R.W. Köster, M. Tacke, E.Y. Tshuva, and I. Ott, Chem. Commun. (Camb). 49, 4785 (2013). *Corresponding author, E-mail: fezh@dtu.dk 7 T.A. Immel, M. Grützke, A. Späte, U. Groth, and P. Öhlschläger, Chem. Commun. 48, pp. 5790-5792(2012). 8 M. Shavit, D. Peri, C.M. Manna, J.S. Alexander and E.Y. Tshuva,  J. Am. Chem. Soc. 129, 12098 (2007). 9 D. Peri, S. Meker, M. Shavit, and E.Y. Tshuva ,  Chemistry 15, 2403 (2009). 10  Z. Zhu, W. Zhang, and C.Y. Cheng,  Hydrometallurgy 105, 304 (2011). 11 K. Gagnon, G.W. Severin, T.E. Barnhart, and J.W. Engle, AIP Conf. Proc. 1509, pp. 211-214 (2012).   12  J.P. Holland, Y. Sheh, and J.S. Lewis, Nucl. Med. Biol. 36, 729 (2009). 13 J. Siikanen, H. Hong, H. Valdovinos, R. Hernandez, Y. Zhang, T. Barnhart, W. Cai, and R. Nickles, Soc. Nucl. Med. Annu. Meet. Abstr. 54, 1095 (2013).  Acknowledgements We gratefully acknowledge Jonathan Siikanen from Lund University for sharing his experiences and notes about 45Ti solvent extraction, gar-nered while visiting the Cyclotron Gang at UW Madison.  We also wish to thank Dr. A.I. Jensen for fruitful discussions.    

Hydrolytically stable titanium-45

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Hydrolytically stable titanium-45

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