Radionuclide generator-based production of therapeutic 177Lu from its long-lived isomer 177mLu

Background In this work, a lutetium-177 (177Lu) production method based on the separation of nuclear isomers, 177mLu & 177Lu, is reported. The 177mLu-177Lu separation is performed by combining the use of DOTA & DOTA-labelled peptide (DOTATATE) and liquid-liquid extraction. Methods The 177mLu cations were complexed with DOTA & DOTATATE and kept at 77 K for periods of time to allow 177Lu production. The freed 177Lu ions produced via internal conversion of 177mLu were then extracted in dihexyl ether using 0.01 M di-(2-ethylhexyl) phosphoric acid (DEHPA) at room temperature. The liquid-liquid extractions were performed periodically for a period up to 35 days. Results A maximum 177Lu/177mLu activity ratio of 3500 ± 500 was achieved with [177mLu]Lu-DOTA complex, in comparison to 177Lu/177mLu activity ratios of 1086 ± 40 realized using [177mLu]Lu-DOTATATE complex. The 177Lu-177mLu separation was found to be affected by the molar ratio of lutetium and DOTA. A 177Lu/177mLu activity ratio up to 3500 ± 500 was achieved with excess DOTA in comparison to 177Lu/177mLu activity ratio 1500 ± 600 obtained when lutetium and DOTA were present in molar ratio of 1:1. Further, the 177Lu ion extraction efficiency, decreases from 95 ± 4% to 58 ± 2% in the presence of excess DOTA. Conclusion The reported method resulted in a 177Lu/ 177mLu activity ratio up to 3500 after the separation. This ratio is close to the lower end of 177Lu/177mLu activity ratios, attained currently during the direct route 177Lu production for clinical applications (i.e. 4000–10,000). This study forms the basis for further extending the liquid-liquid extraction based 177mLu-177Lu separation in order to lead to a commercial 177mLu/177Lu radionuclide generator. Electronic supplementary material The online version of this article (10.1186/s41181-019-0064-5) contains supplementary material, which is available to authorized users.

from its longer-lived mother radionuclide. Lutetium-177 ( 177 Lu) is a radionuclide that could also benefit from the advantages of a generator vastly. 177 Lu is well-known for its theranostic potential and is expected to play a crucial role in fulfilling the global demand of radionuclides for many targeted radionuclide therapy applications (Das & Banerjee, 2016;Das & Pillai, 2013). The [ 177 Lu]Lu-DOTATATE has already been FDA approved for the application in neuroendocrine tumour therapy (https://www.fda.gov/ NewsEvents/Newsroom/ PressAnnouncements/ucm594043.htm,n.d.). Currently, other 177 Lu radiopharmaceuticals have also entered the clinic in the treatment of prostate cancer, lung cancer, non-Hodgkin lymphoma, bone pain palliation and others (Banerjee et al., 2015;Emmett et al., 2017;Hofman et al., 2018;Repetto-Llamazares et al., 2018;Dho et al., 2018). Clearly, the demand of 177 Lu is only going to increase and radionuclide generator can complement the current production routes. The long half-life of 177m Lu (160.44 days) can potentially lead to on-site and on-demand 177 Lu supply for a long period of time without the need of weekly irradiations in nuclear reactor (De Vries & Wolterbeek, 2012;Bhardwaj et al., 2017). However, the development of 177m Lu/ 177 Lu radionuclide generator needs to tackle the great challenge of separating the physically and chemically alike nuclear isomers 177 Lu and 177m Lu.
It has been previously shown that 177 Lu can be separated from 177m Lu due to the chemical effects occurring as a consequence of internal conversion decay of 177m Lu (Bhardwaj et al., 2017). Internal conversion decay may result in the emission of multiple Auger electrons, often accompanied with the loss of valence electrons and leaving the atom in a highly positively charged state which can result in bond rupture (Cooper, 1942). This principle presents a possibility to separate two isomers, provided that a separation process that can quickly & selectively capture the freed ions is feasible. Additionally, from a radionuclide generator perspective, the separation process should also allow the periodic extraction of the produced daughter radionuclide during the lifetime of the generator.
Previously, a column chromatography based 177 Lu-177m Lu separation process has been reported, where the 177m Lu complexed with DOTATATE has been immobilized on a tC-18 silica and the freed 177 Lu ions produced after the decay have been separated using a mobile phase flow (Bhardwaj et al., 2017). The 177 Lu/ 177m Lu activity ratio of 250 has been reached after separation compared to the equilibrium 177 Lu/ 177m Lu activity ratio of 0.25. However, in order to fulfil the clinical demand the separation method should provide 177 Lu having minimum breakthrough of 177m Lu. The current direct production route delivers 177 Lu with 177 Lu/ 177m Lu activity ratio ranging from 4000 to 10, 000 (Dvorakova et al., 2008;Pawlak et al., 2004;Knapp FFJA et al., 1995;Das et al., 2007;Chakraborty et al., 2014), while the indirect production route affords the nocarrier added 177 Lu with almost negligible amount of 177m Lu (Watanabe et al., 2015).
In this work, a radionuclide generator for the production of 177 Lu based on the pair of nuclear isomer 177m Lu-177 Lu is presented. The 177m Lu-177 Lu separation has been performed using liquid-liquid extraction (LLE). LLE has been explored several times before in the development of other radionuclide generators, such as 99 Mo/ 99m Tc, 68 Ge/ 68 Ga, 188 Re/ 188 W, and 90 Y/ 90 Sr radionuclide generators (Le Minh & Lengyel, 1989;Fikrle et al., 2010;Bhatia & Turel, 1989;Boyd, 1982;Ehrhardt & Welch, 1978;Mushtaq et al., 2007;Dutta & Mohapatra, 2013). The present work demonstrates the application of LLE in 177 Lu-177m Lu separation which can potentially lead to a commercial 177m Lu/ 177 Lu radionuclide generator. The metastable isomer, 177m Lu, was complexed with the chelating agents (DOTA and DOTATATE) and the freed 177 Lu ions was extracted in dihexyl ether using Di-(2-ethylhexyl) phosphoric acid (DEHPA) as the cation extracting agent.
The Lutetium-177 m ( 177m Lu) source was provided by IDB-Holland as a 1 mM [ 177m Lu]LuCl 3 solution with about 5 MBq 177m Lu per g of solution.
Methods γ ray spectroscopy analysis All the activity measurements were performed on a well-type HPGe detector for counting time up to 5 h to reduce the error from the counting statistics to less than 5%. The measurement of the samples obtained at the end of LLE was repeated after 3-4 half-lives of 177 Lu to decrease the background and measure the 177m Lu activity with less than 5% uncertainty. The efficiency calibration for different peaks was performed using a known activity of 177 Lu source supplied by IDB Holland. The obtained gamma ray spectra were analysed using an in-house software to calculate the activity of each fraction (Blaauw, 1993). In order to minimize the error, all the vials were weighed before and after the fraction collection.
The complex formation was confirmed using instant thin layer chromatography. Free 177m Lu ions traces were removed using a cation exchange resin (chelex-100). (Details in S1, Additional file 1).

Liquid-liquid extraction (LLE) procedure
The schematic representation of LLE to separate the freed 177 Lu ions from the complexed 177m Lu ions is shown in Fig. 1.
All the LLE experiments were performed in 2 mL Eppendorf by placing them in a shaking incubator at room temperature. The aqueous and the organic phases were mixed in volumetric ratio (1:1) at 1400 rpm for a stirring time of about 10 min. The stirring time of 10 min was optimised by studying the 177 Lu extraction efficiency as a function of extraction time (see Additional file 1 Figure S1(b), S2, supplementary information). At the end of stirring, the layer separation was achieved after a settling time of about one minute. In order to avoid any contamination of the aqueous layer in the organic layer, only the upper 2/3rd organic layer was taken out using a 20-200uL pipette in all the LLE experiments. The pipetted organic layer was transferred to a pre-weighed vial to know the exact amount of organic phase removed in each extraction.
First, free 177 Lu cations were extracted from a 0.3 mL, pH -4, 1 mM [ 177 Lu]LuCl 3 solution as the aqueous phase. The organic phase consists of 0.3 mL dihexyl ether containing different DEHPA concentrations, namely 0.01, 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, 1.0, 1.2 and 1.6 M. At the end of LLE, the 177 Lu activity in the organic and the aqueous layer was measured using γ ray spectroscopy to obtain the 177 Lu extraction efficiency (EE). The EE is defined as the percentage of the 177 Lu activity moving from the aqueous phase in to the organic phase after the extraction. All the experiments were performed in triplicate.
Subsequently, the LLE was performed to extract the freed 177 Lu ions from the aqueous phase containing [ 177m Lu]Lu-DOTATATE, [ 177m Lu]Lu-DOTA complex. For [ 177m Lu]Lu-DOTATATE complex, the 177 Lu extraction was performed successively at varying 177 Lu accumulation periods for a total time period of up to 60 days. For, [ 177m Lu]Lu-DOTA complex, the freed 177 Lu ions were extracted successively at every 7 days for a total time period of 35 days. In between the extractions, the [ 177m Lu]Lu-DOTA and [ 177m Lu]Lu-DOTATATE complexes were left in a liquid N 2 tank to allow for the accumulation of freed 177 Lu ions. The 177 Lu separation was performed by bringing the vial out of the liquid N 2 tank and quickly adding the 0.01 M DEHPA in DHE in a 1:1 volumetric ratio (0.3 mL: 0.3 mL), at room temperature and 10 min of stirring time, as shown schematically in Fig. 1. At the end of LLE, the 177 Lu and 177m Lu activity in the organic layer was measured using γ ray spectroscopy to calculate the amount of 177 Lu and 177m Lu ions extracted in the organic phase and the 177 Lu/ 177m Lu activity ratio. The 177 Lu extraction efficiency is defined as the amount of 177 Lu ions that were extracted into the organic phase divided by the theoretically produced 177 Lu ions (see section S3, eq. S2 in Supplementary Information). The percentage of 177m Lu extracted is defined as the activity of 177m Lu ions measured in organic phase after the LLE divided by the starting activity of the 177m Lu ions in the aqueous phase.

Lu/ 177m Lu separation using [ 177m Lu]Lu-DOTATATE complex
The 177 Lu/ 177m Lu separation was performed using [ 177m Lu]Lu-DOTATATE complex synthesized in the presence of an excess of DOTATATE (Lu:DOTATATE molar ratio of 1:4). The 177 Lu ions production via the decay of 177m Lu is represented by eq. S1, Supplementary Information, S3 and the expected growth of 177 Lu ions with the increase in the 177 Lu accumulation period is shown in Additional file 1 Figure S2, Supplementary Information. The amount of 177 Lu ions produced increases with an increase in 177 Lu accumulation period and reaches a maximum after 32 days of 177 Lu accumulation. In the presented results, the freed 177 Lu ions were extracted from [ 177m Lu]Lu-DOTATATE complex by performing LLE successively after different 177 Lu accumulation intervals.  Figure S1, supplementary information S2). Additionally, along with the 177 Lu ions, 0.0085 ± 0.0015% of the starting 177m Lu activity was also extracted in the organic phase. Figure 2(b), shows the 177 Lu/ 177m Lu activity ratios obtained after different extractions. An increase in the 177 Lu/ 177m Lu activity ratio is observed with an   Fig. 3&4. The LLE was performed successively at time intervals of 7 days. Figure 3(a) shows the effect of Lu: DOTA molar ratios on 177 Lu extraction efficiency. Figure 3(b) displays the percentage of initial 177m Lu activity extracted in the organic phase at the end of LLE for the different Lu: DOTA molar ratios. It can be seen from Fig. 3(a) that the 177 Lu extraction efficiency reaches a maximum value of 95 ± 4% when Lu & DOTA were present in 1:1 M ratio and decreases to 58 ± 2% for 1:4 Lu:DOTA molar ratio. Further, the 177 Lu extraction efficiency remains almost constant for the first three extractions followed by a slight increase during the 4th and 5th extraction for all the three Lu:DOTA molar ratios. Figure 3(b) shows that 0.0061 ± 0.0015% of 177m Lu activity was extracted in the first extraction when Lu and DOTA were present in 1:1 M ratio, which got reduced to 0.0020 ± 0.0010% for the Lu: DOTA molar ratio 1:4. The percentage of 177m Lu activity extracted remains almost constant during the successive extractions in the presence of excess DOTA, and increases from 0.0061 ± 0.0015% to 0.0095 ± 0.0015% in the presence of 1:1 Lu:DOTA molar ratio. The error bars in Fig. 3 represent the standard deviation in the results of three experiments performed in parallel. Figure 4 shows the 177 Lu/ 177m Lu activity ratios observed in the organic phase at the end of LLE for the three different Lu:DOTA molar ratios. It reveals that the 177 Lu/ 177m Lu activity ratio increases with an increase in the molar quantities of DOTA. The highest 177 Lu/ 177m Lu activity ratio of 3500 ± 500 was obtained when DOTA was present in excess (1:4) and decreases to around 1500 ± 600 in the presence of 1:1 Lu: DOTA molar ratio. Further, a slight decrease in the 177 Lu/ 177m Lu activity ratios was observed in every successive LLE performed during the 35 days of experiments. The fifth 177 Lu extraction performed at the end of the experiments resulted in a 40 ± 5% decrease in the 177 Lu/ 177m Lu activity ratios compared to the 177 Lu/ 177m Lu activity ratio obtained in the first extraction. Overall, the 177 Lu/ 177m Lu activity ratios obtained using DOTA as chelating agent were about 5 times higher when compared with 177 Lu/ 177m Lu activity ratios obtained using DOTATATE for a 177 Lu accumulation period of around 7 days. Also, the percentage of 177m Lu activity extracted in the organic phase was about 5 times higher with DOTATATE than that observed with DOTA as the 177m Lu complexing agent.

Discussion
The separation of the isomers 177 Lu and 177m Lu based on the nuclear decay after effects is achieved using liquid-liquid extraction (LLE) as the separation method and the [ 177m Lu]Lu-DOTA, [ 177m Lu]Lu-DOTATATE complexes. The 177 Lu production at 77 K resulted in negligible dissociation of the starting [ 177m Lu]Lu-DOTA based complexes, and increases the quality of extracted 177 Lu remarkably. The freed 177 Lu ions were extracted in the organic phase by performing the LLE at room temperature. The separation was done sufficiently fast resulting in production of limited quantities of free 177m Lu ions. In the present work, the 177 Lu/ 177m Lu activity ratio of 1086 ± 40 is achieved using [ 177m Lu]Lu-DOTATATE complex which is about 4 times higher than the previously reported 177 Lu/ 177m Lu activity ratio of 250 realized using the same [ 177m Lu]Lu-DOTA-TATE complex (Bhardwaj et al., 2017). In the previously reported method, the 177 Lu ion accumulation was performed at 10°C and the temperature could not be decreased further because of experimental limitations. In contrast, the present LLE based separation allows the 177 Lu accumulation at 77 K. At 77 K, the rate constants for the chemical reactions (i.e. association-dissociation kinetics) are extremely low making the 177m Lu contribution coming from the dissociation of the [ 177m Lu]Lu-DOTATATE complex negligible during the 177 Lu accumulation period. The 177m Lu contribution observed in the present work can be accounted to the dissociation of the [ 177m Lu]Lu-DOTATATE complex during the LLE at room temperature. After the dissociation, the 177m Lu and 177 Lu ions are indistinguishable and they will go into the organic phase with equal rate.
The LLE of 177 Lu ions from [ 177m Lu]Lu-DOTATATE complex resulted in co-extraction of 0.0085 ± 0.0015% of initial 177m Lu activity in the organic phase. This leads to an estimated dissociation rate constant of 1.3*10 − 7 ± 0.3*10 − 7 s − 1 . For Lu-DOTATATE complex, a dissociation constant rate 2*10 − 8 s − 1 has been reported at pH -4.3 and 20°C (van der Meer et al., 2013). It has also been shown that the Lu-DOTATATE complex is accompanied by the presence of short-lived unstable, mono-and di-protonated (MHL, MH 2 L) complex species (van der Meer et al., 2013). These species have a dissociation rate constant of 8*10 − 5 s − 1 (MHL) & 2*10 − 4 s − 1 (MH 2 L) at pH -4.3 and 20°C (van der Meer et al., 2013). Therefore, the presently estimated dissociation rate constant does not represent the dissociation of single species, but is rather a combination of the dissociation contribution from three different species i.e. ML, MHL, & MH 2 L. Overall, the [ 177m Lu]Lu-DOTATATE complex behavior clearly highlights the fact that a careful consideration of all the possible species at a certain pH should be given while assessing the role of any complexing agent in 177 Lu-177m Lu separation.
The 177 Lu/ 177m Lu activity ratio obtained during the LLE of 177 Lu ions from [ 177m Lu]Lu-DOTATATE complex was found to be influenced by the length of the 177 Lu accumulation period. The highest 177 Lu/ 177m Lu activity ratio of 1086 ± 40 was obtained after 177 Lu accumulation period of 26 days and decreased to 600 ± 200 for accumulation periods of 5 to 10 days. This was expected as the amount of 177 Lu ions produced from the internal conversion of 177m Lu ions grows as the 177 Lu accumulation period increases. In contrast, the 177m Lu contribution is only due to dissociation of the complex taking place during the extraction. Additionally, a 177 Lu extraction efficiency of 60 ± 10% was observed which can be associated to the loss of free 177 Lu ions due to their re-association with the excess complexing agent, as reported before by Bhardwaj et al. (Bhardwaj et al., 2017).
The crucial role of association kinetics on 177 Lu-177m Lu separation is further emphasised by studying the 177 Lu-177m Lu separation in the presence of varying amounts of DOTA as the complexing agent. The 177 Lu extraction efficiency obtained during the LLE of freed 177 Lu ions was affected by the applied ratio of complexing agent. The 177 Lu extraction efficiency of 58 ± 2% was achieved in the presence of excess DOTA (Lu:DOTA molar ratio, 1: 4), and it increases to 95 ± 4% when Lu:DOTA was present in the molar ratio 1:1, confirming that the association kinetics of freed 177 Lu and the excess of DOTA play an important Bhardwaj et al. EJNMMI Radiopharmacy and Chemistry (2019)  role in the process. Similarly, the extracted 177m Lu activity decreases from 0.0060 ± 0.0015% to 0.0020 ± 0.0010% with the increase in the Lu:DOTA molar ratios from (1:1) to (1:4) respectively, due to the re-association of 177m Lu ions with the excess of DOTA. The 177 Lu/ 177m Lu activity ratios obtained during the LLE of 177 Lu ions from [ 177m Lu]Lu-DOTA complex were also found to be effected by the starting Lu:DOTA molar ratio. A 177 Lu/ 177m Lu activity ratio up to 3500 ± 500 was achieved when the LLE was performed using aqueous [ 177m Lu] LuDOTA complex with Lu:DOTA present in the molar ratio 1:4. Remarkably, the obtained 177 Lu/ 177m Lu activity ratios are very close to the 177 Lu/ 177m Lu activity ratios of 4000-10,000 associated to the direct-route production of 177 Lu supplied to the clinics (Das et al., 2007;Chakraborty et al., 2014). These ratios were found to decrease with the decrease in the amount of DOTA, i.e. an activity ratio of 1500 ± 600 was observed when Lu and DOTA were present in the molar ratio 1:1. The presence of excess DOTA leads to a proportional decrease in the amount of both 177 Lu and 177m Lu ions due to re-association. However, the 177 Lu production from internal conversion of 177m Lu ions adds to a constant positive contribution in the amount of 177 Lu ions, which leads to an overall increase in the 177 Lu/ 177m Lu activity ratios.
Finally, the observed decrease in the 177 Lu/ 177m Lu activity ratio with the increase in time are well in agreement with the theoretically expected ratios based on the 177m Lu and 177 Lu extracted shown in Fig. 3 and incorporating the effect of incomplete organic phase removal on every successive extraction (see Additional file 1 Figure S3, supplementary information). The reported separation method suffers from the drawback of incomplete organic phase removal during the LLE. The residual 1/3rd of the organic phase left unrecovered after every LLE contains un-extracted 177 Lu and 177m Lu ions. The 177 Lu ions will reduce to about a half after accumulation time of 7 days, but the 177m Lu ions will remain almost unchanged as they have a half-life of 160.44 days. They will add to the total amount of free 177m Lu ions in the successive extraction and correspondingly to a decrease the 177 Lu/ 177m Lu activity ratio. In case of a complete organic phase removal, the separation method could lead to a constant value of 177 Lu/ 177m Lu activity ratio of around 3500 on performing periodic 177 Lu extraction every 7 days. Additionally, the use of longer 177 Lu accumulation period of 32 days will lead to 1.7 times more 177 Lu production compared to 7 days 177 Lu accumulation period. This can potentially lead to an activity ratio of 7000 on considering a constant 0.0020 ± 0.0010% 177m Lu contribution due to dissociation and 58 ± 2% 177 Lu extraction efficiency. In such a case, the extracted 177 Lu would contain a 177m Lu contribution as low as 0.01% and would be comparable to the direct route 177 Lu production.
It should be pointed that the specific activity of the produced 177 Lu is not a discussed parameter since the starting 177m Lu source has very low specific activity and therefore also the extracted 177 Lu. Consequently, the values would not represent a fair comparison with the commercially available 177 Lu. Additionally, the extracted 177 Lu ions have not been stripped from the organic phase back into the aqueous phase considering that it is a well-reported process in literature (Trtic-Petrovic et al., 2010).
Overall, the presented work is an important milestone towards the development of a 177m Lu/ 177 Lu radionuclide generator for clinical application. It also establishes the possibility of employing other separation techniques such as micro-fluidic separation (Davide et al., 2014), membrane based liquid-liquid extraction (Pedersen-Bjergaard & Rasmussen, 2008) or an automatized LLE separation devices that can allow the commercialization of LLE based 177m Lu/ 177 Lu radionuclide generator. However, there are several aspects that needs further investigation and optimization. Firstly, the back extraction of 177 Lu from the organic phase and the complete removal of any traces of organic solvents will be crucial for its potential commercialization. Secondly, this work has been performed at lab-scale with low activity levels and excludes the effect of radiolysis on the proposed 177m Lu-177 Lu separation method. The radiolysis can impact the quality of the produced 177 Lu and should be carefully evaluated in the future investigations. Lastly, the described method can be further optimized in terms of shorter extraction time, use of lower temperature to perform the 177 Lu extraction improve the produced 177 Lu quality.

Conclusion
A novel 177m Lu-177 Lu separation method is developed that allows the 177 Lu production via internal conversion of 177m Lu at low temperatures (77 K) and the use of ultrastable 177m Lu complexes with liquid-liquid extraction. For the best conditions, the [ 177m Lu]Lu-DOTA complex and LLE provides a 177 Lu/ 177m Lu activity ratio of 3500 ± 500. A value that is close to the 177 Lu/ 177m Lu activity ratio 4000-10,000 obtained during the 177 Lu production via the direct route and exemplifies the potential applicability of the 177m Lu/ 177 Lu generator in clinical studies. Future research will be focused on further optimization of novel 177 Lu-177m Lu separation technologies aimed to ultimately lead to a clinically applicable 177m Lu/ 177 Lu radionuclide generator. The around the clock availability of 177 Lu via a 177m Lu/ 177 Lu radionuclide generator can significantly accelerate the research on 177 Lu based radiopharmaceuticals and help in realizing its full potential in nuclear medicine.

Additional file
Additional file 1: Figure S1. The 177 Lu extraction efficiency of 0.3mL, 1mM [ 177 Lu]LuCl 3 as a function of a) varying DEHPA concentration in dihexylether and b) as a function of phase stirring time. Data points represent the average and standard deviation for six experiments. Figure S2. The amount of 177 Lu produced from 1 MBq of 177m Lu for different 177 Lu accumulation period as calculated by using equation 1. Figure S3. The 177 Lu/ 177m Lu activity ratio obtained at different elution time when the LLE is performed with [ 177m Lu]Lu-DOTA complex synthesized in a molar ratio 1:4. The data points represent the experimentally observed ratios, while the dotted line represents the expected activity ratios with 60% 177 Lu extraction efficiency and 0.002% 177m Lu ions leakage. (DOCX 87 kb)