Introduction

Therapeutic radiopharmaceuticals comprising β− emitters are increasingly used to target a number of disease conditions, most importantly cancer. Amongst the various β− emitters which are in use currently, 177Lu has received noteworthy attention in the past decade and is one of the extensively used radionuclides in recent times for in vivo targeted therapy [1–4]. The favorable nuclear properties and the well-established co-ordination chemistry of 177Lu have led to the speedy translation of 177Lu radiopharmaceuticals from laboratories to the nuclear medicine clinics. 177Lu labeled receptor-targeted peptides and antibodies are regularly used in the nuclear medicine clinics for targeting a number of cancers, demonstrating its dominant role in cancer therapy. 177Lu emits medium energy β− particles (78.6 % of 497 keV β−) thereby decaying to stable 177Hf [5–7]. Scintigraphic imaging and dosimetric evaluation during therapy is possible due to the emission of two low energy γ radiations with low abundances (11 % of 208 keV and 6.4 % of 113 keV) [5–7]. Radiolabeling of biomolecules such as peptides and antibodies with 177Lu is accomplished through the use of bifunctional chelating agents. The macrocyclic chelator DOTA (1,4,7,10-tetraaza cyclododecane tetraacetic acid) and its analogs are universally employed chelating agents for cross-linking biomolecules with many radiometals, including 177Lu [7–9]. DOTA remains the chelating agent of choice for sequestering many radionuclides including 177Lu due to the achievement of high radiolabeling yields in moderately high specific activities as well as superior stability of the radiolabeled complexes in vivo [7–9]. In pursuit of newer chelating agents which demonstrate excellent thermodynamic and kinetic stabilities on radiolabeling with the diagnostic and therapeutic radioisotopes, a wide range of ligands with varied donor atoms as well as chelating networks have been synthesized and investigated [10–12]. Selection of a ligand for use with a particular radioisotope is based on radiolabeling yields, reaction conditions including temperature, influence of trace metallic impurities in solution, in vitro stability, kinetic inertness in vivo preventing the release of the radiolabel in vivo and the extent of accumulation of the radiolabeled complexes in non-target organs. Although DOTA and its bifunctional derivatives are considered to be very efficient chelators for 177Lu, they reportedly suffer from slow complex formation at mild conditions requiring the performance of the radiolabeling reaction at elevated temperatures [13, 14]. This is definitely a drawback especially while radiolabeling temperature-sensitive biological vectors such as antibodies. Hence, extensive research has been carried out in pursuit of ligands which can enable instantaneous radiolabeling at room temperature along with imparting high kinetic stability in vivo to the radiolabeled complexes. In this respect, the development of backbone substituted derivatives of the acyclic ligand DTPA such as the cyclohexyl derivative of DTPA (CHX-A′′-DTPA) which form stable complexes with 177Lu is an important advancement [15]. However, macrocyclic ligands which can sequester the radiometals at rates faster than DOTA with the additional benefit of kinetic inertness in vivo are highly desirable. Recently, Chong et al. have reported the synthesis of the ligand NETA which consists of a macrocyclic triaza cyclononane ring linked to an iminodiacetic acid moiety while Baranyai and co-workers have reported the synthesis of AAZTA, a cyclic heptadentate ligand forming instantaneous complexes with many metals including radiolanthanides [16, 17]. Tircsó G et al. have reported the synthesis of the ligand [S-5p-(para nitrobenzyl) 3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid] (p-NO2-Bz-PCTA) which forms complexes with several metals of particular interest to nuclear medicine including lutetium, indium and yttrium, at an order of magnitude quicker than DOTA [18, 19]. The authors observed that the acid catalyzed dissociation of the lanthanide-PCTA complexes was slower even though the thermodynamic stabilities of the complexes were low. However, they reportedly exhibit high kinetic inertness analogous to the DOTA complexes [19]. Recently, the potential of this ligand for use with 64Cu was examined by Ait-Mohand et al. [20] while our group has evaluated its 68Ga and 90Y complexation properties [21–23]. The objective of the present study is to investigate the use of para isothiocyanato benzyl 3,6,9,15-tetraazabicyclo [9.3.1]pentadeca-1(15),11,13-triene-3,6,9-triacetic acid (PCTA-NCS) as a BFCA for preparation of 177Lu radiopharmaceuticals with respect to the complexation kinetics, amenability for room temperature labeling, influence of trace metal ions on the radiolabeling yields, in vitro and in vivo stabilities and the biodistribution pattern in normal Swiss mice, in comparison to 177Lu-DOTA-NCS.

Experimental

Materials

177Lu of specific activity 25.7 ± 1.4 Ci/mg (at 24 h after the end of irradiation) was produced by irradiation of enriched Lu2O3 target (82 % in 176Lu) at a thermal neutron flux of ~1.4 × 1014 n/cm2 s for a period of 21 days and further processed at the Radiochemicals Section of Isotope Production and Applications Division, BARC [6, 24]. The bifunctional chelating agents viz. PCTA-NCS (Fig. 1) and DOTA-NCS were purchased from M/s. Macrocyclics, Dallas, USA. Lutetium chloride used as a carrier in the experiments, calcium (II), copper (II), iron (III) and zinc (II) salts as well as sodium acetate were purchased from M/s. Aldrich, USA. HPLC grade water (Merck, India) was used for preparation of all reagents. Radioactivity measurements were performed using a well-type NaI (Tl) detector (ECIL, India) while radioactivity measurements during biodistribution experiments were performed using a flat-geometry NaI(Tl) detector. HPLC analyses were carried out on a dual pump HPLC system (JASCO, Japan) equipped with a reversed phase C-18 HiQ Sil column (5 µm, 4 × 250 mm), coupled to a PU 1575 UV/visible detector (JASCO, Japan) and a NaI (Tl) radioactivity detector (Raytest, Germany). Whatman 3 mm chromatography paper (20 mm width) was purchased from M/s. Whatman International, UK. Approval from the institutional animal ethics committee was obtained before carrying out the biodistribution studies. All the reactions were carried out in acid washed glass vials to minimize the presence of trace metallic impurities.

Fig. 1
figure 1

Chemical structure of PCTA-NCS

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Methods

Preparation and characterization of 177Lu-PCTA-NCS complex

The ability of the ligand PCTA-NCS to sequester 177Lu effectively was ascertained by carrying out its radiolabeling at various Lu to PCTA-NCS molar ratios (1:20, 1:10, 1:4, 1:2 and 1:1) in 0.1 M sodium acetate buffer at pH ~5.5 at 80 °C for 30 min. The reaction volume was kept constant at 1 ml. All the experiments were carried out with trace activity of 177LuCl3 (~37–74 MBq) with deliberate addition of 176Lu carrier equivalent to 100 mCi of 177Lu (~4 µg, based on the specific activity of 177Lu as ~25 Ci/mg). The trace level activity of 177LuCl3 (~37–74 MBq) contained very small amounts of carrier 176Lu (~40–80 ng) which was much less in comparison to the carrier 176Lu deliberately added in the reaction. Complexation of 177Lu with DOTA-NCS was also carried out at the above-mentioned molar ratios, under identical reaction conditions, for comparison. After ascertaining the complexation yields at the above-mentioned Lu to PCTA-NCS molar ratios, complexation kinetics was determined by carrying out the radiolabeling reaction at ambient temperature (25 °C) and at 80 °C at the lowest 177Lu:PCTA molar ratio (1:2) at which >95 % radiolabeling yield was achieved. For comparison, complexation was also carried out with DOTA-NCS by adopting identical reaction conditions using 37 MBq of 177LuCl3 and 4 µg of 176Lu carrier.

Characterization of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS complexes as well as the determination of radiolabeling yields was performed by HPLC and paper chromatography techniques. For HPLC characterization, 20 µl aliquots of the 177Lu labeled complexes were injected into a C18 reverse phase column eluted using water (solvent A) and acetonitrile (solvent B) both containing 0.1 % trifluoroacetic acid in a gradient mode of elution (0–4 min 5 % B, 4–20 min 5–95 % B, 20–30 min 95–5 % B) at a flow rate of 1 ml/min and the radioactivity was monitored using a flow-through NaI(Tl) scintillation detector. Paper chromatography was performed using 50 % aqueous acetonitrile as the mobile phase to differentiate between free and complexed 177Lu.

Role of trace metal impurities on yields of 177Lu-PCTA-NCS

It is well recognized that trace metallic contaminants play a major role in reducing the yields of 177Lu radiopharmaceuticals, particularly the receptor-targeted radiopharmaceuticals synthesized using small amounts of ligands. For instance, Fe(III) ions may be present in trace levels even in highly pure reagent solutions [25, 26]. Hence, the potential role of trace metal ions such as Ca(II), Cu(II), Fe(III) and Zn(II) on the radiochemical yield of 177Lu-PCTA-NCS was studied in comparison to that of 177Lu-DOTA-NCS. For this purpose, the absence of trace metal ions in the 177LuCl3 solution was ascertained by carrying out ICP-AES analysis of decayed 177Lu samples, as reported elsewhere [27]. All the experiments were carried out using 37–74 MBq of 177LuCl3 with deliberate addition of 176Lu carrier (4 µg) equivalent to 100 mCi of 177Lu, as explained previously. Standard solutions of the trace metal metal ions (Ca(II), Cu(II), Fe(III) and Zn(II)) were prepared and diluted to the appropriate concentrations corresponding to trace metal to 176Lu molar ratios of 0.1:1, 1:1 and 10:1 respectively, keeping the Lu:PCTA-NCS molar ratio constant at 1:2 for all the studies). In a typical complexation reaction, 37–74 MBq of 177LuCl3 was taken in 0.1 M sodium acetate buffer to which carrier Lutetium (176Lu) (4 µg/23 µM) equivalent to 100 mCi of 177Lu (3.7 GBq) was added along with the corresponding trace metal solution followed by the ligand PCTA-NCS (46 µM). The reaction volume was kept constant at 1 ml and was buffered at pH 5–5.5. The radiolabeling reaction was carried out for 15 min at ambient temperature. A reference reaction was also set-up simultaneously in the absence of trace metal ions. All the reactions were carried out with DOTA-NCS under identical reaction conditions, for comparison. All the reactions were repeated four times and the results correspond to the mean ± SD of four replicates (n = 4).

In vitro stability studies and determination of octanol/water partition co-efficients (log P)

In vitro stability of 177Lu-PCTA-NCS complex (which has a Lu:PCTA-NCS molar ratio of 1:2) was determined in saline and human serum for 7 days at 37 °C. For determining the in vitro stability in saline, 100 µl of the complex was incubated in 1.9 ml of saline and incubated at 37 °C. Aliquots of ~100 µl were taken at specific time intervals up to 7 days post-incubation and the radiochemical purity of the complex was determined by paper chromatography using 50 % aqueous acetonitrile as mobile phase. For determination of stability in human serum, ~100 µl of the complex was incubated in 1 ml of human serum at 37 °C for 7 days. At end of every 24 h, ~100 µl was taken and 2 ml of acetonitrile was added. The precipitate was removed by centrifugation and the supernatant was analysed by paper chromatography using 50 % aqueous acetonitrile as mobile phase. A similar experiment was carried out with 177Lu-DOTA-NCS for comparison (Lu:DOTA-NCS molar ratio of 1:2).

The octanol to water partition coefficient of 177Lu-PCTA-NCS was determined by adding 100 µl of the complex to a biphasic solution consisting of 900 µl saline and 1 ml of n-octanol. The resulting solution was shaken for ~5 min after which the aqueous and octanol layers were separated. Equal aliquots of both the layers were counted. 100 µl of the octanol phase was again mixed with 900 µl of octanol and 1 ml of saline and the experiment was repeated once again. Logarithm of the ratio of the counts in the octanol phase to that in the aqueous phase gave the partition coefficient values. Similar experiment was also carried out with 177Lu-DOTA-NCS for comparison.

Biodistribution studies

For biodistribution studies, 177Lu-PCTA-NCS complex was prepared at a Lu:PCTA-NCS molar ratio of 1:2 (using 37–74 MBq of 177LuCl3, 4 µg of 176LuCl3 carrier) and injected via the lateral tail vein in normal healthy Swiss mice. In brief, each mice, weighing 20–25 g, was injected with 0.1 ml of 177Lu-PCTA-NCS complex (~370 kBq per animal, n = 4 per time point). The animals were kept in separate cages and were sacrificed by carbon dioxide asphyxiation at 1, 3 and 24 h p.i., blood was collected, the organs of interest (liver, lungs, kidney, intestine, spleen, stomach, muscle and bone) were excised and weighed. The associated radioactivity was measured in a flat-bed NaI (Tl) scintillation counter. The organ activities were corrected for decay and percentage injected activity (%ID) and percentage injected activity per gram (%ID g−1) were determined. Percentage activity excreted (urine and feaces) was determined by counting the cage paper. Blood, bone and muscle uptakes were calculated by assuming their weights to be 6.5, 10 and 40 % of the body weight respectively [28]. Biodistribution study of 177Lu-DOTA-NCS was also carried out under identical experimental conditions (at a Lu:DOTA-NCS molar ratio of 1:2, using 37–74 MBq of 177LuCl3 and ~4 µg of 176LuCl3 carrier) for comparison.

Results

Preparation and characterization of 177Lu-PCTA-NCS complex

For a bifunctional chelating agent to be considered suitable for preparation of radiopharmaceuticals for in vivo targeted therapy, it must preferentially form complexes with the radiometals at low radiometal to ligand ratios thereby reducing the amount of ligand used for preparing the radiopharmaceuticals. Herein, experiments were carried out to determine the amount of PCTA-NCS ligand required for preparation of 177Lu-PCTA-NCS complex at high radiochemical yields using carrier 176LuCl3 equivalent to 100 mCi of 177LuCl3 and ~37 MBq of 177LuCl3 activity. Complexation reactions were carried out at various 177Lu to PCTA-NCS molar ratios. Table 1 depicts the radiolabeling yields at various 177Lu:PCTA-NCS molar ratios, in comparison to 177Lu-DOTA-NCS, when reaction was carried out at 80 °C for 30 min, using trace levels of 177Lu and ~4 µg of Lu carrier equivalent to ~100 mCi of 177Lu. It is evident that at a 1:1 molar ratio of 177Lu to PCTA-NCS, the radiolabeling yield was only 80 ± 6 %. Radiolabeling yields in excess of 95 % could be obtained even at 1:2 (lutetium to PCTA) ratios, similar to that of 177Lu-DOTA-NCS. Based on the results of the above experiments, all further experiments (complexation kinetics, influence of trace metal ions, in vitro stability studies and biodistribution studies) were carried out using 177Lu-PCTA-NCS complex having a Lu:PCTA molar ratio of 1:2. The 177Lu-PCTA-NCS and 177Lu-DOTA-NCS complexes were characterized by paper chromatography and HPLC techniques. In paper chromatography using 50 % aqueous acetonitrile as the mobile phase, R f value of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS complexes was about 0.8–1.0 while uncomplexed 177Lu remained at R f = 0.0. HPLC analysis employing a C18 reverse phase column with a gradient elution of water and acetonitrile both containing 0.1 % TFA using the optimized protocol revealed the formation of 177Lu-PCTA-NCS complex having the major peak at a retention time of 19.1 min (Fig. 2) while unbound 177Lu(III) eluted at 3.2 min. Results of the experiments carried out to determine the complexation kinetics at ambient temperature (25 °C) and 80 °C showed that high radiolabeling yields (>95 %) could be achieved even within 15 min of incubation at ambient temperature for 15 min, for both 177Lu-PCTA-NCS and 177Lu-DOTA-NCS (at Lu:Ligand molar ratio of 1:2). Hence, all subsequent radiolabeling reactions were carried out for 15 min at ambient temperature. This finding demonstrates that it was not essential to perform the radiolabeling reaction at elevated temperature to obtain high radiolabeling yields of 177Lu-PCTA-NCS.

Table 1 Complexation of 177Lu with PCTA-NCS and DOTA-NCS at various Lu to ligand molar ratios (pH ~5.5, added ca. Lu ~4 µg, 30 min at 80 °C, n = 3)
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Fig. 2
figure 2

HPLC radiochromatogram of 177Lu-PCTA-NCS

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Role of trace metal impurities on yields of 177Lu-PCTA-NCS

Results of the studies carried out to determine the role of trace metal ions on the 177Lu labeling yields are summarized in Table 2 in which the percentage yield of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS is given in terms of trace metal to lutetium molar ratios of 0.1, 1.0 and 10 respectively. In control experiments carried out in the absence of any trace metallic contaminant, radiolabeling yields exceeding 95 % could be readily obtained. It is very well evident from the Table 2 that the adverse effects exerted by Fe(III) ions on the yields of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS is similar with reduced yields only at higher trace metal to lutetium ratios (10:1). The yield of 177Lu-PCTA-NCS was reduced only at ten times molar excess of Cu(II) ions over Lu(III) ions. In contrast, the yields of 177Lu-DOTA-NCS were reduced at equimolar ratios of Cu(II) and Lu(III) ions. Zn(II) ions reduced the radiochemical yields of 177Lu-PCTA-NCS at equimolar ratios with Lu(III) ions. A similar trend was also observed in the formation of 177Lu-DOTA-NCS in presence of Zn(II) ions. Ca(II) ions did not have any adverse effect on the radiochemical yields of the 177Lu complexes at lower Ca(II) to Lu(III) molar ratios (0.1 and 1.0).

Table 2 Influence of trace metal ions on the radiochemical yields of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS (Lu:ligand molar ratio of 1:2, n = 4)
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In vitro stability studies and determination of octanol/water partition co-efficients (log P)

The radiochemical purity of 177Lu-PCTA-NCS when incubated in saline and serum respectively was determined at various time intervals up to 7 days (at 37 °C) in comparison to 177Lu-DOTA-NCS. The results are given in Fig. 3. It is evident that the radiochemical purity of 177Lu-PCTA-NCS did not decrease significantly with time and was more or less similar that of 177Lu-DOTA-NCS complex. Even at 7 days post-incubation, >95 % of the 177Lu-PCTA-NCS complex remained intact, both in saline and serum.

Fig. 3
figure 3

In vitro stability of 177Lu-PCTA-NCS and 177Lu-DOTA-NCS in saline and serum (incubated for 7 days at 37 °C)

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Based on the results of the Octanol:water partition experiments, log P values for 177Lu-PCTA-NCS and 177Lu-DOTA-NCS were determined to be −1.4 ± 0.2 and −2.4 ± 0.4. Based on the log P values obtained herein, it is evident that 177Lu-PCTA-NCS is less hydrophilic than the corresponding DOTA-NCS complex.

Biodistribution studies

Biodistribution of 177Lu-PCTA-NCS was carried out in normal Swiss mice in comparison to that of 177Lu-DOTA-NCS, the results of which are shown in Table 3 as the percentage administered activity per gram of tissue/organ (%ID g−1). It is evident from the data that most of the 177Lu-PCTA-NCS complex is excreted by 24 h p.i., similar to that of 177Lu-DOTA-NCS complex. Activity in the blood was not very high in both cases. However, the uptake of 177Lu-PCTA-NCS complex in the liver and intestine was higher than that of the 177Lu-DOTA-NCS complex at 1 h and 3 h p.i. which decreased significantly by 24 h p.i., as shown in Table 4. The absence of noteworthy uptake in the bone affirmed the in vivo stability of the 177Lu-PCTA-NCS complex.

Table 3 Biodistribution pattern of 177Lu-PCTA-NCS in normal Swiss mice (n = 4)
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Table 4 Biodistribution pattern of 177Lu-DOTA-NCS in normal Swiss mice (n = 4)
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Discussion

The successful clinical deployment of 177Lu labeled peptides and antibodies has led to the widespread recognition of 177Lu as an important therapeutic radionuclide. The pursuit for chelating agents which enable near-complete complexation at ambient temperature while imparting high stability to the complex in vivo has resulted in the synthesis and evaluation of a number of ligands in recent times. One such ligand PCTA is reportedly of potential application due to its fast complexation rate with a number of medically important radioisotopes [18, 19]. Although there are a few reports on the use of this ligand for preparation of 177Lu labeled monoclonal antibodies and peptides [29], to the best of our knowledge, detailed studies as regards the influence of trace metal ions, in vitro stability and the biodistribution pattern of 177Lu-PCTA-NCS complex are not yet reported. Herein we report detailed studies carried out to evaluate the suitability of PCTA-NCS as a BFCA for design of 177Lu radiopharmaceuticals. The results of our work reported here show that under the optimized conditions, high radiolabeling yields could be achieved with PCTA-NCS even at 1:2 lutetium to PCTA-NCS molar ratios. However, the radiolabeling yields at equimolar ratios of Lu and PCTA-NCS was less than 90 %, in contrast to 177Lu-DOTA-NCS complex having equimolar ratios of Lu and DOTA-NCS. Experiments carried out to determine the detrimental effects of trace metal contaminants on 177Lu-PCTA-NCS complex formation showed that while Fe(III) and Cu(II) ions had minimum influence on the radiolabeling yields at low concentrations, Zn(II) ions reduced the yields at equimolar ratios with respect to lutetium. A similar trend was observed for DOTA-NCS ligand. However, none of the trace metallic impurities studied reduced the radiochemical yields significantly even at high metal to lutetium molar concentrations, although all the studied cations such as Cu(II), Zn(II), and Fe(III) also form thermodynamically stable complexes with the BFCAs used for complexation with 177Lu due to their suitable ionic size, charge and co-ordination chemistry [18, 30, 31]. Asti et al. and Pawlak et al. have previously reported a detailed study of the influence of trace metal ions on 90Y and 177Lu labeling of DOTA-TATE [26, 32]. They have reported that the radiochemical yields of 177Lu-DOTA-TATE is drastically reduced in presence of trace metal ions such as Cu, Fe and Zn. However, a direct comparison of our results of 177Lu-DOTA-NCS with that reported by them for 177Lu-DOTA-TATE is difficult due to the difference in the experimental conditions. Asti et al. have reported that the exclusion of one carboxylate group as in DOTA-TATE (DO3A) has a major impact on the stability of the complexes with Y3+ or Lu+3 [26]. In addition, the influence of trace metal ions would also depend on the lutetium to ligand molar ratios used for the studies. With higher lutetium to ligand molar ratios, the interference of the trace metal ions is expected to be lower [26]. 177Lu-PCTA-NCS complex also exhibited high in vitro stability, both in saline and serum. Results of the biodistribution studies carried out in Swiss mice show that 177Lu-PCTA-NCS did not show any significant uptake in any vital organs, except the liver and intestine. The dose to the liver and intestine was more with 177Lu-PCTA-NCS than 177Lu-DOTA-NCS at 1 and 3 h p.i. Similar results have been also reported for 90Y and 68Ga labeled PCTA-NCS complexes by other researchers [21, 22]. The higher liver uptake may be attributed to the lower hydrophilicity of 177Lu-PCTA-NCS complex in comparison to the 177Lu-DOTA-NCS complex. The kinetic stability of 177Lu-PCTA-NCS in vivo could be confirmed from the absence of notable uptake in the bone. These results establish the utility of PCTA-NCS ligand as a bifunctional ligand for use with 177Lu, especially while radiolabeling temperature-sensitive biomolecules as the radiolabeling is complete within few minutes at ambient temperature.

Conclusion

In this study, the potential use of PCTA-NCS ligand as a BFCA for 177Lu was evaluated. The high labeling efficiency, in vitro stability and results of the biodistribution studies show that PCTA-NCS ligand could be a suitable BFCA for use with 177Lu.