Studies on the Labeling of Ethylenediaminetetramethylene Phosphonic Acid, Methylene Diphosphonate, Sodium Pyrophosphate and Hydroxyapatite with Lutetium-177 for use in Nuclear Medicine.

For the treatment of skeletal metastasis, a therapeutic radionuclide tagged with a bone seeking ligand is required, while for radiation synovectomy (RS), a therapeutic radionuclide irreversibly attached to pre-formed particles of appropriate size is required. Radio lanthanides are mostly therapeutic, and ligands containing phosphate groups are predominantly bone seekers. Exploiting these facts, number of new therapeutic radiopharmaceuticals could be developed. Labeling of four phosphate containing materials was pursued in the present study. It was hypothesized that various (177)Lu-labeled bone-seeking complexes such as (177)Lu-ethylenediaminetetramethylene phosphonic acid (EDTMP), (177)Lu-methylene diphosphonate (MDP) and (177)Lu-pyrophosphate (PYP) could be developed as agents for palliative radiotherapy of bone pain due to skeletal metastases, and (177)Lu-Hydroxyapatite (HA) could be developed as an agent for radiosynovectomy of small joints. Lyophilized kit vials of EDTMP, MDP and sodium pyrophosphate (Na-PYP) were formulated. HA particles were synthesized locally and purity was checked by high-performance liquid chromatography (HPLC). (177)Lu was labeled with EDTMP, MDP, PYP, and HA and the behavior of all was studied by radio-thin layer chromatography (TLC) radio-HPLC and radio-electrophoresis. Radio-TLC confirmed the labeling. HPLC analysis too verified the labeling. Radio-electrophoresis results depicted peaks for (177)Lu-MDP, (177)Lu-EDTMP and (177)Lu-PYP at 3.37 ± 0.06 cm, 5.53 ± 0.15 cm and 7.03 ± 0.06 cm respectively confirming negative charge on each specie as all migrated toward positive anode. All 3 methods verified the labeling. The study demonstrated that EDTMP, MDP and PYP form stable complexes with (177)Lu in injectable solution form. HA particulates could too be labeled with (177)Lu with high radiochemical yields (>98%) in suspension form. Former three could be utilized as bone-pain palliation agents for the treatment of bone metastases, and the later could be applied for the treatment of Rheumatoid arthritis of small joints. The study has also indicated the possibility of developing other numerous radiolanthanide analogs with the potentials of possible use in radiation therapy.

Introduction

Phosphate containing ligands like Ethylenediaminetetramethylene phosphonic acid (EDTMP), methylene diphosphonate (MDP) sodium pyrophosphate (Na-PYP) all labeled with a radionuclide act as bone-seeking radiopharmaceuticals. Structures of EDTMP, MDP and Na-PYP are shown in Figure 1.

Figure 1

Structure of (a) 177Lu-Methylene diphosphonate, (b) Sodium pyrophosphate and (c) 177Lu-ethylenediaminetetramethylene phosphonic acid

Bone scanning using the 99mTc-phosphate analogs is an established diagnostic modality for a variety of pathologies.[1] Complex of MDP with 99mTc has been widely used as radiopharmaceutical for bone scintigraphy in cases of metastatic bone disease, Paget's disease, fractures in osteoporosis, and henceforth for the last quarter of a century.[2345] Bone scanning with 99mTc pyrophosphate is very useful for the detection of soft-tissue lesions that produce extra skeletal ossification.[6] EDTMP labeled with 153Sm give rise to 153Sm-EDTMP (153Sm labeled EDTMP) a bone-seeking tetraphosphonate, which have been approved by the Food and Drug Administration for the treatment of painful osseous metastases.[7] Synovectomy by an intra-articular application of a β-emitting radioisotope in colloidal form or radiation synovectomy (RS) was introduced in 1952 for treatment of inflamed synovial membrane.[8] An ideal agent for RS would be one in which the radionuclide is irreversibly attached to pre-formed particles of appropriate size. The 177Lu-Hydroxyapatite (HA) [Ca10 (PO4 6)(OH)2] is one of the preferred particulates as it is constituent of bone matrix and natural substance known to be biodegradable.[9]

177Lu (t½ =6.71 d) is an adequate radionuclide for therapy, which has both beta particle emissions with Emax = 497 keV (78.6%), 384 keV (9.1%) and 176 keV (12.2%) for therapeutic effect and gamma emissions 113 keV (6.4%) and 208 keV (11%) for imaging. 177Lu decays to stable 177Hf and its long half-life provides logistic advantage for facilitating the supply to places far away from the reactor.[91011] The major advantage of 177Lu lies in the feasibility of its large-scale production with excellent radionuclide purity and adequate specific activity owing to the high thermal neutron capture cross-section of 176Lu (2100 b) using moderate flux reactors.[12]

For the treatment of skeletal metastasis, a therapeutic radionuclide tagged with a bone seeking ligand is required while, for RS, a therapeutic radionuclide irreversibly attached to pre-formed particles of appropriate size is required. Radio lanthanides are mostly therapeutic, and ligands containing phosphate groups are predominantly bone seekers. Fortunately, lanthanides have a strong affinity towards ligands containing phosphate groups. Exploiting these facts, number of new therapeutic radiopharmaceuticals could be developed. Complex formation of four phosphate containing materials was pursued in the present study. It was hypothesized that various 177Lu-labeled bone-seeking complexes such as 177Lu-EDTMP, 177Lu-MDP and 177Lu-PYP could be developed as agents for palliative radiotherapy of bone pain due to skeletal metastases, and 177Lu-HA could be developed as an agent for radiosynovectomy.

Materials and Methods

Methylene Diphosphonic acid (99.0%), Tetra Na-PYP (95.0% F. Wt. =265.9) and EDTMP all of Aldrich Chemistry were used in the study.

Natural Lu2O3 (99.9% chemically pure, 2.6% 176Lu) powder from A Johnson Mathey Company (UK) was used as a target for the production of 177Lu. 177LuCl3 solution was prepared by dissolving irradiated natural Lu2 O3 powder in 0.1 MHCl with a little heating. Normally, vials containing 10 mg/ml MDP, 28 mg/ml Na-PYP and 35 mg/ml EDTMP (except reported) were used for labeling studies. 177LuCl3 solution was used for labeling of kit vials. HA [Ca10 (PO4)6 (OH)2 ], was synthesized locally. Kits of EDTMP, MDP and Na-PYP were formulated by dissolving appropriate amounts of the ligands in double distilled water with adjustment of relevant pH. These solutions were dispensed in vials. Vials were placed in a freeze dryer. Freeze drying was performed for 24 h with a shelf temperature of −80°C and 0.630 mbar pressure. Vials were caped under vacuum and stored at room temperature. Composition of each freeze dried kit is mentioned in Table 1.

Table 1

Composition of freeze dried kits

Natural Lu2 O3 (10 mg) target was irradiated at a thermal flux ~8.0 × 10 13n/cm2/s for 12 h for the production of 177Lu. The irradiated target was dissolved in 1 MHCl with gentle heating and filtered inside a home-made lead-shielded plant. Specific activity of the product was ~25.3 mCi/mg at EOB. Analysis of the gamma ray spectrum of the irradiated target revealed major γ-peaks at 72, 113, 208, 250 and 321 keV, which correspond to the photopeaks of 177Lu as per literature[13] and the radionuclide purity of 177Lu more than 99%. Analysis of the gamma ray spectrum was carried out by using a p-type coaxial HPGe detector (Eurisys Mesures, France) coupled through a 570 ORTEC made spectroscopy amplifier toTrump PCI, 8 k ADC/MCA card with Gamma Vision-32 ver. 6 software (ORTEC, USA).

Desired volume of 177LuCl3 solution (containing required activity) was taken in the vials containing MDP 10 mg/ml Na-PYP 28 mg/ml and EDTMP 35 mg/ml Resulting solutions were incubated for ½ h at room temperature. 1 ml NaHCO3 (0.5M) and 1 ml normal saline were added to 100 mg of HA in a vial. A volume of 1 ml NaOH was added to make the pH > 7. 177LuCl3 (solution in HCl) was injected to the vial and shaken for 30 min. The shaken mixture was centrifuged at 3500 rpm for 10 min. Supernatant was removed carefully and again saline was added for washing. Centrifugation was carried out for another 5 min. Supernatant was removed again to have 177Lu-labeled hydroxyapatite (177Lu-HA), which was used for further studies.

The centrifuged shaken mixture of 177LuCl3 and HA in 1 ml saline in the form of suspension was used for radiochemical purity check by thin layer chromatography (TLC) system using EDTA as mobile phase. Aliquots from the vial were spotted on Whatman 3 MM paper strips and eluted to develop actigrams.

The kit vials (MDP, PYP and EDTMP) containing 177LuCl3 solution (0.5–1.0 mCi/vial) were shaken and kept at room temperature, and the radiochemical purity check was carried out by TLC system using ammonium hydroxide: Methanol: Water (1:20:20) as mobile phase. Aliquots from the vials were spotted on Whatman 3 MM paper strips of 2 × 14 cm and eluted up to 12 cm. The chromatograms were dried and after drying the strip was subjected to 2π-scanner Berthold coupled with NaI detector to get actigrams depicting the labeling yield.

All the 4 Lu labeled complexes were incubated for >24 h at room temperature. To observe the stability of the complexes, aliquots from the vials containing 177Lu-PYP, 177Lu-MDP, 177Lu-EDTMP and 177Lu-HA complex at different time intervals (1 h-24 h) were also spotted on paper strips, eluted and processed likewise by virtue of which in vitro stability of the labeled preparations were ascertained.

To determine the effect of temperature on labeling yield, 177Lu-MDP, 177Lu-PYP and 177Lu-EDTMP solutions were heated in three vials with temperature monitoring and aliquots from the vials at various temperatures 20, 40, 60, and 80°C (each vial was heated at the specified temperatures other than 20°C for 1 min) were spotted on paper strips and eluted and subjected to 2π-scanner Berthold coupled with NaI detector to get actigrams depicting the labeling yield and hence the effect of temperature on the labeling yield was determined. Vial containing 177Lu-HA particulates in 1ml saline was also subjected to high temperatures likewise and aliquots from the vials at various temperatures were spotted on paper strips and eluted with EDTA and actigrams were developed. The centrifuged shaken mixture of 177LuCl3 and HA in 1 ml saline in the form of suspension was used for radiochemical purity check by TLC system using EDTA as mobile phase. Aliquots from the vial were spotted on Whatman 3 MM paper strips and eluted to develop actigrams.

To verify the complex formation and to determine the retention time of 177Lu-PYP, 177Lu-MDP and 177Lu-EDTMP complexes, the reaction mixtures were analyzed by high performance liquid chromatography (HPLC). First 20 μl of 177LuCl3 (10 mCi) solution was injected (thrice) into the column, and the elution was monitored by observing the radioactivity profile. Similarly, 20 μl of the test solution of each type was injected (thrice) into the column and the elution was monitored. Chromatograms were obtained on Hitachi L6200 HPLC system with NaI crystal detector using C-18 reversed phase (25 × 0.5 cm) column utilizing (1:1) mixture of water and methanol as the mobile phase. Both results of TLCTLC and HPLC were compared for the said reaction mixture.

To determine the charge on the 177Lu-PYP, 177Lu-MDP and 177Lu-EDTMP complex, radio-electrophoresis was conducted. 10.0 μl of each solution (177Lu-PYP, 177Lu-MDP and 177Lu-EDTMP and 177LuCl3) was spotted in the center of 30 cm strip of Whattman 3 MM chromatography strips (30 × 2 cm) at 15 cm from each electrode. Paper electrophoresis was carried out for 1 h under a voltage of 300 V using 0.025M phosphate buffers pH 6.9 and 45 mA current. Wet paper strips were removed and placed on a tissue paper to dry for an hour. Radio electrophoretograms were accomplished by placing the filter paper strip on the 2π-scanner. Paper electrophoresis was carried out with the Delux electrophoresis chamber coupled with the power supply (Gelman Instrument Company USA).

Results

After elution, the dried Whatman 3 MM paper strips were subjected to 2π-scanner. The scanner generated radiochromatograms [Figure 2a–2c] which depicted the labeling of 177Lu-PYP, 177Lu-MDP and 177Lu-EDTMP to be 99.689%, 99.379% and 99.698%, respectively. All the complexes moved towards solvent front while free 177LuCl3 remained at the origin. Radiochemical purity and labeling efficiency was found to be >99%. While the radiochromatogram [Figure 2d] showed peak at RT = 1.14 (98.021%) indicating 177Lu-HA and shoulders at RT = 5.52 indicating 177Lu-EDTA.

Figure 2

Peaks of actigrams (a-c) representing 177Lu-pyrophosphate complex, 177Lu-Methylene diphosphonate complex and 177Lu-ethylenediaminetetramethylene phosphonic acid complex respectively and actigram (d) representing 1777Lu-Hydroxyapatite (% radiochemical purity is depicted as shown by the Printout of 2π-scanner)

Labeling yield at different time intervals 1 h, 6 h, 18 h and 24 h after the initiation of reaction came out to be 99.99 ± 0.01, 99.34 ± 0.89 98.74 ± 0.74, and 98.14 ± 0.67 for 177Lu-PYP complex while 99.14 ± 0.25, 99.31 ± 0.20, 99.19 ± 0.10, 99.35 for 177Lu-MDP, and 99.11 ± 0.21, 99.04 ± 0.29 98.44 ± 0.14, and 98.05 ± 0.37 for 177Lu-EDTMP and 99.88 ± 0.52, 99.74 ± 0.49, 99.44 ± 0.34 and 98.55 ± 0.47 for 177Lu-HA. The complexes retained >98% labeling efficiency even after 24 h hence all the 4 complexes could be considered quite stable. Graphic representation of this result is depicted in Figure 3.

Figure 3

Labeling yield at various time intervals

The labeling yield for 177Lu-PYP at various temperatures 20, 40, 60 and 80°C was determined to be 98.09 ± 0.13, 99.13 ± 0.10, 99.76 ± 0.14 and 100.00 ± 0.00 respectively. The labeling yield for 177Lu-MDP was 65.43 ± 5.05, 81.03 ± 1.29, 90.42 ± 0.61, 99.11 ± 0.21 and was 60.53 ± 4.25, 89.05 ± 1.18, 90.12 ± 0.43, 99.41 ± 0.31 for 177Lu-EDTMP and 99.79 ± 0.15, 99.63 ± 0.19, 99.26 ± 0.24, 99.06 ± 0.04 for 177Lu-HA. The results as depicted in Figure 4 showed that the stability of the complexes remained intact at temperatures higher than room temperature.

Figure 4

Labeling yield at various temperatures

The HPLC chromatogram of the test solutions clearly showed distinct peaks at different retention times thereby confirming the labeling of 177Lu with PYP, MDP and EDTMP. The HPLC chromatograms are shown in Figure 5. It was observed that the retention time of 177Lu-PYP complex was 1.42 ± 0.01 min, 177Lu-MDP 1.35 ± 0.05 min and 177Lu-EDTMP 1.54 ± 0.01 min while that of the free 177LuCl3 was found to be 2.23 ± 0.02 min. On injecting all the three labeled species simultaneously, 4 distinct peaks appeared as shown in Figure 6. Results shown by HPLC were in close agreement with those shown by TLC.

Figure 5

Radio-electrophoratograms (A) free 177LuCl3 (B) 177Lu-pyrophosphate complex (C) 177Lu-methylene diphosphonate complex (D) 177Lu-ethylenediaminetetramethylene phosphonic acid complex

Figure 6

High performance liquid chromatography pattern of (a) Free 177LuCl3 (b) 177Lu-pyrophosphate complex (c) 177Lu-Methylene diphosphonate complex (d) 177Lu-ethylenediaminetetramethylene phosphonic acid complex

In paper radio-electrophoresis (0.025 M phosphate buffers pH 6.9), 177LuCl3 did not show any movement from point of spotting. Peaks for 177LuCl3 appeared at 15 cm (point of spotting). Activity peaks of 177Lu-PYP complex appeared far from point of spotting [Table 2]. 177Lu-PYP complex showed migration toward anode to the extent of 7.03 ± 0.06 cm, indicating the formation of negatively charged complex. Point of spotting for each experiment was 15.0 cm. Data pertaining to all three radiopharmaceuticals is tabulated in Table 3.

Table 2

Labeling yields of 177Lu-PYP 177Lu-MDP and 177Lu-EDTMP complexes for various quantities of Lutetium as a function of fixed quantity of ligands

Table 3

Electrophoresis data of Lu-phosphate complexes

Peaks for 177Lu-MDP and 177Lu-EDTMP appeared at 3.37 ± 0.06 cm and 5.53 ± 0.15 cm respectively. Radio electrophoretograms accomplished by placing the filter paper strip on the 2π-scanner are shown in Figure 5.

Radio-electrophoresis not only confirmed the labeling of 177Lu with PYP, MDP and EDTMP but also confirmed that all thee complexes 177Lu-MDP, 177Lu-PYP and 177Lu-EDTMP are negatively charged as they migrated toward positive anode.

Discussion

Large-scale production of 177Lu with excellent radionuclide purity and adequate specific activity due to high thermal neutron capture cross-section of 176Lu using moderate flux reactors makes it a suitable candidate for therapeutic applications. As much interest is being shown currently in the use of 177Lu for various applications, so pursuing, the evaluation of any of its complexes would be quite reasonable[1415]

From the present study, it is clearly evident that 177Lu could be labeled with MDP, PYP and EDTMP with radiochemical purity higher than 99% at 30 min after the start of the reaction. Also, the preparation of complexes 177Lu-MDP, 177Lu-PYP and 177Lu-EDTMP is very simple, and the complexes are quite stable. Lu + 3ions are oxygen seekers and phosphonic acid groups (containing oxygen) of MDP, PYP and EDTMP are available for co-ordination with 177Lu + 3. HA particulates too, could be labeled with 177Lu with high radiochemical yields (>98%). The lanthanides chemically are very similar, and any ligand that makes complex with one could make complexes with all of them.

This study has indicated that numerous radiolanthanide complexes like 169Er-MDP, 161Tb-MDP, 143Pr-MDP, 159Gd-MDP, 153Sm-MDP, 149Pm-MDP, 165Dy-MDP, 166Ho-MDP, 142Pr-MDP, could be developed as well as their analogs with HA, PYP and EDTMP. All these complexes have potentials to be utilized as palliative agents for bone metastasis. Based on the results obtained, hypothetical Structures of the polyphosphate complexes namely 177Lu-PYP, 177Lu-MDP and 177Lu-EDTMP could be designed as mentioned in Figure 7.

Figure 7

Hypothetical structure of (a) 177Lu-pyrophosphate (b) 177Lu-Methylene diphosphonate and (c) 177Lu-ethylenediaminetetramethylene phosphonic acid complex

Conclusion

The study demonstrated that MDP, EDTMP and Na-PYP form stable complexes with 177Lu in injectable solution form. HA, particulates too could be labeled with 177Lu with high radiochemical yields (>98%) in suspension form. Former three could be utilized as bone-pain palliation agents for the treatment of bone metastases, and the later could be applied for the treatment of Rheumatoid arthritis of small joints. The study has also indicated the possibility of developing other numerous radiolanthanide analogs with the potentials of possible use in radiation therapy.