Production, quality control, and bio-distribution studies of (159)Gd-EDTMP as a palliative agent for bone pain.

INTRODUCTION: Particle-emitting, bone-seeking radiopharmaceuticals have attracted the attention of the nuclear medicine community over the last three decades for the treatment of the pain of osteoblastic metastases. The objectives of this research were to produce quality-controlled (159)Gd-EDTMP in order to provide a new therapeutic radiopharmaceutical for use in clinical applications.
METHODS: The investigation was an experimental study in which (159)Gd (T1/2=18.479 h, Eβ (max)=970.60 keV, Eγ=363.55 (11.4%) keV] was produced by thermal neutron bombardment of natural Gd2O3 at the Tehran Research Reactor (TRR) for a period of 7 d at a flux of 3-4×10(13) neutrons/cm(2).s. It was then quality-controlled and used to radio-label the in-house prepared ethylene diamine tetra acetic acid (EDTM).
RESULTS: Complexation parameters were optimized to achieve maximum yields (>99%). The radiochemical purity of (159)Gd-EDTMP was checked by radio thin layer chromatography RTLC. It was found to retain its stability at room temperature (>95%). Bio-distribution studies of the complexes conducted in wild rats showed significant bone uptake with rapid clearance from blood.
CONCLUSION: The properties of the (159)Gd-EDTMP that was produced suggest then use of a new, efficient, palliative therapeutic agent for metastatic bone pain instead of some other current radiopharmaceuticals.

Introduction

Bone metastases are a frequent complication of cancers such as prostate and breast (70% of patients), lung, colon, stomach, bladder, uterus, rectum, thyroid, or kidney (15–30% of patients) cancers leading to painful and untreatable consequences including fractures, hypercalcemia, and bone pain, as well as reduced performance status and quality of life. The exact incidence of bone metastasis is unknown, but it is estimated that 350,000 people die with bone metastases annually in the United States (1, 2). For all these reasons, bone metastasis is a serious and costly complication of cancer. Currently, the treatment of bone pain remains palliative at best with systemic therapy (analgesics, hormones, chemotherapy, steroids, and bisphosphonates) as well as local treatments (such as surgery, nerve blocks, and beam radiation) (3). Particle-emitting bone-seeking radiopharmaceuticals have attracted the attention of the nuclear medicine community over the last three decades for the treatment of the pain of osteoblastic metastases. For the eight pharmaceuticals including: 188Re (Sn) HEDP, 153Sm-EDTMP, 90Y-Citrate, 186Re(Sn)HEDP, 117mSn-DTPA, 32P-phosphate, 89Sr-chloride, 85Sr-chloride, there are published data on clinical trials in humans. All are reactor produced, and all emit a beta particle except for tin (Sn)-117 pentetate and strontium-85 (Sr-85) which produce low energy conversion electrons. 89SrCl2 and 153Sm-EDTMP are widely preferred for the management of pain arising due to skeletal metastasis. 89SrCl2 has the advantage of comparatively longer half-life of 50.5 days, which makes it possible to supply the radiopharmaceuticals worldwide. 153Sm-EDTMP is preferred by many investigators due to the more favorable radionuclidic properties of 153Sm. However, the relatively short half-life of 153Sm precludes its use from places other than proximal or well connected to the production site (4–12). 177Lu-EDTMP or other phosphonates are also proposed as alternatives to 153Sm-EDTMP as the long half-life (13–18).

The 159Gd radionuclide is a beta (Eβ(max)=970.60 keV(32%)) and gamma (main energy: 363.55 keV) emitter with a half-life of 18. 479 h (19). The physical characteristics of the 159Gd isotope suggest that it has the potential to be used in nuclear medicine research (20–22). The β− energy of 159Gd is lower than that of 89Sr and hence the bone marrow dose is expected to be much lower. The presence of accompanying gamma photons which can be imaged by using widely available gamma camera systems is advantageous in carrying out simultaneous dosimetry and scintigraphy studies. 159Gd can be produced by a relatively easy route involving thermal neutron bombardment on natural Gd2O3 in medium flux research reactors. The requirement for an enriched target does not arise and radionuclidic impurities are not formed by radiative capture during neutron activation. G. J. Beyer introduced the 159Gd-ethylenediaminetetra-methylinephosphonic acid agent for effective palliative treatment of skeletal metastases (23). These types of phosphonate complexes concentrate in the skeleton, in proportion to osteoblastic activity and interrupt the vicious cycle and cause not only a reduction in osteolytic bone lesions, but also decrease the tumour burden in bone (2). In the present study, the preparation, quality control and biodistribution studies of 159Gd-EDTMP is reported in order to provide a new therapeutic radiopharmaceutical to enter in clinical applications in the country.

Material and Methods

Materials

Gadolinium oxide (spectroscopic grade N99.99% pure) was obtained from E. Merck (Darmstadt, Germany). EDTMP was synthesized and characterized in-house as per the reported procedure. All other chemicals were purchased from Sigma-Aldrich Chemical Co. U.K. Whatman 3 MM chromatography paper (UK) was used as the stationary phase. Radiochemical purity of gama-spectroscopy on the base of 363.55 keV peak and beta-spectroscopy were carried out using the HPGe detector and the Wallac 1220 Quantulus liquid scintillation spectrometer, respectively. Radio-chromatography was performed by counting of Whatman No. 2 using a thin layer chromatography scanner, Bioscan AR2000, Paris, France. Animal studies were performed in accordance with the United Kingdom Biological Council’s Guidelines on the Use of Living Animals in Scientific Investigations, 2nd edition.

Synthesis of EDTMP

EDTMP was synthesized by following a Mannich-type reaction (24), using orthophosphorus acid, 1,2-ethylenediamine and formaldehyde in strongly acidic medium. In a typical reaction, 1,2-ethylenediamine (5 g, 0.08 mol) was added slowly to a solution of anhydrous orthophosphorus acid (33.66 g, 0.34 mol), in concentrated HCl (33.44 g, 0.92 mol) and the mixture was allowed to reflux. Formaldehyde 37% (10 g, 0.01 mol) was added drop wise over a period of 15 min to the fluxing mixture. The refluxing was continued for another 2 h and subsequently the mixture was cooled to room temperature overnight. Then the resultant was added to ethanol and EDTMP was precipitated in ethanol. The precipitation then was filtered under vacuum and then was dried in oven in 60 °C. It was purified after recrystallization from water/methanol m.p. 214–215 °C. IR (KBr, ν cm−1): 3308, 2633, 2311, 1668, 1436, 1356. 1H-NMR (D2O, δ ppm): 3.53 (d, J=12.3 Hz, 8H,-N-CH2-P=O), 3.85 (s, 4H, -N-CH2-). 13C NMR (D2O, δ ppm): 51.63, 52.73. 31P NMR (D2O, δ ppm): 10.52 (25).

Production of 159Gd

159Gd was produced by thermal neutron bombardment on natural Gd2O3 at the Tehran Research Reactor (TRR) for a period of 7 d at a flux of 3–4×1013 neutrons/cm2.s. In a typical procedure, 11.52 mg of Gd2O3 was sealed and irradiated in the reactor after placing it inside aluminum can. The irradiated powder was dissolved 1 mL of HCl 0.1M heated until all the powder was completely dissolved. This radiochemical form was used for the subsequent studies. The radionuclidic purity of the solution was tested for the presence of other radionuclides using beta spectroscopy as well as HPGe spectroscopy for the detection of various interfering beta and gamma emitting radionuclides.

Preparation 159Gd -EDTMP complex

A stock solution of EDTMP was prepared by dissolving EDTMP (100 milligram) NaHCO3 buffer (5 ml, pH .9). A portion of this solution containing 37.5 mg of EDTMP, was used for complexation of 159Gd (1 mg, 2 mCi) which results in a complex with specific activity of 12.3GBq/mmol. The pH of the reaction mixture was adjusted to 7 and was incubated at room temperature for 15 min to facilitate complexation. The radiochemical purity of the preparation was determined by paper chromatography using two solvent systems. Ammonia/methanol/water (2:20:40 v/v) and also 1 mM DTPA were used as the eluting solvents for paper chromatography.

Stability of 159Gd -EDTMP in final formulation

The final formulation was stored in 25 °C for two days in order to determine the stability. Radiochemical purity of the complex was studied by frequent ITLC analysis using the mentioned system.

Stability of 159Gd -EDTMP in the presence of human serum

To determine the stability of final formulation in human serum, 200 μCi (200 μl) of complex (159Gd -EDTMP) was incubated in the freshly prepared human serum (300 μl) at 37 °C for 2 d. The stability was determined by performing frequent ITLC analysis using the mentioned system.

Biodistribution studies in rats

Biodistribution studies of the 159Gd -EDTMP complex were carried out in wild-type rats each weighing 180–210 g. A volume of 200 μl containing 200±5 μCi of radioactivity was injected through a lateral tail vein. The animals were sacrificed at the exact intervals of 2 h, 4 h, 6 h, 20h and 40 h post injection. The tissue and the organs were excised and the activity associated with each organ/tissue was measured in a flat-type NaI (Tl) scintillation counter. The uptake in different organs/tissue was calculated from these data and expressed as % Injected Dose (% ID/gram).

Results

Production and quality control of 159Gd

Irradiation of natural Gd2O3 was performed at a thermal neutron flux of 3–4×1013 neutrons/cm2.s for 7 d at TRR and the radionuclide was prepared according to regular methods with a range of specific activity 15–20 mCi/mg for radiolabeling use. Gamma ray spectrum of the appropriately diluted 159GdCl3 solution showed a major peak at 363.5 keV, which are the photo-peak of 159Gd (Figure 1).

Figure 1.

γ-ray spectrum for 159Gd chloride solution

The radioisotope was dissolved in acidic media as a starting sample and was further diluted and evaporated for obtaining the desired pH and volume followed by sterile filtering. The absence of any other photo-peaks in the gamma ray spectrum indicated that the 159Gd was produced with a radio-nuclidic purity of >99.99%. The radiochemical purity of the 159Gd solution was checked in two solvent systems, including DTPA 1 mM and ammonia/methanol/water (2:20:40 v/v) and Whatman 3MM as the stationary phase. In the first solvent system, Gd3+ cations migrated to the higher Rf. The ITL-chromatogram is presented in Figure 2a. In the latter, the Gd3+ cations remained at the point of spotting (lower Rfs), as shown in Figure 2b.

Figure 2.

TLC chromatogram of 159GdCl3 solution in DTPA, Whatman 3 MM system (a) and 159GdCl3 solution in NH4OH/MeOH/HOH: 2/20/40, Whatman 3 MM (b), 159Gd-EDTMP solution in DTPA, Whatman 3 MM system (c), 159Gd-EDTMP solution in NH4OH/MeOH/HOH: 2/20/40

Preparation of 159Gd-EDTMP complex

Various parameters such as ligand concentration, temperature, pH of reaction and time were varied in order to reach the maximum complexation. It was observed that complexation gradually increased with increase in ligand concentration and reached to ∼ 100% at a ratio of [ligand]/[metal] ∼ 15:1. On variation of reaction pH from 4 to 10, it was found that a maximum complexation yield of >99% was achieved in the pH range of 6 to 8. The in vitro stability studies were performed by incubating the complex at room temperature and showed that the radiochemical purity of the complex remained >95% up to 4 days after preparation. In paper chromatography using (DTPA 1 mM as solvent and Whatman 3 MM as stationary phase), it was observed that the un-complexed 159Gd moved towards the solvent front (Rf=0.8–0.9) while the 159Gd -EDTMP complex remained at the point of spotting (Rf=0–0.1) under identical conditions (Figure 2a and 2b). Paper chromatography using another system, ammonia/methanol/water (2:20:40 v/v), was also performed. It was observed that the 159Gd -EDTMP complex moved towards the solvent front (Rf=0.8–0.9) while the un-complexed 159Gd remained at the point of spotting (Rf=0–0.1) under identical conditions (Figure 2c and 2d). The stability of 159Gd -EDTMP complex was checked up to 48 h (Figure 3) using DTPA 1 mM as solvent and Whatman 3 MM as stationary phase after preparation. The complex was stable in final sample and its radiochemical purity was above 99% even 48 h after preparation using Whatman 3 MM eluted with 1 mM solvent of DTPA. The latter stability test was developed for the complex in presence of human serum at 37 °C using ITLC (DTPA 1 mM as solvent and Whatman 3 MM as stationary phase) (Figure 4).

Figure 3.

TLC chromatogram of 159Gd -EDTMP complex after 48 h in DTPA, Whatman 3 MM system

Figure 4.

TLC chromatogram of 159Gd -EDTMP complex in presence of human serum at 37 °C in DTPA, Whatman 3 MM system

The animals were sacrificed by CO2 asphyxiation at selected times after injection (2 h, 4 h, 6 h, 20 h and 40h). Dissection began by drawing blood from the aorta followed by removing heart, spleen, muscle, brain, bone, kidneys, liver, intestine, stomach, lungs and skin samples. The tissue uptakes were calculated as the percent of area under the curve of the related photo peak per gram of tissue (% ID/g). The distribution of injected dose in rat organs up to 40 hours after injection of 159Gd-EDTMP (200 μCi / 200 μl) solution was determined. Based on these results, it was concluded that the major portion of injected activity was extracted from blood circulation into bones. The results of the biodistribution studies are expressed in Figure 5 and revealed significant uptake in skeleton within less than 4 h pi. It was determined in wild-type animals for better comparison for 2–40 h pi. The blood wash-out happens after 4h. 159Gd-EDTMP is rapidly taken up in bones 2 h after administration and retains almost constantly up to 40 hours while the free radio-lanthanides (zzzLns) uptake increases at first as a result of affinity of the lanthanide ions to the bone due to their similarity to the calcium cation. It reaches a maximum value more than that of zzzLn-EDTMP (chelated radio-lanthanide) and then decreases to a value much less than that of zzzLn-EDTMP. 159Gd-EDTMP has almost no accumulation in liver, while as a free cation, being transferred by serum metalloproteins, free Gd-159 would accumulate in liver. Also 159Gd-EDTMP has almost no accumulation in spleen, while free Gd-159 would accumulate in spleen especially after 2 d.

Figure 5.

Percentage of injected dose per gram (ID/g %) of 159Gd -EDTMP in wild-type rat tissues at 2 h, 4 h, 6 h, 20 h and 40h post injection

Discussion

The absence of any other photo-peaks in the gamma ray spectrum indicated that the radio-gadolinium was prepared with a radionuclidic purity of more than 99.99%. As the Gd3+ cations are complexed to the more lipophilic 159Gd-DTPA form, migration to higher Rf occurs. As expected, the data obtained from other solvent systems concluding (ammonia/methanol/water: 2/20/40 v/v) confirmed that there was just one cationic specimen in the sample. In the second system, lower Rf was observed because of the significant difference in polarity between the Gd(III) cation and the solvent system it remains at the origin of the stationary phase.

Preparation of the 159Gd-EDTMP complex

As a result of the similarity between the polarities of the 159Gd-EDTMP complex and the ammonia/methanol/water solvent system, the more lipophilic species 159Gd-EDTMP complex (other than Gd3+) moved towards the solvent front in the (ammonia/methanol/water: 2/20/40 v/v) - Whatman 3 MM stationary phase system and vice versa for the other solvent system.

The affinity of the phosphonate complex is more than that of the free ion to the bone (4–7); therefore the free cation is released from the bone structure faster than zzzLn-EDTMP. As mentioned earlier, 159Gd-EDTMP is rapidly taken up in bones and the trapping continues in a way that almost no blood circulation activity as well as kidney excretion can be observed. As can be seen from Figure 5, and similar to the previous studies (3–17, 25), the washed-out activity of free cation is higher than that of complexed isotope. One of the most important features of the prepared formula was that there was almost no accumulation of 159Gd-EDTMP in the spleen or liver, which is a major advantage for its use as a therapeutic radiopharmaceutical because it would be possible to increase the maximum injectable dose (25). Another advantage of the prepared compound is that its activity was observed to be retained in the skeletal bones until 40 h post injection up to which time the bio-distribution studies were continued.

Conclusions

159Gd-EDTMP preparation (radiochemical purity of more than 99%) was administered to normal rats and related biodistribution data were checked 2 h to 40 hours later showing at least 2.3 ID%/g accumulation of the drug in the bone tissues. Also 159Gd-EDTMP has almost no accumulation in liver and spleen which is main advantage of this radiopharmaceutical. The development of other 159Gd-labeled therapeutic molecules, monoclonal antibodies and also peptides for ultimate radioimmunotherapy is possible.