Three principal diseases associated with synovia hypertrophy are rheumatoid arthritis, hemophilic joint disease (principally related to bleeding into the joint), and some cases of traumatic joint disorders. The number of hemophilic patients is relatively small. In contrast, rheumatoid arthritis affects 1–2% of the world's population with a preponderance of men over women [
1,
2]. Cellular recruitment and proliferation, with subsequent formation of synovial granulation tissue (pannus) and an increased secretion of synovial fluid characterize the inflammatory process. Progression of the disease leads to the destruction of the joint or loss of function. Surgery is the last option as a conventional technique for treating intractable joint disease after pharmacological therapies have failed, but removal of all the tissue is not frequently achieved and so there is high recurrence after 3 to 5 years [
2]. Radiation synovectomy provides an interesting alternative because cartilage is relatively radioresistent, so a radioactive agent with effective soft tissue penetration can be administered directly into the joint, causing no harm to the adjacent cartilage. As beta radiation can penetrate only a few hundred cell diameters, microparticles labeled with beta emitting radionuclides are effective in treating the disease by radiation in confined spaces without endangering nearby normal tissues [
2]. Radiopharmaceutical particle size must be small enough to be phagocyted by the superficial cells of the sinovium, but not so small as to facilitate a fast biological clearance by diffusion from the joint [
2,
3].
Many beta emitters, and many other particulate chemical compounds have been used as radiosynovectomy agents [
4-
6]. In this study
188Re is the chosen radionuclide as it is readily available on routine basis from the
188W/
188Re generator. The radionuclide
188Re, has a β-ray emission of sufficient energy (2.11 MeV) to penetrate 5–10 mm of thickened synovial membrane, and its low-level γ-ray emission (155 keV) makes scintigraphic monitoring possible, without harming patients or practitioners. Its half-life (16.9 hr) is adequate in terms of obtaining an appropriate therapeutic effect or for handling of the agent, avoiding hazardous residual effects.
In this study, particulate chemical compounds such as tin colloid, hydroxyapatite particles and ferric hydroxide macroaggregates were compared from the physico-chemical and biological point of view.
The most important criteria of therapeutically useful radiolabeled microparticles are their physico-chemical characteristics such as size range, surface area and volume, insolubility in aqueous media and irreversible attachment of radionuclide to the particles [
1,
2,
7]. The best method of particle size determination is electron microscopy and more recently laser scattering, both of which provide information on particle number, size and size distribution. The last of these is used in this study.
Radiopharmaceutical leakage from the knee was evaluated by the acquisition of scintigraphic images over 48 hr after intraarticular administration of the radiolabeled preparations to New Zealand rabbits. Biodistribution studies were also performed.
The results were compared in order to establish which was the radiopharmaceutical that best fits the requirements of this kind of therapy.
Methods
Radiopharmaceutical composition
Four radiopharmaceutical kits were prepared according to the following formulations:
• 15 mg SnCl2.2H2O
• 0.5 mL HCl 0.1 N
• N2 atmosphere
188Re-Hydroxyapatite particles (Indirect method) [
1]
First step: preparation of 188Re-HEDP kit
• 10 mg HEDP (free acid)
• 3 mg gentisic acid
• 0.3 mg KReO4
• 3.8 mg SnCl2.2H2O
Second step: preparation of 188Re-Hydroxyapatite (HA) particles kit:
• 40 mg Hydroxyapatite (Ceramed, Type II, 20 um, CAT N° 157-2000)
• 650 μL 0.9% saline solution
• 50 μL of 20% Tween 80 (in water)
• 100 μL of SnCl2.2H2O solution (4 mg/mL)
• N2 atmosphere
188Re-Hydroxyapatite particles (Direct method) [
9]
• 40 mg Hydroxyapatite (Ceramed, Type II, 20 μm, CAT N° 157-2000)
• 20 mg SnCl2.2H2O
• 13.7 mg K2C2O4.H2O
pH was adjusted to 1.5 with HCl 0.75 N
188Re-Ferric Hydroxide Macroaggregates, modified from Castro M, Portilla A. [
10]
First step: preparation of 188Re-Tin colloid:
• 15 mg SnCl2.2H2O
• 0.5 mL HCl 0.1 N
• N2 atmosphere
Second step: preparation of 188Re-Ferric Hydroxide Macroaggregates (FHMA):
• 1.8 mL FeSO4.7H2O (7.36 mg/mL)
• 0.7 mL NaOH 0.1 N
• 1.1 mL 0.9% saline solution
• 0.6 mL PVP K30 (16 mg/mL)
• 3 mL PVP K30 (16 mg/mL) pH 8.5
All reagents used were analytical grade.
Equipment
• Dose calibrator system: Capintec Radioisotope Calibrator CRC 5
• Solid scintillation counter NaI(Tl) 3 × 3": EG&G ORTEC Multichannel Analyzer
• Particle size analyzer: Particle Size Analyzer Coulter ®
• Gammacamera: Sophy Camera DSX
Labeling procedure and quality control
The 188Re used in all formulations were eluted from a 188W/188Re generator (Oak Ridge Laboratories, United States).
188Re-Tin colloid
The 188Re-Sn colloid was labeled by the addition of 500 μCi (18.5 MBq) of 188ReO4- to the kit formulation described above, then it was autoclaved for 1 hour. pH was adjusted to 6.0 with the addition of NaOH 1.0 N.
Radiochemical purity was determined by paper chromatography (Whatmann 1) using 0.9% saline solution as the mobile phase. Radioactivity was measured with a NaI(Tl) solid scintillation counter.
188Re-Hydroxyapatite particles (Direct method)
40 mg of hydroxyapatite particles were mixed in a centrifuge tube with 13.7 mg K2C2O4.H2O and 20 mg SnCl2.2H2O with 0.3 mL of distilled water. 3 mCi (111 MBq) of 188ReO4- (contained in 0.4 mL) was added to the tube. The pH of the reaction mixture was made acidic (pH 1.5) by slow addition of HCl 0.75 N. The suspension was vortexed and incubated at room temperature for 1 hour. 1 mL of ascorbic acid (10 mg/mL) was added to the suspension at the end of the hour and was centrifuged at 2000 rpm for 2 minutes. Activity in supernatant and particles was measured in a dose calibrator system. The particles were washed twice with 2 mL of ascorbic acid. The activity of the particles and washings was also measured. The final suspension was made in ascorbic acid (10 mg/mL, pH 5).
The percentage of bound activity was determined by measuring the activity of both particles and supernatant solution in a dose calibrator. From these data the percentage of labeled particles yield was calculated.
188Re-Hydroxyapatite particles (Indirect method)
First step: preparation of 188Re-HEDP. 188Re-HEDP was prepared by adding 1 mL of a 188ReO4- solution (11 mCi / 414 MBq) to a vial containing the lyophilized HEDP kit formulation described above (pH ≈ 1). The solution was heated in a water bath for 10 minutes at 100°C.
Radiochemical purity of 188Re-HEDP was determined by the paper chromatography method using a solution of HEDP 0.01 M in saline/Whatmann 3 MM and acetone/Whatmann 1 to determine 188ReO4-, 188Re-HEDP and reduced hydrolyzed 188Re species respectively.
Second step: preparation of 188Re-HA. 188Re-Hydroxyapatite particles were prepared by sequential addition of the following materials to a centrifuge tube containing 40 mg of hydroxyapatite particles: 650 μL of N2-purged saline, 200 μL of 188Re-HEDP (4 mCi / 148 MBq), 50 μL of 20% Tween 80 in water and 100 μL of a N2-purged SnCl2.2H2O solution (4 mg/mL). The mixture was incubated for 1 hour at room temperature with occasional stirring. 4 mL of saline solution was added to the contents of the tube and then centrifuged at 2000 rpm for 4 minutes. The supernatant and the particles were separated. The 188Re-Hydroxyapatite particles were resuspended with 3 mL of saline solution.
The percentage of bound activity was determined by measuring the activity of both particles and supernatant solution in a dose calibrator. From these data the percentage of labeled particles yield was calculated.
188Re-Sn-Ferric Hydroxide Macroaggregates
First step: preparation of 188Re-Sn colloid: The 188Re-Sn colloid was labeled by the addition of 500 μCi (18.5 MBq) of 188ReO4- to the kit formulation described above, then it was autoclaved for 1 hour.
Radiochemical purity was determined by paper chromatography (Whatmann 1) using 0.9% saline solution as the mobile phase. Radioactivity was measured with a NaI(Tl) solid scintillation counter.
pH was adjusted to 7 with NaOH 1.0 N.
Second step: preparation of 188Re-FHMA: 1 mL of 188Re-Sn colloid and 1.8 mL of ferrous sulfate solution (7.36 mg/mL) were mixed in a centrifuge tube, 0.7 mL of NaOH 0.1 N and 1.1 mL of saline solution were added. The contents of the tube were vortexed for 10 seconds. 0.6 mL of Polivinylpirrolidone (PVP) solution (16 mg/mL) were added, vortexed and centrifuged at 1400 rpm for 4 minutes.
The precipitate particles were separated and the labeled FHMA were washed as follows.
188Re-FHMA were mixed with 3 mL of a PVP solution (16 mg/mL, pH 8.5), vortexed and centrifuged at 1400 rpm for 4 minutes. The supernatant and the macroaggregate were separated and the activity of both was measured in a dose calibrator.
The 188Re-FHMA was resuspended in 1.5 mL of saline solution and 1.0 mL of phosphate buffer 0.2 M (pH 7.5).
The percentage of bound activity was determined by measuring the activity of both precipitate and supernatant solution in a dose calibrator system. From these data the percentage of FHMA labeled yield was calculated.
Physical characterization of the radiopharmaceuticals
Non-radioactive forms of the radiopharmaceuticals were prepared using tracer quantities of potassium perrhenate in saline solution in volumes and concentrations corresponding to those of generator eluate.
The number, volume and area of the radiopharmaceuticals particles were analyzed with a laser diffraction particle size analyzer. Particles size were grouped in the following ranges, <2 μm, 2–10 μm, 10–40 μm and >40 μm.
Stability studies
In vitro and in vivo stability studies were performed for the 188Re-Sn colloid, 188Re-HA particles (Direct method) and for the 188Re-Sn-FHMA.
In vitro stability studies
Each radiopharmaceutical was kept at room temperature or at 37°C for 2 and 24 hr after labeling. Stability of 188Re-Sn colloid, 188Re-HA particles and 188Re-Sn-FHMA was assessed in saline solution, ascorbic acid (pH 5)/human serum and saline solution/human serum, respectively. In every case the percentage of bound activity was measured.
In vivo stability studies
Urine samples were collected during the first 24 hr post-radiopharmaceutical administration.
Biodistribution in New Zealand adult male rabbits (4 kg weight) was performed. The animals were sacrificed with an overdose of sodium thiopenthal after 48 hr of intraarticular administration of 188Re-Sn colloid, 188Re-HA direct method and 188Re-Sn-FHMA (1 mCi/mL).
Knee joint, thyroid, heart, urinary bladder, gall bladder, liver, spleen, lungs, stomach, intestines, kidney, muscle, bone, blood and urine samples were collected and radioactivity was measured in a scintillation counter.
Scintigraphic studies
The New Zealand rabbits were anesthetized by intramuscular administration of 50 mg/kg of ketamine and 10 mg/mL of xilazine. Scintigraphic images were acquired with a Sophy Camera DSX, with medium energy collimator, at 0, 24 and 48 hr after intraarticular administration of each radiopharmaceutical.
1 María Cristina Ures,1 Patricia Zeledón,2 Victoria Trindade,1 Andrea Paolino,1 Virginia Mockford,1 Antonio Malanga,3 Marcelo Fernández,1 and Javier Gaudiano4
publication history
