Journal of Radioanalytical and Aruclear Chemistry, Articles, Vol. 132, No. 2 (1989) 241-249
FORMAMIDINE SULFINIC ACID AS REDUCING AGENT IN TECHNETIUM'99m RHENIUM SULFIDE LABELLING M. NEVES,* H. FERRONHA,** L. PATRICIO* *Departamento de Radiois6topos, LaboratSrio National de gngenharia e Technolot,ia Industrial Estrada Naeional 10. 2685 Saeav~'m, (portugal) **Servile de MicroScopia Electr6nica, Laborat6rio Nacional de Investigafao Veterindria, Estrada de Benfica 70.1:.t 500 Lisboa (Portugal)
(Received January 17, 1989) Labelling kinetic studies, radiochemical characterization and particle size evaluation of technetium-99m rhenium sulfide colloid using forraamidine sulfinie acid as reducing agent are described. Comparison with the same colloid wMcli makes use of Sn-sodium pyrophosphate complex as reducing agent has shown higher labelling yields, simplification of labelling procedure and a longer shelf life when formamidine sulf'mie acid is used,
Introduction Radionuclide lymphoscintigraphy has major significance in therapeutic irradiation planning and has become an integral part o f clinical practice of nuclear medicine. A relative number of colloids for lymphoscintigraphy were described. Its selection is not easy because in some cases, tracer migration is slow and does n o t reflect lymphatic flow. The phagocytosis o f colloids is related to the physical and chemical properties of the colloidal solution and the biological environment, but remains unclear which is the preponderant parameter. Concerning physical and chemical properties, several authors have suggested that particle size is a critical parameter and STRAND et alJ claimed that the most suitable colloid has a very small range of particle size, in the o~der of a few nanometers. Surface properties of colloidal particles, as surface charge, and the presence o f stabilizers s e e m t o be relevant parameters. In fact, NAGAI et al.2 showed that the positive charged 99mTc-stannous colloid, despite o f having particles size in the range of 7 0 - 8 0 nm, is more easily taken up by macrophages than negative charged 99mTc-colloids. They also showed that colloids preparea *Author for correspondence. This work was presented in part at the 3th European Symposium. on Radiopharmacy and Radiopharmaeeutieals. Elsinore. Denmark. May 1987. Elsevier SequoiaS. A., Lausanne Ak~Idmiai Kill6, Budapest
?/l. NEVES et al.: FORMAMIDINESULFINICACID AS REDUCINGAGENT using stabilizers, such as gelatine and polivinylpyrrolidone (PVP), are incorporated into macrophages to a greater extent than colloids without stabilizers. 19 SAu colloid provided high quality lymphoscintigraphy owing to their optimum particle size but has unacceptably high absorbed radiation dose, especially at the site of injection. Several 99mTc colloids have been used to visualize lymph channels and nodes, such as sulfur, antimony sulfide, rhenium sulfide, stannous phytate, and more or less encouraging results are reported in the literature~ 3 -s In some cases, tracer migration is slow and variable from batch to batch and therefore may not accurately or reproducibly represent true lymphatic flow. 6 Among the 99mTc colloids, rhenium sulfide has particle size in the range of a few nanometers and, despite a negative surface charge, 7 is claimed to be a good lymphoscintigraphy agent of internal mammary and cervical lymphoadenopathies, s ,9 In the present work, we describe the 99 m Tc labelling of rhenium sulfide colloid using formamidine sulfinic acid as reduction agent. Its radiochemical characterization and particle size evaluation were also studied. Formamidine sulfinic acid (FSA) was described as a useful biochemical reducing agent by SHASHOUA 10 in 1964 and was introduced as an alternative reducing agent to stannous chloride for the preparation of 99 m Tc-labelled radiopharmaceuticals by FRITZBERG et al. 1 ~ in 1977. The advantage of FSA is to avoid the tendency of stannous salts to hydrolysis and oxidation. FRITZBERG et al. described FSA as a heating dependent reductant on labelling of HEDSPA (ethane-l-hydroxy-l,l-diphosphonic acid, disodium salt), pyrophosphate, DTPA (diethylenetriaminepentaacetic acid) and bioquin-TCA (8-hydroxyquinoline-7-carboxylic acid), SCOTT et al.12 reported that using FSA as reducing agent on labelling of calcium, glucoheptonate a high radiochemical stability was achieved, even at high levels of 99mTc activities. BALDAS and POJER ~3 found that preparations of 99 m Tc-HIDA (N,[a-2,6-dimethylcarbamoylmethyl]minodiaceticacid), using FSA as reductant, were significantly different from those using stannous chloride, both in chromatographic behaviour and "in vivo" distribution. In fact, the radiochemical and biological behaviour of some 99 m Tc.labelled compounds were significantly different from those using stannous chloride as reductant. In the case of colloidal solutions, the nature of the reducing agent is not relevant since the chemical management and biodistribution are determined ma.inly by the colloidal particles characteristics. Comparative studies of the 99mTc-rhenium sulfide colloid developed by us (LNETI-kit) using FSA as reductant and a commercial 99m Tc-rhenium sulfide colloid (COM-kit) using stannous chloride as reducing agent were performed.
242
M. NEVES et al.: FORMAMIDINESULFINICACID AS REDUCINGAGENT
Materials and methods
Rhenium sulfide colloid The rhenium sulfide colloid was prepared by a similar procedure developed by BARDY et al.14 using 99,9% rhenium metal powder BDH. By gently bubling of H2 S gas through a HReO4 solution in acid medium and in presence of gelatine powder Merck, Re2 $7 was precipitated as a brown colloidal solution. Excess of ionic rhenium was eliminated by basic anionic exchange resin Dowex 50-100 mesh Fluka. The Re2 S~ colloid was stabilized by L(+)-ascorbic acid Merck, at final pH 4.8-5.0.
Labelling kinet& studies To 1 ml of Re2 $7 colloidal solution, was added 1 ml of formamidinesulfinic acid (aminoiminomethane sulfinic acid, Sigma) at Concentration range 0.5-10.0 mg/ml. Each mixture was adjusted to pH4.8-5.0, freeze-drying and kept under nitrogen. 2 ml of a saline solution 99 m TcO4-eluted from a commercial generator was added to the lyophilized mixture, stirred vigorously and heated in a water-bath at 50 + 1 ~ for 10 minutes and allowed to coot at room temperature. The same procedure was performed at 70 + 1 and 99 -+ 1 ~ Labelling yields were evaluated by paper chromatography.
Paper chromatography Labelling yields were monitored by ascending paper chromatography using Whatman No. 1 developed in NaC1 0.9% solution. The colloid remains at the origin and 99 m TcO4 showed Rf = 0.7.99 m Tc.rhenium sulfide colloid labelling yields expressed in percentage of preparations heated at 50 + 1, 70 • 1 and 99 • 1 ~ were studied as a function of FSA concentrationsrange 0.5-10.0 mglml,
Gel chromatography The sample to be analyzed was applied in a volume of OA ml to the top of a column (30 • 1.6 cm ~b) tilled with Sepharose CL-6B gel and eluted with 17 ml of 0.9% NaC1 solution. The column was sealed and scanned in horizontal position with a NaI (TI) collimated detector. The scan profile recorded allows to evaluate the activity-size distribution of radiocolloids. Identical procedure was also applied to Bio-Gel P6 column (17.5 • 1.0 cm ~b). 243
M. N-EVESet al.: FORMAMIDINE SULFINIC ACID AS REDUCING AGENT
Electron microscopy The following colloidal solutions were studied using electron microscopy: - rhenium sulfide, - mixture of 1 ml of rhenium sulfide and 1 ml of FSA 5 mg/ml, _ 99raTe.rhenium sulfide. The samples were spotted on copper grids (400 mesh) coated with carbon-stabilized formvar and allowed to partially dry after removing the excess of colloid. The grids were washed with bidistilled water and then subjected to osmium tetroxide for intensification of particle density. After washing, preparations were screened and photographed with a Jeol 100CXII electron microscope calibrated against a cross grating replica (Polaron).
Results and discussion Labelling kinetic studies presented in Fig. 1 allow us to optimize the experimental conditions leading to high radiochemical yield labelling. So,-a typical 99 m Tc.rhenium sulfide labelling consists of adding 2 - 3 ml Of A
'ooI
~ 80 E
0 99•176 70_+1oC
q 60
9 50•176
40 20 0 0
.
_~ ~ ' T - ,
2
I
4
6
,
I
,
I
8 10 FSA, mglm[
Fig. 1. Labelling yieid function of FSA concentration at 50 -+ 1, 70 -+ 1 and 99 -+ 1 ~
99 m TcO2 tO a freeze-dried mixture of 1 ml of Re2 $7 colloid and 1 ml of FSA (5 mg/ml), followed by 10 minutes heating in a boiling water bath and then cooled to room temperature. This preparation is radiochemical by stable at least during 4 hours after labelling. The amount of reducing agent used is very low 244
M. NEVES et al.: FORMAMIDINE SULFINIC ACID AS REDUCING AGENT COM - kit
*
A'
* 99r"TcO~.
*
lO0~
L N E T I - kit
99mTcO~'
9
lO0OC/lOmin
Fig. 2. Labelling procedure of COM-kit and LNETI-kit: A - Sn-sodium pyrophosphate complex, B - rhenium sulfide colloid, C - freeze-dried mixture of rhenium sulfide colloid and FSA &
99~T(:_rheni u
LN~EII-ki -
io
"!
;OMjkit O
' FS
| Fig. 3. Chromatograms of LNETI and COM kits on Sepharose CL-6B 245
M. NEVES et al.: FORMAMIDINE SULFINIC ACID AS REDUCING AGENT
compared with the FSA toxicity values referred to in the literature} 1 The radiochemical behavibur of 99mTc.rhenium sulfide developed by us (LNETI-kit) was compared with a commercial rhenium sulfide kit, (COM-kit) wich includes two vials, one containing a Sn-sodium pyrophosphate complex and the other a rhenium sulfide colloid/d solution. The 99mTc.rhenium sulfide labelling is
Tc-rhenium e
o
LNETI-kit COM-kit 0
FS
Fig. 4. Chromatograms of LNETI and COM kits on Bio-Gel P6
achieved in two steps. The labelling procedures of both kits are presented in Fig. 2. The radioactive profiles on Sepharose CL-6B of LNETI and COM kits are shown in Fig. 3. On Sepharose CL-6B 99 m Tc.rhenium sulfide colloid was previously eluted, then 9 9 m T c as pertechnetate, which is in agreement with the activity-size distribution of radiocolloids reported by STRAND et al.15 for Sepharose CL-4B. Concerning the COM-kit, an unexpected peak exhibiting approximately the same partition coefficient as the 9 9 r n T c o 4 peak was assigned and identified as 99roTe-sodium pyrophosphate. This peak, as well as the 99 m TcO4 peak, were identified running individual preparations on the gel chromatography column. Identical gel chromatography procedure using Bio-Gel P6 has shown the same elution order and similar profile (Fig. 4.) The radioactive profiles of paper chromatography strips are shown in Fig. 5. The radioactive impurities presented in the COM-kit were also evaluated by paper chromatography and also identified as 99 m TcO4 and 99 raTc.sodium pyrophosphate by running individual preparations in the same experimental conditions used for the col246
M. NEVES et al.: FORMAMIDINESULFINICACID AS REDUCINGAGENT
> U
Tc-rhenium sulfide
o u E
J
~ 99mTc-pyr~176
0
..>
99,n
FS
99m
ium sulfide
o ~J
0
FS'
F!gr 5. Paper chromatograms of COM and LNETI kits
loidat solutions. The radiochemical impurity of 99 mTc.sodiu m pyrophosphate could be explained by that in COM.kit the stannous chloride is replaced by a Sn-sodium pyrophosphate complex, perhaps to overcome the problem of stannous hydrolysis at pH values of the rhenium sulfide colloid (4.8-5.0). A labelling reaction competition between sodium pyrophosphate and rhenium sulfide colloid may OCCUr, and in some preparations the radiochemical impurities could reach about 20%. This led us to search for a more efficient reducing agent, and FSA has fulfilled this aim. In fact, using FSA the labelling yield is higher than 99% and the labelling procedure is clearly simplified. Another relevant improvement of LNET!-kit is the fact that as the mixture of rhenium sulfide colloid and FSA is 247
M. NEVES et al.: FORMAMIDINE SULFINIC ACID AS REDUCING AGENT
b
,.
r
i
t i *J
I
i
n
,O,5.u J
Fig. 6. Electron micrograph of 99 mTc rhenium sulfide
&
~176 / %
(140 particle measured)
50 z,0Min. size ~5,34 Sdev~ 4,61 Vor : 21,11
30
2o[ i
lOL o
10:15
15120 20:25
Z5:30 30:35 35:40 Particle dio.meter, nm
Fig, 7, Parfi~le size distribution
248
M. NEVES et al.: FORMAMIDINE SULFINIC ACID AS REDUCING AGENT freeze.dried, the kit has a validation period longer than a year when stored at 2 - 8 ~C, Electron microscopic analyses of the colloidal samples revealed the presence of spherical ~ a p e particles and a similar particle size and size variability in all solutions under study. An electron micrograph o f 99 m Tc-rhenium sulfide colloidal solutions .is shown in Fig. 6. The size of 93.5% of measured particles ranged from !0 to 25 nanometers, In Fig, 7 is presented.a particle size distribution graphic of !40 measured particles of the same preparation showing a mean particle size of 15.34 nm. A lymphatic pre-clinical evaluation of LNETI-kit has shown significantly nodal uptake, However, further clinical evaluation should be carried out.
References 1. S E, STRAND, B R. R PERSSON, J. Nucl. Med, 20 (1979) 1038. 2. K. NAGA!, Y ITO, N OTSUKA,Int. J. Nuel. Med, Biol., 9 (1982) 108. 3. G. L- DUNSON, J..H. THRALL, J. S. STEVENSON~ S..M. PINSKY, Radiology, 109 (1973) 387. 4, W. D KAPLAN, M. A. DAVIS, C.M. ROSE, J. Nuc! Med., 20 (1979) 933. 5, K NAGA!, !, YASUHIKO, O. NOBUAKI, A. MURANAKA, Eur, J, Nucl. Med., 7 (1982) 66. 6 H.J. DWORKIN, J. Nue!. Med., 23 (!982) 936. 7. M NEVES, L, PATRIC!O, Int. J, App!. Radiation Isotopes, 39 (!988) 183, 8. A, PI~CK!NG, N. MERNER, R. GOBIN~A. BARDY, Y. NAGEAN, J. Fr. Biophys. Med. Nucl., 2 (!978) 1!7. 9. S TZ!LA ZWAS, Y,.RAMON, Int. J. Nuel Med Biol, !3 (1986) 633. 10~ V. E. SHASHOU, Biochemistry, 3 (1964) 1719. !1, A. R FRITZBERG, D M LYSTER, D. H DOLPHIN, J. Nue!, Med., 18 (1977)553. 12. J, R, ~COTT, G, L. GARRET, B~C KENTLY, Int J Nuel. Med. Biol., 7(19'80) 71. !3,. ~. BALDAS, P, M; POGER, Int. J. Nucl. Med. Biol,, 8(!981) !10. 14, A, BARDY, J, BAyDON, M HEGESIPPE, Int. J. App!. Radiation Isotopes, 24 (1973) 57. ! 5. S. E; STRAND, B. R, R. PERSSON, J. Nucl. Med., 20 (!979) 1038.
249