International Journal of Applied Radiation and Isotopes, 1976, Vol. 27, pp. 69-72. Pergamon Press. Printed in Northern Ireland
Electrolytical Labeling of' "Tc Radiopharmaceuticals MARfA
C E C I L I A GIL, T E R E S A P A L M A and R. R A D I C E L L A * Comisi6n Chilena de Energia Nuclear, Santiago, Chile
(P~cei~d 19 ~ptember 1975)
INTRODUCTION IT IS well known that the labeling of several compounds with °~mTc is carried out by reducing 9°'~Tc0C to a lower valence state. Reducing agents such as Sn ~-, Fe z+ and ascorbic acid have a widespread use in the preparation of °°=Tc labeled radiopharmaceuticals. The labeling mechanism and the role of iron and tin ions are not completely understood. Labeling kits prepared using Sn z+ solutions are unstable because of the prompt oxidation of stannous tin. T o prevent this oxidation the solutions must be kept in an inert atmosphere or they must be liofilized. Electrolytical procedures for the 99'~Tc labelhag of proteins and other compounds have been proposed in the last years. Electrolysis is used both for the reduction of pcrtechnetate and for the production of intermediate agents that facilitate in some way the combination of the °°=Tc with the molecule to bc labeled. In this case the chemistry of the labeling is also rather unclear but it seems evident that the nature of the electrodes plays an important role. Different electrodes have been suggested; (x) however, only the labeling with Zr electrodes has been studied and used in some extent. (s-x°> More recently, tin electrodes have been used by SCHNEIDER(11) to prepare a 99'nTc(Sn) citrate complex, and by NARASIMHA~ and ~ ' D ~ (lz) to obtain °gr+Tc-human serum albumin. The electrolytic method has the advantage of producing Sn z+ ions in situ during the labeling procedure. This means, in principle, that the kits would have a considerable longer shelf-life. Moreover the amount of tin in the final preparations is normally lower than in the case 1
69
of the use of stannous tin solutions and well below the toxic level,as) This is particularly important for the production of radiopharmaccutlcals, where the amount of foreign elements must be kept as low as possible. With these advantages inmindwc have studied the "electrolyrical" preparation, with tin electrodes, of some commonly used 9~"Tc radlopharmaccuticals. W c have investigated the labeling of human serum albumin (HSA), H S A macroaggregates (MAA), phytate, gluconate, pyrophosphate and DTPA. High labeling yields have been obtained in all cases, in a very short time. The results show that the procedure is suitable for the preparation of stable, inexpensive and ready-to-use kits. EXPERIMENTAL Electrolysis was carried out in a penicillin type vial The electrodes were two 4 cm length, 0.05 cm dia. tin wires inserted into the vial through the rubber cap. The electrolytic power (3 V) was supplied by two 1.5 V dry batteries connected in series with a milliammeter and a variable potentiometer. During the electrolysis the vim was shaken gently in order to avoid the slow fall of the current that was observed when the solution was kept stationary. The labcliug yield, for the different compounds, was determined as a function of both the pH of the mixture and the total amount of charge transferred through the solution. * Expert of the International Atomic Energy Agency. Permanent address: Comisldn National de Energia At6mica, Buenos Aires, Argentina.
70
M. G. Gil, T. Palma and R. Radicdla
T h e p H of the solutions was adjusted to values between 4 and 6, with HC1 or N a O H aqueous solutions of suitable concentration. In the case of MAA p H was adjusted to values between 4 and 8. T h e total amount of charge (Q.) passed through the solution at a constant p H value, was established by varying the current or the electrolysis time. T h e presence of labeled particulate matter formed during the labeling procedure was investigated in all cases by filtration of the labeled preparation through colloidal membranes 0.22 nm pore size ("Millipore" type). T h e amount of dissolved tin after electrolysis was determined radiometrically by using electrodes previously irradiated in the R E C H - I reactor and by atomic absorption spectroscopy. The general labeling procedure consisted in adding 2 ml of 99"Tc04- physiological saline solution to a mixture of 0.5 ml of the preparation to be labeled and a certain amount of hydrochloric acid or sodium hydroxide, so that the final solution had the desired p H value and passing the required amount of current. Aqueous solutions of the compounds to be labeled were used. T h e macroaggregate suspension was prepared in the following way: ~t4) heat for 1-2 rain in a water bath at 95°C, with continuous stirring, a mixture of 10 ml HSA solution 0.5 mg/ml, 10 ml sodium acetate 10 ~o and 10 ml H C I 0.2 N. Centrifuge, wash the precipitate with saline solution. Centrifuge again and take up the precipitate in 50 ml saline. Autoclave the suspension for 15 rain at 130°C. Radiochemical purity was determined by ascending paper chromatography on W h a t m a n N ° 3 M M using 85 ~ methanol as solvent for pyrophosphate, phytate, gluconate and HSA preparations. Methyl ethyl ketone was used as solvent to determine free 99mTc-pertechnetate in D T P A preparations. U n b o u n d technetium in MAA preparations was determined by filtration through colloidal membranes 0.22 nm pore size and the presence of labeled particulate material less than 8 n m in diameter was determined by filtration through an 8 nm pore size colloidal membrane. Tissue distribution was studied by i.v. injection in the femoral vein of white mice.
After a suitable time the activity of different organs was measured in an automatic gamma spectrometer with a well type scintillation detector. RESULTS
AND DISCUSSION
Labeling yields as a function of p H at a constant value of Q (0.24 Coulombs) for the different labeled compounds were better than 99 ~o for all molecules labeled except HSA in which case free pertechnetate accounted for 3 ~o of the activity. T h e activity retained by the colloidal membrane was less than 1 ~o. Again HSA was an exception, since the activity associated with particulate material was about
5Vo. For this reason we adopted the value of the p H closest to the physiological one, in which the smallest percent of activity bound to particulate matter was found. T h e values chosen for the electrolytic labeling of the different compounds were those shown in Table I. Labeling yields were studied as a function of the amount of current passed through the solution at the p H value chosen for each compound. Results are shown in Table 1. It can be seen that radiochemical purity for Q =- 0.24 is higher than 99 ~ for all compounds except HSA and DTPA, which nevertheless present a radiochemical yield compatible with clinical use
•
T h e amount of tin dissolved during electrolysis under these conditions was found to be 82 pg/ml. This is lower than the amount of tin used in the preparation of commonly available kits. O n the basis of these results the following procedures for labeling the different compounds were established: (1) Use sterile solutions and materials (2) Pass a 2 mA current for 2 rain, in all cases, gently shaking the vial. (3) Shake for 30 see after electrolysis has finished. (4) Allow to stand for 2 rain.
1. Labeling of potassium pyrophosphate 1. Add 2 ml of 98'~Tc04- saline solution to a mixture of 0.5 ml of a 50 mg]ml potassium pyrophosphate solution and 0.7 ml HC1 0.2 N.
Eleetrolytical labeling of t*mTc radiopharmaceuticals
71
TABLE 1. Free pertechnetate as a function of Q (~o) 0,03 Fyrophosphate pH 5 HSA pH 4.5 Gluconate pH 5.5 Phytate pH 5.0 DTPA pH 5.0 MAA pH 5.0
0.09
0.24
0.30
12.1
6.5
1.8
1.1
1.1
Electrolytical Labeling of' "Tc Radiopharmaceuticals MARfA
C E C I L I A GIL, T E R E S A P A L M A and R. R A D I C E L L A * Comisi6n Chilena de Energia Nuclear, Santiago, Chile
(P~cei~d 19 ~ptember 1975)
INTRODUCTION IT IS well known that the labeling of several compounds with °~mTc is carried out by reducing 9°'~Tc0C to a lower valence state. Reducing agents such as Sn ~-, Fe z+ and ascorbic acid have a widespread use in the preparation of °°=Tc labeled radiopharmaceuticals. The labeling mechanism and the role of iron and tin ions are not completely understood. Labeling kits prepared using Sn z+ solutions are unstable because of the prompt oxidation of stannous tin. T o prevent this oxidation the solutions must be kept in an inert atmosphere or they must be liofilized. Electrolytical procedures for the 99'~Tc labelhag of proteins and other compounds have been proposed in the last years. Electrolysis is used both for the reduction of pcrtechnetate and for the production of intermediate agents that facilitate in some way the combination of the °°=Tc with the molecule to bc labeled. In this case the chemistry of the labeling is also rather unclear but it seems evident that the nature of the electrodes plays an important role. Different electrodes have been suggested; (x) however, only the labeling with Zr electrodes has been studied and used in some extent. (s-x°> More recently, tin electrodes have been used by SCHNEIDER(11) to prepare a 99'nTc(Sn) citrate complex, and by NARASIMHA~ and ~ ' D ~ (lz) to obtain °gr+Tc-human serum albumin. The electrolytic method has the advantage of producing Sn z+ ions in situ during the labeling procedure. This means, in principle, that the kits would have a considerable longer shelf-life. Moreover the amount of tin in the final preparations is normally lower than in the case 1
69
of the use of stannous tin solutions and well below the toxic level,as) This is particularly important for the production of radiopharmaccutlcals, where the amount of foreign elements must be kept as low as possible. With these advantages inmindwc have studied the "electrolyrical" preparation, with tin electrodes, of some commonly used 9~"Tc radlopharmaccuticals. W c have investigated the labeling of human serum albumin (HSA), H S A macroaggregates (MAA), phytate, gluconate, pyrophosphate and DTPA. High labeling yields have been obtained in all cases, in a very short time. The results show that the procedure is suitable for the preparation of stable, inexpensive and ready-to-use kits. EXPERIMENTAL Electrolysis was carried out in a penicillin type vial The electrodes were two 4 cm length, 0.05 cm dia. tin wires inserted into the vial through the rubber cap. The electrolytic power (3 V) was supplied by two 1.5 V dry batteries connected in series with a milliammeter and a variable potentiometer. During the electrolysis the vim was shaken gently in order to avoid the slow fall of the current that was observed when the solution was kept stationary. The labcliug yield, for the different compounds, was determined as a function of both the pH of the mixture and the total amount of charge transferred through the solution. * Expert of the International Atomic Energy Agency. Permanent address: Comisldn National de Energia At6mica, Buenos Aires, Argentina.
70
M. G. Gil, T. Palma and R. Radicdla
T h e p H of the solutions was adjusted to values between 4 and 6, with HC1 or N a O H aqueous solutions of suitable concentration. In the case of MAA p H was adjusted to values between 4 and 8. T h e total amount of charge (Q.) passed through the solution at a constant p H value, was established by varying the current or the electrolysis time. T h e presence of labeled particulate matter formed during the labeling procedure was investigated in all cases by filtration of the labeled preparation through colloidal membranes 0.22 nm pore size ("Millipore" type). T h e amount of dissolved tin after electrolysis was determined radiometrically by using electrodes previously irradiated in the R E C H - I reactor and by atomic absorption spectroscopy. The general labeling procedure consisted in adding 2 ml of 99"Tc04- physiological saline solution to a mixture of 0.5 ml of the preparation to be labeled and a certain amount of hydrochloric acid or sodium hydroxide, so that the final solution had the desired p H value and passing the required amount of current. Aqueous solutions of the compounds to be labeled were used. T h e macroaggregate suspension was prepared in the following way: ~t4) heat for 1-2 rain in a water bath at 95°C, with continuous stirring, a mixture of 10 ml HSA solution 0.5 mg/ml, 10 ml sodium acetate 10 ~o and 10 ml H C I 0.2 N. Centrifuge, wash the precipitate with saline solution. Centrifuge again and take up the precipitate in 50 ml saline. Autoclave the suspension for 15 rain at 130°C. Radiochemical purity was determined by ascending paper chromatography on W h a t m a n N ° 3 M M using 85 ~ methanol as solvent for pyrophosphate, phytate, gluconate and HSA preparations. Methyl ethyl ketone was used as solvent to determine free 99mTc-pertechnetate in D T P A preparations. U n b o u n d technetium in MAA preparations was determined by filtration through colloidal membranes 0.22 nm pore size and the presence of labeled particulate material less than 8 n m in diameter was determined by filtration through an 8 nm pore size colloidal membrane. Tissue distribution was studied by i.v. injection in the femoral vein of white mice.
After a suitable time the activity of different organs was measured in an automatic gamma spectrometer with a well type scintillation detector. RESULTS
AND DISCUSSION
Labeling yields as a function of p H at a constant value of Q (0.24 Coulombs) for the different labeled compounds were better than 99 ~o for all molecules labeled except HSA in which case free pertechnetate accounted for 3 ~o of the activity. T h e activity retained by the colloidal membrane was less than 1 ~o. Again HSA was an exception, since the activity associated with particulate material was about
5Vo. For this reason we adopted the value of the p H closest to the physiological one, in which the smallest percent of activity bound to particulate matter was found. T h e values chosen for the electrolytic labeling of the different compounds were those shown in Table I. Labeling yields were studied as a function of the amount of current passed through the solution at the p H value chosen for each compound. Results are shown in Table 1. It can be seen that radiochemical purity for Q =- 0.24 is higher than 99 ~ for all compounds except HSA and DTPA, which nevertheless present a radiochemical yield compatible with clinical use
•
T h e amount of tin dissolved during electrolysis under these conditions was found to be 82 pg/ml. This is lower than the amount of tin used in the preparation of commonly available kits. O n the basis of these results the following procedures for labeling the different compounds were established: (1) Use sterile solutions and materials (2) Pass a 2 mA current for 2 rain, in all cases, gently shaking the vial. (3) Shake for 30 see after electrolysis has finished. (4) Allow to stand for 2 rain.
1. Labeling of potassium pyrophosphate 1. Add 2 ml of 98'~Tc04- saline solution to a mixture of 0.5 ml of a 50 mg]ml potassium pyrophosphate solution and 0.7 ml HC1 0.2 N.
Eleetrolytical labeling of t*mTc radiopharmaceuticals
71
TABLE 1. Free pertechnetate as a function of Q (~o) 0,03 Fyrophosphate pH 5 HSA pH 4.5 Gluconate pH 5.5 Phytate pH 5.0 DTPA pH 5.0 MAA pH 5.0
0.09
0.24
0.30
12.1
6.5
1.8
1.1
1.1
