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Tetracycline chelates of isotopic metal ions
InternationalJournalof AppliedRadiationand Isotopes,1966,Vol. 17, pp. 603-604. PergamonPressLtd. Printedin NorthernIreland
Tetracycline
Chelates Metal
of
Isotopic
Ions
(Received 9 June
1966)
Introduction THIS study
concerns the preparation of tetracycline chelates of radionuclides which may be potentially useful in nuclear medicine. Since tetracyclines have been shown to concentrate in some types oftumors,(1*2) it was thought that some of these radioactive chelates might concentrate in brain tumors and permit external scanning. The distribution in tissues and organs of most metal ions is known but not as much information is available concerning the distribution of chelated compounds in the body. Among the biologically important compounds which can act as chelating agents are the tetracyclines. Tetracyclines have been found to be good chelating agents for a number of metal ions.(3-s) In view of these considerations, it was thought desirable to investigate the chelates of various radioactive metals with tetracycline and oxytetracycline. Methods
and Techniques
In a search for suitable radioisotopes, it was necessary to determine what metal ions are complexed by the tetracyclines. In this area, we have extended the list of known metals which are chelated by tetracyclines to include some previously untried ions. These new metal ions include bivalent tin, cadmium and lead, in addition to vanadium (valences 3 and 4), trivalent chromium, rare earth metals, tetravalent uranium and pentavalent molybdenum. The metal complexing tendencies were determined by preparing a mixture of the metal salt solution (1 m-mole in l-5 ml) with about 3 m-moles of the tetracycline (or oxytetracycline) hydrochloride. This solution was made alkaline gradually until the first formed precipitate redissolved. If the metal formed a hydroxide which was soluble in excess hydroxide ion (as Cr or Pb), then sodium or potassium carbonate was used to render the mixture alkaline. This was done to obviate the formation of chromite or plumbite ions. Otherwise (as in the case of V or U), sodium or potassium hydroxide was employed to make the solution alkaline. If the precipitate redissolved in alkali, it was assumed that complex formation occurred, as otherwise an insoluble
hydroxide cipitate.
or
carbonate
would
remain
as a pre-
SAKAGUCHI et a1.(7ss) and others prepared some chelates in neutral or weakly acidic media but these were mostly sparingly soluble. The detection of chelation of the metal is simpler using our alkaline solubility procedure and soluble products are obtained. It is presumed that a solution of pH slightly higher than that of blood could be prepared which might be suitable for injection. The fact that rare earth metals form complexes may allow the wide use of different isotopes of nuclear characteristics from this large varying number (fourteen) of closely similar elements. The study of the binding of nuclides to the tetracyclines was done somewhat similarly to the cold runs described above. Here, however, a salt of the tetracycline was isolated from the alkaline solution by saturation with sodium or potassium chlorides (salting out). The precipitated salt was washed with saturated alkali metal chloride solution and purified by redissolving and reprecipitating by salting out again. It was finally washed with saturated salt solution and then dissolved and an aliquot counted. In the cases studied, the salt thus obtained had 40 per cent or more of the original radioactivity used, demonstrating that binding of the isotopic ions to the salt of the tetracycline had occurred. If no binding had taken place, the salt would not have been radioactive. At the present time, Cr5t and Fesg have been used but doubtless other radioisotopes could be utilized and it is planned to examine some of these.
Acknowledgments-This study was supported in part by USAEC Contract No. AT (ll-l)-34Project No. 119. Radioisotopes were kindly furnished by Nuclear Consultants Inc. of St. Louis MO. M. TUBIS Radioisotofie Research Veterans Administration Center Los Angeles, Calif. D. C. MORRISON Dept. of Surgery (Neurological) Univ. of California Center for Health Sciences Los Angeles, CaltjY References 1. JOHNSON R. H. J. Oral Ther. Pharm. 1, 190 (1964). 2. MACHADO L., ZAIDMAN I., GERSTEIN J. F., LICHTENBERG F. and GUY S. J. Cancer Res. 24, 1845 (1964). 603
604
Technical notes
3. ALBERT A. and REES C. W. Nature, Land. 177,433 (1956). 4. ALBERT A. Nature, Land. 172, 201 (1953). 5. BAKER W. A. and BROWN P. M. J. Am. them. SOL 88, 1314 (1966). 6. DOLUISIOJ. T. and MARTIN A. N. J. med. Chem. 6, 16 (1963). 7. SAKAGUCHI T. and ISHIDATE M. Pharm. Bull, Japan 3,147 (1955). Chem. Abs. 50,10692 (1956). 8. SAKAGUCHIT., TAGUCHI K., FUKUSHIMA S. and OBI N. Yakagaku Zasshi 78, 177 (1958). Chem. Abs. 52, 10990 (1958).
part absorbed in the foil. It has been shown(l@) experimentally, that for the very thin foils used here, Equation (1) can be written as K FA’ = 2/(2 -
17,
The Correction for Foil Absorption in 4dZounting of Sources Deposited on Thin Foils (Received
1 March
1966)
ALTHOUGH the classical &r-foil method(l-D) cannot compete in accuracy and reproducibility with some modern methods for the absolute determination of radioactivity, it remains very useful if extremely high precision is not required or if it is used in high precision counting as one independent method together with others. The accuracy of the &-foil method is determined by the self absorption of the source and the foil absorption. But accuracies of 0.2 to 1 per cent for p--emitters with E,,, > 300 keV and approximately 0.1 to O-5 per cent for ,!I+- and x-emitters can be reached with suitable sources if the foil absorption correction is known with adequate Therefore this correction has been accuracy. measured previously(lO) for the most common radionuclides and for the usual foil thicknesses of 20-250 The backing technique, where supplepg/cm2. mentary foils are attached to the foil on the opposite side of the source, has been used for the earlier experiments. An extension of these investigations to the sandwich backing technique, where supplementary foils are applied symmetrically on both sides of the source, is reported here. The following expression can be simply derived(rO) for the correction factor KFd’ for foil absorption of “open” B-sources, i.e. sources of which the top is not covered with an absorber foil.
K pA’ = No/Na
= 2/(2 -
a + p”)
(1)
where Nd is the count rate at foil thickness d, N, is the extrapolated count rate at d = 0, p is the part of the radiation backscattered from the foil, and a is the
(2)
where the constants are related to the foil absorption y(b), the backscattering and foil absorption (c). For sources symmetrically sandwiched between thin foils one can assume in a first approximation that the difference between the absorption of the primary and the backscattered radiation is small and since p < 1 the backscattering effect cancels out in the formula. One derives from Equation (2) : Kpd
International Journal ofApplied Radiation and Isotopes, 1966, Vol. pp. 604-606. Pergamon Press Ltd. Printed in Northern Ireland
bd + cd2)
= N,/Na
= l/(1
-
bd)
(3)
where d is the single foil thickness, thus half the total thickness of the sandwich. Equation (3) shows a linear relation between Nd[N,, and the foil thickness d, which simplifies the extrapolation to zero thickness and makes the sandwich backing method attractive. Taking into account the known data on backscattering,(‘*ll) the spectra and the absorption of scattered /I-radiation, this linear relation can be understood as a correct first approximation. The sandwich backing experiments have been done similar to the single backing experiments.(ls) Gold coated VYNS foils, with thicknesses from about 20 to 250 lug/cm2 for the single foils, have been used as absorbers. The measurements have been performed with a routine 477-proportional methane flow counter. Generally the sourcesoO) have been prepared by precipitation, but for Nia3 and Coso also electrodepositedo2) sources have been used. The statistical error and the error due to the usual dead time correction always have been kept below 0.1 per cent. The applied cut-off energy (discrimination) was below 1 keV. From the plots of experimental N, values against the single thickness d, it follows that for Nis3 up to at least d = 100 pg/cm2, for Sss up to at least 150 pg/cm2 and for the other measured radionuclides up to at least 250 pg/cms a linear extrapolation of the count rate to zero thickness can be carried out in order to obtain the extrapolated count rate N,,. This is in agreement with Equation (3). From the plots of N,/N, in dependence of d and Equation (3)) a mass absorption coefficient b for the p-radiation can be derived. The values obtained are given in Table 1. The quoted errors are the standard deviations of the single measurements and lead, as indicated, to errors of the order of 10 per cent in the foil absorption correction. In the last column of Table 1 the b-values calculated from the older backing experiments are For B--emitters the mentioned for comparison. following relation between the mass absorption
Tetracycline
Chelates Metal
of
Isotopic
Ions
(Received 9 June
1966)
Introduction THIS study
concerns the preparation of tetracycline chelates of radionuclides which may be potentially useful in nuclear medicine. Since tetracyclines have been shown to concentrate in some types oftumors,(1*2) it was thought that some of these radioactive chelates might concentrate in brain tumors and permit external scanning. The distribution in tissues and organs of most metal ions is known but not as much information is available concerning the distribution of chelated compounds in the body. Among the biologically important compounds which can act as chelating agents are the tetracyclines. Tetracyclines have been found to be good chelating agents for a number of metal ions.(3-s) In view of these considerations, it was thought desirable to investigate the chelates of various radioactive metals with tetracycline and oxytetracycline. Methods
and Techniques
In a search for suitable radioisotopes, it was necessary to determine what metal ions are complexed by the tetracyclines. In this area, we have extended the list of known metals which are chelated by tetracyclines to include some previously untried ions. These new metal ions include bivalent tin, cadmium and lead, in addition to vanadium (valences 3 and 4), trivalent chromium, rare earth metals, tetravalent uranium and pentavalent molybdenum. The metal complexing tendencies were determined by preparing a mixture of the metal salt solution (1 m-mole in l-5 ml) with about 3 m-moles of the tetracycline (or oxytetracycline) hydrochloride. This solution was made alkaline gradually until the first formed precipitate redissolved. If the metal formed a hydroxide which was soluble in excess hydroxide ion (as Cr or Pb), then sodium or potassium carbonate was used to render the mixture alkaline. This was done to obviate the formation of chromite or plumbite ions. Otherwise (as in the case of V or U), sodium or potassium hydroxide was employed to make the solution alkaline. If the precipitate redissolved in alkali, it was assumed that complex formation occurred, as otherwise an insoluble
hydroxide cipitate.
or
carbonate
would
remain
as a pre-
SAKAGUCHI et a1.(7ss) and others prepared some chelates in neutral or weakly acidic media but these were mostly sparingly soluble. The detection of chelation of the metal is simpler using our alkaline solubility procedure and soluble products are obtained. It is presumed that a solution of pH slightly higher than that of blood could be prepared which might be suitable for injection. The fact that rare earth metals form complexes may allow the wide use of different isotopes of nuclear characteristics from this large varying number (fourteen) of closely similar elements. The study of the binding of nuclides to the tetracyclines was done somewhat similarly to the cold runs described above. Here, however, a salt of the tetracycline was isolated from the alkaline solution by saturation with sodium or potassium chlorides (salting out). The precipitated salt was washed with saturated alkali metal chloride solution and purified by redissolving and reprecipitating by salting out again. It was finally washed with saturated salt solution and then dissolved and an aliquot counted. In the cases studied, the salt thus obtained had 40 per cent or more of the original radioactivity used, demonstrating that binding of the isotopic ions to the salt of the tetracycline had occurred. If no binding had taken place, the salt would not have been radioactive. At the present time, Cr5t and Fesg have been used but doubtless other radioisotopes could be utilized and it is planned to examine some of these.
Acknowledgments-This study was supported in part by USAEC Contract No. AT (ll-l)-34Project No. 119. Radioisotopes were kindly furnished by Nuclear Consultants Inc. of St. Louis MO. M. TUBIS Radioisotofie Research Veterans Administration Center Los Angeles, Calif. D. C. MORRISON Dept. of Surgery (Neurological) Univ. of California Center for Health Sciences Los Angeles, CaltjY References 1. JOHNSON R. H. J. Oral Ther. Pharm. 1, 190 (1964). 2. MACHADO L., ZAIDMAN I., GERSTEIN J. F., LICHTENBERG F. and GUY S. J. Cancer Res. 24, 1845 (1964). 603
604
Technical notes
3. ALBERT A. and REES C. W. Nature, Land. 177,433 (1956). 4. ALBERT A. Nature, Land. 172, 201 (1953). 5. BAKER W. A. and BROWN P. M. J. Am. them. SOL 88, 1314 (1966). 6. DOLUISIOJ. T. and MARTIN A. N. J. med. Chem. 6, 16 (1963). 7. SAKAGUCHI T. and ISHIDATE M. Pharm. Bull, Japan 3,147 (1955). Chem. Abs. 50,10692 (1956). 8. SAKAGUCHIT., TAGUCHI K., FUKUSHIMA S. and OBI N. Yakagaku Zasshi 78, 177 (1958). Chem. Abs. 52, 10990 (1958).
part absorbed in the foil. It has been shown(l@) experimentally, that for the very thin foils used here, Equation (1) can be written as K FA’ = 2/(2 -
17,
The Correction for Foil Absorption in 4dZounting of Sources Deposited on Thin Foils (Received
1 March
1966)
ALTHOUGH the classical &r-foil method(l-D) cannot compete in accuracy and reproducibility with some modern methods for the absolute determination of radioactivity, it remains very useful if extremely high precision is not required or if it is used in high precision counting as one independent method together with others. The accuracy of the &-foil method is determined by the self absorption of the source and the foil absorption. But accuracies of 0.2 to 1 per cent for p--emitters with E,,, > 300 keV and approximately 0.1 to O-5 per cent for ,!I+- and x-emitters can be reached with suitable sources if the foil absorption correction is known with adequate Therefore this correction has been accuracy. measured previously(lO) for the most common radionuclides and for the usual foil thicknesses of 20-250 The backing technique, where supplepg/cm2. mentary foils are attached to the foil on the opposite side of the source, has been used for the earlier experiments. An extension of these investigations to the sandwich backing technique, where supplementary foils are applied symmetrically on both sides of the source, is reported here. The following expression can be simply derived(rO) for the correction factor KFd’ for foil absorption of “open” B-sources, i.e. sources of which the top is not covered with an absorber foil.
K pA’ = No/Na
= 2/(2 -
a + p”)
(1)
where Nd is the count rate at foil thickness d, N, is the extrapolated count rate at d = 0, p is the part of the radiation backscattered from the foil, and a is the
(2)
where the constants are related to the foil absorption y(b), the backscattering and foil absorption (c). For sources symmetrically sandwiched between thin foils one can assume in a first approximation that the difference between the absorption of the primary and the backscattered radiation is small and since p < 1 the backscattering effect cancels out in the formula. One derives from Equation (2) : Kpd
International Journal ofApplied Radiation and Isotopes, 1966, Vol. pp. 604-606. Pergamon Press Ltd. Printed in Northern Ireland
bd + cd2)
= N,/Na
= l/(1
-
bd)
(3)
where d is the single foil thickness, thus half the total thickness of the sandwich. Equation (3) shows a linear relation between Nd[N,, and the foil thickness d, which simplifies the extrapolation to zero thickness and makes the sandwich backing method attractive. Taking into account the known data on backscattering,(‘*ll) the spectra and the absorption of scattered /I-radiation, this linear relation can be understood as a correct first approximation. The sandwich backing experiments have been done similar to the single backing experiments.(ls) Gold coated VYNS foils, with thicknesses from about 20 to 250 lug/cm2 for the single foils, have been used as absorbers. The measurements have been performed with a routine 477-proportional methane flow counter. Generally the sourcesoO) have been prepared by precipitation, but for Nia3 and Coso also electrodepositedo2) sources have been used. The statistical error and the error due to the usual dead time correction always have been kept below 0.1 per cent. The applied cut-off energy (discrimination) was below 1 keV. From the plots of experimental N, values against the single thickness d, it follows that for Nis3 up to at least d = 100 pg/cm2, for Sss up to at least 150 pg/cm2 and for the other measured radionuclides up to at least 250 pg/cms a linear extrapolation of the count rate to zero thickness can be carried out in order to obtain the extrapolated count rate N,,. This is in agreement with Equation (3). From the plots of N,/N, in dependence of d and Equation (3)) a mass absorption coefficient b for the p-radiation can be derived. The values obtained are given in Table 1. The quoted errors are the standard deviations of the single measurements and lead, as indicated, to errors of the order of 10 per cent in the foil absorption correction. In the last column of Table 1 the b-values calculated from the older backing experiments are For B--emitters the mentioned for comparison. following relation between the mass absorption