Labelling of DMSA with 99Tc without exogenous reducing agents

Intrmcltti .Lmmal o/ Applkd Radfetion & lsoropss Vol. 0 Per(tamon Flus Ltd 1980.Fbinted in Great Britain OOW7WXpO/llOl47lSOZ.W-J

31. pp. 115

to

717

of DMSA with p% witbout Exoget~ous Reducing Agents

Labelling

J. GALVEZ, R. GARCfA DOMENECH and J. L. MORENO School of Pharmacy, Paseo al Mar 13 University of Valencia, Spain (Received

18 February

1980; in revisedform

21 April 1980)

designed to accommodate inert atmosphere. For potentiometric titrations, a Crison Digit 74 pH meter was used. Ionic strength was held relatively constant by addition of 0.1 M NOsK. A Job’s plot, (sr as well as iodimetric titrations were made in order to determine the stoichiometry of the reducand DMSA. Different tion reaction between “TcOi ggTc-DMSA solutions, containing from 3: 1 to I:9 molar ratios metal-to-l&and, were prepared. A Job’s plot was also used to establish the stoichiometry of the ggTcSn-DMSA complex. In this case, stoichiometric amounts of Sn(I1) were added to the solutions to assure the complete reduction of Tc(VII), on the basis of a delectron exchange reaction.‘“’ Absorbance measurements were carried out in the absence of Sn(II), at different times, until equilibrium was reached.

htroduction IT IS

accepted that g9”‘TcO~ must be reduced prior to the formation of renal-scanning agents.‘L-3’ In a recent paper, HAGAN indicates that no exogenous reducing agents are necessary in the formation of 99”Tc-TMA complex (TMA = thiomalic acid); however, no suggestions are made in the paper about the TC oxidation state in that radiopharmaceutical. Along these lines, we show in the present work that the formation of the “Tc-DMSA complex (DMSA = dimercaptosuccinic acid) is also possible without using any exogenous reducing agent. Moreover, it is also demonstrated that the Tc oxidation state in the complex formed is +4, acting the same in DMSA as in the reducing agent. The complex formation seems to take place in two steps: first. the reduction of 99TcO; by DMSA, which is very slow, and second, the actual complex formation, which is much faster. The pH influence on the kinetics of the complex formation reaction, as well as the complex concentration at equilibrium, are also studied. For comparison and in order to complete the above results, parallel studies on the ggTc-Sn-DMSA complex have also been undertaken. The different reactions taking place were studied by visible spectroscopy, thin-layer chromatography (with g9”‘Tc),volumetric, and potentiometric techniques. ~~_MMONL.Y

Chemicals

Results and Discussion When a solution containing “TcOi and DMSA (molar ratio Tc:DMSA 1: 1 or lower) is allowed to stand for several hours, a yellow colour develops increasing in intensity with time, until equilibrium is reached (about 190 h; see Fig 1) The maximum in the absorption spectra appears at 403 nm. When the same experiment is made in the presence of stoichiometric Sn(I1) (molar ratio Sn(II):Tc(VII) 1.5: l), the yellow colour appears instantaneously, and the absorption spectra has a maximum at 403 nm. Figure 2 shows the results of the Job’s plot for the pair ‘9TcOi-DMSA without (A) and with (B) Sn(I1). In the first case, the maximum of absorbance occurs at 1:2.5 molar ratio metal-to-ligand; in the second, the maximum appears for a 1:l molar ratio. In the absence of Sn(I1). the higher the pH is, the lower the absorbance. At alkaline pH (starting pH is 3.1 for a 1:2.5 molar ratio), no main peak in the visible region was detected. Complex formation at equilibrium, expressed as a percentage of complexed ggTc as determined by TLC, for different pH values, was found to be 55.92 for pH 1.6, 46.42 for pH 2.3, 27.62 for pH 3.1 and 5.57 for pH 10.4.

Material and Methods

DMSA was obtained from Sigma Chemical Company (St Louis, MO., U.S.A.). g9”TcOi was obtained as eluted from a g9Mo-gg7’c generator (Radiochemical Centre, Amersham, England). “TcOi was used as the ammonium salt, also obtained from Radiochemical Centre. All other reagents used in this study were of analytical grade. Concentrations of “TcOi were determined radiopretritally, with a Nuclear Chicago Isocap/300 beta counter, with standard ‘9TcOi on the basis of a counting efficiency coefficient of 0.83. I2 was used in form of Ii (standardized with SsO;s) and Sn(I1) in the form of Cl,Str2H10. Experimental

procedure

All spectrophotometric determinations were carried out with a Philips sp8-100 u.v.-visible spcctrophotometer. A Picker Nuclear, Autowell 200, gamma counter was used to record 99”‘Tc activity. The pH influence, as well as the complex concentration at equilibrium was studied by thinlayer chromatography (TLC), using acetone as developer on Al-cellulose sheets Volumetric titrations were carried out with a Mettler DV 10 microburet in a 100 ml glass cell 715

FIG. 1. Absorbance versus 9aTcOi molar ratios at “TcOi = 2.85. lo-*M. 0 l = 3:l.

time for different DMSA: 403 nm. Concentration of s 1 :l. A = 2~1, 0 = 2.5~1, A = 5:l.

TechnicalNote

716

On the other hand, if DMSA is oxidized by I, 2 mol of I2 per mol of DMSA are necessary.

According to equations (1) and (2), the reduction reaction of Tc(VII) by DMSA would occur as:

I

COOH

I

1

3 ;[:H+4TcOi

+6Ii,O+4H+e4TcO,.2H,O+3

Further, the potentiometric titration of the final products of the DMSA+ reaction, show the formation of 8 hydrogen ions. These results seem to indicate that the oxidation of DMSA by iodine takes place through a 4-electron exchange, as#against the commonly accepted reaction of mercapto groups by 12.(‘*s) The probable reaction is: COOH

COOH

COOH

I

HCSOH

HCSH

I

HCSOH

(

+21,+2H,O~i HCSH

7 +4IH

(1)

HCSOH

AOOH

I HCSH C!OOH

I

(3)

Ikedaooi has demonstrated that mixed complexes such as Tc-Sn-DMSA are not formed in the presence of the Tc-DMSA complex. On this basis, the stoichiometry of the “Tc-DMSA is l:I, as can be deduced from Fig. 2B. Therefore, the global reaction between DMSA and Tc(VI1) (including complex formation), would be:

COOH

HCSH

+4TcOi*3

I

+ 4 complex

(4)

HCSOH

I

COOH

COOH

When a solution containing ?fcO~ with I- in excess, in the presence of H+. is left to stand for several hours, iodine is formed. The number of iodine moles, as determined by titration with S,O;*. is in a constant ratio of 3/2 with respect to the reduced Tc obtained the concentration of the latter being determined by TLC. Thus it follows that the oxidation state of the reduced Tc must be +4, which is also in accordance with the slowness of the reduction of Tc(VI1) to Tc(IV) by different reducing agents.“’ In our case, that reduction may be expressed as: 2TcO;

“E

+ 61- + 8H’

~2Tc0~.2HzO

+ 31s

(2)

On the other hand, it is quite probable that the yellow complex formed by direct reduction of Tc(VI1) by DMSA would be the same as that formed by stannous reduction, which can be deduced from the spectroscopic results. In fact, both complexes gave the same u.v.-visible spectra. At this point, one must wonder if reaction (4) adequately describes the global process, since, in that case, a maximum in Job’s plot at a 1:1.75 molar ratio Tc:DMSA would be obtained. However, Fig 2A shows that the maximum appears for a 1:2.5molar ratio. It has to be accepted that DMSA is oxidized to a lower oxidation state by the Tc(VI1) than that assumed here. The probable reaction would be:

(5)

r This is in accordance with the results of KLOTZ("' for the oxidation of TMA by Cu(II), with formation of dithio-

0

x)

40

so

J

a0

I(x)

Percent of DMSA

FIG. 2. Equilibrium values obtained from Job’s plot of continuous variation at 403 nm. (A) DMSA-99TcO;; (B) DMSA-Sn(II)-99TcO;. Total concentration of reactants (DMSA + Tc) = 10-5mol. 0 = A, l = B.

malic acid as the oxidized species. The reason why Tc(VII), being more oxidizing than iodine, is suitable to oxidize DMSA is kinetic: in fact, the reaction Tc(VI1) sjt Tc(IV) must be slow, such as suggested by HAMBRIGHT.'~'because there will be, probably, a metal changing from a 4-coordinate tetrahedral to an octahedral configuration, and also because the reduction of Tc(VII) by DMSA is a non-complementary reaction. There is a competitive reaction (complex formation), so that “oxidizable material” is lost and, therefore, successive oxidation steps are hindered. However, our results indicate that this slowness in the reduction of Tc(VI1) to Tc(IV) is closely related to the “reducing strength” of the reducing agent: in fact, in the presence of Sn(I1) the reduction takes place immediately, with I- it takes some hours and with ‘DMSA it is much slower (8 days). The applicability of these results to Radiopharmacy will depend on the behaviour of the quantities of 99”Tc, and no conclusions can be established without more detailed kinetic and thermodynamic studies.

Technical Note Acknowledgement--The author expresses his appreciation to Dr J. Fignernelo, of the Sciences Faculty, for considerable work in repeating and checking the procedures.

6. 7.

1. WINSTONM. A., HALPERNS. E. and Wnss E. R. et al. J. nucl. Med. 12, 171-175 (1971). 2. BOYDR. E., ROEI~ON J. and HUNT F. C. et al. Br. J. Radiol. 46, 604-612 (1973). 3. ECKELMANW. and RICHARDSP. .I. nucl. Med. II, 761 (1970). 4. HAGANP. L. CHAUNCIZY D. M. and HALPERNS. E. et al. J. nucl. Med U&353-359 (1977). 5. MURMANNR. K. Inorganic Complex Compounds. New

8. 9. 10. Il.

111

York, Reinhold Publishing Corp, 1964. (Spanish edition by Selecciones cientifieas, Madrid, p. 26. IKEDA I. INOUE 0. and KURATAK. ht. J. appl. R&at. IsotoDes 27.681-688 (1976). ALLI~GERhI. L., CA