ORIGINAL 
Annals of Nuclear Medicine Vol. 2,No. 2, 55-58, 1988 
Chemical properties of technetium-99m-DL-homocysteine, a possible tumor-imaging agent 
Atsushi TAKEDA, Rensuke GOTO and Shoji OKADA 
Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka 
The chemical properties of 99mTc-DL-homocysteine (99mTc-Hcy) showing high accumulation in several experimental tumors were investigated. The form of tumor-tropic 99mTc-Hcy was a polymeric complex which appeared at void volume on Sephadex G-15 by eluting with 5 mM Hcy. This complex changed into smaller complexes of ca. 600 molecular weight in the presence of 150 mM NaCl and 5 mM Hcy, suggesting that 99mTc-Hcy was a complex composed of smaller polymers which are weakly bound together by an ionic bond. The complex showed a negative charge. The Hcy/Tc molar ratio in the complex was approximately 2 and no Sn was detected. 
Key words: 99mTc-DL-homocysteine, DL-homocysteine, Chemical property, Tumor affinity, Tumor-imaging agent 
INTRODUCTION 
ALTHOUGH 67GA-CITRATE has been clinically used as a tumor detecting agent,1 a new tumor detecting agent needs to be developed because 67Ga-citrate has some disadvantages such as low specificity for tumors and slow clearance from blood. We have reported that among some 99mTc-labeled S-containing amino acids and sugars, 99mTc-DL-homocysteine (99mTc-Hcy) was the most promising agent for tumor detection because it accumulated in the tumor within a short time and showed higher affinity for the tumors than for abscesses.2-4 Furthermore, the behavior on gel chromatography suggested that this compound was a polymeric complex.3,4 Recently, it has been reported that tumor-tropic99mTc (V)-dimer-captosuccinic acid may be a polymeric form.5,6 It is of interest to know whether with respect to 99mTc-labeled compounds, polymeric complexes generally show high affinity for tumors. However, the chemical properties of these polymeric 99mTc-complexes have been little reported.5,7 This paper deals with the 

Received September 7, 1987; revision accepted January 18, 1988.
For reprints contact : Shoji Okada, Department of Radiobiochemistry, School of Pharmaceutical Sciences, University of Shizuoka, 2-2-1, Oshika, Shizuoka 422, JAPAN. 

chemical properties of the tumor-tropic form of 99mTc-Hcy.
MATERIALS AND METHODS 
Chemicals Na 99mTcO4 was obtained from a 99Mo-99mTc generator (Daiichi Radioisotope Laboratories Ltd., Tokyo) by eluting with physiological saline. NH499TcO4 was purchased from Amersham, U.K. DL-homocysteine and ion exchange celluloses [diethylaminoethyl cellulose (DE 52) and carboxymethyl cellulose (CM 52)] were purchased from Sigma Chemical Co., U.S.A. and Whatman International Ltd., U.K., respectively. 4-Phenylspiro [furan-2 (3H), 1'-phthalan]-3,3'-dione (FluramR) was the product of Roche, Switzerland. Other chemicals used were of guaranteed grade. 
Tumor-bearing Mice Male mice of ddY strain (16-18 g) were purchased from Shizuoka Agricultural Cooperative Association for Laboratory Animals, Hamamatsu. Mice bearing Ehrlich solid tumors were obtained as described previously.2 
Preparation of 99mTc-Hcy and (99mTc+99Tc)-Hcy 99mTc-Hcy was prepared according to the procedure described previously.2 When Hcy and Sn contents in the labeled compound were determined, (99mTc+99Tc)-Hcy was prepared with a mixture of Na99mTcO4 and NH499TcO4. 
Analysis of 99mTc-Hcy and (99mTc+99Tc)-Hcy The 99mTc-Hcy charge was estimated by adsorption on ion exchange cellulose at neutral pH. Sn was determined by the SATP (salicylideneamino-2-thiophenol) method.8 Hcy was assayed as follows.9 A mixture of 1 ml sample and 0.5 ml borate buffer (0.2 M, pH 9.0) was added to 0.5 ml FluramR (150 ug/ml) with stirring. The fluorescence was measured at 390 nm (excitation wavelength) and 475 nm (emission wavelength). The radioactivity of 99mTc and 99Tc was counted in a gamma counter (Beckman 5500) and a liquid scintillation counter (Aloka 661), respectively. 
In vivo uptake Four mice bearing Ehrlich solid tumors (approximately 0.5cm in diameter) were intravenously injected with 0.2 ml/head of 99mTc-labeled compound (ca. 1 uCi) in physiological saline and, 3 hr later, sacrificed by bleeding under ether inhalation. Tumors and organs were excised and weighed. The radioactivity was counted in a gamma counter. All the counts were corrected for decay. 
RESULTS 
Molecular size and stability of tumor-tropic form of 99mTc-Hcy Elution profiles of 99mTc-Hcy on Sephadex G-15 are shown in Fig. 1. The profiles in the presence and absence of Hcy in the eluent (H2O) were different. Almost all the radioactivity was eluted in void volume in the presence of 5 mM Hcy, while two peaks were obtained in the absence of Hcy. The radioactive complexes in the first peak at void volume and the second peak were named Complex I and Complex II, respectively. TLC analysis was applied to determine whether both the radioactive complexes were involved in unfractionated 99mTc-Hcy containing excess Hcy in the reaction mixture. The unfractionated 99mTc-Hcy showed a single peak on TLC (Fig. 2d). However, Complexes I and II had a peak with the same Rf value as 99mTcO4- and some undefined peaks besides that of unfractionated 99mTc-Hcy (Fig. 2a, b). These TLC profiles indicated that Complexes I and II were unstable in Hcy-free H2O. 
On the other hand, the fact that most of the radioactivity appeared at void volume in the presence of 5 mM Hcy suggests that 99mTc-Hcy mainly consisted of Complex I. This was confirmed by the finding that Complex I eluted with 5 mM Hcy had the same TLC profile as unfractionated 99mTc-Hcy (Fig. 2c). 99mTcO4- in 5 mM Hcy had the same Rf value as 99mTcO4- (Fig. 2e, f), indicating that 99mTcO4-, without a reducing agent such as SnCl2, did not react with Hcy. 
In order to confirm that Complex I was the tumor-tropic form of 99mTc-Hcy, the distribution of these complexes in mice bearing Ehrlich solid tumors was compared with that of unfractionated 99mTc-Hcy (Fig. 3). Complex I had almost the same distribution pattern as unfractionated 99mTc-Hcy and had higher affinity for tumors than for normal tissues, while Complex II, which might be artificially formed by eluting with H2O, showed the highest affinity for liver, different from that of 99mTc-Hcy. 
Chemical properties of 99mTc-Hcy Since it was found that the tumor-tropic form of 99mTc-Hcy was Complex I, its chemical properties were investigated. When Complex I was eluted with a mixture of 5 mM Hcy and 5, 50, or 150 mM NaCl on Sephadex G-25, it changed into compounds of smaller molecular weight ; in the case of 5 mM Hcy and 150 mM NaCl it was eluted at a position corresponding to a molecular weight of about 600 (Fig. 4). 
The charge of Complex I was examined by means of adsorption on ion exchange cellulose (Table 1). Complex I adsorbed on DE 52, but not on CM 52, and was released from DE 52 by adding 0.2 M NaCl, indicating that it had a weak negative charge at neutral pH. 
To determine the molar ratio of Hcy to Tc in Complex I, Hcy was labeled with a relatively large amount of 99Tc in addition to 99mTc because the amount of 99mTc-Hcy alone was too small to analyze. (99mTc+99Tc)-Hcy exhibited the same Rf value as 99mTc-Hcy (Fig. 5). Complex I was fractionated on Sephadex G-15 using H2O as an eluent in order to remove free Hcy completely. Hydrogen peroxide was used to dissociate Complex I to TcO4- and Hcy for the determination of Hcy with a fluorescent agent, FluramR, that reacted with primary amine. Tc was measured by the radioactivity of 99Tc after the decay of 99mTc. As shown in Table 2, the binding ratio was approximately 2 both before and after the H2O2-treatment. No detectable amount of Sn was found in Complex I by the SATP method. 
DISCUSSION 
99mTc-Hcy was a polymeric complex which eluted with 5 mM Hcy at void volume on Sephadex G-15. This polymer (Complex I) was a tumor-tropic form. In the presence of 5-150 mM NaCl, Complex I changed into smaller complexes. This suggests that Complex I in 5 mM Hcy was a polymer in which smaller complexes were bound together by a weak ionic bond. In a recent study, we found that 99mTc-cysteine also contained a polymeric complex and the polymer had relatively high affinity for tumors as well as 99mTc-Hcy (unpublished data). Glickson et al.10 reported that 67Ga-citrate formed polymeric complexes. Recently it was reported that 99mTc(V)-dimercaptosuccinic acid of which a considerable amount accumulated in tumors was assumed to be a polymer.5,6 The form of the polymeric complex might be one of the important properties in tumor-imaging agents. 
ACKNOWLEDGMENT 
The authors wish to thank Mr. Takashi Aomatsu, Miss Yukari Fushimi, and Miss Naomi Tsukamoto for the technical assistance. 
REFERENCES 
1. Edwards CL, Hayes RL: Tumor scanning with 67Ga-citrate. J Nucl Med 10 :103-105, 1969 
2. Tamemasa O, Takeda A, Goto R : Tumor detection with some 99mTc-labeled S-containing amino acids. Gann 75 : 395-402, 1984 
3. Tamemasa O, Goto R, Takeda A, et al: 99mTc-DL-homocysteine, a potential tumor imaging agent. Radioisotopes 33 : 636-638, 1984 
4. Tamemasa O, Goto R, Takeda A, et al: Accumulation in tumors of 99mTc-labelled sulphur-containing amino acids and sugars. Radiopharmaceuticals and Labeled Compounds, Vienna, 1985, pp.281-290 
5. Yokoyama A, Hata N, Horiuchi K, et al: The design of a pentavalent 99mTc-dimercaptosuccinate complex as a tumor imaging agent. Int J Nucl Med Biol 12: 273-279, 1985 
6. Jeghers O, Puttemans N, Urbain D, et al : Comparison of two 99mTc(V)-dimercaptosuccinic acid preparations. Appl Radiat Isot 38: 13-18, 1987 
7. Libson K, Deutsch E: Structural characterization of a 99mTc-diphosphonate complex. Implications for the chemistry of 99mTc skeletal imaging agents. J Am Chem Soc 102: 2476-2478, 1980 
8. Gregory GRC, Jeffery PG : Salicylideneamino-2-thiophenol-A new reagent for the photometric determination of tin: application to the analysis of ores, rocks, and minerals. Analyst 92 : 293-299, 1967 
9. Weigele M, Bernardo SLD, Tengi JP, et al : A novel reagent for the fluorometric assay of primary amines. J Am Chem Soc 94: 5927-5928, 1972 
10. Glickson JD, Pitner TP, Webb J, et al: Hydrogen-1 and gallium-71 nuclear magnetic resonance. Study of gallium citrate in aqueous solution. J Am Chem Soc 97: 1679-1683, 1975