Development of a Conjugate of 99mTc-EC with ... - ACS Publications

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Bioconjugate Chem. 2002, 13, 16−22

Development of a Conjugate of 99mTc-EC with Aminomethylenediphosphonate in the Search for a Bone Tracer with Fast Clearance from Soft Tissue Kristin Verbeke,†,§ Jef Rozenski,‡ Bernard Cleynhens,† Hubert Vanbilloen,† Tjibbe de Groot,† Nancy Weyns,† Guy Bormans,† and Alfons Verbruggen*,† Laboratory of Radiopharmaceutical Chemistry, University Hospital Gasthuisberg, Herestraat 49, B-3000 Leuven, and Rega Institute for Medical Research, University of Leuven, Belgium. Received December 29, 2000; Revised Manuscript Received August 30, 2001

For the currently used 99mTc-labeled diphosphonates such as 99mTc-MDP and 99mTc-HDP, the required interval of 2.5 to 3 h between injection and the scintigraphic bone imaging is an inconvenience. The present study was set up in an attempt to develop a technetium-99m-labeled diphosphonate with efficient bone uptake and more rapid clearance from blood and soft tissue by renal extraction and excretion so that it would be possible to start imaging as early as 1 h after injection. A conjugate of the new renal tracer agent 99mTc-ethylene dicysteine (99mTc-L,L-EC), covalently bound via one of its carboxylates with aminomethylenediphosphonic acid (AMDP), was synthesized in seven steps. ECAMDP could be labeled easily and efficiently with 99mTc at pH g12 and room temperature. Analysis using ion pair reversed phase high performance liquid chromatography showed the formation of a mixture of two main compounds with reproducible relative ratios, which were stable as a function of time. In a baboon, the scintigraphic images obtained with the new agent showed good quality bone scans, with clear visualization of the skeleton and low soft tissue activity at respectively 1 and 2 h after injection.

INTRODUCTION

Radionuclide bone imaging is the most common clinical investigation in nuclear medicine. Since the mid-1970s, the bone scan has replaced the X-ray skeletal survey because of its 95% sensitivity in detecting metastatic disease and because this radioisotopic technique allows detection of some lesions several months earlier than with X-ray examination. The reason for the latter lies in the fact that the bone scan demonstrates osseous remodeling, which must precede and is the cause of structural changes seen on the X-ray image (1). For more than 20 years, 99mTc-labeled diphosphonates have been the radiopharmaceuticals of choice for radioisotopic bone scanning. The tracer agents most widely used at present are complexes of 99mTc with methylenediphosphonate (99mTc-MDP) and hydroxymethylenediphosphonate (99mTc-HMDP) (2-4). Despite their wide clinical use, 99mTc-diphosphonates suffer from a number of suboptimal properties. First, they are fairly weak chelates and, therefore, tend to degrade with time and produce pertechnetate as an impurity. The use of a suitable antioxidant such as ascorbic acid, gentisic acid, or p-aminobenzoic acid helps to overcome this problem (5). Second, 99mTc-diphosphonates are not single, well-defined chemical species, but mixtures of short- and long-chain polymers in which one or more technetium atoms are bridged by two or several phos* To whom correspondence should be addressed. Phone: 3216-343732. Fax: 32-16-343891. e-mail: alfons.verbruggen@ uz.kuleuven.ac.be. † University Hospital Gasthuisberg. ‡ Rega Institute for Medical Research. § Postdoctoral fellow of the Fund for Scientific Research, Flanders.

phate and hydroxyl ligands. Different polymers can have a different degree of ionization and biological behavior. Analysis by high-pressure liquid chromatography (HPLC) has also revealed that the relative amounts of the different species in a 99mTc-diphosphonate preparation vary as a function of time after reconstitution (6-10). In addition, the diphosphonate group is necessary both for the complexation of 99mTc and the uptake of the complex in the skeleton (11). This may have the disadvantage that a phosphonate group involved in the binding of 99mTc has a reduced ability of binding to the bone surface. Following intravenous administration, the uptake of 99mTc-diphosphonates in bone is almost complete 1 h after injection, but the clearance from soft tissue proceeds more slowly (12). For this reason, a time interval of 2 to 4 h is required between injection of the radiopharmaceutical and the bone scanning. This necessary delay is an important inconvenience for the nuclear medicine personnel and for the patients, especially for children. Therefore, it would be a major improvement for nuclear medicine if the 99mTc-diphosphonates could be modified in such a way that: (a) they still have a high and rapid uptake in the skeleton with the same lesion-to-normal bone activity ratio; (b) the clearance from soft tissue by the kidneys followed by excretion into the urine would be much faster. In an ideal situation, it should be possible to obtain good quality scintigraphic images already 1 h after injection with the same clinical information as now after 3-4 h. Therefore, the aim of the present study was the development of a “new generation” bone-seeking radiopharmaceutical with the above-described characteristics. For this purpose, a strategy was designed in which a diphosphonate is conjugated to a stable and easy to

10.1021/bc0001600 CCC: $22.00 © 2002 American Chemical Society Published on Web 12/27/2001

Bone Tracer with Fast Clearance

Figure 1. Structure of 99mTc-EC and aminomethylenediphosphonate (AMDP).

prepare 99mTc complex with known very rapid active renal excretion. In this conjugate, the diphosphonate would be “free” (i.e., not bound to Tc) and thus retain its full ability for binding to the bone surface. On the other hand, the 99m Tc-labeled renal agent would ensure both a stable binding of the radionuclide (99mTc) and a rapid urinary elimination of the fraction of the tracer agent not bound to the skeleton. For different reasons, we selected the complex of 99m Tc with L,L-ethylene dicysteine (99mTc-L,L-EC, Figure 1) as the 99mTc-labeled renal agent to which the diphosphonate can be bound. It is a very stable and easy to prepare 99mTc complex with the highest plasma clearance by renal extraction ever reported for a 99mTc-compound (13). In humans the 1-h plasma clearance of 99mTc-L,LEC is 75% of the Hippuran clearance as compared to about 60% for 99mTc-MAG3 (14, 15). Moreover, it has in its structure two carboxylic groups of which one can be omitted without significantly affecting the renal excretion characteristics (16). This means that one of the carboxylates can be used for coupling to a diphosphonate bearing an amino group in its structure. Aminomethylenediphosphonate (AMDP, Figure 1) is such a diphosphonate and was selected as the diphosphonate part of our new compound. Preliminary experiments have demonstrated that during labeling of equimolar amounts of EC and a diphosphonate with 99mTC, the radionuclide exclusively binds to the EC ligand and not to the diphosphonate (17), which in this way retains its full ability for binding to the bone surface. MATERIALS AND METHODS

Materials. Thin-layer chromatography (TLC) was carried out using precoated TLC plates (Alugram SIL G/UV254, Macherey-Nagel, Du¨ren, Germany). For purification by column chromatography, silica gel (MN Kieselgel 60, 70-230 mesh ASTM, Macherey-Nagel) was used. L,L-Ethylene dicysteine (1) and the tetraethyl ester of 1-aminomethylenediphosphonic acid (5) were prepared as described elsewhere (18-20). 1 H and 13C NMR spectra were obtained on a Varian Gemini 200 spectrometer (Varian, Palo Alto, CA). Chemical shifts are reported in ppm relative to the standard reference tetramethylsilane. Liquid secondary ion mass spectra (LSIMS) were recorded on a Kratos Concept IH mass spectrometer (Kratos Analytical, Manchester, UK) controlled by a Masspec II data system (MSS, Manchester, UK) and obtained in a negative (-LSIMS) ion mode. Negative mode electrospray ionization (-ESI) mass spectra were acquired on an orthogonal acceleration time-offlight mass spectrometer (qTof-2, Micromass, Manchester, UK). 99mTc-MDP was prepared by reconstitution of a conventional MDP labeling kit with sodium [99mTc]pertechnetate solution from a 99mTc generator (UltratechnekowFM, Mallinckrodt Medical, Petten, The Netherlands). S,S′-Bis-benzyl-L,L-1,2-ethylene Dicysteine (2). 1,2L,L-Ethylene dicysteine (1, 41.37 g, 154 mmol) was

Bioconjugate Chem., Vol. 13, No. 1, 2002 17

dissolved in liquid ammonia (1000 mL) in a two-necked flask with a dewar cooler, and the mixture was stirred vigorously. Sodium (12.8 g, 555 mmol) was added until a blue color persisted for at least 15 min. Ammonium chloride was then added in portions till decoloration. After dilution with liquid ammonia (400 mL), benzyl chloride (40 mL, 320 mmol) was added dropwise. After stirring for 30 min, the liquid ammonia was removed by evaporation at room-temperature overnight. Water (600 mL) was then added, and the solution was washed with ether (3 × 200 mL). The water layer was acidified to pH 2 with 6 M HCl. The precipitate formed was filtered off and washed successively with water, ethanol, acetone, and finally with ether and dried under vacuum to yield 25.99 g (60%) of a white product (Scheme 1). N,N′-Bis-carbobenzyloxy-S,S′-bis-benzyl-1,2-L,Lethylene Dicysteine (3). In a 2000-mL beaker S,S′-bisbenzyl-1,2-L,L-ethylene dicysteine 2 (27.2 g, 60 mmol) was suspended in water (1000 mL) and dissolved by addition of 1 M NaOH up to pH 11. After cooling to 0-5 °C, benzyl chloroformate (27 mL, 180 mmol) was added dropwise over a period of 30 min while the pH was maintained at 11 by addition of 4 M NaOH. After washing with ether (300 mL), the water layer was acidified to pH 2 with 6 M HCl and extracted with EtOAc (3 × 300 mL). The combined organic layers were dried over Na2SO4 and evaporated to yield a yellow oil which was purified by column chromatography using gradient mixtures of methanol in CH2Cl2/acetic acid (100:1, V/V). Evaporation of the fractions containing the pure main compound as shown by TLC (CH2Cl2-methanol-HOAc, 95:5:1, Rf ) 0.7) yielded 26.8 g of a colorless oil. 1H NMR: (CDCl3): δ 2.6-3.1 (4H, m, 2 x CH-CH2-S); 3.3-3.5 (4H, m, 2 x CH2-N-); 3.7 (4H, 2 x s, 2 x S-CH2-Ar); 4.3 (2H, m, 2 x N-CH-COO-); 5.5 (4H, m, 2 x Ar-CH2-O-CO-); 7.3 (20H, m, 4 x CH2-Ar); 9.0 (2H, br s, 2 x CH-COOH). N,N′-Bis-carbobenzyloxy-S,S′-bis-benzyl-1,2-L,Lethylene Dicysteine Monomethyl Ester (4). To a solution of 3 (3.58 g, 5 mmol) and (dimethylamino)pyridine (DMAP, 61 mg, 0.5 mmol) in a mixture of methanol (0.2 mL, 5 mmol) and CH2Cl2 (50 mL) at 5 °C was added dicyclohexylcarbodiimide (DCC, 1.031 g, 5 mmol), and the reaction mixture was stirred overnight at room temperature. After removal of the precipitate (dicyclohexylurea), the filtrate was evaporated and the residual oil purified by column chromatography using gradient mixtures of hexane and CH2Cl2 as the mobile phase. A colorless oil (1.56 g, 42%) was obtained. 1H NMR: (CDCl3): δ 2.5-3.1 (4H, m, 2 x CH-CH2-S); 3.33.8 (4H, m, 2 x CH2-N-; 3H, s, COOCH3); 3.6 (4H, 2 x s, 2 x S-CH2-Ar); 4.1-4.6 (2H, m, 2 x N-CH-COO-); 5.1 (4H, m, 2 x Ar-CH2-O-CO-); 7.3 (20H, m, 4 x CH2Ar); 8.3 (H, br s, CH-COOH). Ethylene-l-(S-benzyl-N-carbobenzyloxycysteine methyl ester)-2-S-benzyl-N-carbobenzyl-oxy-cysteinylmethylenediphosphonic Acid Tetraethyl Ester (6). To a solution of 4 (1.56 g, 2.14 mmol) and 1-hydroxybenzotriazole (HBT, 0.29 g, 2.14 mmol) in acetonitrile (20 mL) at 0-5 °C was added DCC (0.442 g, 2.14 mmol), and the mixture was stirred for 30 min. After addition of 1-aminomethylenediphosphonic acid tetraethyl ester (5) (0.648 g, 2.14 mmol) in one portion, stirring was continued overnight at room temperature. The precipitate was removed by filtration, the filtrate evaporated, and the residue purified by column chromatography using gradient mixtures of methanol in CH2Cl2 to yield 6 as a colorless oil (1.1 g). 1H NMR (CDCl3): δ 1.25 (12H, m, 4 x O-CH2-CH3); 2.5-2.9 (4H, m, CH-CH2S); 3.4 (1H, m, CO-NH-CH); 3.3-3.8 (11H, m, COOCH3,

18 Bioconjugate Chem., Vol. 13, No. 1, 2002

Verbeke et al.

Scheme 1. Synthesis of the Fully Protected EC-AMDP Conjugate (6)

2 x CH2-N, 2 x S-CH2-Ar); 4.2 (8H, m, 4 x O-CH2CH3); 4.4 (2H, m, N-CH-CO); 5.1 (4H, m, 2 x Ar-CH2O-CO); 7.3 (20H, m, 4 x CH2-Ar). 13C NMR (CDCl3): δ 16.3 (O-CH2-CH3); 30.8, 30.9 (2 x CH-CH2-S); 35.5 (CO-NH-CH); 36.1, 36.3 (2 x S-CH2-Ar); 45.8, 46.4 (2 x CH2-N); 52.3 (COOCH3); 58.2,60.1 (2 x N-CH-CO); 63.6 (O-CH2-CH3); 67.6,68.0 (2 x Ar-CH2-O-CO); 127.1, 128.5, 135.9, 137.4 (Ar); 156.8 (Ar-CH2-O-CO); 170.4 (CO-NH-CH); 170.9 (COOCH3). MS LSIMS (nba): [M - H]- 1015.6. Ethylene-l-L-cysteine-2-L-cysteinyl-aminomethylenediphosphonic Acid (7b). To a solution of 6 (1.016 g, 1 mmol) in CH2Cl2 (10 mL) at 0 °C was added trimethylsilyl bromide (TMSBr, 2 mL, 15 mmol). After stirring at room temperature for 72 h, the reaction mixture was evaporated, and to the residue was added methanol (10 mL). After stirring during 1.5 h, the methanol was removed by evaporation, and the residue was dried in a vacuum over P2O5. After addition of a mixture of methanol (10 mL) and 1 M NaOH (6 mL), the mixture was refluxed during 2 h. The solvent was evaporated, and the residual solid was stirred in methanol (20 mL). The white precipitate was filtered off, washed with methanol, and dried under a stream of nitrogen (intermediate 7a, Scheme 2). MS-ESI: m/z calcd for C39H44N3O13P2S2 [M - H]- 888.1790; found 888.1780. The precipitate (50 mg) was dissolved in liquid ammonia (20 mL), and pieces of sodium were added until a blue color persisted for 4 h. After decoloration with NH4Cl, the ammonia was removed under a stream of nitrogen, and the residual solid (7b, Scheme 2) was dried in a vacuum and used as such for labeling experiments.

Labeling with 99mTc and Analysis. Direct labeling of 7b with 99mTc was performed by addition of 100 µg of SnCl2‚2H2O, dissolved in 25 µL of 0.05 M HCl, and 1 mL of eluate of a commercial generator containing 370-3700 MBq 99mTc in the form of sodium pertechnetate to a labeling vial containing 1 mg of 7b (EC-AMDP) dissolved in 1 mL of 0.2 M NaOH. After incubation for a few minutes at room temperature, the labeling reaction was analyzed by ion-pair reversed phase high-pressure liquid chromatography (IPRP-HPLC). The system consisted of a Merck-Hitachi ternary gradient pump (model L-6200 intelligent pump, Merck, Darmstadt, Germany), a Valco N6 injector (Alltech, Deerfield, IL), and a 250 mm × 4.6 mm Hypersil ODS 5 µm column (Shandon Scientific, Maidstone, England). After application of the sample, the column was eluted with gradient mixtures of water, methanol, 0.008 M tetrabutylammonium hydroxide, and 0.0013 M MDP (pH 7.5) (see Table 1). The radioactivity in the column effluent was monitored with a 2-in. NaI(T1) scintillation detector and recorded on an IBM-compatible PC equipped with an integration program (Rachel, Lablogic, Sheffield, England). Biodistribution in Rats. The 99mTc-EC-AMDP labeling mixture was used as such or its main components were isolated using the RP-HPLC described above. The labeling reaction mixture or the HPLC isolated peaks were diluted to a concentration of 0.37 MBq/mL with saline. Male rats (body mass 60-80 g) were sedated by im injection of 0.1 mL of a 1 to 4 diluted solution of Hypnorm (Duphar, Weesp, The Netherlands). Then, 0.2 mL of the diluted tracer solution was injected via a tail



Scheme 2. Deprotection of the Fully Protected Intermediate To Obtain the EC-AMDP Conjugate

Table 1. Composition of the Mobile Phase Used in the HPLC Analysis as a Function of Time (linear gradient)a time (min)

%A

%B

0 5 10 25 30 35

100 50 50 0 0 100

0 50 50 100 100 0

a A: water containing 10% MeOH, 0.008 M tetrabutylammonium hydroxide (TBAH) and 0.0013 M MDP (pH 7.5). B: watermethanol (40:60, V/V), containing 0.008 M TBAH and 0.0013 M MDP (pH 7.5). Flow rate: 1 mL/min.

vein into each of six rats. The rats were sacrificed by decapitation at fixed time intervals (two rats at 30, 60, and 120 min postinjection (p.i.), respectively) and the organs were dissected. The left and right femur were carefully cleaned and weighed. The activity in all organs and other body parts was counted in an automated sample counter (1480 Wizard3”, Wallac, Turku, Finland) and corrected for background radiation and physical decay during counting. Results were expressed as percentage of injected activity, equal to the sum of the net activity in all organs. Blood was assumed to be 7% of the body mass. Biodistribution in Rabbits. 99mTc-MDP or 99mTclabeled EC-AMDP (without HPLC purification) were

diluted with saline to a concentration of 37 MBq/mL. A male albino rabbit (body mass 3.0 kg) was sedated by im injection of 0.75 mL of Imalge`ne (Rhoˆne Me´rieux, Lyon, France), and 1 mL of the diluted tracer solution was injected via an ear vein. After 60 min, whole body images were acquired during 6 min using a gamma camera (BIAD XLT 24, Trionix, Twinsburg, OH), equipped with a low energy ultra resolution parallel collimator (LEURparallel) and connected to a data system SUN (Sparc station 10, Trionix). Evaluation in a Baboon. A male baboon (body mass approximately 21 kg) was anaesthetized by intramuscular injection of 75 mg of ketamine (Imalge`ne). Anaesthesia was sustained with Fluothane. Then, 185 MBq of the radiolabeled preparation (99mTc-MDP or 99mTc-EC-AMDP, both without HPLC-purification), adjusted to pH 7 with 0.2 M phosphate buffer, was injected via a limb vein. Whole body images were taken at different time intervals postinjection using a dual head gamma camera as described above for the rabbit study. At fixed time intervals (2, 5, 7, 10, 15, 20, 30, 45, 60, 80, 100, 120 min p.i.) a 2-mL blood sample was taken in heparinized tubes (Monoject H100, Sherwood Medical, Balleymoney, Ireland). After centrifugation for 10 min at 1000 g plasma samples of approximately 0.5 g were pipetted in tubes, weighed, and counted as described above.


Verbeke et al. Table 3. Femur to Blood Activity Ratios for and 99mTc-EC-AMDP (total preparation and HPLC-isolated peaks) in Rats (n ) 2) 99mTc-MDP 99mTc-EC-AMDP

Figure 2. Ion pair RP-HPLC chromatograms of 99mTc-ECAMDP and 99mTc-MDP. For conditions: see Materials and Methods. RESULTS AND DISCUSSION

Synthesis of the EC-AMDP Conjugate. A multistep procedure has been designed to obtain the conjugate of ethylene dicysteine with aminomethylenediphosphonate in reasonable yields (Schemes 1 and 2). Since EC contains in its structure six functional groups (2 thiols, 2 amines, 2 carboxyl functions), a complex strategy of protection and deprotection of the respective functional groups had to be applied. The thiols were protected with a benzyl group, the amines with a carbobenzyloxy (CBO) group, and one of the carboxylates as a methyl ester (Scheme 1). The preparation of the monomethyl ester was performed by reaction of the diacid with 1 equiv of methanol in the presence of DMAP and DCC, followed by purification of the reaction product. In this way, a reasonable yield (42%) of the monoester was obtained. In addition, the four phosphonate groups of AMDP had to be protected as their ethyl ester to allow purification and confirmation of the identity of the precursors, the intermediates, and the fully protected EC-AMDP conjugate. Removal of the different protective groups was performed successfully in three steps using trimethylsilyl bromide for the phosphonate esters, alkaline hydrolysis of the methyl ester of the EC-moiety, and finally treatment with sodium in liquid ammonia to remove the S-benzyl and N-CBO groups (Scheme 2). Characterization of the intermediates by either NMR or mass spectrometry was possible up to intermediate 7a, but was unsuccessful for the final fully deprotected conjugate 7b because of high salt concentrations in the reaction product. In fact, the compound obtained after deprotection of the S-benzyl and N-CBO groups by treatment of 7a with sodium in liquid ammonia was not further purified due to the small amounts available and the difficulty of chromatographic desalting of the highly polar conjugate 7b and it was used as such for labeling with technetium-99m. Table 2. Biodistribution in Rats of

99mTc-MDP

(n ) 2) and

total peak A peak B

99mTc-MDP

30 min

60 min

120 min

12.4 19.9 23.9 26.9

35.8 55.1 106.3 50.2

48.4 98.0 363.5 745.9

Labeling of EC-AMDP with 99mTc and Analysis. Labeling of the conjugate with 99mTc was performed in the same conditions as described for EC, i.e., by a direct labeling procedure at alkaline pH and room temperature. As mentioned higher, previous experiments had indicated that the complexation strength of the N2S2-ligand EC is much higher than that of the diphosphonate. Therefore, there is no doubt that technetium is bound only by the N2S2-ligand and not by the diphosphonate moiety. Analysis of the labeling reaction product by ITLC showed that the solution did not contain measurable amounts of pertechnetate or 99mTc in colloidal form. Further analysis by IP-RP-HPLC showed the formation of a mixture of three compounds, of which the relative amounts were reproducible and constant as a function of time after preparation (Figure 2). The different peaks could easily be separated by HPLC and were stable after isolation. The HPLC-chromatogram of 99mTc-MDP clearly shows different complexes of which the relative amounts varied as a function of time after reconstitution and which were not reproducible. When one of the components of 99mTcMDP was isolated by HPLC, it gradually converted to the other components as a function of time. In the biodistribution studies, 99mTc-MDP was always used as the complete preparation. Biological Evaluation in Rats. The results of the biodistribution studies in rats of 99mTc-MDP and 99mTcEC-AMDP (total preparation) at 30, 60, and 120 min p.i. are shown in Table 2. The activity in the femur is almost identical for 99mTc-MDP and 99mTc-EC-AMDP at 30 and 60 min p.i., whereas at 120 min p.i. 99mTc-EC-AMDP has a clearly higher uptake in bone than 99mTc-MDP. At each time point, the residual activity in the blood is lower for the new 99mTc-labeled conjugate, and as a result, the femur to blood activity ratios found with the total preparation are significantly higher for 99mTc-EC-AMDP than for 99mTc-MDP (Table 3). The femur to blood activity ratio of the 99mTc labeled EC-diphosphonate conjugate at 1 h p.i. is higher than the same ratio for 99mTc-MDP at 2 h p.i. In biodistribution studies with the isolated peaks A and B, the femur to blood activity ratios are significantly higher than for the total preparation. This indicates that the total preparation contains some constituents, possibly not eluted from HPLC, with lower affinity for the bone. In the muscles and the hepatobiliary system, the activity is low at all tested time points for each of the preparations tested. The activity not taken up in the bone is excreted rapidly via the kidneys to the urine, and its percentage is almost similar for the two preparations. 99mTc-EC-AMDP

(n ) 6) at 30, 60, 120 min p.i.

% of injected dose 99mTc-MDP

99mTc-EC-AMDP

time p.i. (min)

kidneys+urine

liver+intestines

blood

%/g muscle

%/g femur

30 60 120 30 60 120

22.0 24.4 24.2 26.7 22.4 26.4

1.8 1.2 1.2 1.7 1.3 0.8

4.5 1.9 1.3 2.7 1.2 0.5

0.2 0.4 0.1 0.2 0.1 0.0

12.9 15.1 15.4 12.7 14.8 29.4



Figure 5. Whole body images 2 h after injection of 99mTc-MDP and 99mTc-EC-AMDP in a baboon.

a slightly faster renal extraction or to a faster distribution to the extravascular space. Whole body scintigraphic images obtained 2 h after injection of both agents are shown in Figure 5. As in the rabbit study, nearly identical images are obtained with the two radiopharmaceuticals. This means that the newly developed 99mTc-diphosphonate conjugate has the characteristics of a suitable bone scanning agent, with almost the same contrast between soft tissue and bone as obtained with 99mTc-MDP. CONCLUSION

Figure 3. Whole body images of a rabbit 1 h after injection of 99mTc-EC-AMDP (A) and 99mTc-MDP (B) (total preparation).

Figure 4. Plasma disappearance curves in a baboon for 99mTc-MDP and 99mTc-EC-AMDP.

Evaluation in a Rabbit. The scintigraphic images obtained in a rabbit at 1 h p.i. are shown in Figure 3. Both tracer agents allow a clear visualization of the skeleton and a net distinction between the respective vertebrae and ribs. Uptake in soft tissue is low, and it is clear that the activity not bound to the skeleton is excreted through the kidneys. The image quality is quite similar for both compounds. Biological Evaluation in a Baboon. Figure 4 shows the plasma disappearance curve in a baboon for 99mTcMDP and 99mTc-EC-AMDP (total preparation) during 2 h after injection. Both tracer agents behave in nearly the same way, although the clearance of 99mTc-EC-AMDP from plasma is somewhat faster. This may be related to

The results shown in this paper are part of a research project with the aim to develop a “new generation” bone scanning agent with improved characteristics as compared to the currently used 99mTc-diphosphonates. The intended improvements concern the stability of the tracer agent, its clearance rate from soft tissue, and its uptake in the bone. For this purpose, a 99mTc-labeled renal tracer agent with known rapid active renal excretion (99mTcethylene dicysteine) was conjugated to a diphosphonate. In this concept, the EC moiety of the new tracer agent is assumed to be responsible for both the stable binding of the 99mTc radionuclide and the rapid urinary excretion of the fraction not bound to the bone. The diphosphonate moiety in this compound is not involved in the binding of 99mTc and thus fully available for binding to the skeleton. Up to now, the most favorable results have been obtained with the 99mTc-complex of the conjugate between ethylene dicysteine and aminomethylenediphosphonate, coupled by amide formation between a carboxylate of EC and the amine of AMDP. We have synthesized several other conjugates and evaluated them as their 99mTccomplexes (21) (to be submitted for publication). In rats, 99mTc-EC-AMDP showed very promising characteristics with clearly higher femur to blood and femur to muscle activity ratios than 99mTc-MDP. Especially when the HPLC-isolated peaks A and B were used, these ratios were favorably high. This suggested that an optimized preparation containing only peak A and peak B of 99mTc-EC-AMDP would constitute a bone-seeking radiopharmaceutical with more rapid clearance from soft tissue and higher uptake in the skeleton than the currently used 99mTc-diphosphonates. In the rabbit and the baboon, the scintigraphic images obtained with the new agent (total preparation) showed good quality bone scans with clear visualization of the


skeleton and low soft tissue activity at respectively 1 and 2 h after injection. The fact that the plasma activity disappearance in the baboon was only slightly faster for 99m Tc-EC-AMDP than for 99mTc-MDP indicates that the new tracer is a good bone-scanning agent, but in this species not a significant improvement as compared to the currently available 99mTc-diphosphonates. On the other hand, the new 99mTc-labeled EC-AMDP conjugate has the clear advantage over the common 99mTc diphosphonates of a high stability and a constant composition as a function of time. The development and evaluation of further derivatives on the basis of the new concept may eventually result in a 99mTc-labeled tracer agent with the intended biological characteristics. LITERATURE CITED (1) Subramamian, G., McAfee, J. G., O’Mara, R. E., et al. (1971) Tc-99m polyphosphate PP46: A new radiopharmaceutical for skeletal imaging. J. Nucl. Med. 12, 399. (2) Yano, Y., McRae, J., Van Dyke, D. C., and Angor, H. 0. (1973) Technetium-99m labeled stannous ethane-l-hydroxyl1,1-diphosphonate: a new bone scanning agent. J. Nucl. Med. 14, 73-78. (3) Castronovo, F. P., and Callahan, R. J. (1972) New bone scanning agent: Tc-99m labeled 1-hydroxyethylidene-1,1disodium diphosphonate. J. NucI. Med. 13, 823-827. (4) Subramanian, G., McAfee, J. G., Blair, R. J., Mehler, A., and Connort, T. (1972) Tc-99mEHDP: a potential radiopharmaceutial for skeletal imaging. J. Nucl. Med. 13, 947950. (5) Cleynhens, B., Bormans, G., Van Nerom, C., De Roo, M., and Verbruggen, A. (1991) Evaluation of the efficacy of different antioxidants in Tc-99m MDP preparations, in Nuclear Medicine. The state of the art of Nuclear Medicine in Europe (H. A. E. Schmidt, and J. B. van der Schoot, Eds.) pp 133-135, Schattauer, Stuttgart-New York. (6) Baldas, J., and Bonnyman, J. (1985) Substitution reactions of 99mTcNCl4- - A route to a new class of 99mTc-radiopharmaceuticals. Int. J. Appl. Radiat. Isot. 36, 133-139. (7) Pinkerton, T., Heineman, W., and Deutch, E. (1980) Separation of technetium hydroxyethylidene diphosphonate complexes by anion-exchange high performance liquid chromatography. Anal. Chem. 52, 1106-1110. (8) Srivastava, S., Bandyopadhyay, D., Meinken, G. et al. (1981) Characterization of 99mTc-bone agents (MDP, EHDP) by reversed phase and ion exchange high performance liquid chromatography. J Nucl. Med. 22, P69. (9) Tanabe, S., Zodda, J., Deutsch, E., and Heineman, W. (1983) Effect of pH on the formation of Tc(NaBH4)-MDP radiopharmaceutical analogues. Int. J. Appl. Radiat. Isot. 34, 15771584.

Verbeke et al. (10) Hoch, D., and Pinkerton, T. (1986) Reversed-phase HPLC of 99mTc methylenediphosphonate bone imaging kits with quantification of pertechnetate. Appl. Radiat. Isot. 37, 593598. (11) Subrahamian, G., McAfee, J. G., Blair, R. J, Kollfelz, F. A., and Thomas, F. P. (1975) Technetium-99m methylene diphosphonate - a superior agent for skeletal imaging: comparison with other technetium complexes. J. NucI. Med. 16, 744-755. (12) Fogelman, I. (1982) Diphosphonate bone scanning agents - current concepts. Eur. J. Nucl. Med 7, 506-509. (13) Verbruggen, A. M., Nosco, D. L., Van Nerom, C. G., Bormans, G. M., Adriaens, P. J.; DeRoo, M. J. (1991) Technetium-99m-L,L-ethylene dicysteine: a renal imaging agent 1. Labeling and evaluation in animals. J. NucI. Med. 33, 551557. (14) Van Nerom, C., Bormans, G., De Roo, M., and Verbruggen, M. (1993) First experience in healthy volunteers with technetium-99m L,L-ethylene dicysteine, a new renal imaging agent. Eur. J. Nucl. Med. 20, 738-746. (15) Van Nerom, C., Bormans, G., Bauwens, J., Vandecruys, A., De Roo, M., and Verbruggen, A. (1991) Comparative evaluation of 99mTc-L,L-ethylene dicysteine and 99mTc-MAG3 in volunteers, in Nuclear Medicine. The state of the art of Nuclear Medicine in Europe (H. A. E. Schmidt, and J. B. van der Schoot, Eds.) pp 290-292, Schattauer, Stuttgart-New York. (16) Cleynhens, B., Vanbilloen, H., Seuntjens, E. et al. (1994) Comparison of labeling characteristics of 99mTc-ethylene dicysteine and its mono-acid derivative 99mTc-ethylene cysteamine cysteine. J. Labeled Compd. Radiopharm. 36, 1922. (17) Crombez, D., Cleynhens, B., Bormans, G., De Roo, M., and Verbruggen, A. (1991) Comparison of the chelating strength of different tetraligand systems. Eur. J. Nucl. Med. 18, 605. (18) Blondeau, P., Berse, C., and Gracel, D. (1967) Dimerisation of an intermediate during the sodium in liquid ammonia reduction of L-thiazolidine-4-carboxylic acid. Can. J. Chem. 45, 49. (19) Hormi, O., Pajunen, E., Avall, A., and Pannaren, P. (1990) A cheap approach to tetraethyl-methylenediphosphonates. Synth. Commun. 20, 1865-1667. (20) Sturz, G., and Guervenau, J. (1991) Synthesis of novel gembiphosphonates. Synthesis 8, 661-662. (21) Cleynhens, B., Vanbilloen, H., Delvaux, C., and Verbruggen, A. (1999) Synthesis and evaluation in rats of a bisconjugate of Tc-99m-EC with aminomethylene-diphosphonic acid. Eur. J. Nucl. Med. 26, 1193.

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