ORIGINAL ARTICLE 

Annals of Nuclear Medicine Vol. 7, No. 4, 239-244, 1993 

Basic evaluation of 67Ga labeled digoxin derivative as a metal-labeled bifunctional radiopharnraceutical 

Yasuhisa FUJIBAYASHI,** Yasutaka TAKEMURA,* Hideyuki TANlUCHI,* Naoko IIJIMA,* Junji KONISHI** and Akira YOKOYAMA* 

*Faculty ofPharmaceutical Sciences and **School of Medicine, Kyoto University, Kyoto, Japan 

To develop metal-labeled digoxin radiopharmaceuticals with affinity with anti-digoxin antibody as well as Na+,K+_ATPase, a digoxin derivative conjugated with deferoxamine was synthesized. The derivative had a high binding affinity with 67Ga at deferoxamine introduced to the terminal sugar ring of digoxin. The 67Ga labeled digoxin derivative showed enough in vitro binding affinity and selectivity to anti-digoxin antibody as well as Na+,K+_ATPase. The 67Ga labeled digoxin derivative is considered to be a potential metal-labeled bifunc-tional radiopharmaceutical for digoxin RIA as well as myocardial Na+,K+_ATPase imaging. Key words: digoxin, radiopharmaceutical, deferoxamine, radioimmunoassay, Na+ K+ ATPase 

INTRODUCTION

THE DEVELOPMENT Of metallic radionuclide-1abeled bifunctional radiopharmaceuticals with high affinity with and specificity for corresponding antibodies or receptors has long been desired in nuclear medicine. Short-lived radioactive metals, such as 67Ga, 99mTc, and Inln, have numerous advantages in the design ,of radiopharmaceuticals over 125I, because of their superior specific activity, simplicity of labeling kit formation, and availability at reasonable cost. So various trials have been performed in this field, and are still progressing. 
Recently we reported the potential usefulness of 123I-labeled digoxin radiopharmaceutical for myo-cardial Na+,K+_ATPase imaging.1 That study sug-gested that the first and second sugars at the C-3 position of digoxin, as well as the steroid aglycone, were essential for myocardial accumulation, but the third sugar could be a site capable of modifi-cations for radiolabeling. These findings introduced a new approach for the design of digoxin-based bifunctional radiopharmaceuticals. That is to say, the introduction of a metal-chelating site to the third sugar residue of digoxin may have less effect on the binding ability and specificity of digoxin for the anti-digoxin antibody and/or Na+,K+_ATPase. 
In the present study, we selected the 67Ga-defer-oxamine (67Ga-DFO) system2 as a metallic radio-nuclide-bifunctional chelating agent system and syn-thesized digoxin-deferoxamine[bis(o-carboxymethyl-oxime)] (DFO-digoxin conjugate). Its 67Ga-1abeling with high specific activity and in-vitro binding assay to the anti-digoxin antibody as well as Na+,K+_ ATPase were performed. A biodistribution study in guinea pigs was then attempted in comparison with 67Ga-DFO and 67Ga-citrate. 
MATERIALS AND METHODS 

Digoxin and desferal (deferoxamine mesylate) were obtained from Aldrich (Milwaukee, WI), Ciba Geigy (Basel, Switzerland), respectively. Iron-free HCI was purchased from Nacalai Tesque (Kyoto, Japan). 67Ga-citrate was obtained from Nihon Mediphysics (Takarazuka, Japan). "Amerlex digoxin", digoxin radioimmunoassay (RIA) kit, was obtained from Amersham (Buckinghamshire, England). All other reagents and solvents were of reagent grade. 
The radioactivity of 67Ga and 125I was measured with an autogamma counter (ARC300, Aloka, Japan). Analytical thin-1ayer chromatography (TLC) was performed with Merk TLC plates (silica gel 60 F254 pre-coated, 0.25 mm layer thickness). The TLC plate was stained with iodine vapor to visualize spots on the plate. HPLC was performed in a Shimadzu LC-5A high performance liquid chro-matograph (Shimadzu, Japan) equipped with a UV detector and a reverse-phase column (cosmosil 5 C 18, 8 x 250 mm; Nacalai Tesque, Japan). IH NMR at 300 MHZ Was performed with a Bruker AC-300 apparatus for DFO-digoxin conjugate, dissolved in dimethyl sulfoxide-D6. Tetramethylsilane was used as the internal standard. 

Chemical synthesis 
N-hydroxysuccinimide ester of digoxin[bis(o-car-boxymethyloxime)]. The synthetic reaction sequence from digoxin dialdehyde to N-hydroxysuccinimide ester of digoxin[bis(o-carboxymethyloxime)] was followed according to the method reported previously in detail.3,4 In brief, as shown in Fig. 1, the terminal digitoxose in digoxin was cleaved to give digoxin dialdehyde by using sodium metaperiodate. The condensation reaction of digoxin dialdehyde and carboxymethoxylamine hemihydrochloride pro-ceeded rapidly in sodium acetate/ethanol and a quantitative yield of digoxin[bis(o-carboxymethyl-oxime)] was obtained. The digoxin-dioxime deriv-ative was immediately reacted with N-hydroxysuc-cinimide in the presence of dicyclohexylcarbodi-imide to give N-hydroxysuccinimide ester of di-goxin[bis(o-carboxymethyloxime)]. 
DFO-digoxin conjugate. A mixture of desferal (656 mg, I mmol) and N-hydroxysuccinimide ester of digoxin[bis(o-carboxymethyloxime)] in 60 ml of dry pyridine was stirred and heated at 95-105'C for 36 hr under nitrogen. The reaction was monitored by TLC in chloroform/methanol/water (80/25/3 by volume); Rf 0.75=dioxime active ester, 0.43=N-hydroxysuccinimide. 0.26-0.37=DFO-digoxin con-jugate, 0.12=DFO. The reaction mixture was evaporated in a high vacuum. Pure DFO-digoxin conjugate was isolated from the crude reaction mixture by HPLC, eluted with methanol and distilled water (6/4 by volume) at a flow rate of I .4 ml/min with deferoxamine mesylate and DFO-digoxin con-jugate which were eluted at 6.5 and 61.2 min, respec-tively. DFO-digoxin conjugate was obtained as a white powder (22% yield) and had a melting point of 167-169'C. IH NMR (sigma, ppm): 0.65 (singlet, 3H, 18-CH3), 0.85 (singlet, 3H, 19-CH3), 1.02-1.92 (complex multiplet), 1.97 (singlet, 6H, terminal methyl group in DFO), 2.90-4.97 (complex multi-plet), 5.95 (singlet, IH, Iactone, C=CH). Elemental anal. Calcd for C95H160032Nl4'3H20: C, 55.27; H, 8.10; N, 9.50. Found: C, 55.12; H, 7.92; N, 9.63. 

Determination of the conjugation ratio of DFO-digoxilt conjugate 
To test the progress of DFO-digoxin conjugate formation, a solution of the conjugate (Chelating ligand: 316 pM, DMSO) was mixed with a Fe3+ solution (Metallic ion: 316 pM of FeN03, at pH 3) in various ratios (Metal/Metal+Ligand=0, 0.2, 0.4, 0.6, 0.7, 0.8, 1.0 by volume), and the concentrations of the Fe complex were measured after 3 min with a spectrophotometer (330s. Hitachi, Japan). The amount of deferoxamine bidning to DFO-digoxin conjugate was determined by Job's method of continuous variation.5 

Antigenicity of DFO-digoxin conjugate Antigenicity of the newly synthesized digoxin derivative was determined with a commercially available digoxin RIA kit. Fifty microliters of DFO-digoxin conjugate solution {O M (control), 0.219 nM. 2.19 nM, 21.9 nM, 219 nM, 2.19 pM; 1% BSA/ saline}, 200 pl of 1251 Iabeled digoxin derivative solution, and 200 pl of anti-digoxin antibody coated beads were mixed and incubated for 30 min at 37'C. After centrifugation (1500 x G, 15 min), the super-natant was decanted. The 1251 radioactivity bound to the beads was measured and the ratios of binding counts to total counts were calculated. The amount of digoxin in the DFO-digoxin conjugate sample was estimated from the standard curve by simple extrapolation and the equivalent of digoxin in DFO-digoxin conjugate was calculated. 

Radiolabeling of DFO-digoxin conjugate 
The 67Ga solution was purified as reported by Furukawa et al.6 with some modifications in regard to the final extraction of 67Ga as 67Ga-citrate instead of 67Ga-chloride by the method introduced by Horiuchi et al. (unpublished). Briefly, 67Ga-citrate (292 MBq, 584 MBq/ml), 0.1 M ascorbic acid, and concentrated HBr were mixed at a volume ratio of 4 : 1 : 7 and extracted with the same volume of butyl acetate. After evaporation of the organic solvent. the clear residue was extracted with a solution containig 100 pe/ of 0.1 N iron-free HC1, 25 ul of 0.1 M ascorbic acid, and 175 ul of concentrated HBr. The solution was then extracted with 500 pJ of butyl acetate, separated, and evaporated in the same way as above. Finally, 50 pl of sodium citrate (2 mg/ml) was added to the clear residue. The radiolabeling of DFO-digoxin conjugate was carried out by adding 50 pl of the purified high specific activity 67Ga solution along with 50 pl of the previously prepared DFO-digoxin conjugate (50 ug/ml, DMSO). The addition was followed by simple mixing and standing for I hr. The labeling efficiency was analyzed by cellulose acetate electro-phoresis (EP) in veronal buffer (1=0.05, pH 8.6) with a stationary current of 0.8 mA/cm for 40 min. The labeling efficiency percentage was estimated as the ratio of the radioactivity associated with the DFO-digoxin conjugate to the total radioactivity in each strip. As a control, both the 67Ga-citrate solution and the 67Ga-DFO were also analyzed under the same conditions. The radiolabeled 67Ga-DFO-digoxin conjugate was diluted 40,000 times with I ~ BSA/saline solution for use in the RIA. 

RIA using 67Ga labeled DFO-digoxin conjugate 
The RIA with 67Ga-DFO-digoxin conjugate (67Ga RIA) was adapted from the 1251 RIA, using 67Ga-DFO-digoxin conjugate instead of 1251 labeled digoxin derivate. The standard digoxin (0.35-5.0 ng/ml) used was provided by a commercial RIA kit. The cross-reactivity of 67Ga RIA with the DFO-digoxin conjugate to ouabain (1 ng-10 pg/ml saline) was analyzed following the above manufacturer's instructions, but using ouabain solution instead of standard digoxin solution. 
In-vitro Na+,K+_ATPase binding studies The effect of ouabain on the binding of 67Ga-DFO-digoxin conjugate to Na+,K+_ATPase known as digitalis receptor, was studied according to a method ¥described previously,1,7 using the crude Na+,K+_ 
ATPase fraction separated from guinea-pig kidney cortex.8 As a reference, 3H-digoxin binding was also determined. 

Biodistribution studies 
Male guinea pigs weighlng 300 g were injected with 67Ga-DFO-digoxin conjugate, 67Ga-DFO, or 67Ga-citrate (200 pl, 131 KBq) via the femoral vein and then killed by cervical decapitation at 60 min after the injection. Tissue accumulation was calculated as the percentage injected dose/gram of tissue. 

RESULTS 

Chemical synthesis 
The synthetic sequence is outlined in Fig. 1. Isolated DFO-digoxin conjugate was characterized by IH NMR measurement and elemental analysis. From these data, it was demonstrated that DFO-digoxin conjugate had two deferoxamine molecules attached (Fig. 2). As a solid, DFO-digoxin conjugate was stable at 4'C. In solution, the conjugate was stable in methanol and water (6/4 by volume) for several days. 

Determination of the conjugation ratio of DFO-digoxin conjugate 
It is well known that desferal (or deferoxamine) has high binding affinity with Fe3+ (10g K=30.6) and Ga3+(log K=28.0). Figure 3 shows the line graph obtained by plotting absorbance against the ratio of metal concentration to metal plus ligand concen-tration. The maximum occurred at a ratio of about 0.66, indicating that Fe3+ and DFO-digoxin con-jugate made a complex with Fe3+/ligand ratio of 2/1 , This showed that there were two binding sites for Fe3+ in DFO-digoxin conjugate. 

Antigenicity of DFO-digoxin conjugate The antigenicity of the newly synthesized DFO-digoxin conjugate was then determined. Table 1 shows that as the amount of the DFO-digoxin conjugate increased, a clear decrease in the radio-iodinated binding fraction was detected. The im-munoactivity was the same for 2.19 nmol of DFO-digoxin conjugate and 2.05 nmol of digoxin. 

Radiolabeling of DFO-digoxin conjugate The specific activity of the purified gallium solution used for the radiolabeling was 4.4 GBq/ml. DFO-digoxin conjugate was easily radiolabeled with the purified iron-free 67Ga extracted as 67Ga-citrate by a simple mixing reaction. The average labeling yield was 90% (85-94%) calculated after electrophoretic analysis (Fig. 4) and the specific activity of 67Ga-DFO-digoxin conjugate reached 79 GBqlmg (159 MBq/nmol). Electrophoretic analysis did not detect the presence of free 67Ga-DFO and 67Ga-citrate in the reaction mixture. 67Ga-DFO-digoxin conjugate diluted 102 times was chemically stable in BSA solution within one half-life of the radionuclide. 

RIA using 67Ga labeled DFO-digoxin conjugate The digoxin RIA was performed with 200 pl per sample of 67Ga-DFO-digoxin conjugate, providing approximately 20,000 counts per minute (cpm) per tube as described under MATERIALS AND METHODS. The 67Ga RIA gave a good linearity between the percent bound/total counts ( % B/T) and the concentration of digoxin (Fig. 5). Figure 5 shows the specificity of 67Ga RIA to ouabain. The cross-reactivity to ouabain was below 0.1% of the digoxin binding, and was as low as in 1251 RIA (below 1.0%)-In-vitro Na+,K+_ATPase binding studies Based on the antigenicity of DFO-digoxin conjugate to anti-digoxin antibody, the in-vitro binding to Na+,K+_ATPase was compared with that of 3H-digoxin (Fig. 6). Studies with ouabain showed that the IC50 value for the binding of 67Ga-DFO-digoxin conjugate to Na+,K+_ATPase was very similar to that of 3H-digoxin. 

Biodistribution studies Table 2 shows the biodistribution of 67Ga-DFO-digoxin conjugate, 67Ga-DFO, and 67Ga-citrate in guinea pigs. 67Ga-DFO-digoxin conjugate showed higher myocardial accumulation than 67Ga-DFO or 67Ga-citrate. DISCUSSION 

Because digoxin is too small a molecule to be anti-genic by itself, digoxin and its derivatives as haptens were caused to conjugate with a large-molecular carrier. Based on the reaction sequence of Butler and Chen,9 the carboxy-groups of digoxin dioxime3 were reacted with the amino-group of deferoxamine, using the activated ester method. 
1H NMR spectra and the line graph by continuous variation method indicated that the chelating ability of two DFO sites bound in the DFO-digoxin con-jugate was preserved. The results of the binding-test to the anti-digoxin antibody suggested that DFO-digoxin conjugate maintained binding affinity to the antibody. 
Since the sensitivity of RIA is dependent on precise measurement of the binding as radioactivity, the improvement in the specific activity of radiolabeled antigen increases the assay sensitivity. 67Ga has a short half life, and considering the theoretical half life alone an 18 fold increase is possible (67Ga : 3.3 vs. 1251 : 60 days). In our study, the 67Ga labeled com-pound had a higher specific activity (1 59 MBq/nmol) than the 1251 Iabeled compound from the commer-cially available RIA kit (80 MBq/nmol). Further improvement in the specific activity could be achieved if a generator producing 68Ga with a half life of 68.3 minutes were available. Moreover, the short half-life of radiogallium (67Ga, 68Ga) contributes to the reduction of radioactive waste disposal problems. The standard cruve of 67Ga-RIA was obtained with the synthesized 67Ga-DFO-digoxin conjugate, and this suggested that 67Ga-DFO-digoxin conjugate retained immunoreactivity to anti-digoxin antibody. 
In guinea-pig kidney Na+,K+_ATPase, ouabain displacement studies suggested that 67Ga-DFO-digoxin conjugate retained its binding ability to Na+,K+_ATPase in spite of the chelation of 67Ga at the carrier level with DFO. 
Previously, Misra et al.10 synthesized three 99*Tc labeled cardiac glycoside derivatives with cymarin, convallotoxin, and strophanthidin-D-glucoside to develop a potential myocardial imaging agent. In their work, thiosemicarbazone as the metal binding site for 99mTc labeling was attached to an aldehyde group placed in the C-19 position of the steroid aglycone in cardiac glycosides. Their biodistribution studies of three 99~Tc-1abeled compounds in guinea pigs showed a lower heart/non-target (except kidney)  
ratio than that of 67Ga-DFO-digoxin conjugate. We suspected that the metal binding site had little chelating ability with 99mTc and the modification of the steroid ring for radiometallic labeling would be a big problem in preserving the binding affinity.  
In conclusion, 67Ga-DFO-digoxin conjugate re-tained enough affinity with and specificity for anti-digoxin antibody as well as Na+,K+_ATPase, even thought deferoxamine (M.W. =560.71) is a compara-tively large molecule (digoxin : M.W.=708.95). The 67Ga-DFO-digoxin conjugate with high specific activity was applicable to digoxin RIA, and the 67Ga-RIA for routine practice may provide a solution to some of the problems inherent in 1251-RIA. 67Ga-DFO-digoxin conjugate would be also a candidate for in-vivo Na+,K+_ATPase imaging agent. In addition, our studies are applicable for the 67Ga labeling method at high specific activity in other compounds with sugar residues. 

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