Tricatecholamide Analogs of Enterobactin as Gallium- and Indium-Binding Radiopharmaceuticals Stephen M. Moerlein, Michael J. Welch, Kenneth N. Raymond, and Frederick L. Weitl The Edward Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, Missouri; University of California, Berkeley; and Lawrence Berkeley Laboratory, University of California, Berkeley, California Isopropyl N-substituted tricatecholamide analogs of enterobactin have been found to form gallium and indium complexes with very high stability constants and to exhibit in vivo characteristics significantly different from gallium- or indium- transferrin and EDTA. The 3,4-DiP-LICAMS and TiP-MECAMS complexes were found to clear primarily through the kidneys, whereas the less polar 3,4-DiP-LICAM complex was eliminated through the liver. The rationale for developing new metal- binding analogs with larger organic groups attached to the amide nitrogens is dis cussed. J NucíMed 22: 710-719, 1981 Enteric bacteria such as Escherichia coli and Sal have investigated the applicability of synthetic trica monella typhimurium are known to synthesize iron- techolamide enterobactin analogs as gallium- and in chelating agents of low molecular weight, called sider- dium-binding radiopharmaceuticals. The three ligands ophores, to satisfy their nutritional demands for iron studied are (a) 3,4-DiP-LICAMS [N,N"-bis(isopropyl)- (7,2). Enterobactin is a siderophore and is the most N,N',N"- tris(5-sulfo-2,3-dihydroxybenzoyl)-1,5,10- powerful ferric-ion-chelating agent known (3,4). Al triazadecane]; (b) 3,4-DiP-LICAM [N,N"-bis(iso- though its high log K of 52 is able to solubilize ferric iron propyl)-N,N',N"-tris (2,3-dihydroxybenzoyl)-l,5,10- and facilitate the transport of the metal into cells, en triazadecane]; and (c) TiP-MECAMS [1,3,5-N,N', terobactin is a cyclic ester that hydrolyzes readily at N"-tris(isopropyl)-N,N',N/'-tris-(5-sulfo-2,3-dihyd- physiological pH, and hence is not useful as a metal- roxybenzoyl)triaminomethylbenzene] (Fig. 1). 3,4- complexing radiopharmaceutical. Development of DiP-LICAMS and TiP-MECAMS were studied to see 2,3-dihydroxybenzoylamide analogs of enterobactin has whether the flexibility of the linkage between the three led to iron-binding ligands characterized by both high catecholamide groups of the former ligand would show stability constants and resistance to hydrolysis in solution biological behavior differing from that of TiP-ME (5,6). These synthetic tricatecholate ligands are also CAMS with its more constrained platform structure. kinetically and thermodynamically capable of removing The sulfonic acid groups attached to the aromatic rings iron from transferrin under physiological conditions (7), were expected to alter the pharmacological character as well as from the iron-storage protein ferritin in the istics of the complexes, since sulfonation increases the presence of ascorbic acid. acidity of the phenolic oxygens, the stability to oxidation, Nuclides of gallium and indium have found wide and the water-solubility of catechols. The isopropyl spread use in nuclear medicine (8). Because these are substitution on the amide nitrogens was expected to Group III B elements with charges and complexing decrease water-solubility and increase lipophilicity. characteristics similar to those of ferric ion (9 JO), we MATERIALS AND METHODS Received Sept. 2, 1980; revision accepted April 10, 1981. For reprints contact: Michael J. Welch, PhD, Dept. of Radiology, Determination of stability constants. Stability con Washington Univ. School of Medicine, 510 S. Kingshighway, St. stants for these ligands with gallium and indium were Louis, MO 63110. determined. Each ligand was synthesized, characterized, 710 THE JOURNAL OF NUCLEAR MEDICINE ••r' BASIC SCIENCES RAIMOCHEMISTRY AND RADIOPHARMACEUT1CALS HC 3.4DÌP-LICAM : 3.4DÌP-LICAMS TiP-MECAMS FIG. 1. Chemical structures of tricatecho- lamide analogs of enterobactin. and analyzed for purity according to reported techniques studied, including those of Ga-67 catecholamide used (/ / ), and the stability constants of the gallium complexes separately to determine elution volumes. were determined using Ga-67 in competitive exchange The stability constants of the indium complexes were between the ligand and EDTA. Gallium-67 citrate was determined by Sephadex G-10 separation after com added to 0. l M citrate buffer containing catecholate li petitive equilibration of In-111 in a solution of EDTA gand and EDTA in varying ratios, and the pH adjusted and ligand, as was done with the Ga-67 complexes. In to 7.0 using l M citrate. From sampling studies up to 10 addition, the decay characteristics of In-111 allow the days after mixing, it was found that exchange equilib use of perturbed angular correlation (PAC) experiments rium was achieved within 48 hr. Therefore, after 48 hr to study the complexing of this metal. This second of equilibration in sealed Pyrex test tubes at room tem method of analysis was used to study the agreement of perature, the Ga-67 catecholamide and Ga-67 EDTA the results from this technique with those obtained from products that formed were separated by gel-permeation gel-permeation chromatography, where exchange on the chromatography using a Sephadex G-IO column (1 X column may occur. The integral perturbation factor 145 cm) eluted with 0.075 M Tris-acétatebuffer (pH (G22(a>)}is related to the rotational correlation times 7.0). Fractions were collected (accounting for 95 ±5% of the environment of the In-111 nucleus, such that a of the administered activity) and counted in a Nal(Tl) lower (G22(°°))indicates a slower rotational correlation well counter, and the relative concentrations of Ga-67 time (15,16). The <G22(°°))was determined during EDTA and Ga-67 catecholamide were determined from complex formation using equipment described earlier the elution volumes for each complex. From the mid- (17,18). Indium-! 11-tagged 3,4-DiP-LICAMS, 3,4- exchange concentration ratio (the ligand concentration DiP-LICAM, and TiP-MECAMS were studied by ratio that gave equal yields of Ga-67 EDTA and Ga-67 adding mInCl3 to 0.1 M citrate solutions (pH 7.0) catecholamide), the stability constants were calcu containing various ratios of EDTA to catecholamide. lated. The point of mid exchange was found from a plot of In these competitive equilibrium experiments, citrate (G22(°°))against concentration ratio (Fig. 3) and the buffer was used because this organic acid forms soluble stability constants were calculated as in the gel-perme Ga3+ complexes up to pH 8 without hydrolysis to form ation separations. a gallium hydroxide precipitate (/2,13). Sodium citrate Biodistribution data. Biological distributions for the is necessary to maintain constant pH as well as ionic three gallium complexes were determined using 150-g strength, so that activity coefficients can be ignored in Sprague-Dawley rats with 10-20 ¿¿Ciof the respective the calculation of formation constants. In addition, the Ga-67 complex injected into the femoral vein. The dis log stability constant for Ga citrate is equal to 10.02(/4), tribution of In-111 3,4-DiP-LICAMS was studied and which is low enough so as not to interfere with the equi Ga-67 citrate was used for purposes of comparison. The libria of the exchange processes occurring between Ga- animals were killed in groups of three or more at various EDTA (log K = 20.3) (14) and ligands having even times after injection (1,3,5,18, and 24 hr) and the major greater gallium-binding affinity. Complete absence of organs were removed, weighed, counted, and the percent Ga-67 citrate was seen in the gel filtration of all solutions injected dose/gram of tissue calculated. Volume 22, Number 8 711 MOERLEIN, WELCH, RAYMOND, AND WEITL 045 040 1 035 030 I IO'1 10' 10 ; MOLARITY OF EDTA MOLARITY OF TRICATECHOLAMIDE FIG. 2. Variation of integral perturbation factor of ln-111 as a function of total ligand concentration ratio (in 0.1 M citrate, pH 7.0). Biological clearance data. To investigate further the incomplete exchange between Ga-67 or In-111-labeled pharmacokinetics of these complexes in the rat, clear EDTA and catecholamide occurred sharply (within a ance data were collected based on the techniques of range of log [EDTA]/[ligand] = ±1),so the concen Konikowsky et al. (19). Male 150-g Sprague-Dawley tration ratio at which mid exchange had been reached rats underwent penile ligation, and 10-20 ¿¿Ciof Ga- was easily found. This was true for the PAC exchange 67-labeled EDTA, citrate, or tricatecholate were injected process, as seen by the relatively sharp change from into the femoral vein. The animals were then killed, in <G22(°°)>^ 0.44 for In-111 EDTA to <G22(°°)>=*0.27 groups of at least three, at 10, 20, 30, 60, and 120 min for the In-111-labeled ligand (Fig. 2). At concentration after injection. Blood concentration was determined for ratios of EDTA-to-ligand greater or less than that of the each time, and the cumulative urinary activity was found exchange region, the (G22(°°)shows a flat response by quantitatively removing the intact bladder and con corresponding to either the In-111 EDTA or In-111 tents. Similarly, the kidneys, liver, and intestines (plus catecholate values. Similar exchange curves were found contents) were removed and counted to give the cumu using the gel-permeation data, as seen in Fig. 3 for the lative activity per organ. In-111 case and Fig. 4 for the Ga-67 case. Our value for (G22(°°)>of In-111 EDTA (0.44 ± RESULTS 0.02) differs from others reported in the literature for Determination of stability constants. The region of EDTA and its derivatives: <G22(°°)>= 0.58 ±0.02 10 06 04 io-' MOLARITY OF EDTA MOLARITY OF TRICATECHOLAMIDE FIG.