Bioconiugate Chem. 1001, 2, 117-123
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Hepatobiliary Delivery of Polyaminopolycarboxylate Chelates: Synthesis and Characterization of a Cholic Acid Conjugate of EDTA and Biodistribution and Imaging Studies with Its Indium-111 Chelate David A. Betebenner? Patrick L. Carney,t A. Michael Zimmer,* Joanne M. Kazikiewicz,: Ern0 Brucher,gvll A. Dean Sherry,g and David K. Johnson*J Abbott Laboratories, Department 90M, Abbott Park, Illinois 60064, Department of Nuclear Medicine, Northwestern University Medical Center, Chicago, Illinois 60601, and Department of Chemistry, The University of Texas at Dallas, Richardson, Texas 75083. Received December 20, 1990
A conjugate in which the steroid nucleus of cholic acid was linked to EDTA via an ll-atom spacer was obtained by reacting the succinimidyl ester of cholic acid with the amine formed by reaction of a benzyl isothiocyanate derivative of EDTA with N-(tert-butoxycarbony1)ethylenediamineand subsequent deprotection. Potentiometric titration studies with model complexes showed that the EDTA moiety retained the ability to form 1:l chelates of high thermodynamic stability, although formation constants were some 3-4 log K units lower for complexes of the conjugate than for the analogous chelates with underivatized EDTA. A complex formed between the cholic acid-EDTA conjugate and l11InI1*was clearly rapidly into the liver when injected iv into mice, with subsequent excretion from the liver into the gastrointestinal tract being complete within 1 h of injection. Radioscintigraphic imaging studies conducted in a rabbit given the ll11n-labeledconjugate also showed early liver uptake followed by rapid clearance from the liver into the intestine, with good visualization of the gallbladder in images obtained at 20-25 min postinjection. It is concluded that conjugation to cholic acid provides a useful means for the hepatobiliary delivery of EDTA chelates that otherwise exhibit predominantly extracellular distribution and renal clearance.
INTRODUCTION Metal complexes of polyaminopolycarboxylate chelators find use in diagnostic radiology both as radiopharmaceuticals and as MRIl contrast agents. Chelates of DTPA with the y-emitting radiometals technetium-99m and indium-111 are used as radiopharmaceuticals in renal clearance studies and cisternography (1-4). The gadolinium(II1) complex of DTPA has been widely studied as an MRI contrast agent (5, 6) and is finding clinical use, particularly in delineating brain lesions that result in altered capillary permeability (7). Characteristics that make these chelates useful for such applications include the thermodynamic stability of the complexes, the inertness of the polyaminopolycarboxylate framework to metabolic degradation, and the primarily extracellular distribution of these agents that results from their highly hydrophilic nature. Because of these properties, such chelates are largely restricted to the vascular compartment when given intravenously and are rapidly excreted, intact, into the urine. While metal complexes that distribute extracellularly are useful in studying blood flow, perfusion, and disease states that cause changes in capillary transit, there are instances where an agent must be able to distribute intracellularly in order to provide useful physiological information. One such case is the study of liver disease and hepatobiliary function. Agents for detecting tissue + Abbott
Laboratories.
* Northwestern University Medical Center.
The University of Texas at Dallas. Present address: Lajos Kossuth University, H-4010 Debrecan, Hungary. Abbreviations used: DTPA, diethylenetriaminepentaacetic acid; EDTA, ethylenediaminetetraacetic acid; EHPG,N,N'-ethylenebis(2-hydroxyphenylglycine);MRI, magnetic resonance imaging; TMS, trimethylsilane; TSP, 3-(trimethylsilyl)-l-pro11
panesulfonic acid. 1043-180219 7 12902-0 117$02.50/0
abnormalities in the liver and for evaluating the status of the bile ducts and gallbladder must be able to cross the hepatocyte membrane and eventually be excreted via the bile. The design of chelates that fulfill this requirement has generally involved the use of hydrophobic ligands. For example, recent work on the development of liver MRI contrast agents (5, 8, 9) has concentrated on high-spin iron(II1) complexes of EHPG, as these are substantially more hydrophobic than the analogous EDTA chelates by virtue of the two phenolic substituents that function as metal binding groups. We set out to examine an alternative approach to the hepatobiliary targeting of chelates, in which hydrophilic polyaminopolycarboxylatesthat do not normally cross the hepatocyte membrane to any great extent are conjugated to endogenous compounds that possess the intrinsic property of circulating through liver parenchyma. The first such endogenous compound chosen for study was cholic acid. Cholic acid (1, Chart I) is one of a number of acidic steroid derivatives that are produced by cholesterol catabolism and are secreted by hepatocytes into the bile (10). A significant fraction of secreted bile acids is resorbed in the ileum and returned via the plasma compartment to the liver, in a process termed the enterohepatic circulation (11). Cholic acid radiolabeled with selenium75 has previously been used for hepatobiliary radioimaging (12),and efforts to develop therapeutic chelators for the decorporation of toxic metals have included the synthesis of several metal-binding derivatives of cholic acid, among them a hydroxamate (2, Chart I) (13) and analogues containing thioether and carboxymethyl thioether substituents ( 3 , 4 , Chart I) ( 1 4 ) . However, the latter compounds contain only bidentate metal binding sites and the sulfur donors would be expected to bind only weakly to many metals of interest. We undertook the synthesis of conjugates in which polyaminopolycarboxylate chelators, which have a well-defined coordination 0 199 1 American Chemical Society
118 Bioconjugate Chem., Vol. 2, No. 2, 1991
Betebenner et al.
Chart I OH
,
0
HO'
'OH
HO'
2
1 0
PJ 3
chemistry, are coupled to cholic acid via the C-24 carboxylic carbon atom. This report describes the preparation and characterization of an EDTA conjugate of this type (9, Scheme I) and its ability to deliver indium-111 to the hepatobiliary system in animals. EXPERIMENTAL PROCEDURES
Materials and Methods. N-(Carboxymethyl)-N-[2[bis(carboxymethyl)amino]ethyl]-3-(4-isothiocyanatopheny1)alanine (5) was synthesized as previously described
(15). All other starting materials and reagents were obtained from Aldrich Chemical Co., Milwaukee, WI, unless otherwise specified, and were used without further purification. AGl-X4 anion-exchange resin (formateform, Bio-Rad Laboratories, Richmond, CA) was slurried with 5 M HCOzH for 30 min then filtered and washed with deionized water until the pH of the washings reached 5. The resin was then swelled in deionized water for 90 min and poured into a glass chromatography column. Acidwashed glassware, reagent-grade buffer salts and deionized water from a MILLI-Q system (Millipore Corp., Bedford, MA) were employed in preparing all buffers for use with chelator derivatives. 'H NMR and 13C NMR spectra were recorded on a General Electric QE-300 spectrometer, operating at 300.23 and 75.5 MHz, respectively. Chemical shifts are reported in ppm relative to TMS or TSP and coupling constants (J)are given in hertz. Mass spectra were obtained by either the fast atom bombardment technique using a glycerol/thioglycerol matrix or by direct chemical ionization using ammonia. Elemental analyses were performed by Galbraith Laboratories, Knoxville, TN. Metal chloride stock solutions used in formation constant determinations were prepared from the corresponding oxides (Aldrich,99.9 % ) by dissolution in concentrated HC1 and subsequent evaporation of the excess acid. Concentrations of these stock solutions were determined by complexometry using xylenol orange as indicator. Buffered solutions of indium-111 for labeling conjugates were obtained by diluting carrier-free research-grade indium-111 chloride (Nordion International, Inc, Kanata, Ontario, Canada) with 0.5 M HC1 to a specific activity of of 13 mCi/mL then adding an equal volume of 1.0 M aqueous NaOAc, pH 6. Radioscintigraphic studies were performed with a Raytheon y-camera equipped with a medium-energy parallel-hole collimator.
//
0
4
N-(Carboxymethyl)-N-[2-[ bis(carboxymethyl)amino]-3-[4-[N-[2-[(tert-butoxycarbonyl)amino]ethyl](thioureido)]phenyl]alanine (6). A solution of the dihydrochloride salt of 5 (0.52 g, 1.01 mmol) and triethylamine (0.39 g, 3.84 mmol) in DMF (10 mL) was stirred in an argon-purged flask at 0 "C as a solution of N-(tertbutoxycarbony1)ethylenediamine(0.59 g, 3.69 mmol, prepared as described in ref 16) in DMF (8 mL) was added. The resulting solution was stirred at 0 "C for a further 15 min then at room temperature for 48 h. Water (3mL) was added and stirring at room temperature continued for a further 6 h, after which time the solution was evaporated to dryness under vacuum to yield a brown oil. This was dissolved in HzO (30 mL) and extracted with CHzC12 (3 X 35 mL) and the aqueous layer lyophilized to give a yellow powder which was dissolved in HzO (10 mL), adjusted to pH 9, and applied to an AGl-X4 anion-exchange column (formate form, bed volume 65 mL). The column was eluted successively with HzO (300 mL), 3.5 M HCOzH (300 mL), and 7.0 M HCOzH (300 mL), fractions being monitored for the presence of the desired product by TLC on silica gel plates developed in 40% concentrated aqueous NH4OH/60% EtOH (95%) ( R f of 6 = 0.33). Fractions containing the desired product, which eluted in 7 M HCOzH, were combined and evaporated to dryness under vacuum to afford 0.49 g (815%) of 6 in the form of a yellow solid: 'H NMR (DMSO-&, TMS) 6 1.38 (9, 9 H), 2.683.15 (m, 8 H), 3.28-3.58 (m, 8 H), 3.63 (dd, 1 H, J = 8.5, 6.25),6.90(t,lH),7.18(d,2H,J=8.5),7.19(s,lH),7.25 (d, 2 H, J = 8.5), 7.72 (m, 1H), 8.42 (9, 1H),9.55 (s, 2 H), 9.78 (s, 1 H); mass spectrum (FAB, EtOH/HzO), m / e 600 (M + H)+; high-resolution mass spectrum calcd for C25H38N5010S 600.2339, found 600.2346. N-(Carboxymethyl)-N-[2-[bis(carboxymethy1)aminolet hyll-3-[4-[ W -(2-aminoethyl)(thioureido)]phenyllalanine Trihydrochloride (7). A solution of 6 (0.48 g, 0.80 mmol) in trifluoroacetic acid (15 mL) was stirred at room temperature for 6 h then evaporated to dryness under vacuum to give a yellow oil. This was dissolved in 2 M HC1 (25 mL) and reevaporated to dryness and the residue redissolved in HzO (5 mL). The pH of the resulting solution was adjusted to 9 then it was applied to an AG1X4 anion-exchangecolumn (formate form, bed volume 40 mL). The column was eluted first with HzO (250 mL) then with 1 M HCOzH (1 L) with collection of 15-mL fractions, which were monitored for the presence of the
An EDTA-Cholic Acid Conjugate
Bimonjugate Chem., Vol. 2, No. 2, 1991
119
Scheme I
1
S HO'
desired product by TLC on silica gel plates developed in 40% concentrated aqueous NH40H/60% EtOH (95%) @for 7 = 0.60, ninhydrin positive). Fractions containing the product were combined, evaporated to dryness, redissolved in 4 M HC1 (150 mL), and reevaporated to dryness. Drying of the resulting residue under vacuum over P205 afforded 0.45 g (92%) of 7 in the form of an off-white solid: lH NMR (D20, TSP) 6 3.06 (dd, 1 H, J = 14.1, 8.2), 31.6-3.27 (m, 5 H), 3.40-3.52 (m, 2 H), 3.68 (d, 1 H, J = 18.7), 3.87-3.96 (m, 4 H), 7.27 (d, 2 H, J = 8.0), 7.37 (d, 2 H, J = 8.0); 13C NMR (D20, TSP) 6 37.36, 42.09,44.45,52.23,54.04,56.78,56.94,68.94,129.03,133.25, 138.33, 139.43, 171.34, 177.59, 178.83, 183.72; mass spectrum (FAB, DMF), m / e 500 (M + H)+; high-resolution mass spectrum calcd for C ~ O H ~ O N500.1815, ~ O ~ S found 500.1809. Anal. CaIcd for C ~ O H ~ ~ C ~ ~C, N 37.24; S O ~ H, S: 5.63; N, 10.86. Found: C, 37.16; H, 5.36; N, 11.06. 3a,7a,l2a-Trihydroxy-5-cholan-24-oic Acid, N-SUCcinimidyl Ester (8). Cholic acid (Sigma Chemical Co.,
9
St. Louis, MO) was recrystallized from absolute EtOH prior to use. This material was then converted to the active ester following the procedure of Okahata et al. (13, employing 4.27 g (10 mmol) of the recrystallized cholic acid. Recrystallization of the resulting ester from CHCls/ hexane afforded 3.17 g (60%)of 8 in the form of a white crystallinesolid: mp 118-120°C (lit. (17)119-120 "C); 'H NMR (DMSO-& TMS) 6 0.60 (s, 3 H), 0.69-1.06 (m, 9 H), 1.13-1.50 (m, 10 H), 1.56-1.86 (m, 6 H), 1.92-2.30 (m, 3 H), 2.54-2.75 (m, 2 H), 2.81 (s,4 H), 3.12-3.25 (br m, 1H), 3.61 (br s, 1 H), 3.80 (br s, 1 H), 4.03 (d, 1 H),4.15 (d, 1 H), 4.32 (d, 1 H); 13CNMR (CDCl3, TMS) 6 12.38,17.18, 22.38,22.58,25.54,26.23,27.41,28.09,30.22,30.50,33.86, 34.62,34.72,35.01,35.24,39.41(two atoms), 41.41,41.53, 46.41, 46.70, 68.35, 71.79, 72.94, 169.16, 169.29; mass spectrum (DCI, NH3) m / e 523 (M + NH4)+, 506 (M + H)+;high-resolution mass spectrum calcd for C28H~N07 506.3118, found 506.3124. Anal. Calcd for C28H43N0,:
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Betebenner et al.
Bioconjwte Chem., Vol. 2, No. 2 , 1991
C, 66.51; H, 8.57; N, 2.77. Found: C, 66.77; H, 8.89; N, 3.29. N-(Carboxymethyl)-N-[2-[ bis(carboxymethy1)aminolethyll-3-[ 4-[N-[24(3a,7a, 12a-trihydroxy-5-cholan24-oyl)amino]ethyl](thioureido)]phenyl]alanine Dihydrochloride (9). A solution of 8 (0.245 g, 0.48 mmol) in DMF (1.0 mL) was added dropwise to a stirred solution of 7 (0.268 g, 0.44 mmol) and triethylamine (0.313 g, 3.09 mmol) in DMF (4.0 mL). The resulting reaction mixture was stirred at room temperature, the contents being monitored periodically by TLC on silica gel plates developedin 25% concentratedaqueous NH40H/75% EtOH (95%) (Rffor 7 = 0.15, Rf for 9 = 0.35). After 3 days of stirring at room temperature, the reaction mixture was evaporated to dryness under vacuum to give a yellow oil which was dissolved in H2O (5 mL). The pH of the resulting solution was adjusted to 9.5 by addition of 6 M NaOH then it was applied to an AGl-X4 anion-exchange column (formate form, bed volume 90 mL). The column was eluted sequentially with H20 (200 mL), 1M HCOzH (200 mL), 4 M HCOzH (200 mL), 5 M HCOzH (800 mL), and 6 M HC02H (400 mL), collecting 10-mL fractions which were monitored by TLC. The desired product eluted in the 6 M HC02H fractions, and these were combined and evaporated to dryness under vacuum. The residue was dissolved in 4 M HCl (100 mL) and the resulting solution evaporated to dryness to give an oil. This was dissolvedin HzO (100mL) and evaporated to dryness under vacuum. Further drying under vacuum over PzO5 afforded 0.22 g (52%) of 9 in the form of a pale beige solid: 'H NMR (DMSO-& TMS, 60 "C) 6 0.59 (9, 3 H), 0.78-1.05 (m, 9 H), 1.10-1.51 (m, 10 H), 1.53-1.91 (m, 6 H), 1.932.37 (m, 5 H), 2.87 (dd, 1 H, J = 13.7,5.7), 2.94 (dd, 1H, J = 13.7,8.9),3.03-3.15 (m,2H),3.17-3.39 (m,6H),3.433.56 (m, 4 H), 3.60-3.68 (m, 3 H), 3.73 (dd, 1 H, J = 8.9, 6.1), 3.77-3.83 (m, 1H), 4.10 (s,4 H), 7.18 (d, 2 H, J = 8.1), 7.39 (d, 2 H, J = 8.1), 7.78 (t, 1 H, J = 4.8), 7.91 (br s, 1 H), 9.69 (s, 1H); massspectrum (FAB, EtOH/DMF), m / e 890 (M HI+; high-resolution mass spectrum calcd for C44H6&012S 890.4585, found 890.4584. Anal. Calcd for C44H&12N50&: C, 54.87; H, 7.22; N, 7.27. Found: C, 55.52; H, 7.49; N, 7.16. Potentiometric Measurements. A 0.5 mM stock solution of 9 was prepared by suspending the dihydrochloride salt in deionized water and adding 6 equiv of KOH to completely dissolve the conjugate. Immediately prior to titration, 5 equiv of HCl were added to an aliquot of the stock solution to yield the pentaprotonated ligand. The concentration of the standard 0.1 M KOH solution used in the pH potentiometric titrations was determined by titration against a 0.01 M potassium hydrogen phthalate solution. The pH potentiometric titrations were performed with a Corning Ion Analyzer 250 pH meter, an Orion 8103 combination electrode, and a Metrohm 665 Dosimat automatic burette. Titrated solutions (5-10 mL) were covered by a cyclohexane layer to exclude C02 and the cell was thermostated at 25 f 1"C. All solutions were maintained at constant ionic strength with 0.1 M KCl. The hydrogen ion concentration was obtained from the measured pH values by the method of Irving et al. (18). Stability constants for the Zn2+ and Lu3+ chelates of 9 were determined by potentiometric titration of 1:l mixtures of the metal ion and the conjugate. Radiolabelingof 9 withhdium-111. A stocksolution of 9 was prepared by dissolving 2 mg of the conjugate in 0.1 M aqueous NaOAc, pH 6 (2 mL). An aliquot of this solution (40 ,uL) was then mixed with a buffered solution of indium-111 (20 pL) and incubated at room tempera-
+
ture for 30 min. Uncomplexed indium-111 was then removed by centrifugation through a spin column containing Chelex resin (Sigma, bed volume 0.3 mL) equilibrated in 0.1 M aqueous NaOAc. The resulting eluate had a specific activity of ca. 85 pCi/mL and was used in subsequent animal studies without further purification. Biodistribution Studies. BALB/c mice (n = 4 per group) were injected intravenously with indium-llllabeled 9 (100 ,uL per mouse) via a tail vein. At various time points postinjection, groups of animals were sacrificed by cervical dislocation and the internal organs were removed, weighed, and counted in a gamma-counter (Clinigamma 1272,PharmaciaLKB Biotechnology, Inc., Gaithersburg, MD). A weighed aliquot of blood was also counted and the tail was counted separately to check for extravasation at the injection site. The residual carcass was counted so as to obtain an estimate of the whole-body retention of radioactivity, by totaling the carcass counts and all individual organ counts. A 100-pL aliquot of the injectate was counted a t the same time as the tissues, and the radioactivity in each tissue expressed as a percentage of this injected dose per gram of tissue. Radioscintigraphy Studies. A female New Zealand rabbit was fasted for 24 h then anesthetized by intramuscular injection of nembutal and positioned on the head of the y-camera so as to provide anterior views of the whole body. A commercially available hepatobiliary imaging agent (technetium-99m disofenin, NEN-Du Pont, N. Billerica, MA) was administered via a marginal ear vein and serial scintigraphic images were taken beginning at 5 min postinjection. Subsequently, the rabbit was revived, rested, and fed for 24 h then fasted for a further 24 h. An identical radioscintigraphic study was then performed, by administering indium-111-labeled 9 (1.0 mCi) via an ear vein and placing the animal on the y-camera head in the same position as before. RESULTS
Benzyl isothiocyanate derivatives of EDTA have been used in the past to covalently link the chelating moiety to proteins (15,19,20). The same reaction was used here to prepare an EDTA derivative containing a side chain that terminated in an aliphatic amino group (71, by reaction of 5 with a monoprotected ethylenediamine derivative and subsequent deprotection. Both steps proceeded in good yield, with anion-exchange chromatography being the preferred method for purification of both 6 and 7. Reaction of 7 with an active ester of cholic acid (8), prepared by standard methods, proceeded rather slowly at room temperature, but was complete after 3 days. Anion-exchange chromatography was again the method of choice for purification of the final product (9), which featured the EDTA moiety linked by an 11-atom spacer to the (2-24 carbon atom of cholic acid. As it was of interest to evaluate the effect of this derivatization of the EDTA framework on the thermodynamic stability of its chelates, potentiometric titration studies were undertaken (Figure 1). Protonation constants for 9, defined as Ki = [HiL]/[Hi-lL][Hf] (i = 1,2, or 3; L = 9) and calculated from the titration data using a Simplex/ Marquardt algorithm (21),were found to be logK1= 9.84, log K2 = 5.44, and log K3 = 3.44. The corresponding values for EDTA are 10.23, 6.13, and 2.69, suggesting that conjugation to cholic acid affected the basicity of both nitrogen atoms (assuming that the two highest protonations occur at the nitrogens, as in EDTA). Complexes of
Bloconjugate Chem., Vol. 2, No. 2, 1991
An EDTA-Cholic Acid Conjugate
0
I
I
I
I
0.1
0.2
0.3
0.4
I
0.5
I
0.6
1
0.7
KOH added (mL) Figure 1. Potentiometric titration curves for (A) 0.42 mM 9, (B)0.42 mM 9 + 0.4 mM ZnClz, (C)0.42 mM 9 + 0.4 mM LuC13. All measurements were made at 25 O C in the presence of 0.1 M KCl. Table 1. Biodistribution of Indium-111-Labeled 9 in BALB/c Mice mean % injected dose/g of tissue (SD) tissue 15 mina 60 minn 120 mina blood 5.5 (1.5) 1.0 (0.6) 0.2 (0.1) 1.5 (0.9) 1.8 (1.0) liver 10.2 (2.6) GI tract 14.9 (7.8) 14.5 (2.9) 17.6 (3.0) gallbladder 164.6 (42.3) 69.3 (57.0) 10.3 (8.2) kidney 4.0 (1.3) 1.0 (0.5) 0.3 (0.1) lungs 3.1 (1.9) 0.9 (0.4) 0.5 (0.2) 0.8 (0.2) 4.6 (4.4) spleen 2.6 (2.4) (I
Time postinjection.
9 with lanthanum(III), gadolinium(III), and copper(I1) were too insoluble to permit potentiometric measurements. It was however possible to obtain data for chelates of 9 with zinc(I1) and lutetium(II1) (Figure l), the titration curves giving an excellent fit to a model that assumes the formation of 1:l complexes. Stability constants ( K = [ML]/ [MI [L]),derived from the potentiometric data using a Simplex algorithm, were determined to be log K = 15.9 for the [Lu(9)]- complex and log K = 13.9 for the [Zn(9)I2chelate. The [In(9)]- chelate used in biodistribution studies showed rapid clearance from the blood when injected iv into normal mice, with early localization in the liver, gallbladder, and gastrointestinal (GI) tract (Table I). Approximately 90% of the injected radiometal had left the circulation by 15 min postinjection, while all of the dose was still present in the animals at that time (108 f 15%). Even at this early time point, significant excretion into the gut had occurred and this level remained virtually constant throughout the study. The intense early localization in the gallbladder decreased rapidly with time and liver activity had essentially cleared by 1h postinjection. Whole-body retention of radioactivity after 2 h (55 f 3%) was virtually unchanged from that at 1 h (57 f 1% ). It is doubtful that these whole-body losses of radioactivity represented fecal excretion, given the relatively short time span involved, loss of some 40% over the dose of the first
121
60 min being best explained by clearance through the urine of that fraction of injected activity not taken up by the liver. The relatively high initial kidney activity seen, and the order of magnitude decrease observed over the subsequent 2 h, are also indicative of early renal clearance of a proportion of the injected dose. Taken together, these data suggest rapid extraction by the liver of a substantial fraction of the injected dose of indium-111, with the radiometal subsequently leaving the liver and being excreted into the GI tract within 1 h of administration. These findings were confirmed in radioscintigraphic imaging studies conducted in a rabbit (Figure 2). To facilitate anatomic identification and provide a basis for comparison with subsequent images obtained with 9, a clinically used hepatobiliary radioimaging agent (technetium-99m-disofenin) was first administered (Figure 2A). Early images showed prominent uptake of 9 9 m T activity ~ in the liver, with some excretion into the intestinal tract apparent even at 5 min postinjection. Clearance of activity from the liver into the gut proceeded continuously over the following 30 min and was essentially complete by 40 min postinjection. The gallbladder was most easily visualized in images obtained between 25 and 35 min after giving the radiopharmaceutical. Images obtained in the same animal after administration of indium-111-labeled 9 (Figure 2B) showed essentially the same pattern of uptake and clearance as had been seen with the Tc-99m agent. In the initial image, obtained at 5 min postinjection, only the liver was visualized. Clearance of In-111 activity from the liver into the GI tract had begun by 10 min postinjection and proceeded rapidly, such that most of the radioactivity was in the gut after 20 min. The gallbladder was clearly visualized in the 20- and 25-min images, with all subsequent views showing only intestinal activity. Although these data suggest that hepatic clearance of radioactivity may be faster for indium-111-labeled 9 than for technetium99m-disofenin, this observation would have to be repeated several times before drawing any such conclusion, as the difference seen here may simply reflect metabolic variability in the particular animal studied. DISCUSSION
Clinical experience over the past 5 decades with the systemic administration of exogenous chelators and chelates has been limited to a very small number of compounds. Except for radiologic applications, the only other area of clinical practice in which such agents have been given with any frequency is the treatment of metal intoxications and, here again, the polyaminopolycarboxylates have been prevalent among the handful of chelators used for such purposes (22). Efforts to improve upon the limited tissue penetration and, hence, efficacy of compounds such as EDTA and DTPA have for the most part focused on the design and synthesis of alternative, frequently biomimetic, chelators that in many cases employ aromatic metal-binding moieties (8, 9, 23-27). While such systems often display improved thermodynamic stability and greater tissue penetration, they have frequently proven to be more toxic and, despite much preclinical development, they have to date rarely been used clinically. In contrast, the polyaminopolycarboxylates have toxicities that are generally both low and wellunderstood, and there exists an extensive body of data on their use in man that dates back to the 1950s. An attractive alternative strategy for the development of high-affinity metal-binding entities with enhanced ability to target selected tissues and metabolic processes in vivo is to conjugate known polyaminopolycarboxylate structuresto molecules that alter their biodistribution.
122 Bloconjugate Chem., Vol. 2, No. 2, 1991
Betebenner e t at.
A
B
Figure 2. Anterior whole-body radioscintigraphic images of a rabbit given (A) -Tdisofenin, (B) lllIn-labeled 9. Images were obtained at 5 (l),10 (2), 15 (3), 20 (4), 25 (5), 30 (6), 35 (7), and 40 min (8) postinjection. To provide anatomic orientation, the 5-min image obtained with the %Tc agent is shown at contrast settings that result in visualization of a whole-body contour. All other images show only the organs of interest (liver and GI tract).
Such a strategy was used in the past to prepare an EDTA derivative with tumor-targeting properties, by linking the chelator to the anti-tumor antibiotic bleomycin (28). Similarly, we employed a bifunctional chelator to link the EDTA moiety to a bile acid that targets hepatocytes. Examples of hybrid molecules that contain both a steroid nucleus and a high-affinity metal-binding site are rare, although a series of compounds in which high affinity FeI1 and FelI1chelators were linked to various 21-aminosteroids was recently described by Braughler et al. (29). Linkage of a steroid moiety to EDTA through one of the carboxymethyl arms of the chelator proved to have a measurable impact on the thermodynamic stability of chelates formed by the resulting conjugate. The stability constants for 1:l complexes of EDTA with LulI1and Zn", measured under similar conditions to those employed in studies of 9, are ca. 19.8 and 16.5, respectively. Thus, conjugation reduced these values by ca. 4 log K units for LulI1and 3 log K units in the case of Zn". Although the stability constant for the [In(9)]- complex that was used in the biological studies has not been determined, it would seem reasonable to assume a log K value on the order of 20, as the stability constant for [In(EDTA)]- is variously reported to be 25.3 (30) or 25.8 (31). Whatever the actual reduction in thermodynamic stability of the In"' chelate of 9 relative to free EDTA may be, it is apparently not sufficient to produce any evidence of chelate instability in vivo. The transient pulse of radioactivity through the liver that was documented in both the mouse biodistribution study and the rabbit
imaging procedure is consistent with retention of the radiometal within the chelate throughout the course of these studies. If the complex had been unstable, liver levels of radioactivity would have been expected to remain elevated for many hours, and possibly days, as it is well-established that intracellular dissociation of indium(II1) chelates results in prolonged retention of the radiometal within the cell (32). When an indium-111chelate of the aminobenzyl-DTPA analogue of 5 was injected intravenouslyinto mice, virtually all of the radiolabel was cleared rapidly through the urine (unpublished data). This suggests that introduction of a hydrophobic benzyl group into a polyaminopolycarboxylate structure is not, in and of itself, sufficient to shift excretion to the hepatobiliary route. The cholic acid moiety is thus essential for efficient hepatic uptake, although it remains unclear whether this reflects entry into the hepatocyte via specific mechanisms of bile acid transport or whether the observed behavior of 9 in animals simply represents uptake by nonspecific anionic transport processes. Bile acid transport into liver cells is a poorly understood process that is known to require sodium ions and metabolic energy (11). Studies in isolated rat hepatocytes (33) have suggested that there may be separate carrier-mediated transport systems for cholic acid itself and for the amide formed from its reaction with taurine (2-aminoethanesulfonic acid), which is a prominent metabolite. Although the relatively bulky chelate formed between a trivalent metal and the benzyl-EDTA group in 9 does not sterically resemble the ethanesulfonic acid side
An EDTA-Cholic Acid Conjugate
chain of taurocholic acid, the net uninegative charge and hydrophilic character that both moieties share may enable the chelate conjugate to enter hepatocytes through the taurocholate transport system. The preparation of analogues of 9 that contain chelates of differing net electronic charge and studies of the pathways and kinetics of intracellular processing of these species will be needed to fully define the extent to which chelate conjugates of cholic acid are able to mimic the underivatized molecule. Although details of the mechanism of hepatic uptake remain to be elucidated, the data presented here indicate that conjugation to cholic acid provides a useful means for the controlled delivery of polyaminopolycarboxylate chelates to parenchymal liver cells. LITERATURE CITED (1) Klopper, J. F., Hauser, W., Atkins, H. L., Eckelman, W. C., and Richards, P. (1972) Evaluation of mTc-DTPA for the measurement of glomerular filtration rate. J.Nucl. Med. 13, 107-110. (2) Gates, G. F. (1982) Glomerular filtration rate: estimation from fractional renal accumulation of mTc-DTPA (stannous). Am. J. Roentgenol. 138, 565-570. (3) Som, P., Hosain, F., Wagner, H. N., Jr., and Scheffel, U. (1972) Cisternography with chelated complex of%Tc. J. Nucl. Med. 13, 551-553. (4) Hosain, F., and Som, P. (1972) Chelated 111In: an ideal radiopharmaceutical for cisternography. Br. J.Radiol. 45,677679. (5) Lauffer, R. B. (1987) Paramagnetic metalcomplexes as water proton relaxation agents for NMR imaging: theory and design. Chem. Reo. 87, 901-927. (6) Runge, V. M., Clanton, J. A., Herzer, W. A., Gibbs, S. J., Price, A. C., Partain, C. L., and James, A. E. (1984) Intravascular contrast agents suitable for magnetic resonance imaging. Radiology 153, 171-176. (7) Carr, D. H., Brown, J., Bydder, G. M., Weinmann, H.-J., Speck, U., Thomas, D. J., and Young, I. R. (1984) Intravenous chelated gadolinium as a contrast agent in NMR imaging of cerebral tumors. Lancet 1, 484-486. ( 8 ) Lauffer, R. B., Greif, W. L., Stark, D. D., Vincent, A. C., Saini, S., Wedeen, V. J., and Brady, T. J. (1988) Iron-EHPG as an hepatobiliary MR contrast agent: initial imaging and biodistribution studies. Nucl. Med. Biol. 15, 47-49. (9) Lauffer, R. B., Vincent, A. C., Padmanabhan, S., Villringer, A., Saini, S., Elmaleh, D. R., and Brady, T. J. (1987) Hepatobiliary MR contrast agents: 5-substituted iron-EHPG derivatives. Magn. Reson. Med. 4 , 582-590. (10) Bjorkhem, 1. (1985) Mechanism of bile acid biosynthesis in mammalian liver. Sterols and Bile Acids (H. Danielsson, and J. Sjovall, Eds.) pp 231-278, Elsevier, New York. (11) Elliott, W. H. (1985) Metabolism of bile acids in liver and extrahepatic tissues. Sterols and Bile Acids (H. Danielsson, and J. Sjovall, Eds.) pp 303-329, Elsevier, New York. (12) Merrick, M. V., Eastwood, M. A., Anderson, J. R., and Ross, H. M. (1982) Enterohepatic circulation in man of a gammaemitting bile-acid conjugate, 23-selena-25-homotaurocholic acid (SeHCAT). J. Nucl. Med. 23, 126-130. (13) Hershko, C., Grady, R. W., and Cerami, A. (1978) Mechanism of iron chelation in the hypertransfused rat: definition of two alternative pathways of iron mobilization. J.Lab. Clin. Med. 92, 144-151. (14) Jones, M. M., Pratt, T. H., Mitchell, W. G. (1976) Enterohepatic chelatin gagen@-1. General principles and examples. J. Inorg. Nucl. Chem. 38, 613-616. (15) Westerberg,D.A., Carney,P. L., Rogers,P. E.,Kline,S. J., and Johnson, D. K. (1989) Synthesis of novel bifunctional chelators and their use in preparing monoclonal antibody conjugates for tumor targeting. J. Med. Chem. 32, 236-243. (16) Essian, H., Lai, J. Y.; and Hwang, K. J. (1988) Synthesis of diethylenetriaminepentaacetic acid conjugated inulin and utility for cellular uptake of liposomes. J. Med. Chem. 31, 898-901.
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