Bioconjugate Chem. 1995, 6, 219-225
219
Yttrium-90 Chelation Properties of Tetraazatetraacetic Acid Macrocycles, Diethylenetriaminepentaacetic Acid Analogues, and a Novel Terpyridine Acyclic Chelator? Julie B. Stimmel, Marie E. Stockstill, a n d Frederick C. Kull, J r . * Division of Cell Biology, Wellcome Research Laboratories, 3030 Cornwallis Road, Research Triangle Park, North Carolina 27709. Received November 17, 1994@
Realization of the potential of yttrium-90 for the radioimmunotherapy of cancer depends on rapid and kinetically stable chelation. Conditions were evaluated that influenced the chelation efficiency of these select chelators for yttrium-90: the macrocyclic chelators 2-(~-nitrobenzyl)-l,4,7,lO-tetraazacyclododecane-N,”,”JV”’-tetraacetic acid (nitro-DOTA);a-(2-(~-nitrophenyl)ethyl)-1,4,7,lO-tetraazacyclododecane-l-acetic-4,7,l0-tris(methylacetic) acid (nitro-PADOTA); 2-(~-nitrobenzyl)-l,4,7,10tetraazacyclotridecane-N,W,”JV”-tetraacetic acid (nitro-TRITA); the acyclic chelator diethylenetriaminepentaacetic acid (DTPA);its analogues N-[2-amino-3-(~-nitrophenyl)propyll-truns-cyclohexane1,2-diamine-N,”JV”-pentaacetic acid (nitro-CHX-A-DTPA) and 2-methyl-6-(e-nitrobenzyl)-1,4,7triazaheptane-NJV,WJV”J’-pentaacetic acid (nitro-1B4M-DTPA or nitro-MX-DTPA); and a novel acyclic terpyridine chelator, 6,6”-bis[[N~,”,”-tetra(carboxymethyl)amino]methyl]-4‘-(3-amino-4methoxyphenyl)-2,2’:6’,2”-terpyridine (TMT-amine). The chelators fell into two distinct classes. The acyclic chelators, DTPA, nitro-CHX-A-DTPA, nitro-MX-DTPA, and TMT-amine, chelated instantaneously in a concentration-independent manner. Chelation efficiency was affected minimally when the concentrations of trace metal contaminants were increased. In contrast, the macrocyclic chelators, nitro-DOTA, nitro-TRITA, and nitro-PADOTA, chelated yttrium-90 more slowly in a concentrationdependent manner where efficiency was maximal only when the chelatormetal ratio was greater than 3. Their chelation efficiency diminished in a concentration-dependent fashion as the concentrations of trace metal contaminants were increased. Optimum labeling efficiencies were obtained through application of these principles. Additionally, the kinetic stabilities of these chelator-yttrium-90 complexes were evaluated a t low pH, where the order of stability was nitro-DOTA, nitro-PADOTA > nitro-CHX-A-DTPA,nitro-MX-DTPA > nitro-TRITA, TMT, DTPA. pH lability stratified the chelators to a conveniently measurable degree and, interestingly, correlated with their in vivo stabilities where known.
INTRODUCTION
N,N’,N“-pentaacetic acid (nitro-CHX-A-DTPA,Figure 1) (14, 15, 40). A novel terpyridine chelator, 6,6”-bis[N,N,N”,N”-tetra(carboxymethyl)amino]methyl]-4’-( 3amino-4-methoxypheny1)-2,2’:6’,2”-terpyridine(TMTamine, Figure 11, has demonstrated ability as a highly efficient, aqueous-stabilized fluorescent label that can chelate europium (16). The acyclic chelators are not as kinetically stable as the macrocyclic chelator 1,4,7,1O-tetraazacyclododecaneN,N’,N“,N”’-tetraacetic acid (DOTA, Figure 1)(17, 18). DOTA possesses unique chemistry that makes it an ideal chelator for yttrium (19). The kinetic stability of yttrium-DOTA complexes has been well documented and proven to be critical for minimizing toxicity to bone (17,
Radioimmunotherapy utilizing yttrium-90 has shown encouraging results in hematopoietic malignancies (11. Yttrium-90 is a pure @-emitterand has a of 64.1 h and a n average range in tissue of 3.9 mm. Yttrium-90 may be attached to antibodies via chelation by any of a number of chelators that are covalently conjugated to the antibodies. Like other systemic radiotherapies, in vivo results utilizing acyclic yttrium-90-labeled ethylenediaminetetraacetic acid (EDTA) or diethylenetriamine pentaacetic acid (DTPA) conjugates demonstrated doselimiting toxicity to bone (2-11). It has been speculated that some of this toxicity was due to dissociation of the chelator-metal complex (7-11), and it is generally 18,20-24). believed that more stable chelation improves therapeutic value. Although yttrium-90-DOTA is kinetically stable, chelation has proved problematic. For example, the maxiRecently, we have seen the introduction of a third mum efficiency of chelation that was achieved with a generation of acyclic chelators. These chelators have DOTA conjugate was < 50% (22). Practically, inefficient demonstrated increased stability in vivo compared with DTPA. They include 2-methyl-6-[(~-nitrobenzyl)diethyl- chelation is not attractive for the development of a radioimmunotherapeutic. It necessitates incorporation enel-N,N,iV’,N”,N”-pentaacetic acid (nitro-lB4M-DTPA, of another manipulation to remove unchelated yttriumor nitro-MX-DTPA, Figure 1)(12, 13) and N-[2-amino3-(~-nitrophenyl)propyl]-trans-cyclohexane-l,2-diamine- 90, and this radioactivity is unrecoverable. Therefore, we sought to examine chelation conditions with the goal of improving labeling efficiency. Utilizing the macrocyclic + Presented in part a t the 5th Conference on Radioimmunchelators illustrated in Figure 1(top), 2-(~-nitrobenzyl)detection and Radioimmunotherapy of Cancer, Princeton, NJ. DOTA (nitro-DOTA), a-[2-(e-nitrophenyl)ethy1]-1,4,7,10* To whom correspondence should be addressed. Tel.: (919)tetraazacyclododecane-l-acetic-4,7,lO-tris(methylacetic)315-4215. Fax: (919)315-0286. acid (nitro-PADOTA) (25-281, and 1,4,7,10-tetraazaAbstract published in Advance ACS Abstracts, March 15, 1995. cyclotridecane-N,N’,N”,N“’-tetraacetic acid (TRITA)(291, @
1043-1802/95/2906-0219$09.00/0
0 1995 American Chemical Society
Stimmel et al.
220 Bioconjugate Chem., Vol. 6, No. 2, 1995
n o 2 c 1nr c o 2 n
n o 2 c 7 nP C O ~ H
N
'N
fH02C
HO2C
n itro-DOTA
COzH COzH
N
& )NO2
co2n
nitro-PADOTA
CO2H
+
CHI
CHI
I
HO2C
COzH
HOnC
COzH
TMT-amine
OzN
COzH COzH
COzH
nitro-CHX-A-DTPA
COzH
1
COzH
I
C02H
nitro-1B4M-DTPA
Figure 1. (Top) structures of the macrocyclic chelators compared in this study. (Bottom) structures of the acyclic chelators compared in this study.
and the acyclic chelators illustrated in Figure 1(bottom), DTPA, nitro-CHX-A-DTPA,nitro-MX-DTPA, and an acyclic novel terpyridine derivative, TMT-amine, we evaluated the following parameters and their effect on chelation efficiency: metal and chelator concentration dependence, the minimum time interval necessary to achieve maximum labeling efficiency, and potential interference by trace metals. Our results indicate that under the appropriate conditions, efficient labeling of DOTA can be achieved in a few minutes. Having achieved efficient chelation, we also compared the kinetic stabilities of the yttrium-90-chelator complexes a t low pH. EXPERIMENTAL PROCEDURES
Materials and Reagents. Metal-free plasticware (polypropylene) o r plasticware that had been soaked in 3 M HC1 overnight and rinsed thoroughly with Milli-Q (18 MW) water was used throughout this work to reduce metal contamination (30). All buffers and reagents were dissolved in Ultrex water (JT Baker). The reagents were analytical grade or better. All prepared buffers were treated with Chelex-100 resin (Na+ form, 100-200 mesh, Bio-Rad Laboratories) according to the manufacturer's instructions and passed through a 0.22 pM filter (Corning) prior to use. Diethylenetriaminepentaacetic acid (DTPA)was purchased from Sigma. Zn(I1) acetate dihydrate, Ca(I1) acetate hydrate, Fe(I1) acetate, and yttrium-89 (cold yttrium) were purchased from Aldrich. Yttrium (89YC13)was solubilized in 0.1 M ammonium acetate, pH 6.0. All chelations were per-
formed in 1.5 mL polypropylene microcentrifuge tubes that were capped. Nitro-DOTA, nitro-PADOTA, and nitro-TRITA were synthesized in the Wellcome Research Laboratories according to literature protocols with minor modifications. Analytical evaluation confirmed the identity of the final structures. Nitro-CHX-A-DTPA and nitro-MX-DTPA were provided by Dr. Martin Brechbiel of the National Institutes of Health. TMT-amine was provided by Sterling Winthrop, Inc. Radioactivity. Carrier-free 90YC13was purchased from DupontJNew England Nuclear (5 mCi in 10-30 pL of 0.5 N HC1, specific activity 5.6 x lo5 Ci/g, goSrPoYratio < 1x Prior to use, the was buffered with 6 M ammonium acetate to an approximate pH of 5.8. Stock solutions of radiolabeled yttrium were prepared by trace-labeling cold yttrium with goYoAc. Thin-Layer Chromatography (TLC). The TLC method was used as developed by Meares et al. (31). Briefly, the TLC plate (Whatman no. 4865 821) was prepared as follows: the origin was indicated by a line drawn 2 cm from the bottom of the plate. Another line was drawn 7 cm from the bottom of the plate, and this line was scraped free of silica gel to stop the solvent front. When separation of radiolabeled metal-chelator complex from free metal was necessary, samples were added to 0.1 M sodium phosphate, pH 6.5 containing an excess of cold yttrium. A 5 pL aliquot was applied to the origin and dried with a stream of air. The samples were eluted with 10% ammonium acetate:methanol (1:l). A radioanalytical image of the plate was obtained using an
Bioconjugate Chem., Vol. 6,No. 2, 1995 221
Chelation Properties of Select Chelators
A-MK2 scanner (Automated Microbiology Systems, Inc.). Free metal remained a t the origin while labeled chelator migrated to the solvent front. Data were expressed as percent metal chelated. Radiolabeling Efficiency. Stock (200 mM) solutions of each chelator were prepared in 0.1 M ammonium acetate, pH 6.0 and two times the indicated concentrations of yttrium-90 were prepared in the same buffer. Equivolume amounts of chelator and yttrium-90 were mixed and incubated a t room temperature for 2 h. The chelation was stopped by the addition of 20 pL of blocking solution, and a 5 pL aliquot was analyzed by TLC. Blocking solution was 0.1 M sodium phosphate, pH 6.5 that contained a 5-fold excess of cold yttrium over the chelator concentration and was prepared immediately before analysis to minimize precipitation of the cold yttrium. The addition of cold yttrium was necessary to prevent rechelation of yttrium-90. Duration of Yttrium-90Chelation. Stock solutions of chelator and yttrium-90 were prepared in 0.1 M ammonium acetate, pH 6.0. Equal volumes of 100 mM chelator and 20 mM yttrium-90 were mixed at room temperature. Five p L aliquots were removed from the mixtures immediately (time zero) and a t the indicated time intervals. To terminate chelation, the samples were added directly into 10 pL of 0.1 M sodium phosphate, pH 6.5 containing an excess of cold yttrium to terminate the chelation. A 5 pL aliquot was analyzed by TLC. Effect of Trace Metals on Yttrium-90 Chelation. Stock solutions of chelator (4mM) and yttrium-90 (800 pM) were prepared in 0.1 M ammonium acetate, pH 6.0. In addition, the following stock concentrations of Fe(I1) acetate, Ca(I1) acetate hydrate, and Zn(I1) acetate dihydrate were prepared in the same buffer: 20 mM, 10 mM, 2 mM, 1600, 1200, 800, 400, and 200 pM. To each polypropylene tube was added 5 p L of the indicated trace metal and 2.5 pL of the yttrium-90 stock. Finally, 2.5 pL of the chelator stock was added. The chelations were performed for 2 h a t room temperature and were stopped by addition of 20 p L of sodium phosphate, pH 6.5. A 5 pL aliquot was analyzed by TLC.
Dissociation of Yttrium-90-LabeledChelators at pH 2.0. A 2 mM stock solution of each chelator and a 400 mM stock of yttrium-90 were prepared in 0.1 M ammonium acetate, pH 6.0. Equivolumes of chelator and yttrium-90 were mixed and allowed to chelate at room temperature for 2 h. An initial time point was obtained by removing 1 p L from the chelation solution for TLC analysis. Four pL aliquots of chelation solution was diluted into 56 pL of 0.1 M glycine-HC1, pH 2.0 in triplicate and incubated at 37 "C. The final concentration of chelator was 67 pM. Five pL aliquots were removed a t the indicated time and added to 10 p L of 0.1 M sodium phosphate, pH 6.5 containing a 60-fold excess of cold yttrium to stop chelation and to prevent rechelation. A 5 pL aliquot was analyzed by TLC. RESULTS AND DISCUSSION
Preliminary work indicated that neither the tetraaza macrocyclic chelators (Figure 1(top)) nor DTPA chelated yttrium-90 efficiently a t chelator concentrations below 10 pM. Thus, this concentration appeared to be our threshold for chelation, and only chelator concentrations above 10 pM were used in the following studies. Relatedly, a minimum ligand concentration for efficient chelation of indium-111 by DTPA conjugates has been reported (221. The effect of metal concentration on chelator labeling efficiency was examined in Table 1. In this experiment, chelator was mixed with increasing concentrations of
Table 1. Effect of Yttrium-90 Concentration on Chelator Labeling Efficiencp [gOYoAcl, 10-6M
DOTA
PADOTA
100 50 33 25 17 10
49.7 93.3 97.4 97.8 97.2 97.2
38.3 70.0 92.6 97.0 97.6 97.4
%
chelated TRITA 53.4 89.6 95.8 96.3 94.1 96.2
TMT
DTPA
97.4 98.4 97.9 97.9 97.8 97.3
97.8 96.3 94.8 92.9 79.4 86.4
a [Chelator] = 100 pM; reaction period = 2 h; pH 6.0; temperature = 22 "C; final volume of chelation = 10 pL.
Table 2. Time Interval of Yttrium-90 Chelationa %
time(min)
DOTA
PADOTA
0 15 30 60 90 120
5.1 96.1 98.5 98.7 99.0 99.1
1.5 79.7 89.9 95.0 96.8 97.6
chelated TRITA TMT 3.2 74.4 90.6 96.5 97.4 97.7
97.0 98.6 98.8 98.8 98.8 98.7 .
DTPA 91.4 89.2 88.7 88.5 88.5 90.6
[Chelator] = 50 yM; [goYoAcl= 10 yM; temperature = 22 "C; volume of chelation = 10 pL.
yttrium-90, and the extent of chelation, expressed as a percentage, was determined. Maximum labeling efficiency with the macrocyclic chelators was achieved only when the concentration of chelator was in excess of the yttrium-90 concentration. A 3-fold chelatormetal molar ratio was sufficient to achieve labeling in excess of 90%. In contrast, the chelation of TMT-amine, nitro-CHX-ADTPA (data not shown), nitro-MX-DTPA(data not shown), and DTPA with yttrium-90 was efficient a t a metal: chelator molar ratio of 1:l. When similar experiments were performed with different concentrations of chelator ('10 pM, data not shown), maximum labeling efficiency was achieved only when the chelatormetal molar ratio > 3 for the macrocyclics and 21 for the acyclics. Curiously and in contrast to yttrium-90, the macrocyclic chelators chelated divalent cobalt-57 in a chelatormetal molar ratio of 1 : l (data not shown). Thus, the chelator: metal molar ratio required for optimum efficiency may be unique for different radiometals and chelators. In Table 2, we investigated the time course of yttrium chelation. In this experiment, yttrium-90 was incubated in the presence of a 5-fold excess of chelator, and the progress of the chelation was monitored a t the time intervals indicated in Table 2. Expectedly, DTPA achieved > 85% chelation instantaneously as observed previously (22). Rapid chelation was also evident with TMT-amine. We also observed this with nitro-CHX-A-DTPAand nitroMX-DTPA (data not shown). In contrast, the macrocycles were slower to complex yttrium-90. This retardation in the progress of chelation has been documented previously with DOTA (29). Interestingly, nitro-DOTA was slightly faster than nitro-PADOTA and nitro-TRITA. Since the final concentrations of chelator ('100 pM) and yttrium-90 (< 10 pM) in a typical conjugate chelation are very low, it is not surprising that the presence of trace metals can potentially influence the efficiency of conjugate labeling (23).Therefore, we investigated the influence of divalent cations that are commonly found in commercial preparations of yttrium-90 on chelation labeling efficiency (Figure 2). Nitro-MX-DTPA was not evaluated in this study. Yttrium-90 was incubated with a 5-fold excess of chelator in the presence of increasing amounts of Zn(I1) acetate (A), Fe(I1) acetate (B), or Ca(11)acetate (C). The labeling efficiency of DTPA was not affected by the presence of Ca2+or Fez+. However, at
:
222 Bioconjugate Chem., Vol. 6,No. 2, 1995
Stimmel et al.
100
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100 90 80 70 60 50 40 30 20 10 0
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8
9
+ TMT
10SO100
[Ca(II)Acetate] , x 10-5M
I -A-
PADOTA -2-
DOTA t TRITA t DTPA
+ TMT I
Figure 2. Effect of the presence of trace metals on chelation efficiency of macrocyclic chelators and DTPA with yttrium-90: [chelator] = 1 mM; [goYoAcl= 100 mM; reaction period = 2 h; buffer = 0.1 M ammonium acetate pH 6.0; temperature = 22 "C; final volume of chelation = 10 mL; (A) Zn(I1) acetate; (B) Fe(I1) acetate; (C) Ca(I1) acetate.
high concentrations of Zn2+,there was a slight decrease in efficiency. TMT-amine and nitro-CHX-A-DTPA exhibited properties similar to those of DTPA. The labeling efficiency of TMT-amine and nitro-CHX-A-DTPA (data not shown) was not affected by the presence of increasing amounts of Ca2+. In contrast, high concentrations of Zn2+
and Fe2+affected the labeling efficiency of TMT-amine and nitro-CHX-A-DTPA (data not shown) to a greater degree. This experiment was repeated with equimolar concentrations of DTPA, nitro-CHX-A-DTPA, TMTamine, and yttrium-90, and the effect of Ca2+on chelation efficiency was minimal. In contrast, there was a concentration dependent decrease with Fez+and Zn2+(data not shown). Thus, it appeared that the contaminantinduced loss of efficiency of DTPA, nitro-CHX-A-DTPA, or TMT-amine increased as the chelator concentration decreased. The influence of trace metals on macrocyclic chelation was more pronounced. A 1- to %fold contaminant concentration increase over the yttrium-90 concentration diminished labeling efficiency. In addition, yttrium-90 labeling efficiency decreased as the contaminant concentration increased in a dose-dependent manner. The extent of the influence varied with the particular chelator, but in general, nitro-PADOTA chelation demonstrated the most sensitivity followed by nitro-DOTA and nitro-TRITA. Surprisingly, nitro-TRITA labeling was not affected by the presence of Ca2+. We also observed that the presence of trace metals substantially retarded the time necessary to achieve maximum efficiency (data not shown). These results clearly demonstrated the necessity of a metal contaminant-free environment to enhance chelation efficiencies. A similar conclusion has been reached relatedly in a study of the influence of Zn2-, Mg2+, and Ca2" on the chelation efficiency of several chelators, including DOTA, for 67Cu(32). When comparing DOTA's chelation efficiency for with that for 6 7 C ~ 2(32), + the chelation efficiency for yttrium-90 was inhibited by 10-fold less contaminent; thus, the comparison underscores the fastidious requirements for chelation. The kinetic stability of yttrium-90-labeled chelator complexes was evaluated a t low pH. These studies were undertaken for two reasons. Firstly, acid-assisted dissociation is a well-documented catabolic phenomenon that contributes to in vivo physiologic stability. Secondly, pH lability (in the laboratory) was a convenient means allowing for ready discrimination among this group of chelators. We observed that the rates of dissociation increased with lowering pH and were chelator dependent. Furthermore, when comparing this group of chelators, the order of stability was maintained as pH was lowered. The degree of discrimination was most evident a t pH 2-3. The results a t pH 2.0 are illustrated in Figure 3. The chelators were labeled with yttrium-90 (> 90%) and diluted in triplicate into a glycine-HC1 buffer a t pH 2.0 under conditions where reassociation was negligible. At the indicated time intervals, the yttrium-90 remaining in the chelator was quantitated. Chelator stability fell into three distinct classes. Nitro-DOTA and nitro-PADOTA exhibited exceptional stability over the course of 7 days, and then small differences between the two chelators became apparent. Nitro-CHX-A-DTPA and nitro-MX-DTPA exhibited moderate kinetic stability. Yttrium-90-labeled DTPA, TMT-amine, and nitro-TRITA were unstable a t this pH. After 3 days, no radioactivity remained complexed in these latter chelators. The kinetic stability of yttrium-90-DOTA complexes a t low pHs has been demonstrated recently (23)but was implied in the early 1980's (19). Nuclear magnetic resonance analysis of lanthanide-DOTA complexes demonstrated that the steric requirements of the 12-membered tetraaza cavity resulted in unusual rigidity that retarded protonation. Even though PADOTA is in the same class of stability as DOTA, minor differences were observed. It can be inferred that functionalization a t the 2-position alters the conformation enough to allow pro-
Bioconjugate Chem., Vol. 6,No. 2, 1995 223
Chelation Properties of Select Chelators
I
3
1
1
+ PADOTA
-C-
DOTA
-A-
5
7 9 Time, days
TRITA t TMT
-D DTPA
I1
13
+ CHX-A-DTPA
15
4- MX-DTPA
~~
Figure 3. Stability of $OY-chelator complex at pH 2.0: [chelator] = 67 mM; buffer = 0.1 M glycine-HC1 pH 2.0; temperature = 37 "C.
tonation to occur more easily for PADOTA than with DOTA. However, both nitro-DOTA and nitro-PADOTA complexes exhibited exceptional stability a t low pH. Nitro-MX-DTPA and nitro-CHX-A-DTPAdemonstrated moderate kinetic stability a t pH 2.0 that was similar to one another. Additionally, these third-generation DTPA analogues displayed biphasic stability a t this pH. This could suggest the presence of diastereomeric yttriumchelate complexes that demonstrate different kinetic stabilities. Both nitro-CHX-A-DTPA and nitro-MXDTPA are mixtures of enantiomers (14, 33). YttriumMX-DTPA complexes have been observed to form diastereomeric mixtures that exhibited unique behavior (33). It is reasonable that diastereomeric mixtures also form from yttrium-CHX-A-DTPA complexes. Recent work has demonstrated that yttrium complexes of these third generation DTPA analogues are more stable in vivo than DTPA but not as stable as DOTA (12, 34,35). The kinetic stability of yttrium-90 complexes of these chelators a t pH 2.0 thus appears to correlate with other analyses including in vivo stability. The kinetic instability of DTPA-metal complexes at low pH has been well established (36-39), and our results confirmed past experience. The novel terpyridine derivative, TMTamine, exhibited pH stability properties similar to those of DTPA. It is interesting that the macrocycle, nitroTRITA, also exhibited stability similar to DTPA a t pH 2.0. It has been postulated that the 13-membered ring forms a metal complex conformation with metals that possess larger radii such as yttrium that is considerably more flexible and less stable than that of DOTA (4043). The absence of steric constraint may have resulted in more facile protonation and resultant kinetic instability. SUMMARY
In this study, we evaluated the relationship of metal and chelator concentration, time interval of chelation, and the presence of trace metal contaminants on the chelation efficiency of a select group of chelators for yttrium-90. We also report the kinetic stability of the chelator-yttrium-90 complexes a t pH 2.0. All of these chelators have clinical utility and certain advantages for different radiometals and applications. Our principle aim was to appreciate conditions that influenced chelation so
that we could later evaluate the behavior of immunoconjugates. We saw merit in head-to-head evaluation since the results could have been influenced by reagent purity. Within this select group, we observed two distinct classes. The acyclic chelators, TMT-amine, nitro-MXDTPA, nitro-CHX-A-DTPA,and DTPA, chelated yttrium instantaneously in a chelatormetal molar ratio of 1:l. Also, the labeling efficiency of these chelators was affected minimally by the presence of trace metal contaminants when the chelator concentration was 5-fold greater than the yttrium-90 concentration. However, when the contaminant concentration was equivalent to or greater than the chelator concentration, the labeling efficiency diminished in a concentration-dependent manner. Both TMT-amine- and DTPA-yttrium-90 complexes demonstrated minimal stability a t pH 2.0, whereas nitro-MXDTPA and nitro-CHX-A-DTPA demonstrated moderate kinetic stability a t pH 2.0 and exhibited biphasic characteristics. These latter compounds are mixtures of enantiomers that form diastereomeric mixtures when coordinated to yttrium. The biphasic nature of the kinetic stability a t pH 2.0 suggests that the diastereomers may have different kinetic stabilities. The macrocyclic chelators, nitro-DOTA, nitro-PADOTA, and nitro-TRITA, chelated yttrium-90 a t a slower rate. Nitro-DOTA was the fastest and achieved maximum labeling efficiency in less than 30 min. NitroPADOTA and nitro-TRITA required less than 60 min to achieve labeling efficiencies comparable to nitro-DOTA. All three macrocycles required minimally a %fold excess of chelator over yttrium concentration to achieve maximum labeling efficiencies. Curiously, excess chelator was not required to chelate divalent cobalt efficiently. The labeling efficiencies of the macrocyclic chelators (5-fold excess of chelator compared to yttrium-90 concentration) were inhibited in a concentration-dependent manner by the presence of increasing amounts of trace metals (Ca2+, Fez-, ZnZf) that are commonly found in commercial yttrium-90 preparations. Nitro-TRITA was an exception. The labeling efficiency of the 13-membered ring chelator was not affected by increasing concentrations of CaZf. Finally, nitro-DOTA- and nitro-PADOTA-yttrium-90 complexes exhibited exceptional kinetic stability a t pH 2.0. In contrast, the nitro-TRITA-yttrium-90 complex
224 Bioconjugafe Chem., Vol. 6,No. 2, 1995
demonstrated kinetic instability a t pH 2.0 similar to TMT-amine and DTPA. In conclusion, we demonstrate the relationship between metal and chelator concentration, time to chelation, and the necessity of maintaining a metal contaminant-free environment to achieve optimum labeling efficiencies. We also show that low pH stability stratifies these chelators. The order of stratification correlates with published observations on their physiological kinetic stabilities. We hope these observations will be especially useful for immunoconjugate chelation. ACKNOWLEDGMENT
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