Comparison of rac- and meso-2,3-Dimercaptosuccinic Acids for

Jan 15, 1996 - Comparison of rac- and meso-2,3-Dimercaptosuccinic Acids for Chelation of Mercury and Cadmium Using Chemical Speciation Models. Xiaojun...
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Chem. Res. Toxicol. 1996, 9, 284-290

Comparison of rac- and meso-2,3-Dimercaptosuccinic Acids for Chelation of Mercury and Cadmium Using Chemical Speciation Models Xiaojun Fang,†,‡ Fengmei Hua,§ and Quintus Fernando*,† Departments of Chemistry and Pharmaceutical Sciences, University of Arizona, Tucson, Arizona 85721 Received June 7, 1995X

The formation constants of various mercury and cadmium chelates of the stereoisomers of 2,3-dimercaptosuccinic acid (DMSA)1 have been determined from potentiometric titrations in the presence of the competing ligand EDTA. The mercury chelates formed at pH 7.4 are the monomeric HgL of the DMSA diastereoisomers and HHgL2 of rac-DMSA. Mercury is completely complexed at pH greater than 3.0 in solutions containing more than 1 equiv of either rac- or meso-DMSA. At high concentrations (10 µM and above) mercury tends to bind to a greater extent to rac- than to meso-DMSA. At pH 7.4, the predominant cadmium meso-DMSA chelate species in solution is CdL, and HCdL is present at a much smaller concentration. With racDMSA, however, the predominant cadmium chelate species is HCdL at a low concentration of the ligand, and at a high concentration of the ligand the species CdL2 predominates. Cadmium is completely chelated at pH 7.4 in solutions containing more than 1 equiv of either rac- or meso-DMSA. At pH around 5.5, which corresponds to the pH of the kidney, however, a significant amount of free cadmium is present in solutions containing 1 equiv or less of either DMSA stereoisomer. From the results of an analysis of speciation models, probable kidney damage, that may result from free cadmium ion release in the kidney during chelation therapy, is inferred when meso-DMSA is used for mobilizing cadmium. In contrast, the release of free cadmium ion is negligible in the pH range in the kidney when rac-DMSA is used. On the basis of the speciation models, rac-DMSA is found to be far superior to meso-DMSA in the treatment of acute cadmium poisoning.

Introduction Effects of mercury toxicity manifest themselves primarily in the central nervous system and in kidneys, where mercury accumulates after exposure. Studies with experimental animals showed that meso-2,3-dimercaptosuccinic acid (meso-DMSA)1 (1, 2) and 2,3-dimercaptopropanesufonic acid (DMPS) (1) are very effective in the treatment of mercury intoxication. The lack of knowledge of the chemistry of mercury meso-DMSA chelation, however, still hampers our understanding of the effectiveness of meso-DMSA in comparison with other chelating agents in the treatment of acute and chronic mercury poisoning. Cadmium is highly nephrotoxic (3) and has an extremely long biological half-life (4). An assessment of a number of chelating agents for their abilities in removing cadmium after injection of 115mCdCl2 revealed that mesoDMSA was most effective for the treatment of acute cadmium poisoning (5). Comparison of meso-DMSA with other chelating ligands by Jones et al. (6), using computer modeling of chelation equilibria, showed that mesoDMSA is likely to be one of the most useful ligands for * To whom correspondence should be addressed. † Department of Chemistry. ‡ Present address: Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, AZ 85721. § Department of Pharmaceutical Sciences. X Abstract published in Advance ACS Abstracts, December 15, 1995. 1 Abbreviations: FDA, Food and Drug Administration; meso-DMSA, meso-2,3-dimercaptosuccinic acid; DMPS, 2,3-dimercaptopropane-1sulfonic acid; EDTA, ethylenediaminetetraacetic acid; rac-DMSA, rac2,3-dimercaptosuccinic acid; RPMI, relative plasma mobilizing index; NIST, National Institute of Standards and Technology.

0893-228x/96/2709-0284$12.00/0

the treatment of acute cadmium intoxication, and this correlates very well with the results of an in vivo study (5). DMSA exists in two diastereoisomeric forms, meso and racemo. Unlike its meso isomer, rac-2,3-dimercaptosuccinic acid (rac-DMSA), which is an equimolar mixture of (R,R)-2,3-dimercaptosuccinic acid and (S,S)-2,3-dimercaptosuccinic acid, is very soluble in water, in strongly acidic solutions, and in organic solvents such as ethyl acetate and ethyl ether. These striking differences in the hydrophilicities and lipophilicities of the two DMSA diastereoisomers suggest that rac-DMSA is a more effective antidote than meso-DMSA for the treatment of heavy metal poisoning. A computer modeling of lead chelation equilibria at physiological pH as well as a calculation of the relative plasma mobilization index (RPMI) of DMSA stereoisomers for lead also indicated that rac-DMSA is more effective than its meso isomer in removing in vivo lead (7). Egorova et al. (8) and Okonishnikova (9, 10) reported in the early 1970’s, in their toxicological studies with the isotopes Hg203 and Cd115, that with identical doses of the rac- and meso-DMSA, the rac-DMSA invariably increased the elimination of mercury and cadmium in comparison with the meso-DMSA. Despite this important observation that rac-DMSA was superior to meso-DMSA in the treatment of mercury and acute cadmium poisoning, the chelating properties of rac-DMSA, especially with mercury and cadmium, have received little attention. The formation constants of the mercury and cadmium chelates of rac-DMSA have not been determined, probably because of the difficulties encountered in the synthesis © 1996 American Chemical Society

Mercury and Cadmium Chelates of meso- and rac-DMSA

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of rac-DMSA and the low solubilities of the mercury and cadmium chelates of rac-DMSA. It is important to rationalize these observed differences of rac- and mesoDMSA in mobilizing in vivo mercury and cadmium ions. The objectives of this paper are (i) to postulate the compositions of the various mercury and cadmium chelates of meso- and rac-DMSA that are formed in aqueous solution, (ii) to determine their formation constants, (iii) to calculate the distributions of the various mercury and cadmium species as a function of pH, (iv) to assess the relative effectiveness of meso- and rac-DMSA in the chelation of mercury and cadmium at physiological pH, and finally, (v) to provide a rationale for the therapeutic use of DMSA stereoisomers in the treatment of mercury and cadmium poisoning.

previously siliconized with siliconizing liquid and calibrated with double-deionized water. The titration vessel was also siliconized before use.

Experimental Section Materials. rac-DMSA was synthesized in our laboratory as previously described (11); meso-2,3-dimercaptosuccinic acid was a gift from Johnson & Johnson Baby Products Co. (Skillman, NJ); buffer solutions were purchased from Fisher Scientific (Fair Lawn, NJ); siliconizing fluid was purchased from Princeton Applied Research (Princeton, NJ); all other inorganic compounds used were purchased from Mallinckrodt, Inc. (Paris, KY), and were of analytical reagent grade. Caution: Gloves are necessary in the handling of all the above chemicals to avoid direct contact with skin. Potentiometric Determination of Formation Constants of the Cadmium and Mercury Chelates of DMSA Stereoisomers. Standard 0.1 M KOH solutions were prepared from 45% (w/w) KOH solution and CO2-free double-deionized H2O. The prepared base solutions were kept under a nitrogen atmosphere to prevent the absorption of CO2. The carbonate content, determined as described by Martell et al. (12), was found to be 0.5% in the KOH solution; the exact molarity of KOH solution was determined with potassium acid phthalate with phenolphthalein as indicator. A stock 0.1 M HNO3 solution was prepared from concentrated HNO3 and CO2-free double-deionized H2O and standardized by KOH titration. A standard 0.1 M ethylenediaminetetraacetic acid (EDTA) solution was prepared from the disodium salt of EDTA and CO2-free doubledeionized H2O and standardized by titration with a standard zinc solution prepared from zinc oxide (13). A stock EDTA solution (0.01 M) was prepared from the standard 0.1 M EDTA solution. Stock Cd2+ and Hg2+ solutions (0.01 M) were prepared from Cd(NO3)2 and HgCl2 and CO2-free double-deionized H2O, and their concentrations were obtained by titration with EDTA. The diastereoisomeric DMSA stock solutions (0.01 M) were prepared by dissolving about 91.1 mg of the solid DMSA in 20 mL of CO2-free double-deionized H2O containing 2 equiv of KOH, and diluting the solution to 50 mL with CO2-free doubledeionized H2O. The stock solution of DMSA was transferred to a small polystyrene bottle, deaerated with argon for 1-2 min, and stored in a freezer. The concentrations of the DMSA solutions were calculated from the initial weights of the ligands and then confirmed by KOH titration with methyl red as indicator. The apparatus and procedures used for potentiometric measurement of hydrogen ion concentration have been described previously (11). The titration solutions were prepared according to the following procedure for the determination of the formation constants of the lead-DMSA complexes. Thirty milliliters of CO2-free deionized water was placed in a titration vessel into which 6 mL of the stock EDTA solution was transferred, followed by addition of 6 mL of the stock lead nitrate solution and 6 mL of the stock DMSA solution. Finally, a desired amount of KNO3 was weighed and added into the titration vessel to maintain the ionic strength equal to 0.10 ( 0.01 throughout the titration. The accuracy of volume transfer in the preparation of the stock solutions and the titration solutions was ensured by the exclusive use of volumetric pipets which were

Results Determination of Formation Constants. The potentiometric titration data were used to calculate the formation constants of the cadmium and mercury chelates of EDTA and DMSA stereoisomers with the aid of the BEST program (12). The BEST program calculates the formation constants of the species which are assumed to be present in the titrated solution, minimizing the discrepancies between the calculated values of -log[H+] and the experimental values of -log[H+] obtained from a potentiometric titration. Since the presence of the species which were included in the calculation by the BEST program cannot be proved by the potentiometric titration itself, the formation constants were determined only on the basis of statistics. A statistically favorable model is a set of chemical species assumed to be present in the titrated solution, which, with the aid of BEST program, yields a best fit for the experimental titration data. Because of the low solubilities of the mercury and cadmium chelates of the DMSA stereoisomers, the potentiometric titrations of the mercury- and cadmiumDMSA solutions were performed in the presence of a competing ligand, EDTA. Although the formation constants of various mercury and cadmium complexes of EDTA are available from the database of the National Institute of Standards and Technology (NIST) (14), they were verified independently in our laboratory to ensure the accuracy of the data as well as to validate our potentiometric method. (A) Formation Constants of Mercury and Cadmium Chelates of EDTA. The statistically favorable model for the titration of solutions containing equimolar amounts of Hg2+ and H2EDTA2- consisted of HgEDTA, HHgEDTA, H2HgEDTA, H3HgEDTA, and HgEDTAOH. The mean log formation constants calculated from two titrations are listed in Table 1. The formation constants of H3HgEDTA and HgEDTAOH were not available for comparison. The mean log formation constant of HgEDTA and its stepwise log protonation constants along with their standard deviations determined from two repetitive experiments in our laboratory are 21.3 ( 0.3, 3.80 ( 0.01, and 2.75 ( 0.02, respectively. These values are in good agreement with those selected from the NIST database (14), 21.5 ( 0.1, 3.2 ( 0.1, and 2.1, respectively. Our value of the log formation constant of HgEDTA also agrees very well with that determined by Casas and Jones (15), 22.14 ( 0.15. This demonstrates the reliability of using a statistically favorable model to calculate the formation constants of metal chelates by using the BEST program. All five Hg-EDTA complexes were later included in the models for calculations of the formation constants of the mercury complexes of DMSA stereoisomers. The statistically favorable model for the titration of solutions containing equimolar amounts of Cd2+ and H2EDTA2- consisted of CdEDTA, HCdEDTA, H2CdEDTA, and CdEDTAOH. The average log formation constants calculated from two titrations are listed in Table 1. The difference in the reported value of the log formation constant of CdEDTAOH (14) and that determined in our laboratory is 1.5, which may be attributed in part to the

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Table 1. Formation Constants of Mercury and Cadmium EDTA Chelates at µ ) 0.10 and T ) 25.0 °C species log βpqra

p

q

r

Hg:EDTA ) 1:1

1 1 1 1 1

1 1 1 1 1

0 1 2 3 -1

21.3 ( 0.3b 25.1 ( 0.3b 27.8 ( 0.2b 30.2 ( 0.3b 11.7 ( 0.3b

21.5 ( 0.1 (14) 24.7 ( 0.2c (14) 26.8d (14)

Cd:EDTA ) 1:1

1 1 1 1

1 1 1 1

0 1 2 -1

16.57 ( 0.01b 19.40 ( 0.01b 20.7 ( 0.4b 4.81 ( 0.05b

16.5 ( 0.1 (14) 19.4e (14) 21.0f (14) 3.3g (14)

22.14 ( 0.14 (15)

a β (4q-2p-r)-]/[Hg2+]p[L4-]q[H+]r or [Cd L H (4q-2p-r)-]/[Cd2+]p[L4-]q[H+]r; L4- represents completely deprotonated EDTA pqr ) [HgpLqHr p q r ligand. b The values were determined in our laboratory, and the estimated errors were calculated from the log formation constants obtained by the BEST program from two sets of titration data with σ(pH)fit of 0.0047 and 0.0092, respectively, for Hg-EDTA system and with σ(pH)fit of 0.0017 and 0.0025, respectively, for Cd-EDTA system (12). c The value was calculated from the reported log protonation constant of HgL of EDTA, 3.2 ( 0.1. d The value was calculated from the reported log protonation constant (2.1) of HHgL of EDTA measured at an ionic strength 1.0. e The value was calculated from the reported log protonation constant (2.9) of CdL of EDTA. f The value was calculated from the reported log protonation constant (1.6) of HCdL of EDTA measured at an ionic strength 1.0. g The value was calculated from the reported log formation constant (13.2) of CdLOH of EDTA, which was defined by the following: log KCdLOH ) log([CdL2-]/[CdLOH3-][H+]), measured at an ionic strength 1.0.

ionic strength of 1.0 that was employed by the previous investigators (14). The log formation constant of CdEDTA and its stepwise log protonation constants determined in our laboratory are 16.57 ( 0.01, 2.83 ( 0.01, and 1.3 ( 0.6, respectively, which are in good agreement with those selected from the NIST database (14), 16.5 ( 0.1, 2.9, and 1.6 ( 0.1, respectively. This again demonstrates the reliability of calculating the formation constants of metal chelates on the basis of a statistically favorable model. All four Cd-EDTA complexes were always included later in the models for calculation of the formation constants of the cadmium complexes of DMSA stereoisomers. The potentiometric titration points of solutions containing equimolar amounts of Hg2+ and H2EDTA2- and equimolar amounts of Cd2+ and H2EDTA2- are plotted in Figure 1, and so are their corresponding simulated titration curves by the BEST program (solid lines). (B) Formation Constants of Mercury Chelates of meso- and rac-DMSA. The formation constants of mercury chelates of DMSA stereoisomers were calculated from two repetitive titrations of solutions containing equimolar amounts of Hg2+, H2EDTA2-, and H2DMSA2stereoisomers. The presence of three mercury complexes (HgL, HHgL, and HgL2) of meso-DMSA was deduced from the refinements by the BEST program, and their corresponding log formation constants, listed in Table 2, are 27.5 ( 0.2, 32.4 ( 0.2, and 34.2 ( 0.2, respectively. The presence of five mercury complexes (HgL, HHgL, H2HgL, HgL2, and HHgL2) of rac-DMSA was deduced from the refinements by the BEST program, and their corresponding log formation constants, listed in Table 2, are 28.5 ( 0.1, 33.0 ( 0.1, 35.8 ( 0.2, 36.7 ( 0.6, and 46 ( 1, respectively. The values of the standard deviation for the formation constants of HgL2 and HHgL2 of rac-DMSA are greater than those for the other chelates because the maximum distributions of the two chelates in the entire course of the titration are comparatively small, 15% and 7%, respectively, of the total amount of mercury ions present in solution. The sample titration curves obtained with the solutions containing equimolar amounts of mercury, EDTA, and the DMSA stereoisomer are plotted in Figure 1, and so are the corresponding simulated titration curves by the BEST program (solid lines). (C) Formation Constants of Cadmium Chelates of meso- and rac-DMSA. The formation constants of

Figure 1. Potentiometric titration curves of the solutions containing the following: (9) 1.296 mM Na2H2EDTA and 1.264 mM Hg2+ (Hg2+:H2EDTA2- ) 1:1); (b) 1.255 mM Na2H2EDTA, 1.247 mM Hg2+, and 1.290 mM Na2H2(rac-DMSA) (Hg2+:H2EDTA2-:H2(rac-DMSA)2- ) 1:1:1); (2) 1.255 mM Na2H2EDTA, 1.247 mM Hg2+, and 1.259 mM Na2H2(meso-DMSA) (Hg2+:H2EDTA2-:H2(meso-DMSA)2- ) 1:1:1); (0) 1.315 mM Na2H2EDTA and 1.268 mM Cd2+ (Cd2+:H2EDTA2- ) 1:1); (O) 1.255 mM Na2H2EDTA, 1.249 mM Cd2+, and 1.290 mM Na2H2(rac-DMSA) (Cd2+:H2EDTA2-:H2(rac-DMSA)2- ) 1:1:1); (4) 1.255 mM Na2H2EDTA, 1.249 mM Cd2+, and 1.259 mM Na2H2(meso-DMSA) (Cd2+:H2EDTA2-:H2(meso-DMSA)2- ) 1:1:1). The solid lines are corresponding titration curves simulated by the BEST program.

cadmium chelates of EDTA determined in our laboratory were used in the calculations. The formation constants of cadmium chelates of DMSA stereoisomers were calculated from two repetitive titrations of solutions containing equimolar amounts of Cd2+, H2EDTA2-, and H2DMSA2- stereoisomers. The presence of four cadmium complexes (CdL, HCdL, CdL2, and HCdL2) of meso-DMSA was deduced from the refinements by the BEST program, and their corresponding log formation constants, listed in Table 2, are 16.6 ( 0.4, 22.8 ( 0.1, 22.1 ( 0.1, and 32.2 ( 0.1, respectively. The presence of three cadmium complexes (HCdL, H2CdL, and CdL2) of rac-DMSA was deduced from the refinements by the BEST program, and their corresponding log formation constants, listed in Table 2, are 25.1 ( 0.2, 28.1 ( 0.1, and 29.3 ( 0.2,

Mercury and Cadmium Chelates of meso- and rac-DMSA Table 2. Formation Constants of Mercury and Cadmium Chelates of DMSA Stereoisomers at µ ) 0.10 and T ) 25.0 °C species log βpqra

p q r Hg:EDTA:rac-DMSA ) 1:1:1b

1 1 0 28.5 ( 0.1

1 1 1 1 Hg:EDTA:meso-DMSA ) 1 1:1:1c 1 1 Cd:EDTA:rac-DMSA ) 1 1:1:1d 1 1 Cd:EDTA:meso-DMSA ) 1 1:1:1e 1 1 1

1 1 2 2 1

1 2 0 1 0

33.0 ( 0.1 35.8 ( 0.2 36.7 ( 0.6 46 ( 1 27.5 ( 0.2 17.3 (22) 39.4 (23)

1 1 32.4 ( 0.2 2 0 34.2 ( 0.2 1 1 25.1 ( 0.2 1 2 28.1 ( 0.1 2 0 29.3 ( 0.2 1 0 16.6 ( 0.4 17.11 (6) 1 1 22.8 ( 0.1 23.50 (6) 2 0 22.1 ( 0.1 2 1 32.2 ( 0.1

a β (4q-2p-r)-]/[Hg2+]p[L4-]q[H+]r; L4- represents pqr ) [HgpLqHr completely deprotonated DMSA. b The values were determined in our laboratory, and the standard deviations were calculated from the log formation constants obtained by the BEST program from two sets of titration data with σ(pH)fit of 0.011 and 0.016, respectively (12). c The values were determined in our laboratory, and the estimated errors were calculated from the log formation constants obtained by the BEST program from two sets of titration data with σ(pH)fit of 0.059 and 0.065, respectively (12). d The values were determined in our laboratory, and the estimated errors were calculated from the log formation constants obtained by the BEST program from two sets of titration data with σ(pH)fit of 0.035 and 0.0095, respectively (12). e The values were determined in our laboratory, and the estimated errors were calculated from the log formation constants obtained by the BEST program from two sets of titration data with σ(pH)fit of 0.010 and 0.010, respectively (12).

respectively. The formation constant of CdL of racDMSA is not reported because the complex has a high log protonation constant (greater than 10) and the observed maximum distribution of the complex in the entire course of the titration is small (less than 0.2% of the total amount of cadmium ions present in solution). The sample titration curves obtained with the solutions containing equimolar amounts of cadmium, EDTA, and the DMSA stereoisomers are plotted in Figure 1, and so are the corresponding simulated titration curves by the BEST program (solid lines).

Discussion rac-DMSA Is More Effective Than meso-DMSA in Mobilizing High Concentrations of Mercury at Physiological pH. rac-DMSA was found to be superior to meso-DMSA for the treatment of acute mercury poisoning in laboratory animals (9). The formation of complexes between mercury(II) and rac-DMSA, however, has not been studied, and their formation constants have not been reported. meso-DMSA has been used for the treatment of heavy metal poisoning by researchers in the former Soviet Union (16) since 1958 and in China (17, 18) since 1965. In the U.S., meso-DMSA was approved by the Food and Drug Administration (FDA) in 1991 (19) for the treatment of children with blood lead levels greater than 45 µg/dL, and it is used at present for the detoxification of children with blood lead levels greater

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than 25 µg/dL. meso-DMSA is a useful antidote for acute and chronic inorganic mercury poisoning as well as for methylmercury poisoning (1, 2, 9, 20, 21). There is, however, very little information about the reaction of mercury compounds with this ligand. The formation constant of the complex of meso-DMSA, HgL, determined in our laboratory is 1028.5, which is substantially larger than the value, 1017.3, reported by Okonishnikova et al. (22), but smaller than the value, 1039.4, suggested by Jones (23). Mercaptan compounds are far better complexing agents than EDTA for the removal of mercury(II) and organomercury compounds from the mammalian body (23). It can be inferred, therefore, that the formation constants of Hg-mercaptan compounds are probably larger than the formation constant of HgEDTA. The formation constant determined by Okonishnikova et al., however, is much smaller than the formation constant, 1021.5, of HgEDTA (14), and most likely is underestimated. The formation constant suggested by Jones (23) was not determined experimentally, but was estimated on the basis of the formation constants of mercury complexes of other mercaptan compounds. Casas and Jones pointed out (15) that the formation constants of the mercury complexes of several mercaptan compounds determined in their laboratory could be overestimated because of the systematic errors associated with their experimental procedures. The relative efficacy of rac- over meso-DMSA in mobilizing mercury ions was evaluated, with the aid of the computer program SPE (12), by calculation of the distributions of various mercury species as a function of pH in the presence of both DMSA stereoisomers. It was assumed in the calculations that only the mercury chelates of rac- and meso-DMSA listed in Table 2 are present in aqueous solution. The distributions of mercury species were calculated at various total mercury concentrations ranging from 0.2 mg/dL (10 µM) to 30 µg/ dL (1.5 µM) and at various concentrations of ligand up to 3.3 mM to simulate the conditions of mercury poisoning and the in vivo treatment with DMSA. The distribution curves, calculated for solutions containing 60 µg/dL mercury, for various mercury species which contributed more than 0.1% of total mercury in the pH region 3.010.0 are plotted in Figure 2. The behavior of rac- and meso-DMSA in the complexation of mercury is similar, as shown by the titration curves obtained with the two isomers (filled circles and filled triangles, respectively, in Figure 1). Both stereoisomers are very effective in mobilizing mercury, because mercury is completely complexed by the two DMSA stereoisomers in the entire pH region when the ratio of total ligand to total mercury in solution is equal to or greater than 1, as shown in Figure 2. At a molar ratio of total DMSA and mercury in solution equal to 1, meso-DMSA is as effective as racDMSA in binding mercury, with 50% of the mercury being complexed by meso-DMSA and the other 50% by its racemic isomer (Figure 2(a)). The mercury chelates formed in this solution can be represented as HgL, where L is one of the stereoisomers of DMSA. When the concentration of the stereoisomers of DMSA is elevated (Figure 2(b)-(d)), HHgL2 of rac-DMSA starts to form at pH 7.4 and the distribution pattern at pH 7.4 undergoes a gradual change, with an increase and decrease in the concentrations of HgL of rac- and meso-DMSA, respectively. Despite its formation, the contribution of HHgL2 of rac-DMSA is not significant unless the ligand:mercury ratio exceeds 30.

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Figure 3. Relative plasma mobilizing index (RPMI) of rac- over meso-DMSA for Hg2+ versus the molar ratio of the total amount of DMSA stereoisomers to the total amount of mercury at total mercury concentrations of (9) 1.5 µM, (2) 3 µM, and (b) 10 µM; the total concentrations of rac-DMSA are kept equal to the total concentrations of meso-DMSA, i.e., half of the total concentrations of the DMSA stereoisomers, in the calculations.

Figure 2. Distribution curves of all mercury species present in solutions containing 3 µM mercury ion and rac- and mesoDMSA at (a) 1.5 µM each, (b) 3 µM each, (c) 30 µM each, and (d) 300 µM each.

The concept of metal RPMI was developed by Fang et al. (24) to quantitatively assess the relative efficacy of two chelating agents in mobilizing a metal at physiological pH. For mercury mobilization, the RPMI of rac- and meso-DMSA is defined as:

RPMI ) (total amount of mercury bound in rac-DMSA)pH)7.4 (total amount of mercury bound in meso-DMSA)pH)7.4 The RPMI was calculated for the solutions containing mercury and the stereoisomers of DMSA using the concentrations of the mercury species obtained with the aid of the SPE program (12). The results are plotted in Figure 3 in which it is apparent that the RPMI is always greater than 1. This indicates that rac-DMSA is more effective than its meso isomer in mobilizing mercury at pH 7.4. The RPMI curves corresponding to solutions containing various amounts of mercury are almost superimposable at ligand:mercury ratios below 20. At ratios above 20, they deviate from each other. At a constant ligand:mercury ratio, the higher the concentration of mercury present in solution, the higher the RPMI. On the basis of our complex formation studies with mercury, we conclude that rac-DMSA is superior to mesoDMSA in the treatment of mercury poisoning, especially in the case of acute poisoning when a high mercury

concentration is found in blood and a high dose of DMSA is administered. This prediction has been verified by early investigators (9, 10, 12) with the results obtained from animal experiments. In addition, the high hydrophilicity and lipophilicity of rac-DMSA imply that, unlike meso-DMSA, it will be readily absorbed and transported to sites where there are high levels of mercury. We believe, therefore, that rac-DMSA is a better antidote than meso-DMSA for acute mercury poisoning. In cases of chronic mercury poisoning, the mercury concentration in the body is comparatively low. As shown in Figure 3, the effect of ligand:Hg ratio on RPMI decreases when the concentration of mercury decreases. Hence, the doserelated enhancement of mercury chelation by rac-DMSA may not be significant when rac-DMSA is used for the treatment of chronic mercury poisoning. rac-DMSA Is More Effective Than meso-DMSA in Mobilizing Cadmium at Physiological pH. racDMSA was found to be more effective than its meso isomer in the elimination of cadmium from rats when the drug was administered simultaneously with a single subcutaneous tracer dose of Cd115Cl2 solution (10). The stoichiometry of some cadmium complexes of DMSA stereoisomers was studied by Egorova et al. (25); however, the cadmium complexation of rac-DMSA has not been quantitatively studied, and the formation constants of the cadmium complexes of rac-DMSA have not been reported. Jones et al. (6) compared the relative effectiveness of several chelating agents in removing cadmium from humans using chemical speciation models and showed that meso-DMSA and DMPS are likely to be the most efficacious ligands. Formation constants of several meso-DMSA cadmium chelates have been reported by Jones et al. (6) and are the only such constants that are available in the literature. The formation constants of meso-DMSA cadmium chelates determined in our laboratory and by Jones et al., listed in Table 2, are in fair agreement in view of the experimental difficulties encountered in the measurement of the formation constants of the complexes of heavy metals with chelating agents containing mercaptan groups.

Mercury and Cadmium Chelates of meso- and rac-DMSA

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Figure 5. Relative plasma mobilizing index (RPMI) of rac- over meso-DMSA for Cd2+ versus the molar ratio of the total amount of DMSA stereoisomers to the total amount of cadmium at the total cadmium concentrations of (9) 1.5 µM, (2) 3 µM, and (b) 10 µM; the total concentrations of rac-DMSA are kept equal to the total concentrations of meso-DMSA, i.e., half of the total concentrations of the DMSA stereoisomers, in the calculations.

Figure 4. Distribution curves of all cadmium species present in solutions containing 3 µM cadmium ion and rac- and mesoDMSA at (a) 1.5 µM each, (b) 3 µM each, (c) 30 µM each, and (d) 300 µM each.

The relative efficacy of rac- and meso-DMSA in mobilizing cadmium ions was evaluated in the same manner as the relative efficacy of the two DMSA isomers in mobilizing mercury ions. The distributions of cadmium species were calculated at various total cadmium concentrations ranging from 112 µg/dL (10 µM) to 17 µg/dL (1.5 µM) and at various concentrations of ligand up to 3.3 mM to simulate the conditions of cadmium poisoning and the in vivo treatment with DMSA. The distribution curves, calculated for solutions containing 34 µg/dL cadmium, for various cadmium species which contributed more than 0.1% of the total cadmium in the pH region 3.0-10.0 are plotted in Figure 4. The behavior of racand meso-DMSA in complexing with cadmium is drastically different from each other, as can be clearly seen from their titration curves (empty circles and empty triangles, respectively, in Figure 1). Neither rac- nor meso-DMSA is as effective in mobilizing cadmium as it is in mobilizing mercury, because unlike in Figure 2, a significant concentration of free cadmium ions was observed at pH below 7, in the solution containing 1:1 ligand:cadmium, and at pH below 5, in the solution containing 2:1 ligand:cadmium (Figure 4(a),(b)). In a solution containing equimolar concentrations of ligand and cadmium, meso-DMSA is as effective as racDMSA in mobilizing cadmium at pH 7.4, with 50% of the cadmium being complexed by meso-DMSA and the other 50% by its racemo isomer. The major types of cadmium

chelates formed in this solution with the two DMSA stereoisomers, however, are different, since rac-DMSA forms HCdL and meso-DMSA forms mainly CdL (Figure 4(a)). Formation of the protonated cadmium chelate, HCdL, of rac-DMSA may facilitate the transport of intracellular cadmium because of the charge reduction in the cadmium chelate. When the concentration of the stereoisomers of DMSA is increased, rac-DMSA exhibits a higher cadmium mobilizing ability than meso-DMSA. As shown in Figure 4(b)-(d), with an increase in ligand concentration the contribution of the cadmium complex, CdL, of meso-DMSA at pH 7.4 decreases, while the contribution of HCdL of rac-DMSA first increases and then decreases as a consequence of the domination of the cadmium complex, CdL2, of rac-DMSA at a high ligand concentration. The relative cadmium mobilizing ability of rac- and meso-DMSA was also assessed quantitatively using RPMI, which is defined in this case as:

RPMI ) (total amount of cadmium bound in rac-DMSA)pH)7.4 (total amount of cadmium bound in meso-DMSA)pH)7.4 The values of RPMI for cadmium were calculated in the same manner as the RPMI for mercury, and the results are plotted in Figure 5. Although both rac- and mesoDMSA are less effective in mobilizing cadmium than in mobilizing mercury, rac-DMSA alone is much more effective than meso-DMSA in its ability to mobilize cadmium. This is demonstrated in Figure 5 by an increase of ten to several hundredfold in the RPMI observed for solutions containing a wide range of cadmium concentrations. Also shown in Figure 5 is the substantial increase in the RPMI with an increase in cadmium concentration, thereby demonstrating the superiority of rac-DMSA as an effective antidote for acute cadmium poisoning. Implicit in Figure 4(a),(b) are two important features which have a significant bearing on the use of rac- and meso-DMSA in chelation therapy. It is reasonable to

290 Chem. Res. Toxicol., Vol. 9, No. 1, 1996

assume from the experimental evidence that cadmium is mobilized by meso-DMSA in vivo as CdL and HCdL. In the absence of excess ligand (this may happen shortly after a sudden cessation of chelation therapy with high dose administrations), these chelates will reestablish equilibrium in the tissues where they are transported, while maintaining a 1:1 ligand:cadmium ratio. The free cadmium distribution in this situation was calculated and found to be similar to that shown in Figure 4(a), with about 13% of the cadmium being dissociated from the transported chelates as free cadmium ion at pH around 5.5, which corresponds to the pH in urine. The free cadmium ion released may accumulate in the kidney during urinary excretion and cause additional damage to the kidney. In contrast, cadmium is mobilized by racDMSA mainly in the form of CdL2 and the reestablishment of equilibrium by the transported chelates is governed by the distribution diagram corresponding to a solution in which a 2:1 ligand:cadmium ratio is maintained, and in which the free cadmium distribution is similar to that shown in Figure 4(b). In this case, the free cadmium ion contribution around pH 5.5 is essentially zero, and this indicates that the potential kidney damage that is likely caused by cadmium chelation therapy can be minimized if rac- instead of meso-DMSA is administered. We conclude that rac-DMSA is superior to meso-DMSA for the treatment of cadmium poisoning and far superior to meso-DMSA for the treatment of acute cadmium poisoning, on the assumption that thermodynamic equilibrium is attained in solution at various pH values in the presence of the chelating agents and the various cadmium species.

Acknowledgment. This work was supported in part by NIEHS Grant ES 03356.

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