Chem. Res. Toxicol. 1994, 7, 367-373
367
Design of in Vivo Cadmium-Mobilizing Agents: Synthesis and Properties of Monobenzyl meso-2,3-Dimercaptosuccinate Mark M. Jones,* Pramod K. Singh, and Mark A. Basinger Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235
Glen R. Gale and Alayne B. Smith Veterans Affairs Medical Center and Department of Pharmacology, Medical University of South Carolina, Charleston, South Carolina 29403
Wesley R. Harris Department of Chemistry, University of Missouri at St. Louis, S t . Louis, Missouri 63121-4499 Received October 25, 1993'
A novel method for the synthesis of monoesters of meso-2,3-dimercaptosuccinicacid (DMSA) is presented which utilizes the reaction of the vicinal sulfhydryl-protected anhydride with the corresponding alcohol in the presence of a base. The product is then treated successively with mercuric chloride to remove the protecting group and form the mercuric complex, and hydrogen sulfide to regenerate thiol groups by removal of mercury as HgS. This strategy was exploited (MBzDMS), CsH&H20(0)CCH(SH)to synthesize monobenzyl meso-2,3-dimercaptosuccinate CH(SH)C(O)OH, and demonstrates a feasible synthesis of monoesters difficult to obtain by direct esterification, via the use of the reactive anhydride. The resultant compound was found to be an effective cadmium-mobilizing agent when used with cadmium-exposed rats or mice and when administered by any one of several routes (ip, iv, PO). This monobenzyl ester (MBzDMS) of DMSA was found to be somewhat less effective than the corresponding monoisoamyl ester (Mi-ADMS) in mobilizing cadmium from such cadmium deposits. The ability of MBzDMS to mobilize cadmium into the urine is significantly decreased by the coadministration of p-aminobenzoic acid, in support of the hypothesis that MBzDMS enters renal cells via an anion transport system. An analysis of the structural features of vicinal dithiols examined as antagonists for chronic cadmium intoxication allows a hypothesis to be formulated indicating essential features required for the design of effective new cadmium antagonists of this type.
Introduction Chronic cadmium intoxication is characterized by the presence of long-lived deposits of cadmium, especially in the liver and the kidney where they ultimately produce symptoms such as progressive renal failure (1-3). The possibilities for the development of a clinical treatment for chronic cadmium intoxication have been increased in recent years by the development of chelating agents specifically for the purpose of removing this metal from its intracellular deposits in the liver and the kidney (414). These compounds have all been either dithiocarbamates or dithiols. The benzyl monoester of meso-2,3-dimercaptosuccinic acid (MBzDMW was selected as a promising candidate for detailed examination because of the apparent advantage of the benzyl group over alkyl groups as the
* Address correspondence to this author at Box 1583, Station B, Vanderbilt University, Nashville, TN 37235. * Abstract published in Aduance ACS Abstracts, April 1, 1994. 1 Abbreviations: MBzDMS, monobenzyl meso-2,3-dimercaptosuccinate; Mi-ADMS; monoisoamyl meso-2,3-dimercaptosuccinate;MPhOEDMSA, mesoDMS,mono-Bphenoxyethyl meso-2,3-dimercap~uccinate; 2,3-dimercaptosuccinic acid; BAL, 2,3-dimercapto-l-propanol; DMPS, sodium 2,3-dimercaptopropanesulfonate;DMPA, N-(2,3-dimercaptopropybphthalamic acid;MOAT,multiple organicanion transportsystem; MT, metallothionein; CdMT, cadmium-metallothionein complex. QS93-228~/94/27Q7-Q367$04.50 f0
hydrophobic moiety in the amphipathic dithiocarbamates, which have been found to be effective as cadmiummobilizing agents (3-5, 7). Thus, sodium N-benzyl-Dglucamine-N-carbodithioate(dithiocarbamate) has been shown by Kojima and his co-workers (3-7)to be superior to analogous compounds bearing aliphatic groups in this position (15). A feature of these dithiocarbamate compounds of special interest is the fact that they can remove cadmium from its deposits in renal and hepatic cells. This has been hypothesized to occur via the use of one of the systems used to transport singly charged anions into such cells in vivo (16,17). There are three well-characterized anions whose transport in the kidney has been studied in considerable detail: p-aminohippurate, sulfate, and succinate (18, 19). The effects of these three anions on the ability of MBzDMS to mobilize cadmium into the urine are examined in this study. Any information obtained on the molecular mechanisms of cadmium mobilization should assist in refining the design process for the development of newer, superior antagonists for chronic cadmium intoxication.
Materials and Methods Materials. meso-2,3-Dimercaptosuccinicacid (l), acetyl chloride,triethylamine, cadmium chloride (99.999% 1, Gold Label,
1994 American Chemical Society
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368 Chem. Res. Toxicol., Vol. 7, No. 3, 1994
1. DMSA
2
"2:
3
I
PhCH20H Et3N. 3,
Mei
HS
0 6
L" 5
4
Figure 1. Synthetic method for the preparation of the monobenzyl ester of DMSA. and benzyl alcohol were obtained from Aldrich Chemical Co. (Milwaukee,WI), nitric acid was Baker Instru-Analyzed from J. T. Baker Inc. (Phillipsburg, NJ), and the mercuric chloride was from Fisher Scientific Co. (Fairlawn, NJ). Hydrogen chloride gas was freshly generated by the dropwise addition of concentrated HzSO4 on NaCl in an assembly and then passed through a HzSO4 bubbler for drying. The H2S gas (99% purity) was supplied by A-L Compressed Gases, Inc. (Nashville, TN). (Mi-ADMS)which Monoisoamyl meso-2,3-dimercaptosuccinate served as standard drug was prepared as described previously (12). Radioactive cadmium (1Wd) was obtained from DuPont NEN Products (North Billerica, MA) and had a radiochemical purity of 99% with a specific activity of 2.56 rCi/mg. Melting points were determined on a Thomas-Hoover stirred-liquid apparatus. lH NMR spectra were recorded on a Brucker AC300 FT NMR spectrometer in deuterated solvents using solvent peaks as internal reference (e.g., a septet at 6 2.04 for acetone-&, a quintet at 6 3.3 for MeOH-d4, a singlet at S 4.77 for DsO, and a singlet at 6 7.24 for CDCl3). The chemical shifts are reported in parts per million (6). Thin-layer chromatography was performed on silica gel 60 A plates (Whatman catalog no. 4861 620 with a proprietary fluorescent indicator) using visualization under UV illumination,or in an iodinevapor chamber. Elemental analyses were done on a Carlo Erba Strumentazione model 1106 elemental analyzer, except for the liquid sample (4)which was analyzed by Galbraith Laboratories, Inc. (Knoxville, TN). The synthesis of monobenzyl ester (6,MBzDMS) from meso2,3-dimercaptosuccinicacid (1) was carried out in five steps using the reaction sequence illustrated in Figure 1, as follows. 2,2-Dimethyl-l,3-dithiolane-4,5-cis-dicarboxylic Acid (2). This compound was prepared by adapting the procedure of Gerecke et al. (20) with certain modifications during workup. meso-2,3-Dimercaptosuccinicacid (1, DMSA) (30.00 g, 164.64 mmol) was suspended in anhydrous acetone (600 mL), and dry HC1 gas was bubbled through at 0-5 "C for 2 h or until all the DMSA went into solution while stirring (caution:this operation
must be performed in a well-ventilated hood to avoid any inhalation of corrosive HC1 gas.) The reaction mixture turned clear yellow and was stirred at 22 "C overnight. Solvent was pulled off at reduced pressure, leaving ca. 90 mL which was cooled at 5 OC , neutralized with saturated aqueous NazC03, and then washed with EbO (3 X 100 mL). The aqueous layer was cooled and treated with concentrated HC1 almost to neutrality (slightly acidic) when a massive crystalline solid separated out. The solid was collected on a Buchner funnel and dried. Further purification was effected by dissolution of the crude product in acetone and discarding the undissolved impurity, if any. Concentration of the acetone solution to ca. 25 mL followed by the addition of hexane (100 mL) afforded the dithiolane 2 as a white crystalline solid: yield 30.10 g (82.3%);mp 155-157 "C dec (lit. mp 156-157 "C; 20); 'H NMR (DzO) 6 4.77 (8, 2 H), 1.82 (8, 3 H), 1.75 (8, 3 H). Anal. Calcd for C7H1004S2: C, 37.82; H, 4.52. Found C, 39.73; H, 4.89. 2,2-Dimethyl-l,3-dithiolane-4,5-cis-dicarboxylic Acid Anhydride (3). Acetyl chloride (45 mL) was added slowly to the anhydrous powdered diacid 2 (25.00 g, 112.48 mmol) at 5-10 "C over 30 min under Nz. When the exothermic reaction subsided,
the contents were refluxed for 1 h and then cooled to 22 "C, following which nitrogen was bubbled through the mixture to purge most of the remaining acetyl chloride. Petroleum ether (250 mL) was added, shaken well, and let stand at 0-5 "C for 2 h. The white hazy crystalline solid was collected and washed with cold petroleum ether (2 X 30 mL). Further purification was achieved by dissolving the crude solid in acetone (300 mL), discarding any suspended impurity (NaCl), concentrating the filtrate to ca. 50 mL, and reprecipitating the product by the addition of petroleum ether (250 mL) as a white crystalline solid: yield 20.00 g (87.9%);mp 142-144 "C (lit. mp 142-144 "C; 20); 1H NMR (acetone-de)6 4.90 (s, 2 H), 1.90 (s,3 H), 1.81 (s,3 H). Anal. Calcd for C7Hs03Sz: C, 41.16; H, 3.95. Found: C, 40.96; H, 3.87. The crude dithiolane dicarboxylicacid 3 could be used directly without further purification, since excess NaCl could be easily removed during the anhydride purification by the use of acetone. 2,2-Dimethyl-1,3-dithiolane-4,5-cis-dicarboxylic Acid Monobenzyl Ester (4). A mixture of benzyl alcohol (2.16 g, 19.58mmol)and triethylamine (2.14g, 19.58mmol)inanhydrous methylene chloride (40 mL) was added dropwise (30 min) to a stirred solution of anhydride 3 (4.00 g, 19.58 mmol) in dry methylene chloride (100 mL) under Nz at 5-10 "C. The reaction mixture was stirred for an additional 1 h at room temperature and then washed with 2 M aqueous hydrochloric acid (4 X 40 mL), and finally with water (4 X 40 mL). The organic extract was dried over anhydrous magnesium sulfate and evaporated to give a highly viscous yellowish oil: yield 5.00 g (81.7 % ): TLC Rf 0.46,10% MeOH in CHZC12; lH NMR (CDCls) S 7.36-7.26 (m, 5 H), 6.15 (s, 3 H), 1.80 ( 8 , 3 H). Anal. Calcd for C14H1704Sz: C, 54.00; H, 5.18. Found C, 53.86; H, 5.18. Removal of IsopropylideneGroup from 4 by Conversion into the Corresponding Hg Complex (5). This procedure was based on the methods for the removal of isopropylidene group from a thioketal(21-23) and was carried out as described below. (Caution: Extreme care must be taken during handling of
mercury compounds since they are highly toxic.) Mercuric chloride (6.00 g, 22.09 mmol) solution in a 4 1 acetonitrilewater mixture (15mL) was added (30 min) to astirred solution of the thioketal ester 4 (4.60 g, 14.73 mmol) in the same solvent (100 mL) under Nz at 22 "C and stirred overnight. The reaction mixture initially turned turbid then a white precipitate was formed which finally cleared up. Solventwas removed under reduced pressure, and the thick viscous residue was dried under vacuum for 2 h. The resulting mass was thoroughly washed with water and dried under vacuum to give crude mercury complex. No further purification was effected at this stage. Yield 5.39 g (64.2% based on structure 5), mp >300 "C. Anal. Calcd for CzzHlsO&Hg,: C, 23.17; H, 1.59. Found: C, 23.32; H, 2.11. Monobenzyl meiw2f-Dimercaptosuccinata(6,MBzDMS). A slow stream of hydrogen sulfide gas was carefully bubbled through asuspension of the mercury complex 5 (4.0 g, 3.51 mmol) in methanol (100 mL) at 22 "C over a period of 1h while stirring (caution: H$ is a highly toxic gas of pungently disagreeable
odor, and therefore this operation must be conducted strictly in a highly efficient hood). The progress of the reaction was evident by the disappearance of the complex with simultaneous formation of a black HgS precipitate. After 2 h of additional stirring the black precipitate was removed by suction filtration through a cellulose filter aid, and the clear colorless filtrate was concentrated under reduced pressure to give crude dithiol6 as a slightly yellowish-white solid. This was stirred with toluene (20 mL) to get rid of colored impurities, leaving pure 6 as a perfectly white solid: 1.40 g (73.3% yield); mp 135-137 "C; TLC R10.25,20% MeOH in CHzC12; 'H NMR (acetone-de)6 7.44-7.34 (m, 5 H), 5.26 (s,2 H), 3.74-3.63 (m, 2 H), 2.94-2.51 (2d, J = 10.5 Hz, 2 H). Anal. Calcd for CllH1~04S~: C, 48.51; H, 4.44. Found: C, 47.97; H, 4.03. Direct Esterification of DMSA (Figure 2). (A) Unusual Formation of 2-Phenyl-l,3-dithiolane-4,5-cis-dicarboxylic Acid Monobenzyl Ester (7) by the Direct Esterification of DMSA with Benzyl Alcohol. A heterogeneous mixture of
In Vivo Cd-Mobilizing Agents DMSA (1.00 g, 5.49 mmol), benzyl alcohol (25 mL), and concentrated HC1 (1 mL, 36% w/w) were heated in a mantle under a reflux condenser for 30 min, at which time the reaction mixture cleared up and turned yellow. The contents were further stirred for 18 h. The excess benzyl alcohol was removed under vacuum (at 115 "C bath temperature, 60-70 "C distillation temperature) until 5 mL of the oil remained. Cyclohexane (50 mL) was added, and the mixture was shaken well and set aside. The creamy-white crystalline solid (mp 145-150 "C) which separated was collected and recrystallized from toluene as white crystals: yield 1.4 g (70.8%); mp 157-159 OC; TLC single-spot Rf 0.53, 12.5% MeOH in CHzClz; 1H NMR (CDC13) 6 7.50-7.17 (m, 10 H), 5.70 (8, 1H), 5.21 (q, 2 H), 4.65 (very br, 1H, COOH), 4.65 (8 flanked by two very small peaks at the base, 2 H).Anal. Calcd for C&Ila04Sz: C, 59.98; H, 4.48. Found C, 60.14; H, 4.40.
(B) 2-Phenyl-1,3-dithiolane-4,5-cis-dicarboxylic Acid (8). Hydrochloric acid gas was passed through a heterogeneous suspension of DMSA (1) (2.5 g, 13.72 mmol) in benzaldehyde (20 mmol) at 22 OC for 1 h, and the mixture was then stirred at 120 OC for 12 h under nitrogen. The clear pinkish-yellow reaction mixture was cooled at 22 "C, and hexane (50 mL) was added to precipitate the hazy-white product, which after washing with chloroform afforded light yellowish-white crystals of the benzylidene 8: yield 2.6 g (70.0 % ); mp 225-227 "C; 1HNMR (acetonede) 6 7.72-7.69 (m, 2 H, Ar), 7.46-7.36 (m, 3 H, Ar), 6.00 (8, 1 H, PhCg), 4.98 (s,2 H). Anal. Calcd for CllH1004SZ: C, 48.88; H, 3.72. Found C, 48.99; H, 3.20. Use of aqueous HCl(3 mL, 36% w/w) instead of gaseous HCl resulted in the formationof the same product in good yield without any difficulty. Animal Studies. (A) Mobilization of Cadmium in Mice. Male mice of the (C57BL/6 X DBA/2)F1 strain were obtained from Charles River Laboratories (Raleigh,NC) and weighed 2123 g at the beginning of the experiment. They were housed in an AAALAC-accredited facility maintained at, 22.2 "C with a 5040% relative humidity. There were 15 complete air changes/ h, and the photoperiodicity was 12 h (0600-1800) light and 12 h ( 1 8 0 0 6 0 0 )dark. Wayne Rodent Blox (Teklad Diets, Madison, WI) and tap water were provided at all times. All mice received a single ip injection of 0.03 mg of CdC1~2.5Hz0 in 1.0 mL of 0.9% NaCl solution containing 1.0 pCi of ' W d (specific activity 2.56 pCi/mg) and were divided into 4 groups. Treatment with chelating agents was initiated 5 days later. One untreated group served as controls. The second group received MGADMS, given PO (by intragastric instillation), as a positive control. The third and fourth groups received MBzDMS PO or ip, respectively. Mi-ADMS was dissolved in a 1% NaHCO3 solution, while MBzDMS was suspended in 0.9% NaCl solution for administration. The dose of each compound administered was 1.0 mmol/kg, and it was given in a volume of 0.5 mL/30 g of mouse weight. One day later, whole-body radioactivity was measured with the apparatus described earlier which compares the whole body levels in mice with that of a mouse phantom containing 1.0 pCi of "Wd of the specific activity 2.56 pCi/mg (24). Mice were then sacrificed (by cervical dislocation) for the removal of selected organs, and the radioactivity in these was measured in a Beckman Biogamma I well counter. Statistical evaluation of data was done by use of Duncan's multiple range test (SAS/STAT SAS Industries, Cary, NG). (B) Biliary and Urinary Excretion of Cadmium in the Rat. Female Sprague-Dawleyrats, weighing approximately 200 g, were used in the experiments involving biliary cannulations. The animals were obtained from Sasco (Omaha, NB). They were housed in a AAALAC-approved facility at 22.2 "C with 5040% relative humidity and were provided with Purina Lab Chow obtained from Purina Mills (St. Louis, MO) and water at all times. Cadmium loading for the biliary excretion studies and preliminary studies of urinary excretion were achieved by ip administration of CdC12.2.5Hz0 using the following schedule: on day 1 0.5 mg of Cd/kg was given, and 1.0 mg of Cd/kg was
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 369 administered on days 2,3,4,5,6, and 7 for a total of 6.5 mg Cd/kg body wt. The biliary cannulations were performed 4 days after the last cadmium injection. A peritonitis was observed subsequent to the ip administration of cadmium chloridewhose severity decreases with time, and which did not interfere with the action of the chelating agent. Bile duct cannulations were performed while the animals were maintained under sodium pentobarbital (Nembutal) anesthesia. Access to the bile ducts was gained through a ventral midline incision which was closed by sutures after the cannulation. The animals were maintained under anesthesia for the duration (5 h) of the bile collection period. At the end of the experiment the rats were sacrificed by cervical dislocation. The animals used for the studies of cadmium excretion in urine were loaded with cadmium using iv injections of 1 mg of Cd/kg/day for 3 days; the experiments were started 4 days after the last cadmium injection. The iv injections were given in the tail vein of the rats. The appropriate treatments were administered, and urine samples were collected for a 15-hperiod. The use of two successive collection periods allowed each animal to serve as its own positive control, and a set of separate positive controls was also run. At the end of the experiment the rats were sacrificed by cervical dislocation. (C) Anionic Transport Competition Experiments. (1) 'Cocktail" Experiment. To test the hypothesis that anionic transport of the chelating agent through the renal tubule was responsible for the mobilization of the cadmium in the urine, the chelating agent was coadministered with a mixture containing a 10-foldexcess of each of three anions whose transport systems were considered to be of potential importance for renal transport of the chelating agent. Under such conditions the ability of the MBzDMS to enhance the urinary excretion of cadmium should be reduced by competition for the anionictransport system which it used. Female Sprague-Dawley rats were loaded with iv injections of 1 mg of Cd/kg/day for 3 days. Three days later at 5 p.m. the animals were administered 0.2 mmol/kg MBzDMS plus a 10-foldmolar excess of each potential transport competitor iv, and the urine was collectedfor 15 h and analyzed for cadmium. On the following day at 5 p.m. the animals were administered MBzDMS at 0.2 mmol/kg iv, and the urine was again collected for 15 h and analyzed for cadmium. (2) Succinate, Sulfate, and pAminohipppurate Competition Experiments. These experiments were run in a similar manner to the "cocktail"experiment, but only one of the possible competing species was present in any experiment. (D) Cadmium Determinations. Cadmium determinations were performed on urine and bile samples which were subjected to nitric acid digestion. Urine or bile samples (200 pL) were heated at 140 OC with an equal volume of concentrated nitric acid until dryness. An additional 20 pL of nitric acid was added to each, and the samples were heated to dissolve the residue. Deionized water was used to bring the samples to volume. Cadmium analyses were then performed using a Perkin-Elmer 4000 atomic absorption spectrometer equipped with a PerkinElmer 400 graphite furnace and operated using standard conditions with deuterium background correction. (E) Statistical Analyses. The data were analyzed using an analysisof variance program followed by Duncan's multiple range test. The data are given as means standard deviations.
*
Results Monobenzyl meso-2,3-dimercaptosuccinate(6)is a white crystalline solid possessing fairly good stability in ambient conditions under an inert atmosphere. The synthesis was achieved via the anhydride route as shown in Figure 1. The vicinal dithiol groups of DMSA were protected by a n iaopropylidene group before transformation to the cyclic anhydride 3 by a published procedure (20).Anhydride could not be obtained if the SH groups were individually protected as acetylthio groups, owing to the unfavorable
370 Chem. Res. Toxicol., Vol. 7,No. 3, 1994
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Ph
I
0 7
PhCH,OH
Figure 2. Products obtained by the direct reaction of benzyl alcohol and DMSA at elevated temperatures. arrangement of the carboxylic acid groups. Opening up of the anhydride 3 with 1 mol of benzyl alcohol in the presence of 2 mol of triethylamine as base in CH2C12 afforded the SH-protected monobenzyl ester 4 in quantitative yield. The deprotection of the mercapto groups was accomplished by converting 4 into a mercury complex, 5. The removal of the mercury was effected by bubbling H2S into a methanolic suspension of the mercury complex. The overall yield of 6 from the anhydride 3 was ca. 50%. No further attempts were made to optimize the yield. Direct esterification has been extensively used for the synthesis of a variety of dialkyl and monoalkyl esters of DMSA (under different conditions) (12,13, 16, 25, 26). Surprisingly, our efforts to prepare the monobenzyl ester 6 from DMSA (1) by the direct esterification using benzyl alcohol in acidic media (Figure 2) always resulted in the formation of an unexpected product which was characterized to be 7, where the two vicinal -SH groups were protected by a benzylidene group. It is proposed that the formation of 7 from DMSA via an anhydride 9 occurs as follows. Initially, DMSA forms a benzylidene dicarboxylic acid 8 by reacting with benzaldehyde (probably formed by the oxidation of benzyl alcohol in acidic medium at high temperature). The diacid 8 then undergoes dehydration at high temperature to form the anhydride 9, which finally opens up with 1 mol of benzyl alcohol to yield 7. We attempted the synthesis of 7 following the steps mentioned above. The dithiolane dicarboxylicacid 8 from DMSA and benzaldehyde was successfully prepared in the presence of HC1 in high yield (70%),but dehydration of this compound to give 9 using acetyl chloride as in Figure 1 for isopropylidene anhydride 3 did not succeed. Information on the ability of MBzDMS to mobilize cadmium from mice is shown in Table 1, where it is compared to Mi-ADMS. It is apparent that orally administered MBzDMS is surprisingly ineffective in removing cadmium from the liver, but is equivalent to Mi-ADMS in removing cadmium from the kidneys. The ip administration of MBzDMS results in a significant enhancement of the cadmium mobilization from the whole body, kidney, and liver, with no redistribution of the metal to the brain. The time course of the biliary excretion of cadmium following the administration of MBzDMS to cadmiumloaded animals is shown in Table 2. The amount of cadmium in the bile of cadmium-loaded rata increases by a factor of over 100-fold following the administration of 1 mmol of MBzDMS/kg iv. The ip administration of 1 mmol/kg of MBzDMS to cadmium-loaded rata also induces a marked increase in
the urinary excretion of cadmium. During a control period in a preliminary study, female Sprague-Dawley rata loaded with 6.5 mg/kg CdCl2 over a 7-day period, 3 days prior to chelating agent treatment, excreted 0.031 f 0.002 pg of cadmium during the 4 h subsequent to an injection of 2 mL of saline. The same animals excreted 1.87 f 0.40 pg of cadmium during the 4-h period following the ip administration of 1mmol/kg MBzDMS in 2 mL of saline. These differences are significant, p I 0.02. The results of the "cocktail experiment" and those for the experiments in which a single competing anion was present are shown in Table 3. The control data show that (a) the rate of excretion of cadmium in the untreated cadmium-loaded rat is very low, of the order of 6 ng of Cd/4 h, and (b) the amount of cadmium excreted drops significantlyon the second day when a cadmium-mobilizing agent is administered at the same dose on two successive days. Group 2 contains data on the urinary excretion of cadmium after an initial injection of MBzDMS; group 3 contains data on the same group of rata when they are subsequently given another injection of MBzDMS on the second day. Together these two groups represent what would be the normal course of cadmium mobilization in the absence of complicating factors. The cocktail causes a significant decrease in the amount of cadmium excreted in the urine during the initial injection, while the administration of the same amount of MBzDMS on the second day leads to a significant increase in the cadmium excreted in the urine (compare groups 4 and 5 with groups 2 and 3 in Table 3). The coadministration of succinate or sulfate leads to a higher value for urinary cadmium on the first day and a lower value on the second day. This is a similar pattern to that of the control data where there is no interference with the action of the MBzDMS on the first day. The coadministration of p-aminohippurate leads to a significant decrease in urinary cadmium excretion on the first day and a significant increase on the second day (compare groups 8 and 9 with groups 2 and 3 in Table 3). The results show that the coadministration of p-aminohippurate clearly interferes with the mobilization of cadmium into the urine by MBzDMS. Taken as a whole, the data presented indicate that this compound is effective in the mobilization of intracellular cadmium when administered by all of the routes used and is effective in both mice and rats.
Discussion While many drugs are designed to pass through the lipid portion of the cellular membranes (23,cells also possess transport systems for charged species such as the cations Na+, K+, Ca2+, and Mg2+ , amino acids, acetate, and benzoate as well as highly polar molecules such as glucose. Drugs can use such transport systems, as the folic acid antagonist methotrexate undergoes active transport into cancer cells (28). There is no a priori reason why such transport systems may not also be utilized to carry chelating agents into cells. This possibility is especially promising when toxic metals accumulate in the kidney and the liver, where anion transport systems play a special role. The main limitation in achieving such a result is in the design of the chelating agent to "fit" the requirements of the transport system. The MBzDMS molecule will bear a single negative charge from its ionized carboxylate group at physiological pH, and one may hypothesize that it is capable of using an anion transport system on the
In Vivo Cd-Mobilizing Agents
Chem. Res. Toxicol., Vol. 7, No. 3, 1994 371
Table 1. Comparison of MBzDMS and Mi-ADMS as Cadmium-Mobilizing Agents in Male (C57BL/6 X DBA/2)Fl Mice (Nof Cdlg Tissue Wet Wt). mOUD whole body kidney liver brain 13.89 f 0.18 3.25 f 0.12 6.64 f 0.19 0.027 f 0.002 control ( N = 8) 2.39 f 0.17b9d 2.88 f 0.466d 0.027 f 0.001 Mi-ADMS, PO ( N = 7) 7.30 f 0.23M 2.50 f 0.25bd MBzDMS, PO ( N = 6) 11.87 f 0.5Fibd 6.07 f 0.60bsd 0.025 f 0.002 8.67 f 0.30b*c 1.70 f 0.3lb& 3.74 f 0.44b.C 0.027 f 0.002 MBzDMS, ip (N = 6) 0 The cadmium-loaded mice were given 1.0 mmol/kg of the indicated compound via the route shown. One day later, the whole-body count of each animal was recorded. The animals were then sacrificed, and the organs were dissected and counted. N indicates the number of animals per group. b Significantly different from control values, p 5 0.05. Significantly different from MBzDMS,po, p I0.05. d Significantly different from MBzDMS, ip, p 5 0.05. Table 2. Biliary Excretion of Cadmium Prior to and following MBzDMS Administration at 1 mmol/k@_______ concentrations concentrations totals (rea of W a ) totals (fig of Cd) ( l a of Cd/a) (fig of Cd) 0.013 f 0.006 3rd hour basal period 0.015 f 0.03 0.87 f 0.38 0.73 f 0.32 7.17 f 3.23 1st hour 7.57 f 2.35 4th hour 0.83 f 0.42 0.43 f 0.06 2.20 f 1.02 2nd hour 3.50 f 1.91 5th hour 0.23 f 0.06 0.10 f 0.02 induced totals 10.90 f 2.11 0 Three female Sprague-Dawleyrata were loaded with cadmium by the ip administration of a total of 6.5 mg of Cd/kg given as a series of daily injections at the levels of 0.5 mg of Cd/kg for the first day and at 1.0 mg of Cd/kg for each of the following 6 days. Four days after loading, the bile ducts of the animals were cannulated and bile was collected as shown above. The basal period was 1 h long, after which the animals were administered MBzDMS iv at 1 mmol/kg. ~
Table 3. Urinary Cadmium Excretion of Cadmium-Loaded Rats under Various Conditions. urinary cadmium group no. group (ng of Cd/15 h) 1 untreated control 5.8 f 4.gb 2 MBzDMS day 1 control 186.0 f 39.2 3 MBzDMS day 2 control 115.5 f 34.gC 4 MBzDMS + cocktail (day 1) 107.6 f 34.W 5 MBzDMS (day 2) 224.9 10l.ldse 6 MBzDMS + succinate (day 1) 168.6 f 85.8 7 MBzDMS (day 2) 135.5 f 41.8 8 MBzDMS + p-aminohippurate 106.1 f 36.7c (day 1) 9 MBzDMS (day 2) 200.4 33.8‘ 10 MBzDMS + sulfate (day 1) 180.7 f 38.4 11 MBzDMS (day 2) 124.5 k 32.6 There were 6 animals in each group, loaded with cadmium as described in the Materials and Methods. On day 3 after the completionof the cadmium loading,the urine of the untreated control group was collected for 15 h and analyzed for cadmium. On day 3 after the completion of the cadmium loading, the ‘cocktail” group animals were given an injection of MBzDMS (0.2 mmol/kg, iv) which also contained 10 mmol/kg each of sodium succinate, sodium sulfate, and sodiump-aminohippurate. In the remaining groups, each animal was removed from ita food supply and received an iv injection (0.2 mmol of MBzDMS/kg + 2.0 mmol/kg of the sodium salt of the anion indicated) at 5 p.m. on day 1 and was placed in a metabolic cage where the urine was collected for 15 h. At 8 a.m. the next day the urine samples were analyzed and the animals were returned to their food. At 5 p.m. on day 2, each animal received an injection of 0.2 mmol of MBzDMS/kg iv and was then placed in a metabolic cage, and ita urine was collected for 15 h. A t 8 a.m. the following day, the urine samples were removed for analysis and the animals returned to their food. On the following day the animals were again placed in metabolic cages after they were were given an iv injection of 0.2 mmol/kg MBzDMS alone, and again urine was collected for 15 h and analyzed for cadmium. The animals were allowed water ad libitum during the entire experiment. b This group differs significantly from all other groups in the table, p I0.05. Differs significantly from the MBzDMS day 1 control group, p 5 0.05. Differs significantly from the MBzDMS + cocktail group,p I0.05. e Differs significantly from the MBzDMS day 2 control group, p 5 0.05.
membrane of renal or hepatic cells to gain access to intracellular cadmium inside such cells. The ability of MBzDMS to mobilize intracellular hepatic and renal cadmium when administered to rata or mice via various routes indicates that this particular structure has general cadmium-mobilizing properties.
~~~
Compounds of this sort are designed primarily to mobilize a larger portion of intracellular cadmium via the biliary route. In a recent report from this laboratory on a structurally related compound, i.e., mono-2-phenoxyethyl meso-2,3-dimercaptosuccinate(MPhOEDMS), it was discovered that fecal excretion is the predominant route of cadmium mobilization which eliminates approximately 8-fold excess cadmium than through the urine (29). The study also revealed that the majority of cadmium excretion occurred within 5 h of the initial administration of the aforementioned compound. In the kidney, three of the anionic transport systems which have been well characterized are those designated the succinate, the sulfate, and the p-aminohippurate transport systems (18, 19, 30). For the liver, which transports a number of anions into hepatocytes and excretes a variety of chemical species into the bile (31), these transport systems include some less specific ones, such as the MOAT (multiple organic anion transport system) (32). The liver and the kidney are the principal sites of cadmium accumulation, and this does not appear to change with changes in the route of administration (33). The data from the competition experiments presented in Table 3 are consistent with hypothesis that the transport of MBzDMS into the renal cells is via the p-aminohippurate system. The fact that the MBzDMS is significantly more effective in removing cadmium from ita hepatic than from ita renal deposist is an advantage. There is considerable evidence to suggest that the renal toxicity of cadmium is due to the movement of CdMT from liver into the blood stream and then into the proximal tubule following glomerular filtration. The injection of CdMT into experimental animals causes renal proximal cell damage (34). There is, not surprisingly, a similar relationship involving CdMT and renal dysfunction in cadmium-exposed humans. Thus studies on cadmium-exposed populations have demonstrated a dose-response relationship between dietary cadmium intakes and the amounts of CdMT in the urine (35)and a similar relationship for cadmium intakes and renal dysfunction (36). The structures of the various vicinal dithiols which have been examined as potential mobilizing agents for the
Jones et al.
372 Chem. Res. Toxicol., Vol. 7, No. 3, 1994 Table 4. Vicinal Dithiols as Cadmium-Mobilizing Agents compound 1,2-ethanedithiol 1,2-propanedithiol 1,2,3-propanetrithiol BAL 2,3-dimercaptopropanoicacid 2,3-dimercapto-l,4-butanediol (DMBD)
effectiveness‘ slightly effective slightly effective effective effective very slightly effective effective
refs 41 41 41 42,38 43 44
compound DMSA DMPS DMSA diesters DMSA monoesters DMPA BAL-glucoside
effectivenessa ineffective ineffective effective effective effective effective
refs 45-47 41,4548 13,25 12 49 48
a The structures of the compounds cited above are shown in Figure 3. The term ‘effective” as used here refers to the ability of the compound to cause a significant decrease in the cadmium burden (in whole body, liver, and/or kidney) of an animal which has been loaded with cadmium at least 24 h prior to the administration of the chelating agent.
I ,2-Ethanedithiol
BAL
HS &OH
1.2-Propanedithiol
DMPS
H S h o H 0
2.3-Dimcrcapto- 1,4butanediol (DMBD)
DMFA
DMSA
1,2,3-PropanetrithioI
2,3-Dimercaptopropanoic acid
H S h o H 0
DMSA-Monoesters
H S h o R 0
DMSA-Diesters
BAL-Glucoside
Figure 3. Structures of vicinal dithiols which have been examined for their ability to mobilize cadmium from its intracellular deposits.
removal of cadmium from its intracellular deposits are presented in Table 4, and the structures of these compounds are shown in Figure 3. It must be noted that uicinal dithiols appear to be required, as monothiols or dithiols with nonvicinal SH groups appear to be ineffective (39, 44,45). An examination of the structures shows that first, simple nonionic dithiols, such as 2,3-dimercapto-l-propanol (BAL), BAL-glucoside, alkyl vicinal dithiols, and the diesters of DMSA, all can mobilize such cadmium. This is due to the ability of such compounds to pass readily through the cellular membranes of those cells in which the cadmium is deposited. These compounds can also pass through the membranes of organelles, such as the mitochondria, which can be sites for intracellular cadmium deposition. DMSA, the only compound which is expected to carry a +2 charge at physiological pH values, is ineffective. DMSA, with its two carboxylate groups, is very hydrophilic and is also rapidly cleared from the mammalian body. Its failure to remove cadmium can be due either to an inability to gain access to intracellular sites where such cadmium is deposited or to its rapid clearance which does not allow enough time for the reaction between the DMSA and the metallothionein-bound cadmium to proceed to an appreciable extent. An examination of the remaining compounds shows that all are expected
to carry a singlenegative charge a t physiological pH values. Of these, sodium 2,3-dimercaptopropanesnesulfonate(DMPS) has been repeatedly shown to be ineffective. On the other hand, the structurally related DMSA monoesters and N-(2,3-dimercaptopropyl)phthalamicacid (DMPA) have been shown to be effective. The main difference between the effective and the ineffective compounds in this group would appear to be the relative degree of hydrophilicity. DMPS is too hydrophilic, while the DMSA monoesters and DMPA have large non-polar groupings which would be expected to alter their pharmacological behavior. These results indicate that, in the design of new vicinal dithiols to act as cadmium-mobilizing agents, they may (1) be neutral or (2) bear a single negative charge a t physiological pH. If they are neutral, structures which also possesspolar nonionic groups such as BAL-glucoside will be less toxic and perhaps equally effective. If they bear a single negative charge, they also need to have groups of sufficient hydrophobicity to provide a partial offset to any negative charge. It is of interest to note that there is excellent evidence that DMPS is transported through renal tubular cells (37,38),but is unable to remove cadmium from them. In view of the fact that a large amount of the cadmium in such cells is present in the cytoplasm (391,it would appear that the ultimate cause for the inability of the DMPS to remove cadmium from the kidneys is its very rapid passage through this organ. DMPS is able to remove mercury (47)and lead from the kidneys (48). The reason for the great differences in the ability of DMPS to mobilize renal cadmium as compared to renal mercury and lead may well lie in the differences in the rates of the reactions of intracellular DMPS with the normal intracellular complexes of these metals. Of the various monoesters which have been examined as cadmium-mobilizing agents, the most effective on a molar basis is the monoisoamyl ester, Mi-ADMS, with a distinctly reduced effectiveness for analogs with larger and smaller alkyl groups. This maximum in effectiveness with an intermediate member of a group is very common and has been explained by Hansch on the basis a random walk model (40). In conclusion, it seems likely that there are a large number of as yet unknown vicinal dithiol structures which will be capable of mobilizing intracellular cadmium. However, if these are to be reasonable candidates for clinical application, they must also contain structural features which will reduce their toxicity and facilitate their distribution to those organs in which the cadmium is concentrated.
Acknowledgment. The project described was supported by Grant 2 RO1 ES02638-13 from the National Institute of Environmental Health Sciences, NIH, and by funds from the Department of Veterans Affairs.
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In Vivo Cd-Mobilizing Agents
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