DRAFT for review - do not cite or quote 1 2 3

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IPCS EVALUATION OF ANTIDOTES 7 8

IN POISONING BY METALS AND METALLOIDS 9 10

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15 16 17

Unithiol 18

(2,3-Dimercapto-1-propanesulphonic acid, DMPS) 19 20 21 22 23 24 25 26 27

April 2009 28 29 30 31 32

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1. Introduction 33 34 Unithiol (2,3-dimercapto-1-propanesulphonic acid, DMPS) was developed and first 35 used in Russia in the 1950s (Petrunkin, 1956; Klimova, 1958), and later used in China 36 (He et al., 1984). It only became more widely used in America and Western Europe 37 since the mid-1970s (Hruby & Donner, 1987), and particularly since the late 1970s 38 when the Heyl Company in Germany began production (Aposhian, 1982; Aposhian et 39 al., 1984). 40 41 Both unithiol and succimer (2,3-dimercaptosuccinic acid, DMSA) are derivatives of 42 dimercaprol (2,3-dimercapto-1-propanol, British Anti-Lewisite, BAL), and they are 43 replacing dimercaprol as the main antidote used in the management of heavy metal 44 poisoning (Hruby & Donner, 1987; Aposhian et al., 1995; Andersen, 1999). These 45 derivatives have several advantages over dimercaprol including lower toxicity, 46 increased solubility in water and lower lipid solubility. It is due to these properties that 47 they are effective by oral administration (Hruby & Donner, 1987). Succimer is less 48 toxic than unithiol and where these two drugs appear to have similar efficacy as an 49 antidote for a particular metal, succimer is generally preferred. 50 51 Unithiol has been used in the management of acute and chronic poisoning with a 52 number of different metals and metalloids, and is particularly useful for arsenic, 53 bismuth and mercury. Unithiol can be given parenterally or orally depending on the 54 clinical situation and severity of poisoning. It is well tolerated and adverse effects are 55 relatively rare. Most common adverse effects are skin reactions such as rashes, 56 pruritis and blistering which are allergic in origin. Most resolve within a few days and 57 generally no treatment is required, but antihistamines and/or corticosteroids may be 58 given if necessary. 59 60 61 2. Name and chemical formula 62 63 International non-proprietary name: Unithiol 64 65 Synonyms: DMPS, sodium (DL)-2,3-dimercaptopropane-1-sulphonate, sodium 2,3-66 dimercaptopropanesulphonate 67 68 IUPAC name: Sodium D,L-2,3-dimercapto-1-propanesulphonic acid 69 70 CAS No.: 4076-02-2 71 72 Chemical formula: H2C(SH)-HC(SH)-H2CSO-3Na.H2O 73 74 75 76

SH

SO3HSH

77

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Relative molecular mass: 228.28 (monohydrate) 78 79 Commercial Names: Dimaval® 80 81 Conversion: 1 g = 4.4 mmol 82 1 mmol = 228.3 mg 83 1 g/L = 4.4 mmol/L 84 1 mmol/L = 0.228 g/L 85

86 87

3. Physico-chemical properties 88 89 Physical condition: White crystalline powder 90 91 Melting point: 2350C (decomposes) 92 93 Boiling point: Not applicable 94 95 Solubility: Readily soluble in water (350 mg/ml); not readily soluble in 96

ethanol; not soluble in apolar solvents 97 98 Optical properties: Not applicable as the racemate is used 99 100 Acidity: pH 4.5-5.5 of an 1% aqueous solution 101 102 pKa: Not known 103 104 Stability in light: No specific advice with respect to storage is necessary 105 106 Thermal stability: Stable (e.g., aqueous solution may be sterilized and the 107

substance may also be heated for drying) 108 109 Refractive index and 110 specific gravity: Not applicable 111 112 Loss of weight on drying: 6-8% when dried to constant weight at 1000C 113 114 115 4. Pharmaceutical formulation and synthesis 116 117 4.1 Routes of Synthesis 118 119 Procedures for the synthesis of unithiol were first described in the 1950s (Johary & 120 Owen, 1955; Petrunkin, 1956). A short description of possible ways of synthesis for 121 unithiol is also given in Hopkins (1981): sodium 2-propanesulphonate is brominated in 122 acetic acid with bromine which gives sodium 2,3-dibromopropanesulphonate. The 123 latter may either be treated with sodium hydrosulphide to give unithiol or may be 124 treated with acetylthiopropane sulphonate which is then hydrolysed with hot aqueous 125 acetic acid thus leading to unithiol. 126 127

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4.2 Manufacturing Process 128 129 The Heyl company uses a patented manufacturing process which involves 130 precipitation of unithiol as the lead salt, after which unithiol is released by addition of 131 hydrogen sulphide. The unithiol is subsequently recrystallised from alcohol (Ruprecht, 132 1997). 133 134 4.2.1 Parenteral Solution 135 136 The crystalline unithiol is diluted in freshly distilled water suited for injection. The 137 sterile filtered solution is then filled into ampoules. All steps have to be performed in 138 an atmosphere of sterile nitrogen in order to protect the sensitive compound against 139 oxidation. For the same reason only non-metallic working materials should be used. 140 A complex-forming agent such as sodium edetate (1% of the amount of unithiol) may 141 be added to the solution (water for injection) in order to bind ions eventually released 142 from working materials. The ampoules are then sterilized. 143 144 4.2.2 Capsules 145 146 The active compound is thoroughly mixed with the filling aid until homogeneity is 147 achieved. Thereafter the mixture is filled into commercially available hard gelatine 148 capsules. 149 150 4.3 Presentation and formulation 151 152 At an analytical grade unithiol is available from several manufacturers. The 153 pharmaceutical product is available from Heyl Chemisch-pharmazeutische Fabrik 154 GmbH & Co. KG, Berlin, Germany, both for oral and parenteral administration. The 155 sodium salt of a racemic mixture is used for medical purposes. 156 157 Unithiol is available in a pharmaceutical preparation as capsules and a parenteral 158 injection. The capsules contain 100 mg unithiol and the ampoules 250 mg as a 5% 159 solution. The injection can be administered either intravenously or intramuscularly. 160 161 162 5. Analytical methods 163 164 5.1 Quality control procedures for the antidote 165 166 The quality control procedures listed below are oriented towards national and 167 supranational pharmacopoeial standards. Quality control parameters for the antidote 168 include 169

• Identity 170

• Purity 171

• By-products (mainly disulphides) are assayed by high performance liquid 172 chromatography (HPLC) and should not amount to more than 5% of total 173 peak area. 174

• Bromide content: maximum 0.5% (as potassium bromide). 175

• Heavy metals: maximum 20 ppm (as lead). 176

• Loss of weight on drying: 6.0-8.0%. 177

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• pH of an 1% aqueous solution: 4.5-5.5. 178

• The content may be assayed by iodometric titration. The assay by HPLC is 179 preferred however, because of its specificity. At least 95% is set for 180 requirements. The method has been validated formerly possessing a 181 variation coefficient of 1.5% and a recovery rate of 99.7%. 182

183 The pharmaceutical preparations are additionally controlled for 184

• Ampoules 185

• Sterility. 186

• Filling volume. 187

• Optical appearance (colour, clarity of the solution, particulate matter). 188

• Testing for leaks. 189 190

• Capsules 191

• Uniformity of filling weight. 192

• Disintegration time (maximum 30 minutes in water). 193

• Optical appearance. 194 195 5.2 Methods for identification of the antidote 196 197 Several methods are available to identify the antidote including HPLC, infrared 198 spectroscopy, colour reaction with sodium nitroprusside and flame spectroscopy 199 specifically for the sodium in the molecule. 200 201 5.3 Methods for identification of the antidote in biological samples 202 203 Methods for qualitative and quantitative determination of unithiol and its metabolites 204 are published by Maiorino et al. (1987; 1988; 1991) and Hurlbut et al. (1994). The 205 urine is treated with sodium borohydride and analysed by HPLC with fluorescence 206 detection. 207 208 5.4 Analysis of the toxic agent in biological samples 209 210 Heavy metals should be analysed in blood and urine before, during and after antidotal 211 therapy. Sensitive methods, such as atomic absorption spectroscopy (AAS) or 212 inductively coupled plasma-atomic emission spectroscopy (ICP-AES), can be used 213 (Berman, 1980; Bertram, 1983). 214 215 216 6. Shelf-life 217 218 The shelf life for the commercial available pharmaceutical preparations Dimaval® is 5 219 years for the capsules and 4 years for the ampoules. The expiry date is stated on 220 each package. Although no special advice for storage is given, it is recommended 221 that capsules are stored in a dry place. 222 223 224 7. General properties 225 226

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Unithiol, like succimer and dimercaprol, owes it metal-binding properties to the 227 presence of two adjacent thiol groups. Unithiol is a water-soluble dithiol, a derivative 228 of dimercaprol and is capable of forming complexes with a number of metals and 229 metalloids. The advantages of unithiol over dimercaprol are: 230

• Lower local and systemic toxicity. 231

• Better solubility in water. 232

• Active by oral administration. 233 234 It should be noted that unithiol is not a true chelating agent; a chelator is a molecule 235 which binds a metal or metalloid ion by at least two functional groups to form a stable 236 ring complex known as a chelate. For mercury, it has been shown that unithiol (and 237 succimer) do not form a true chelate and as such both could be considered 238 suboptimal as metal antidotes (George et al., 2004). However, there are currently 239 no other substances available with the advantages of these two drugs (high water 240 solubility with relatively low toxicity). 241 242 The mechanism of action of unithiol has not been fully elucidated. Most studies on 243 this subject have focused on its interaction with arsenic and mercury. In addition to 244 increasing urinary elimination of arsenic, unithiol has also been shown to alter the 245 relative urinary concentrations of organoarsenic metabolites by interfering with 246 arsenic methylation (Aposian et al., 1997; Gong et al., 2002; Heinrich-Ramm et al., 247 2003). 248 249 Unithiol also promotes mercury excretion and is effective in inhibiting mercury 250 accumulation in renal proximal and distal tubular cells, and protecting against mercury-251 induced renal damage. Studies in chickens (Stewart & Diamond, 1987) and rat 252 kidneys (Klotzbach & Diamond, 1988) have demonstrated that urinary excretion of 253 unithiol is blocked by both the substrate p-aminohippurate (PAH) and the inhibitor 254 probenecid of the organic transport process. In the in vitro study by Zalups et al., 255 (1998) using isolated perfused segments of rabbit proximal tubules the removal of 256 mercury from proximal tubules by unithiol was blocked by the addition of PAH. 257 Islinger et al. (2001) postulated on the transport of unithiol in renal proximal cells. 258 Unithiol enters the cells across the basolateral membrane from the blood, via the 259 organic anion transporter (OAT). Both oxidised and reduced unithiol interact with the 260 transporter, but the majority of unithiol entering cells is probably oxidised since this is 261 present in the blood in a greater concentration. Once in the cell the unithiol is reduced 262 by a glutathione-dependent thiol-disulphide exchange reaction in the proximal tubule 263 cell (Stewart & Diamond, 1988). Unithiol then binds mercury within the cell and the 264 complex exits the cell, presumably via an export pump. The unithiol-mercury complex 265 is not reabsorbed and is excreted in the urine. 266 267 Owing to its high hydrophilicity unithiol is relatively ineffective at clearing metals from 268 the brain (unlike its lipophilic parent compound, dimercaprol) and excretion by the 269 organic anion transporter (expressed in the apical membrane of the choroid plexus) 270 may further reduce the efficacy of unithiol in the brain (Islinger et al., 2001). Of the 271 organic anion transporters expressed in the basolateral proximal tubule cells 272 determined in animal studies (Kojima et al., 2002), recent work has identified (OAT1) 273 as the transporter involved in movement of unithiol into cells; it is a comparatively poor 274 substrate of OAT3 (Koh et al., 2002). 275 276

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Recently, multidrug resistance protein 2 (MRP2 or ATP-binding cassette, sub-family 277 C [ABCC2]) has been showed to be involved in the renal proximal tubular elimination 278 of unithiol complexes of methylmecury (Zalups & Bridges, 2009). 279 280 It is often assumed, that clinical effectiveness of a metal-binding agent is linked to the 281 stability constant in vitro where the greater the stability constant of a metal-binding 282 agent, the greater the mobilisation of that ion following administration of the metal-283 binding agent. However, Jones et al. (1980) found no correlation in an in vivo study in 284 mercury-poisoned mice. Therefore data on stability constants are not given in this 285 monograph, but may be found elsewhere (Casa and Jones, 1979). 286 287 288 8. Animal studies 289 290 8.1 Pharmacodynamics 291 292 There are numerous studies on the effect of unithiol in animals with experimental 293 metal poisoning. Many studies compare the effect of a number of different metal-294 binding agents and in some cases the antidotes are given immediately or sometimes 295 before dosing with the metal. Administration of the antidote with or before exposure 296 does not reflect the clinical situation in human metal poisoning. In addition, the effect 297 of antidotes are measured in a variety of ways including changes in survival rates, 298 urinary excretion, faecal excretion, metal concentrations in organs, body burden and 299 biochemical parameters in target organs. Many studies give conflicting results and 300 this is probably a reflection of a variety of factors including differences in doses of 301 metal and antidote, routes and times of administration and methods of determining 302 elimination and retention. Furthermore, direct extrapolation from animal studies to 303 humans is not possible because of the potential differences in the kinetics of both 304 heavy metals and metal-binding agents such as unithiol between animals and 305 humans. 306 307 8.1.1 Antimony 308 309 Unithiol has been shown to reduce antimony toxicity in animals. 310 311 In a study comparing survival rates of different antidotes in antimony poisoning, mice 312 were given intraperitoneal antimony potassium tartrate 120 mg/kg (LD50 54.6 mg/kg). 313 The antidotes were given by the same route 1 hour later at a dose of 10:1 mole ratio 314 of antidote to antimony (except for dimercaprol which was given at a 1:1 ratio). 315 Succimer and unithiol were found to be the most efficacious antidotes for antimony 316 potassium tartrate poisoning, with succimer the superior of the two (Basinger & 317 Jones, 1981a). 318 319 In an earlier study administration of unithiol was shown to reduce the LD50 of 320 subcutaneous antimony potassium tartrate by a factor of 8 compared to controls 321 (Chih-Chang, 1958). 322 323 8.1.2 Arsenic 324 325 Unithiol has been shown to be of benefit in poisoning with several arsenic 326

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compounds including lewisite (β-chlorovinyl-dichloroarsine). Mückter et al. (1997) 327 argue that unithiol and succimer have advantages over dimercaprol in the treatment of 328 arsenic poisoning since they are more effective in preventing arsenic from crossing 329 epithelial boundaries and entering cells and they enhance the excretion of arsenic 330 more rapidly and completely. In addition, unithiol and succimer are less toxic than 331 dimercaprol. However, dimercaprol appears to be more effective in restoring cellular 332 function to tissues which are poorly penetrated by unithiol or succimer. Dimercaprol 333 also has the disadvantage of increasing arsenic concentrations in the brain; this is not 334 the case with unithiol or succimer. 335 336 Aposhian et al. (1982) demonstrated the effectiveness of unithiol in rabbits exposed to 337 subcutaneous lewisite. Unithiol increased survival when given orally or 338 subcutaneously. Similarly, in mice injected with sodium arsenite (0.14 mmol/kg 339 subcutaneously), intraperitoneal unithiol (0.25 mmol/kg) was a potent antidote, even 340 when given 2 hours later (Tadlock & Aposhian, 1980). 341 342 In rabbits poisoned with dermal lewisite dimercaprol, succimer and unithiol were all 343 shown to reduce the incidence and severity of liver changes. There was no difference 344 between the three agents at the dose of 40 µmol/kg. Compared to dimercaprol, 345 succimer and unithiol may have prolonged survival time and the relatively low toxicity 346 of unithiol and succimer allowed high doses (160 µmol/kg) to be given (Inns & Rice, 347 1993). In a study of intravenous lewisite poisoning in rabbits there was no difference 348 between the level of protection provided by the three antidotes (Inns et al., 1990). 349 350 In mice poisoned with sodium arsenite (0.129 mmol/kg subcutaneously) unithiol (0.8 351 mmol/kg intraperitoneally) given 90 minutes later was found to increase the LD50 by 352 4.2 fold (Aposhian et al., 1981). 353 354 In rabbits poisoned with sodium arsenite (1 mg subcutaneously) given either succimer, 355 unithiol or N-(2,3-dimercaptopropyl) phthalamidic acid (DMPA; 0.2 mmol/kg 356 intramuscularly) 1 hour later, the urinary excretion of total arsenic between 0 and 24 357 hours was elevated after antidote administration. However, urinary excretion of total 358 arsenic between 24 and 48 hours was significantly lower than controls. The three 359 antidotes differed in the proportion of arsenic metabolites in the urine. All increased 360 arsenite excretion by decreased dimethylarsinate excretion. Unithiol and DMPA 361 increased methylarsonate excretion but succimer did not. Both succimer and unithiol 362 increased arsenate excretion. Of the three antidotes used, unithiol was the most 363 effective at removing arsenic from the body (Maiorino & Aposhian, 1985). Unithiol and 364 succimer (both at 50 mg/kg) also significantly increased renal arsenic excretion in rats 365 with chronic arsenic poisoning (sodium arsenate 1 mg/kg orally 6 days a week of 3 366

weeks). Both also restored arsenic-induced inhibition of δ-aminolevulinic acid 367 dehydratase activity and hepatic glutathione concentrations. Although both antidotes 368 reduced arsenic-induced histopathological lesions, succimer was more effective (Flora 369 et al., 1995a). 370 371 Kreppel et al. (1989) compared the effectiveness of D-penicillamine, dimercaprol, 372 unithiol and succimer as antidotes in acute arsenic intoxication using different 373 controlled experimental settings. In one study mice and guinea pigs were injected 374 subcutaneously with 8.4 mg/kg arsenic trioxide (containing a tracer dose of arsenic-375 74). An antidote (0.7 mmol/kg intraperitoneally) was given 30 minutes later. As 376

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determined 4 and 12 hours after the arsenic injection, D-penicillamine was unable to 377 reduce the arsenic-74 content in any organ investigated (blood, liver, kidneys, lungs, 378 heart, brain, testes, spleen, skeletal muscl, and skin). In contrast, dimercaprol, 379 unithiol and succimer markedly reduced the tissue content of arsenic-74 compared 380 to controls. Finally, the ability of the antidotes to reverse biochemical effects of 381 arsenic was investigated in vitro using suspensions of isolated renal tubule cells. 382 The marked inhibition of gluconeogenesis induced by 30 μmol/L arsenic trioxide was 383 almost completely reversed upon addition of 90 μmol of dimercaprol, unithiol or 384 succimer. In this experimental model, too, D-penicillamine was ineffective. 385 386 In mice given arsenic trioxide, intraperitoneal administration of unithiol (0.7 mmol/kg) 387 0.5 minutes later was less effective than the same dose of succimer. When the 388 antidote was given 30 minutes after the arsenic, succimer and unithiol showed 389 reduced but similar efficacy. The efficacy of the antidotes for reducing arsenic 390 organ concentrations was investigated in mice and guinea pigs. Animals received 391 8.4 mg/kg (0.043 mmol/kg) of radiolabelled arsenic trioxide subcutaneously and an 392 antidote (0.7 mmol/kg intraperitoneally) 30 minutes later. Both unithiol and succimer 393 and the two in combination were more effective as reducing organ concentrations of 394 arsenic than dimercaprol. In addition, dimercaprol increased arsenic concentrations 395 in the brain, whereas succimer and unithiol did not. Succimer increased the arsenic 396 content of bile but unithiol and the two in combination did not (Kreppel et al., 1990). 397 398 In rabbits administered radiolabelled arsenic (1 mg/kg subcutaneously as sodium 399 arsenite), dimercaprol (0.2 mmol/kg intramuscularly) 1 hour later was shown to double 400 the arsenic-74 concentration in the brain. In contrast, unithiol (0.2 mmol/kg 401 intramuscularly) was found to decrease the arsenic-74 concentration to about one-fifth 402 of that observed with dimercaprol administration (Hoover & Aposhian, 1983). Schäfer 403 et al. (1991) demonstrated that arsenic depots after injection of arsenic trioxide into 404 mice could be mobilised by oral administration of unithiol or succimer without 405 increasing the brain deposition, however, oral administration of dimercaprol 406 extensively increased the brain deposition or arsenic. 407 408 In mice given arsenic (5 mg subcutaneously as arsenic trioxide) immediately followed 409 by unithiol (100 mg/kg intraperitoneally) arsenic excretion in the faeces exceeded that 410 in the urine (Maehashi & Murata, 1986). In guinea pigs poisoned subcutaneously with 411 arsenic trioxide (2.1 mg/kg) the combination of unithiol (0.1 mmol/kg both 412 intraperitoneally and orally) and cholestyramine (0.2 g/kg orally) significantly enhanced 413 the faecal elimination of arsenic suggesting that interruption of enterohepatic 414 circulation of arsenic may be a valuable adjunct in the treatment of arsenic poisoning. 415 Increased faecal excretion was not observed with intraperitoneal unithiol plus oral 416 cholestyramine or oral plus intraperitoneal unithiol (Reichl et al., 1995). 417 418 Flora et al. (2005) compared the efficiacy of succimer, unithiol and monoisoamyl-419 succimer in rats with chronic arsenic exposure (100 ppm sodium arsenite in drinking 420 water for 10 weeks). Antidotes were given after arsenic exposure at a dose of 50 421 mg/kg for 5 days. Succimer was not effective at resolving arsenic-induced oxidative 422 damage in cells or in reducing the arsenic burden. Unithiol was moderately effective 423 against generation of reactive oxygen species due to intracellular access. However, 424 monoisoamyl-succimer was the most effective antidote in reducing reactive oxygen 425 species in the blood and brain. It was also marginally better at restoring the activity 426

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of antioxidant enzymes. 427 428 An in vitro study of guinea-pig liver treated with arsenic trioxide demonstrated that 429 administration of unithiol (and other antidotes) resulted in a shift to faecal elimination 430 by increasing biliary excretion of arsenic. Unithiol was more effective than succimer 431 or dimercaprol but was not as effective as 2,3-bis-(acetylthio)-propanesulphonamide 432 (BAPSA) (Reichl et al., 1990). 433 434 8.1.3 Beryllium 435 436 Unithiol has been shown to enhance beryllium excretion and reduce beryllium-437 induced toxic effects in experimental animals. 438 439 Unithiol administration (0.7 mmol/kg intraperitoneally) appeared to increase the lethality 440 of beryllium chloride in mice, but the result was not significant (Pethran et al., 1990). 441 442 In rats treated with beryllium (2.5 mg/kg intraperitoneally, as beryllium nitrate), 443 immediate administration of unithiol (50 mg/kg intraperitoneally) or succimer (same 444 dose) was shown to prevent most beryllium-induced biochemical alterations and 445 reduce tissue beryllium concentrations. Unithiol was relatively more effective and 446 resulted in significantly less marked lesions in the liver and kidneys (Mathur et al., 447 1994). 448 449 In another study beryllium nitrate was given to rats for 21 days (0.5 mg/kg, orally 450 daily for 5 days/week) and unithiol or succimer (25 or 50 mg/kg, twice daily for 5 451 days) was administered 24 hours after the last dose of beryllium. Unithiol was 452 effective at reducing beryllium in the liver, spleen and kidneys. The higher dose 453 marginally elevated the faecal excretion of beryllium but also resulted in 454 redistribution of beryllium into blood. Unithiol also reduced hepatic and renal lesions 455 compared to succimer (Flora et al., 1995b). 456 457 In studies comparing antidotal therapy combined with an antioxidant (sodium 458 selenite) supplementation, D-penicillamine with sodium selenite was found to be 459 more efficacious at reducing beryllium toxicity (as measured by glycogen and protein 460 concentrations in the liver, kidneys, lungs and uterus, with concentrations of liver 461 enzymes and beryllium) than unithiol and sodium selenite (Johri et al., 2002; Johri et 462 al., 2004). 463 464 8.1.4 Bismuth 465 466 Several studies comparing different antidotal agents have found unithiol to be an 467 effective antidote in bismuth poisoning. 468 469 In a study of several antidotes comparing efficacy in bismuth poisoning, mice were 470 given intraperitoneal bismuth citrate followed by an antidote 20 minutes later in a 10:1 471 molar ratio antidote:bismuth. All animals treated with unithiol survived and all in the 472 control group died (Basinger et al., 1983). 473 474 In a study of several antidotes comparing efficacy in bismuth poisoning, rats were 475 injected intraperitoneally with colloidal bismuth subcitrate (50 µmol/kg/day, for 14 476

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days). The antidotes were given twice daily (250 µmol/kg/day) for 3 days. The 477 animals were killed on the fourth day and tissue samples analysed. Unithiol, succimer 478 and dimercaprol were most effective in lowering bismuth concentrations in most 479 organs, particularly the kidney and liver, resulting from higher elimination in urine by 480 unithiol and dimercaprol. Dimercaprol was the only antidote effective in lowering 481 bismuth concentrations in brain tissue. It was concluded that unithiol and succimer 482 were the antidotes of choice with dimercaprol reserved for very severe bismuth 483 poisoning because of its own toxicity (Slikkerveer et al., 1992). 484 485 Unithiol was shown to reduce the whole-body burden of bismuth in mice given an 486 intraperitoneal injection of bismuth acetate. The unithiol was given in drinking water 487 (100, 300 or 600 µg/mL) two days prior to and three days after the bismuth. The 488 renal concentration of bismuth was reduced by up to 90% and unithiol also 489 significantly reduced deposition of bismuth in the femur (Jones et al., 1996). 490 491 8.1.5 Cadmium 492 493 Antidotal therapy for cadmium is particularly problematic because the absorbed 494 metal rapidly becomes strongly bound to metallothionein, a low-molecular weight 495 metal-binding protein, whose synthesis is induced by cadmium. Several studies 496 have compared antidote efficacy in cadmium poisoning and concluded that, although 497 unithiol is effective, other metal-binding agents appear to be more efficacious (Eybl 498 et al., 1984; Eybl et al., 1985; Andersen & Nielsen, 1988; Srivastava et al., 1996). 499 500 Unithiol administration (0.7 mmol/kg intraperitoneally) increased the LD50 of cadmium 501 chloride in mice from 9.1 mg/kg to 15.2 mg/kg (Pethran et al., 1990). Aposhian (1982) 502 also demonstrated that unithiol increases survival in mice injected with cadmium 503 chloride. 504 505 In a study comparing antidote efficacy, mice were given intraperitoneal cadmium 506 chloride at the previously determined LD50 (0.0267 mmol/kg). This was followed by 507 the antidote at a dose of 1:2 or 1:5 (cadmium:antidote) molar ratios by the same route. 508 The animals were killed after 14 days. Succimer and unithiol were found to be most 509 effective at reducing lethality and the cadmium burden of the liver, kidneys and brain 510 tissue. The therapeutic index and therapeutic efficacy was highest for succimer 511 followed by unithiol (Srivastava et al., 1996). 512 513 In a similar study mice were given radiolabelled cadmium chloride (0.53 mmol/kg by 514 stomach tube) followed by the antidote (2.12 mmol/kg) 15 minutes later. The 515 animals were killed after 10 days. Unithiol provided some protection against lethality 516 (whereas succimer provided complete protection). Penicillamine, succimer and 517 unithiol were all able to reduce peristaltic toxicity of cadmium chloride and all 518 reduced whole-body retention of cadmium; succimer was most effective at reducing 519 body retention. Succimer, and particularly unithiol, decreased hepatic deposition of 520 cadmium and increased relative deposition in the kidneys and lungs. It was 521 concluded that succimer was the most effective antidote for cadmium poisoning 522 (Andersen & Nielsen, 1988). 523 524 Another study in mice compared antidote efficacy when given intravenously 10 525 seconds, 1 or 3 hours after intravenous cadmium chloride (3 µmol/kg) administration. 526

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When given immediately after cadmium administration, all agents reduced the body 527 burden of cadmium but efficacy declined when dosing occurred at 1 or 3 hours after 528 administration. Unithiol was among the least effective of the antidotes investigated 529 (Planas-Bohne & Lehman, 1983). 530 531 Unithiol (50 mg/kg intraperitoneally) administered to rats 24 hours after injection of 532 radiolabelled cadmium (1 mg/kg intraperitoneally as cadmium chloride) did not have 533 any effect on the excretion or tissue distribution of cadmium. By the time the 534 antidote was given the cadmium was bound to metallothionein. Only dimercaprol 535 was effective in mobilising cadmium from metallothionein into bile (Cherian, 1980). 536 537 Unithiol (3.61 mmol/kg) was as effective as succimer (same dose) in promoting 538 survival in cadmium poisoned mice (1 mmol/kg cadmium chloride orally) when given 539 immediately after administration of cadmium. However, unithiol administration 540 resulted in a concentration of cadmium in the kidneys and liver that was approximately 541 four times greater than those in succimer-treated animals (Basinger et al., 1988). 542 543 Unithiol did not affect faecal excretion of cadmium and the increase in urinary 544 excretion was too small to affect body burden in rats given 0.4 mmol/kg of unithiol 545 following dosing with radiolabelled cadmium (3 µmol/kg as cadmium chloride). The 546 cadmium was given once; administration of the metal-binding agent started on the 547 third day and was given daily, 5 times a week for 2 weeks (Rau et al., 1987). 548 Administration of unithiol (0.1 mmol/kg) did not affect biliary excretion of cadmium in 549 rats 3 days after intravenous exposure to cadmium (Zheng et al., 1990). 550 551 Unithiol had no effect on survival rate in cadmium-poisoned mice when administered 552 intraperitoneally immediately after subcutaneous cadmium (20 mg/kg as cadmium 553 chloride) at antidote to metal molar ratios of 1:1 or 2:1. At a ratio of 5:1 the survival 554 rate was only 20%, whereas the survival rate with succimer was 100%. In another 555 study where cadmium (0.5 mg/kg intravenously) was immediately followed by 556 unithiol at a dose of 10:1, there was no increase in cadmium excretion or reduction in 557 body burden. Unithiol did, however, decrease the cadmium concentration in the liver 558 and gastrointestinal tract (Eybl et al., 1984). 559 560 In a study of antidotal efficacy in chronic cadmium poisoning in mice (2 mg of 561 cadmium chloride intraperitoneally at 48 hours intervals for 5 doses) unithiol 562 administration (225 mg/kg; 1.06 mmol/kg intraperitoneally) resulted in a significant 563 increase in liver cadmium concentrations (39%). However, interpretation of this 564 result is difficult since this group of mice were the only group to have a significant 565 loss in body weight. Therefore the apparent increase in cadmium concentration 566 could be due to changes in organ size rather than a reflection of the effect of unithiol 567 on cadmium distribution. Similarly there was also a slight increase in kidney 568 cadmium concentrations (Shinobu et al., 1983). 569 570 Unithiol had only a slight effect in reducing the cytotoxicity of cadmium in mammalian 571 cell culture (Fischer, 1995). In another in vitro study using human platelets, unithiol 572 was shown to protect against cadmium-induced stimulation of glutamate binding 573 (Borges & Nogueira, 2008) but unithiol increased cadmium-induced inhibition of δ-574 aminolevulinate dehydratase (ALAD) in rat lung in vitro (Luchese et al., 2007). 575 576

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577 8.1.6 Chromium 578 579 There is limited information on unithiol in chromium poisoning. Chromate-induced 580 cytotoxicity (as measured by the chromate content of cells and inhibition of cell 581 growth) was reduced in the presence of unithiol. This was only the case when cells 582 were incubated with unithiol and chromate. If the unithiol was added before or after 583 treatment with chromate it failed to restore chromate-induced cytotoxicity or reduce 584 cellular chromate concentrations (Susa et al, 1994). 585 586 Lethality was reduced in mice injected with chromate (40 mg/kg intraperitoneally as 587 potassium chromate) and then unithiol (500 mg/kg intraperitoneally) immediately 588 afterwards. At half the chromate dose and 300 mg/kg of unithiol the liver and kidney 589 content of chromium was reduced compared to controls. There was also increased 590 renal excretion of chromium and suppression of chromate-induced increase in serum 591 ornithine carbamyl transferase activity (Susa et al, 1994). 592 593 8.1.7 Cobalt 594 595 There is limited information on unithiol in cobalt poisoning. It has been shown to 596 reduce the lethality of cobalt in some studies (Cherkes & Braver-Chernobulskaya, 597 1958; Eybl et al., 1985). In the latter study, although unithiol increased survival it also 598 increased cobalt concentrations in the liver, gastrointestinal tract and carcass in mice 599 receiving 1 mmol/kg of cobalt chloride. The unithiol was given in a dose of 5:1, 600 antidote to metal ratio (Eybl et al., 1985). 601 602 8.1.8 Copper 603 604 Unithiol appears to be of benefit in copper-poisoned experimental animals. 605 606 Unithiol administration (0.7 mmol/kg intraperitoneally) increased the LD50 of copper 607 chloride in mice from 59 mg/kg to 143 mg/kg (Pethran et al., 1990). 608 609 Mice receiving copper sulphate (10 mg/kg intraperitoneally, the approximate LD50) and 610 then unithiol (132 mg/kg, 20 minutes later by the same route) were morphologically 611 free of hepatic or renal evidence of toxicity, whereas control animals have extensive 612 renal tubular necrosis (Mitchell et al., 1982). 613 614 In contrast unithiol has been shown to increase copper-induced haemolysis of 615 human red blood cells in vitro. At a concentration of 0.3 mM unithiol increased the 616 copper-induced haemolysis from 15% to approximately 25%. At 0.1 mM it was 617 ineffective (Aaseth et al., 1984). 618 619 8.1.9 Gold 620 621 Unithiol has been shown to be of benefit in experimentally induced gold toxicity. It is 622 able to reduce kidney gold concentrations and increase urinary excretion of gold. 623 624 In rats given 2 mg gold/kg intravenously (as Auro-Detoxin) and then oral unithiol (0.15-625 3 mmol/kg) 30 minutes later, it was demonstrated that unithiol reduced the gold 626

14

concentration in all organs except the liver and spleen. Similar effects were observed 627 when unithiol was administered 24 hours after injection of gold. In contrast to the 628 immediate treatment with unithiol delayed treatment with the lowest dose (0.15 629 mmol/kg) resulted in significantly decreased gold concentrations in the red blood cells, 630 plasma, femur, muscle and skin. However, the concentration in the kidneys was 631 significantly higher compared to controls (Gabard, 1980). 632 633 In another study rats were given 2 mg gold/kg intraperitoneally daily for 10 days then 634 unithiol 0.75 mmol/kg daily from days 11 to 20. Unithiol administration decreased the 635 gold concentration in the kidneys and in the skin, and increased it in the plasma. The 636 concentrations in the other organs remained unchanged (Gabard, 1980). 637 638 In rats injected with gold sodium thiomalate (up to 0.198 mmol/kg intravenously), 639 treatment with D-penicillamine, unithiol or succimer reduced renal toxicity, measured 640 by urinary concentrations of protein, aspartate aminotransferase and glucose, and 641 the blood urea nitrogen concentration. In addition all three antidotes increased 642 urinary excretion of gold and significantly decreased liver and renal gold 643 concentrations. Unithiol was the most effective antidote tested (Kojima et al., 1991). 644 Characterisation of the gold in urine following treatment with gold sodium thiomalate 645 and then unithiol has shown that it is present as a gold-unithiol complex. In the bile it 646 was present as a gold-unithiol complex, high molecular weight compounds (probably 647 proteins) and gold-L-cysteine (Kojima et al., 1992). 648 649 Takahashi et al. (1994) also demonstrated that administration of unithiol reduced 650 renal toxicity (using the same parameters as above) in rats given an intraperitoneal 651 dose (1.2 mmol/kg) immediately after intravenous injection of gold sodium thiomalate 652 (0.026 mmol/kg). Compared to the other gold antidotes tested (bucillamine, captopril 653 and tiopronin), only unithiol was able to significantly reduce the renal gold 654 concentration at the lowest dose used (0.2 mmol/kg). None of the antidotes reduced 655 the hepatic gold concentrations at doses of 0.2 or 0.4 mmol/kg. 656 657 Succimer and unithiol were the most effective antidotes at increasing survival in mice 658 given gold sodium thiosulphate (200 mg/kg intraperitoneally, the approximate LD99). 659 The antidotes were given by the same route 20 minutes after dosing at a ratio of 3:1, 660 antidote to gold (Basinger et al., 1985). 661 662 8.1.10 Lead 663 664 Unithiol has been shown to increase lead excretion, reduce lead tissue 665 concentrations (except in the brain) and to reduce lead-induced biochemical toxic 666

effects, such as inhibition of δ-aminolevulinic acid dehydratase, in experimental 667 animals. 668 669 In a study comparing dimercaprol and unithiol, rats received lead (2 mg/kg as lead 670 acetate) intraperitoneally daily for 7 days, and then antidotal treatment (50 µmol/kg) 671 daily for 3 days beginning 48 hours after the last lead dose. Bone and blood lead 672 concentrations were not significantly different from controls. The concentration of lead 673 in the kidneys was significantly reduced by both antidotes. In addition, both antidotes 674

reduced δ-aminolevulinic acid excretion and appeared to reactivate δ-aminolevulinic 675 acid dehydratase (Twarog & Cherian, 1983). 676

15

677 In another study rats received lead (2 mg/kg as lead acetate) intraperitoneally daily for 678 7 days and 6 days after the last dose unithiol was administered (25-200 µmol/kg). The 679 highest dose of unithiol removed lead from kidneys, liver and bone, while the lower 680 doses (25 and 50 µmol/kg) decreased only the kidney concentrations. Urinary 681 excretion of lead was higher in animals given 100 or 200 µmol/kg of unithiol. Urinary 682

excretion of δ-aminolevulinic acid was unchanged by unithiol administration in this 683 study (Twarog & Cherian, 1984). 684 685 Administration of unithiol (0.5 mmol/kg subcutaneously daily for 4 days) was of benefit 686 in rats poisoned with oral lead (10 mg/kg 6 days a week for 6 weeks). The effect of 687 the antidotes was measured by changes in the concentrations of indicators of lead 688

toxicity (blood δ-aminolevulinic acid dehydratase, zinc porphyrin, haemoglobin and 689

haemocrit and urinary δ-aminolevulinic acid). Unithiol was shown to reduce lead-690

induced inhibition of δ-aminolevulinic acid dehydratase and increase blood zinc 691 porphyrin and haemoglobin concentrations. Unithiol decreased lead-induced urinary 692

excretion of δ-aminolevulinic acid. Unithiol also decreased blood, hepatic and renal 693 lead concentrations, but did not mobilise lead from the brain (Sharma et al., 1987). 694 Another study in lead-poisoned rats (20 mg/kg intraperitoneally for 5 days) 695 demonstrated that unithiol (25, 50 or 100 µmol/kg intraperitoneally daily 5 days a 696 week for 7 weeks, starting 3 days after last lead dose) increased urinary excretion of 697

lead (due to mobilisation of lead in bone), and reduced δ-aminolevulinic acid 698 excretion but did not have a beneficial effect on lead-induced anaemia. In addition, 699 the lethality of lead was unaffected by antidote administration (Hofmann & Segewitz, 700 1975). In the study by Llobet et al. (1990) unithiol administration at a dose of 2.90 701 mmol/kg given 10 minutes after intraperitoneal lead (0.58 mmol/kg of lead acetate 702 trihydrate) reduced lethality in mice from 55% to 40%. Unithiol also caused a 703 significant increase in urinary lead and a decrease in kidney lead concentrations. 704 705 In rats with chronic lead poisoning (lead acetate in drinking water, 50 mg/L for 86 706 days) administration of unithiol (0.27 mmol/kg intraperitoneally for up to 4 days) 707 failed to alter lead concentrations in the brain (Aposhian et al., 1996). In a study on 708 acute parenteral lead intoxication in mice antidotes (2 mmol/kg) were injected during 709 3 consecutive days after 7 daily injections of lead (50 mg/kg). Unithiol, sodium 710 calcium edetate (EDTA, calcium disodium edetate, calcium disodium versenate, 711 calcium EDTA) and D-penicillamine did not protect against lethality. Unithiol was, 712 however, more efficient than succimer and several other antidotes including sodium 713 calcium edetate in removing lead from the brain and kidneys (Xu & Jones, 1988). 714 715 Tandon et al. (1994) investigated the efficacy of combined metal-binding agents in 716 lead-poisoned rats (lead acetate 0.1% in drinking water for 8 weeks). Animals were 717 treated with calcium disodium ethylenediaminetetraacetic acid (EDTA sodium calcium 718 edetate, succimer, unithiol, sodium calcium edetate and succimer or sodium calcium 719 edetate and unithiol. All metal-binding agents were given in the same dose (0.3 720 mmol/4 mL/kg intraperitoneally) for 5 days, followed, after a 5 day break by another 5 721 day course. Efficacy was measured by metal concentrations in tissues and 722

biochemical changes (blood δ-aminolevulinic acid dehydratase activity, zinc 723

protoporphyrin, urinary δ-aminolevulinic acid and total urinary proteins). The 724 administration of sodium calcium edetate or succimer resulted in more urinary lead 725 excretion than unithiol. In addition, the combination of sodium calcium edetate and 726

16

succimer was more effective than sodium calcium edetate and unithiol. Both sodium 727 calcium edetate and succimer were more effective than unithiol in reducing lead 728 concentrations in blood, liver, kidney and femur. Only succimer reduced brain lead 729

concentrations. All the metal-binding agents reversed lead-induced inhibition of δ-730 aminolevulinic acid dehydratase activity and increase in zinc protoporphyrin and 731

urinary excretion of δ-aminolevulinic acid, but the effect was greater with combined 732 therapy. Again the combination of sodium calcium edetate and succimer was more 733 effective than sodium calcium edetate and unithiol. 734 735 A study in rats demonstrated that antidotal therapy in combination with zinc and 736 copper supplementation was more effective at lowering blood lead concentrations 737 than the antidote alone. However, supplementation with unithiol administration (0.3 738 mmol/kg intraperitoneally, daily for 5 days) did not result in an increase in urinary 739 lead concentrations and unithiol was relatively less effective at promoting lead 740 excretion than sodium calcium edetate and succimer. The lead was administered at 741 a dose of 0.05 mmol/kg daily, 6 days/week for 6 weeks (Flora, 1991). 742 743 In mouse cortical cell cultures unithiol was shown to increase the toxicity of lead 744 chloride (Rush et al., 2009). In another in vitro study using human platelets, unithiol 745 was shown to protect against lead-induced stimulation of glutamate binding (Borges & 746 Nogueira, 2008). Unithiol increased lead-induced inhibition of ALAD in human blood 747 in vitro. In addition the inhibition of ALAD activity in the blood and liver of lead-748 exposed mice was also increased by unithiol treatment (Santos et al., 2006). 749 750 8.1.11 Mercury 751 752 8.1.11.1 Inorganic mercury 753 754 Unithiol has been shown to increase mercury excretion and decrease tissue 755 concentrations in experimental animals. Most studies found that unithiol does not 756 increase faecal excretion of mercury (Aaseth et al., 1982; Kachru & Tandon, 1986). 757 However some studies have shown the opposite (Wannag A. & Aeaseth J., 1980), 758 but this may be an effect of decreased body burden of mercury with unithiol 759 treatment (Gabard, 1976a). In addition, although some studies have found that 760 unithiol mobilises mercury from the brain, others have found that unithiol is relatively 761 ineffective in reducing brain mercury concentrations. The former observation may be 762 due to contamination of brain tissue with blood which has a higher mercury 763 concentration (Buchet & Lauwerys, 1989). 764 765 In rats with mercury toxicity (5 µmol/kg as mercuric chloride) unithiol (400 μmol/kg 766 intramuscularly) was found to be the most effective antidote compared to sodium 767 diethyldithiocarbamate (DDC) and pentetic acid (diethylenetriaminepentaacetic acid, 768 DTPA) when given prior to exposure. Unithiol was shown to mobilise mercury in 769 the urine and reduce concentrations in the liver and spleen. It did not increase 770 faecal excretion (Kachru & Tandon, 1986). Treatment with unithiol (500 µmol/kg 771 intravenously) 24 hours after mercury administration (2 μmol/kg intravenously as 772 mercuric chloride) in mice reduced the mercury content of the kidneys to 60-70% of 773 controls by 48 hours after administration of the antidote (Wannag & Aaseth, 1980). 774 775 Immediate treatment with unithiol (500 µmol/kg intravenously) after mercury 776

17

administration (5 µmol//kg intravenously as mercuric chloride) in rats prevented 777 pathological changes in the kidneys and increased mercury excretion in the urine. 778 When administration was delayed 24 hours, unithiol was relatively ineffective in 779 reversing mercury-induced anuria. The kidney mercury concentration was significantly 780 reduced, but pathological changes could not be prevented. Mercury concentrations in 781 the blood, kidney and brain were reduced irrespective of whether the administration of 782 unithiol was immediate or delayed. Immediate unithiol treatment did not change faecal 783 mercury elimination whereas delayed administration resulted in increased faecal 784 excretion (Wannag & Aaseth, 1980). 785 786 Administration of oral unithiol (30 µmol/200 g) was shown to normalise renal excretion 787 of alkaline phosphatase in mercury-poisoned rats (0.75 mg/kg as intravenously 788 mercuric chloride). The dose of unithiol was given at 6 or 24 hours after mercury and 789 then once a day until the fifth day. Early administration of unithiol also abolished the 790 effect of mercury on renal excretion of leucine aminopeptidase, but there was no effect 791 on this enzyme if administration of unithiol was delayed until 24 hours after mercury 792 exposure. Furthermore, early treatment with unithiol reduced mercury-induced 793 lethality, whereas delayed treatment had no effect (Planas-Bohne, 1977). 794 795 In a study reported by Aaseth (1983), succimer or unithiol (1 mmol/kg daily for 4 796 days) given after a single intravenous dose of mercuric chloride (2 μmol/kg) to mice 797 reduced the renal mercury concentration almost by a factor of three. Brain mercury 798 concentrations were also reduced. Dimercaprol, in contrast, has long been known to 799 increase mercury deposition in the brain after exposure to methyl mercury in mice 800 (Berlin & Ullberg, 1963). 801 802 The efficacy of unithiol (220 mg/kg) and succimer (180 mg/kg) were compared in rats 803 exposed to mercury (0.5 mg/kg as mercuric chloride intraperitoneally 5 times a week 804 for 3 weeks). The antidotes were administered 7 days after the last mercury dose. 805 Both antidotes were ineffective in removing mercury from the brain and unithiol was 806 more effective in increasing urinary mercury excretion. This was also the case when 807 the same dose of mercury was administered as phenyl mercury (Buchet & Lauwerys, 808 1989). In a study of various antidotes given in mercury-poisoned mice (as mercuric 809 chloride 10 mg/kg), where the antidotes were given in doses of antidote:mercury molar 810 ratios of 10, 15, 20 and 30:1, unithiol reduced lethality and was significantly more 811 effective than succimer or dimercaprol. This was true for unithiol even at the smallest 812 dose ratio of 10:1 (Jones et al., 1980). 813 814

Comparing succimer (100 µmol/kg orally) and unithiol (300 µmol/kg orally) in mercury-815 poisoned rats (0.5 mg as mercuric chloride) Planas-Bohne (1981) found that the rise in 816 urinary mercury excreted in the succimer treated animals corresponded to the mercury 817 content of the kidneys. In contrast, the urinary mercury concentration in the unithiol 818 treated group was higher indicating removal of mercury from other organs. The dose 819 of unithiol was three times that of succimer because only 30% of the unithiol was 820 absorbed from the gut compared with 100% of the succimer. However, Cherian et al. 821 (1988) reported that the increase in urinary excretion induced by unithiol (0.2-2.0 822 mmol/kg intraperitoneally) was almost equal to the amount of mercury lost from the 823 kidneys in mercury-poisoned rats (0.1-2 mg/kg intraperitoneally or mercury vapour 0.5-824 2 mg/m3). 825 826

18

In rats with chronic mercury poisoning (0.5 mg/mercury/kg, as mercuric chloride, 827 intraperitoneally 5 days a week for 32 or 41 days) unithiol (0.27 mmol/kg 828 intraperitoneally for up to 4 days) failed to alter mercury concentrations in the brain 829 (Aposhian et al., 1996). 830 831 Both renal and biliary excretion of mercury were increased after administration of 832 unithiol (12.5 mg intramuscularly) in mercury-poisoned rats (120 µg intravenously as 833 203mercuric chloride). The mercury content was decreased in all tissues, especially in 834 the kidneys and the brain. Treatment with the diuretic spironolactone before 835 administration of mercury further increased the biliary excretion of mercury observed 836 with unithiol. However, the overall excretion of mercury was unchanged (Cikrt, 1978). 837 In a similar study comparing unithiol (15 mg/kg intramuscularly) and unithiol combined 838 with spironolactone and oral polythiol resin in mercury-poisoned rats (same dose as 839 above) Cikrt & Lenger (1980) found that urinary excretion was higher with unithiol 840 alone but the combination therapy resulted in increased faecal excretion. Both 841 regimens significantly decreased the mercury content of all tissues. 842 843 Of 15 antidotes studied in mercury-poisoned mice (0.5 mg mercury intravenously 844 mercuric chloride) only unithiol (50 µmol/kg) was shown to have a favourable effect 845 with increased urinary mercury excretion and reduced tissue concentrations 846 (erythrocytes, plasma, liver, kidneys, brain, femur, muscle, spleen and intestine). 847 Unithiol reduced the mercury concentration of the kidneys to approximately 40% of 848 controls and the other organs to 50-80%. Faecal mercury excretion was decreased 849 but this was due to a decreased body burden, and overall elimination was increased 850 due to a large rise in the urinary mercury concentration (Gabard, 1976a). Similarly, 851 oral unithiol (1 mmol/kg/day for 4 days) reduced the mercury concentration in the 852 kidney to about 30% of controls in mercury-poisoned mice. Dosing with unithiol was 853 started immediately after intravenous injection of mercury (2 µmol/kg as mercuric 854 chloride). There was increased urinary excretion of mercury but faecal excretion 855 was unchanged (Aaseth et al., 1982). 856 857 In a study in mercury-poisoned mice (5, 200, 300 or 400 µmol/kg of mercuric chloride 858 orally) unithiol (100, 800, 1200 or 1600 µmol/kg orally) given 15 minutes later was 859 most effective at reducing lethality. In addition oral administration was more effective 860 than parenteral, probably because of reduction in gastrointestinal absorption of 861 mercury. Unithiol also reduced mercury concentrations in the brain; intraperitoneal 862 unithiol reduced mercury brain concentrations to about one third of controls whereas 863 oral administration reduced concentrations to less than 15% of controls (Nielson & 864 Andersen, 1991). 865 866 Simultaneous administration of sodium selenite and mercuric chloride decreased the 867 efficacy of unithiol (300 µmol/kg orally) on mercury elimination in rats. The metal salts 868 were given at a dose of 1.5 µmol/kg by intraperitoneal injection. When both metal 869 salts were given together there was redistribution of mercury with reduced 870 accumulation in the kidneys (decreased by more than 94%) and an increased 871 concentration in the liver (increased about 6 times). Urinary excretion of mercury was 872 also reduced compared to elimination in the absence of selenium (Jureša et al., 2005). 873 874 Kostial et al. (1984) investigated the influence of age on unithiol efficacy in rats (aged 875 2, 6 and 28 weeks old) with mercury toxicity (50 μg/kg intraperitoneally). Unithiol (50 876

19

mg/kg 3 times, 1 day after mercury administration and then at 24 hour intervals) 877 decreased the body retention of mercury in all age groups, and was about twice as 878 effective in adults compared to suckling rats. The reduced effectiveness was due to 879 the reduced efficacy of unithiol in lowering kidney retention in young animals. This 880 age difference was also confirmed in a later study (Kostial et al., 1991). In addition 881 this study found that early treatment with oral unithiol in older rats given oral mercury 882 increased mercury retention. However, this was in contrast to succimer, where early 883 treatment while mercury was still in the gut decreased mercury retention. 884 885 An in vitro study on isolated perfused segments of rabbit proximal tubules exposed to 886 inorganic mercury found that mercury was rapidly taken up by the tubular epithelial 887 cells and resulted in cellular necrosis. The addition of unithiol provided complete 888 protection against this effect. This appeared to be due to a negligible rate of net 889 absorption of inorganic mercury ions from the lumen and low levels of ion 890 accumulation. Unithiol-mercury complexes are not readily transported into proximal 891 tubular cells and it is thought that unithiol reduces the renal mercury burden by 892 extraction of mercury during the trans-epithelial transport of unithiol. The therapeutic 893 efficacy of unithiol in mercury-induced renal damage may be linked to its transport at 894 the basolateral membrane by the organic anionic transporter (OAT) system and to 895 prevention of significant uptake of mercury by the proximal tubular cells due to the 896 formation of unithiol-mercury complexes (Zalups et al., 1998). Another in vitro study 897 confirmed that unithiol was effective at inhibiting mercury accumulation in renal 898 proximal and distal tubular cells, and protecting against mercury-induced renal 899 damage (Lash et al., 1998). 900 901 Unithiol, when incubated with mercuric chloride, partially restored cellular morphology, 902 viability, intracellular adenosine triphosphate (ATP) concentrations and mitochondrial 903 membrane potential in opossum kidney cells in vitro. Unithiol also had a protective 904 effect on mitochondrial morphology and showed potent antioxidant activity (Carranza-905 Rosales et al., 2007). Similarly, unithiol was shown to reduce mercuric chloride 906 toxicity in mouse cortical cell cultures (Rush et al., 2009). In another in vitro study 907 using human platelets, unithiol was shown to protect against the inhibitory effect of 908 mercury on glutamate binding (Borges & Nogueira, 2008). 909 910 8.1.11.2 Organic mercury 911 912 Oral unithiol administration (1 mmol/kg) to rats with methyl mercury poisoning (0.23 913 mg/kg intravenous as methyl mercury) reduced the biological half-life of the mercury 914 body burden from 23.0 days to 4.3 days compared to controls. The mercury 915 concentration was decreased in all tissues, particularly in the kidneys and the brain 916 (Gabard, 1976b). 917 918 The effect of unithiol on motor impairment and cerebellar toxicity has been studied in 919 mercury-exposed mice. Methylmercury was given in drinking water (40 mg/L) for 17 920 days with unithiol given on days 15 to 17 (150 mg/kg by intraperitoneal injection). The 921 mice were tested for signs of toxicity 24 hours after the last dose of unithiol. The 922 mercury-induced motor deficit was reduced in unithiol treated mice and unithiol also 923 reduced mercury-induced lipid peroxidation in the cerebellum but did not prevent 924 mercury-induced reduction of cerebellar glutathione peroxidase. Unithiol significantly 925 reduced mercury deposition in the cerebellar cortex (Carvalho et al., 2007). 926

20

927 Unithiol is able to increase elimination of both organic and inorganic mercury from 928 tissue, however with organic mercury the efficacy of unithiol is affected by the 929 relative capacity of tissue for dealkylation of organic to inorganic mercury. In rats 930 with chronic methyl mercury poisoning (10 ppm in drinking water for 9 weeks in a 931 single dose study and 6 weeks in a repeated dose study) a single dose of unithiol 932 (100 mg/kg intraperitoneally) was shown to reduce kidney inorganic and organic 933 mercury concentrations by 38% and 59%, respectively. In addition, urinary inorganic 934 and organic mercury concentrations increased by 7.2 and 28.3 fold, respectively, 935 compared with pre-treatment concentrations. A single dose of unithiol was relatively 936 ineffective at removing mercury from the brain and the mercury in this tissue was 937 predominantly in the organic form due to slow dealkylation to inorganic mercury. In 938 contrast, repeated doses of unithiol (100 mg/kg every 72 hours for 1, 2 or 3 doses) 939 significantly decreased mercury concentrations in the brain, kidney and blood, but 940 the decrease in the brain and blood was restricted to the organic mercury 941 component. This was thought to reflect the slow dealkylation of methyl mercury in 942 these tissues. This may be why unithiol is relatively ineffective at improving organic 943 mercury-induced neurotoxicity (Pingree et al., 2001). 944 945 Unithiol no effect on methylmercury and increased the toxicity of ethylmercury in 946 mouse cortical cell cultures (Rush et al., 2009). 947 948 8.1.12 Nickel 949 950 Unithiol has been shown to be efficacious in experimental nickel poisoning in 951 animals, in terms of increased survival and reduced nickel-induced toxic effects. 952 953 Unithiol administered intraperitoneally at a 10:1 mole ratio of antidote to nickel, 954 increased survival rate in mice poisoned with intraperitoneal nickel acetate (62 955 mg/kg). Antidotes were administrated 20 minutes after injection of nickel. Unithiol 956 was less effective than D-penicillamine or sodium calcium edetate, although the 957 small sample size prohibited any significant differentiation between them (Basinger 958 et al., 1980). 959 960 Administration of unithiol (0.5 mmol/kg subcutaneously daily for 4 days) significantly 961 enhanced the urinary excretion of nickel in rats poisoned with intraperitoneal nickel 962 sulphate (4 mg/kg 6 days/week for 4 weeks). In unithiol treated animals there were 963 significant reductions in nickel concentrations in renal and hepatic tissue and 964 decreases in evidence of nickel-induced kidney and liver damage (as measured by 965 plasma concentrations of ceruloplasmin and amino acids, blood glucose and 966 glutathione, urinary amino acids, and renal, hepatic and cardiac concentrations of 967 mitochondrial malic dehydrogenase, which is inhibited by nickel). Unithiol reduced the 968 increase in plasma and urinary amino acids and blood glucose, but did not change the 969 decreases in plasma ceruloplasmin and blood glutathione concentrations. There was 970 no significant effect on mitochondrial malic dehydrogenase concentrations. Faecal 971 nickel excretion was unchanged and unithiol was ineffective at mobilising nickel from 972 the brain (Sharma et al., 1987). 973 974 Rats given nickel (1.5 mg/kg intraperitoneally as nickel sulphate daily, 6 days a week 975 for 30 days) then unithiol (0.3 mmol/kg intraperitoneally daily for 5 days) had their 976

21

organs harvested 24 hour after the last injection. Unithiol reduced the nickel 977 concentrations in the liver, blood, heart and kidney but not in the brain. Unithiol also 978 reversed nickel-induced biochemical changes (decreased ceruloplasmin 979 concentration and blood glucose; increased plasma and urine concentrations of 980 amino acids) and increased faecal nickel excretion (Tandon et al., 1996). 981 982 8.1.13 Palladium 983 984 There is limited information on the effect of unithiol on palladium toxicity. It did not 985 influence toxicity or reduce lethality in mice with acute palladium chloride poisoning 986 (586 µmol/kg intraperitoneally). Unithiol was given subcutaneously at the same time 987 as palladium at a dose of 2.93 mmol/kg (5 times the molar dose of metal) (Mráz et 988 al., 1985). 989 990 8.1.14 Platinum 991 992 There is limited information on the effect of unithiol on platinum toxicity. A single 993 injection of unithiol (1 mmol/kg) produced no significant change in renal platinum 994 concentration in rats treated with cisplatin (4 or 6.5 mg/kg) 24 hours previously. 995 After four daily treatments unithiol and succimer caused a significant increase in 996 urinary excretion of platinum, but this was low, and represented only about 3% of 997 the injected dose of platinum. It was concluded that none of the antidotes studied, 998 unithiol, succimer or pentetic acid, were likely to be of benefit in the management of 999 cisplatin-induced renal toxicity (Planas-Bohne et al., 1982). 1000 1001 8.1.15 Polonium 1002 1003 Several animal studies have shown that although unithiol can remove polonium-210 1004 from most tissues it results in concentration of polonium in the kidneys, with the risk 1005 of renal damage. In addition, unithiol has been shown to cause renal damage when 1006 administered to polonium-poisoned animals (Poluboiarinova & Streltsova, 1964). 1007 1008 Rats were given approximately 0.3 µCi of polonium-210 intravenously followed 1.5 1009 minutes later by intraperitoneal or oral administration an antidote (1 mmol/kg). The 1010 α-activity of tissue was determined 48 hours later. Administration of an antidote 1011 produced a marked decrease in polonium-210 retention in the blood, spleen and 1012 bone and, to a lesser extent, in the plasma. Unithiol but did not decrease polonium-1013 210 retention in the kidneys and overall retention of polonium-210 in the blood, liver, 1014 spleen, skeleton and kidneys was increased by 40%. The effect of oral unithiol was 1015 approximately one third of the intraperitoneal dose, and following administration by 1016 this route the overall retention of polonium-210 was increased by approximately 1017 50%. Retention of polonium-210 was particularly seen in the kidneys where it was 1018 transported but not excreted. In view of these findings it was concluded that 1019 although unithiol increased survival of rats given lethal doses of polonium-210, it was 1020 not a suitable antidote for polonium because it could potentiate the toxic effects of 1021 polonium on the kidneys (Volf, 1973). 1022 1023 In a study of rats given a lethal dose (40 µCi) of polonium-210 by intraperitoneal 1024 injection, administration of an antidote (0.2 mmol/kg) 1 minute, 90 minutes, 360 1025 minutes and twice daily on days 2, 3, 4, 12, 22 and 32 increased the mean survival 1026

22

time from 39 days to 106 days. Unithiol significantly decreased the polonium-210 1027 content of all tissues studied except the kidneys. However, DMPA was found to be a 1028 more effective antidote and able to decrease polonium-210 in all tissues (Aposhian 1029 et al., 1987). 1030 1031 The study by Rencová et al. (1993) comparing a number of antidotes for polonium-1032 210 also found that unithiol reduced retention in the bone, spleen and blood of rats 1033 but increased it in the kidneys. Indeed, the total body retention of polonium-210 1034 could not be reduced to less than 85% of controls with any of the 9 antidotes used. 1035 1036 The same investigators (Volf et al., 1995) looked at the use of antidotes in rats with 1037 simulated wounds contaminated with polonium-210. After 2 weeks of unithiol 1038 treatment (intramuscularly at injection site at 1 hour and 5 days, and systemic 1039 treatment with intramuscular injection on days 1, 7, 9 and 12) polonium-210 at the 1040 wound site was reduced to 12% of controls. The retention in the liver, spleen, 1041 muscle and skeleton was reduced to 14-40%, but the blood content was unchanged 1042 and retention in the kidneys was increased to 340% of controls. Unithiol was 1043 effective at removing polonium-210 from the wound site when given either locally or 1044 systemically, but it was not effective at removing polonium-210 from the body as a 1045 whole. In a series of other experiments it was concluded that treatment with unithiol 1046 combined with another antidote (particularly sodium N,N’-di-(2-hydroxyethyl)-1047 ethylenediamine-N’N’-biscarbodithioate; HOEtTTC) was the most effective method of 1048 removing polonium-210 from the body. 1049 1050 8.1.16 Selenium 1051 1052 There is limited information on the effect of unithiol on selenium toxicity. Unithiol 1053 administration (60 mg/kg intraperitoneally) had no effect on selenium-poisoned rats 1054 (2.24 mg of selenium/kg by subcutaneously). The concentration of selenium in urine 1055 and faeces was unchanged (Paul et al., 1989). 1056 1057 8.1.17 Silver 1058 1059 There is limited information on the effect of unithiol on silver toxicity. Unithiol 1060 administration (0.7 mmol/kg intraperitoneally) increased the LD50 of silver chloride in 1061 mice from 13.6 to 74 mg/kg (Pethran et al., 1990). In dogs given intravenous silver 1062 nitrate unithiol prevented the development of toxic pulmonary oedema and death 1063 (Romanov, 1967). 1064 1065 In an in vitro study of silver inhibition of Na,K-ATPase, which was probably due to 1066 deposition of the metal on sulphydryl groups in the enzyme, it was found that the 1067 process was completely reversed by administration of unithiol (Hussain et al., 1994). 1068 1069 8.1.18 Strontium 1070 1071 There is limited information on the effect of unithiol on strontium toxicity. Unithiol did 1072 not affect survival rate in mice poisoned with strontium chloride (Domingo et al., 1990; 1073 Pethran et al., 1990). 1074 1075 8.1.19 Thallium 1076

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1077 Unithiol is not of benefit in thallium poisoning in experimental animals. 1078 1079 In a study comparing the efficacy of Prussian blue (potassium ferric hexacyanoferrate 1080 II) and unithiol, rats were given an oral dose of 20 mg thallium (as thallium sulphate) 1081 and antidotal therapy was started 24 hours later. The rats were divided into 3 1082 treatment groups: Prussian blue 50 mg/kg for 4 days, unithiol 5 mg/kg 6 times a day on 1083 day 1, 4 times on day 2 and 3 times on days 3 and 4. In the third group the animals 1084 were given both antidotes. Animals were killed on day 5 and the thallium content of 1085 organs determined. Prussian blue administration limited thallium distribution into 1086 tissues, whereas unithiol did not decrease thallium concentration in any organ, 1087 although it did decrease thallium concentrations in blood. The two drugs in 1088 combination decreased the thallium content in all organs but no more than Prussian 1089 blue alone. It was concluded that unithiol is not a useful antidote in thallium poisoning 1090 (Mulkey & Oehme, 2000). 1091 1092 Similarly, unithiol did not affect survival rate in mice given thallium sulphate (Pethran et 1093 al., 1990). 1094 1095 8.1.20 Tin 1096 1097 There is limited information on the effect of unithiol on tin toxicity. Unithiol and 1098 succimer have been investigated as antidotes in rats following a single intravenous 1099 dose dibutyltin dichloride (27 µmol/kg). The antidotes were given at two doses, 100 1100 and 500 µmol/kg, orally and by intraperitoneal injection. Several parameters of 1101 organ toxicity were monitored from 6 hours to 8 weeks. Both drugs reduced 1102 dibutyltin dichloride-induced lesions of the bile duct, pancreas and liver. Unithiol was 1103 more effective than succimer in most measured parameters and the drugs were 1104 though to exert their protective effects on these organs by reducing biliary organotin 1105 excretion (Merkord et al., 2000). 1106 1107 In vitro studies with human erythrocytes incubated with tributyl tin have 1108 demonstrated that unithiol is unable to prevent the tributyl tin-mediated haemolysis of 1109 the cells (Gray et al., 1986; 1987). 1110 1111 8.1.21 Vanadium 1112 1113 There is limited information on the effect of unithiol on vanadium toxicity. However, it is 1114 unlikely to be useful since antidotes containing oxygen, rather than thiol groups such as 1115 unithiol, are more effective at binding vanadium. 1116 1117 Unithiol had no effect on lethality in mice poisoned with sodium vanadate (50 mg/kg 1118 intraperitoneally) or vanadyl sulphate (110 mg/kg intraperitoneally). Unithiol was 1119 given intraperitoneally 20 minutes after administration of vanadium, at a dose of 5:1 1120 antidote to metal compound (Jones & Basinger, 1983). 1121 1122 Unithiol had no significant effect on the death rate, body weight reductions, or 1123 reduction in weights of legs and toes in chick eggs incubated with vanadium 1124 (Hamada, 1994). 1125 1126

24

A recent study, however, has shown that oxovanadium (VO2+) is capable of forming a 1127 [VO(DMPS)2]4- complex with unithiol in aqueous solution (Williams & Baran, 2008). 1128 The significance of this in vivo remains to be elucidated. 1129 1130 8.1.22 Zinc 1131 1132 Unithiol can increase excretion of zinc and reduced lethality in zinc-poisoned animals, 1133 but more effective antidotes are available. 1134 1135 In a comparison of several antidotes against the effects of acute parenteral zinc 1136 intoxication in mice, unithiol efficiently reduced acute lethality. Succimer (10:1 1137 antidote:zinc ratio) was given 20 minutes after a fatal dose of zinc (50 mg/kg) was 1138 administered. Survival was 73% with unithiol but other antidotes were equally or 1139 more effective (Basinger & Jones, 1981b). 1140 1141 In mice given zinc acetate (0.49 mmol/kg intraperitoneally) unithiol (2:1 or 5:1 molar 1142 ratios of antidote to metal) was the least effective of the six antidotes tested. Disodium 1143 calcium cyclohexanediaminetetraacetate (CDTA), sodium calcium edetate and 1144 pentetic acid and were the most effective (Llobet et al., 1988). 1145 1146 In a study comparing the efficacy of several antidotes mice were given intraperitoneal 1147 zinc acetate (66-330 mg/kg; LD50 108 mg/kg). Antidotal therapy was given 10 minutes 1148 later, also by intraperitoneal injection. Unithiol reduced the lethality in animals given 1149 66-241 mg/kg of zinc acetate. In those given 330 mg/kg of zinc acetate lethality was 1150 30% compared to 100% in control animals. Unithiol also increased renal excretion of 1151 zinc and reduced blood and heart tissue concentrations compared to controls. 1152 However, pentetic acid and CDTA were found to be more effective zinc antidotes in 1153 this study (Domingo et al., 1988). 1154 1155 8.2 Pharmacokinetics 1156 1157 There is information on unithiol pharmacokinetics in various species of experimental 1158 animals. 1159 1160 Following oral administration between 60% (dogs; Wiedemann et al., 1982) and 30% 1161 (rats; Gabard, 1978) of the dose is absorbed and plasma peak concentrations are 1162 reached after 30 to 45 minutes. Plasma protein binding was measured at 70% in dogs 1163 by equilibrium dialysis (Wiedemann et al., 1982). This is in agreement with the figure 1164 of 65-75%, determined in rats (Planas-Bohne & Lehmann, 1983). 1165 1166 After intravenous administration unithiol is mainly distributed in plasma and kidneys, 1167 only minor concentrations were measured in the brain and other organs. The 1168 apparent volume of distribution in dogs was calculated to be 0.160 mL/kg (Wiedemann 1169 et al., 1982). Most unithiol excreted in the bile of rats is in its altered form and 1170 recovery in bile was 40% of the administered dose (Zheng et al., 1990). 1171 1172 Although unithiol is mainly distributed to the extracellular compartment, studies have 1173 demonstrated uptake by cells. Planas-Bohne & Olinger (1981) studying the 1174 interaction of antidotes with methyl mercury bound to erythrocytes demonstrated that 1175 unithiol is lost from the extracellular fluid. Wiedemann et al. (1982) found that the 1176

25

distribution volume of radiolabelled unithiol exceeded the extracellular volume in dogs. 1177 In vitro studies have demonstrated that unithiol is taken up by cells of the basolateral 1178 membrane of the kidney and can bind mercury within cells (see section 7). 1179 1180 Unithiol is rapidly eliminated from the body. The serum half life is approximately 20 to 1181 60 minutes and plasma clearance is approximately 2.6 mL/min/kg. In dogs given 1182 radiolabelled unithiol 93% was eliminated within 3 days, with the bulk (98%) eliminated 1183 in the urine and the rest in the faeces (Wiedemann et al., 1982). 1184 1185 Gabard & Walser (1979) stated that unithiol is not involved in important metabolic 1186 pathways and a quantity is excreted unchanged; Maiorino et al. (1988) demonstrated 1187 the presence of several acyclic and cyclic oxidized metabolites in the urine of rabbits. 1188 1189 8.3 Toxicology 1190 1191 Unithiol is of low acute and chronic toxicity. Studies have determined the LD50 for 1192 various species and examined the effect of acute or chronic unithiol administration on 1193 trace element concentrations. 1194 1195 In order of decreasing sensitivity to unithiol, animal species can be ranked as follows: 1196 cat, dog, guinea pig, rabbit, rat and mouse (Klimova, 1958; Aposhian, 1983). The 1197 optical isomers of unithiol do not differ in their toxicity (Hsu et al., 1983). 1198 1199 8.3.1 Acute toxicity 1200 1201 Unithiol is of relatively low toxicity. By the parenteral route the acute LD50 of unithiol 1202 for various species is about 1 to 2 g/kg (Planas-Bohne et al., 1980; Aposhian et al., 1203 1981; Aposhian, 1982; Hruby and Donner, 1987; Pethran et al., 1990). After 1204 intraperitoneal injection of lethal doses rats are highly irritable for some minutes before 1205 they become apathetic followed by cessation of breathing and death within 12 hours. 1206 The LD50 for a single dose in rats was 5 mmol/kg (1.14 g/kg) and after dosing on 10 1207 consecutive days was 30.8 mmol/kg (Planas-Bohne et al., 1980). No acute toxicity 1208 was observed in mice receiving oral unithiol (100, 300 or 600 µg/mL) for 5 days in 1209 drink water. These doses are equivalent to 18, 55 and 109 mg/kg/day or 0.1, 0.3 1210 and 0.5 mmol/kg/day (Jones et al., 1996). 1211 1212 In rats treated with a single intraperitoneal dose of 1 mmol/kg of unithiol, urinary zinc 1213 and copper excretion was increased whereas the excretion of iron and manganese 1214 was unchanged (Gabard et al., 1979). 1215 1216 A single subcutaneous injection of unithiol (1.6 mmol/kg) in mice caused a significant 1217 increase in δ-aminolevulinic dehydratase activity in the blood and a decrease in the 1218 kidney, with no change in the liver or brain. Unithiol also significantly increased the 1219 zinc concentration of the kidneys but did not change liver or brain concentrations. 1220 There was also a significant increase in liver and kidney lipid peroxidation (Santos et 1221 al., 2005). 1222 1223 8.3.2 Chronic toxicity 1224 1225 Unithiol is of relatively low toxicity even in chronic administration. Hrdina et al. (1998) 1226

26

studied the effects of repeated injection of unithiol on the heart of rabbits. The animals 1227 were given intravenous unithiol, 50 mg/kg once a week for 10 weeks. There was no 1228 change in iron or selenium concentrations. There was a slight decrease in myocardial 1229 concentrations of calcium, potassium and magnesium, but only the later was 1230 significantly different. These changes were not associated with any haematological, 1231 histological or physiological changes. 1232 1233 Mice receiving unithiol 300 µg/mL in drinking water for 3 months showed no signs of 1234 toxicity. Haematological and biochemical parameters were also unchanged (Jones 1235 et al., 1996). 1236 1237 Rats receiving 600 µmol/kg/day orally (126 mg/kg/day) on 5 days per week for 66 1238 weeks did not show any adverse effects. Treatment for 36 weeks led to a reduction in 1239 copper-concentrations in the kidneys, liver and skin (Planas-Bohne et al., 1980). 1240 Beagle dogs treated for 6 months with doses up to 15 mg/kg/day intravenously or 45 1241 mg/kg/day orally showed no significant changes in blood-concentrations of glucose, 1242 uric acid, creatinine, total protein, sodium, potassium, calcium, magnesium and iron, 1243 and activity of liver enzymes and cholinesterase in the serum. In addition, the red and 1244 white blood picture as well as gain in body weight remained unchanged. A dose-1245 dependent decrease in the copper content was found in the serum, liver, kidney and 1246 spleen. The macroscopic and microscopic examination of several organs revealed no 1247 pathological changes. After treatment for 10 weeks at a dose of 2 x 75 mg/kg/day 1248 intravenously the following changes were noted: a depletion of copper in the serum 1249 and in various organs, an increase of the iron content of the liver and spleen, and a 1250 decrease in haemoglobin, haematocrit, red blood cells, alkaline phosphatase activity 1251 and zinc content in the blood (Szincicz et al., 1983). 1252 1253 8.3.3 Reproductive toxicity and teratogenicity 1254 1255 Unithiol does not appear to produce reproductive toxicity or teratogenicity. 1256 1257 No teratogenic effects were reported in the offspring of rats given 600 µmol/kg/day 1258 orally (126 mg/kg/day) on 5 days per week. Female rats were mated with untreated 1259 males after 14, 26 or 60 weeks of treatment with unithiol. Treatment was continued 1260 during pregnancy and nursing. The number of pregnant animals and the litter size 1261 was smaller in the treated group but the difference was not significant (Planas-Bohne 1262 et al., 1980). 1263 1264 No adverse effects were observed in mothers or offspring in mice given up to 630 1265 mg/kg/day in two dosing regimens: from gestation day 14 until birth or from gestation 1266 day 14 until post-natal day 21. The no observed effect level (NOEL) of 630 1267 mg/kg/day is much higher than that used in the treatment of human heavy metal 1268 poisoning (Domingo et al., 1990). 1269 1270 Mice given unithiol, up to 300 mg/kg, on days 6 to 15 of gestation showed no 1271 maternal or reproductive effects. Unithiol had a minor effect on maternal and fetal 1272 mineral metabolism which was variable and not dose related. These effects did not 1273 produce maternal or embryofetal toxicity (Bosque et al., 1990). 1274 1275 No teratogenic effects were reported in rabbits given unithiol (up to 100 mg/kg 1276

27

intravenously daily) from days 6 to 18 of gestation (Anon, 1992/1993). 1277 1278 Unithiol has been shown to protect against the developmental toxicity of arsenic 1279 (Domingo et al., 1992) and mercury (Gomez et al., 1994) in experimental animals. 1280 Mice were given a single intraperitoneal injection of sodium arsenite on day 9 of 1281 gestation followed by immediate injection of dimercaprol or unithiol with further doses 1282 at 24, 48 and 72 hours. Dimercaprol did not protect against arsenic-induced 1283 developmental toxicity, whereas unithiol was protective at 150 and 300 mg/kg/day 1284 and was able to prevent embryotoxicity and fetotoxicity. The higher dose also 1285 prevented maternal arsenic toxicity (Domingo et al., 1992). Pregnant mice were 1286 given a single oral dose of 30 mg/kg of methyl mercury chloride on day 10 of 1287 gestation followed by dimercaprol (by subcutaneous injection) or unithiol (by gavage) 1288 at 24, 48 and 72 hours. Dimercaprol administration did not prevent maternal or 1289 developmental toxicity, whereas unithiol in doses up to 360 mg/kg/day significantly 1290 reduced maternal lethality. Treatment with the higher doses, 180 and 360 1291 mg/kg/day, also protected against mercury-induced embryotoxicity and teratogenicity 1292 (Gomez et al., 1994). 1293 1294 8.3.4 Genotoxicity 1295 1296 Unithiol has been evaluated for mutagenicity in the Ames test with negative results 1297 (Aposhian et al., 1983; Ruprecht, 1997). Normal DNA synthesis was maintained with 1298 unithiol concentrations up to 8 µg/mL in an in vitro study using 3 murine tumour cell 1299 lines. Above 8 µg/mL unithiol inhibited DNA synthesis, and above 30 µg/mL inhibition 1300 exceeded 80% (Jones et al., 1996). 1301 1302 An in vitro study found that unithiol increased the incidence of nickel-induced DNA 1303 breaks in a human leukaemia cell line. There was also an increase in DNA breaks in 1304 bacterial plasmids (a simpler system). Succimer and dimercaprol also increased 1305 DNA breaks in plasmids in the presence of nickel, but the effect was strongest with 1306 succimer. These metal binding agents all generate hydrogen peroxide in solution 1307 but succimer is the most potent. Free radicals are throught to be involved in the 1308 DNA damage observed. For the most potent compound, succimer, the breakage of 1309 DNA was completely prevented by the presence of mannitol and partially reduced by 1310 antioxidants. This protective effect was not investigated for unithiol (Lynn et al., 1311 1999). 1312 1313 1314 9. Volunteer studies 1315 1316 There are three main studies of unithiol pharmacokinetics, all conducted by the same 1317 group, consequently some volunteers took part in more than one study. The 1318 characteristics of each study are as follows: 1319

• The study by Maiorino et al. (1991) involved 10 male volunteers, aged 24-34 years, 1320 weighing 68-98 kg. 1321

• The study by Hurlbut et al. (1994) involved 5 volunteers (4 male, 1 female), aged 24 1322 to 32 years, weighing 49-93 kg. 1323

• The study by Maiorino et al. (1996) involved 4 male volunteers, aged 23 to 27 1324 years, weighing 86-91 kg. 1325

1326

28

9.1 Absorption 1327 1328 Unithiol is rapidly absorbed. In volunteers given 3 x 100 mg capsules, unithiol was 1329 detected in blood within 0.5 to 4 hours after ingestion. Maximal concentrations were 1330 reached within 3 to 4 hours (Maiorino et al., 1991). 1331 1332 In 4 male volunteers the oral bioavailability of unithiol was calculated to be 39%, with a 1333 range of 19-62% (Hurlbut et al., 1994). 1334 1335 9.2 Distribution 1336 1337 In volunteers given 3 x 100 mg capsules, metabolites (altered unithiol) were confined 1338 to the plasma portion of the blood suggesting that they were bound to plasma 1339 proteins (Maiorino et al., 1991). Plasma protein binding of unithiol was approximately 1340 90% when measured by equilibrium dialysis in human plasma samples from 3 1341 volunteers (Wiedemann et al., 1982). However in a further study by Maiorino et al. 1342 (1996), less than 1% of unithiol was present in an unaltered form in the plasma 5 1343 hours after a single oral dose of 300 mg. The protein-bound unithiol and non-protein-1344 bound unithiol disulphides were present as 62.5% and 36.6% of the total unithiol, 1345 respectively. The protein-bound unithiol was present as a unithiol-albumin complex 1346 (84%) and a higher molecular weight complex (16%). 1347 1348 In volunteers given unithiol 3 mg/kg intravenously over 5 minutes the volume of 1349 distribution varied from 2.67 to 15.4 L/kg (Hurlbut et al., 1994). 1350 1351 Although unithiol is mainly distributed to the extracellular compartment, studies have 1352 demonstrated uptake by cells. An in vitro study investigating the interaction of 1353 antidotes with methylmercury bound to erythrocytes demonstrated that unithiol is lost 1354 from the extracellular fluid (Planas-Bohne & Olinger, 1981) and uptake by human 1355 erythrocytes has been demonstrated in vitro (Wildenauer et al., 1982). Uptake of 1356 unithiol by the renal proximal cells is thought to be mediated by the organic anion 1357 transporter (Islinger et al., 2001; see section 7). 1358 1359 9.3 Elimination 1360 1361 Unithiol is rapidly metabolised and subject to renal elimination. 1362 1363 In volunteers given 3 x 100 mg unithiol capsules the elimination half-life in blood of 1364 unithiol and metabolites (altered unithiol) was 4.4 and 9.6 hours, respectively 1365 (Maiorino et al., 1991). Maiorino et al. (1996) determined the half-lives of unithiol and 1366 metabolites to be 1.8 and 20 hours, respectively. The long half-life of altered unithiol 1367 was thought to reflect the stability of the unithiol-albumin complex and since unithiol is 1368 released slowly, albumin may act as a reservoir for unithiol. 1369 1370 In volunteers given unithiol 3 mg/kg intravenously over 5 minutes blood concentrations 1371 declined rapidly with an apparent elimination half-life of 1.8 hours. By 96 hours 12% of 1372 the total unithiol found in the urine was excreted as the parent compound, 1373 representing 10% of the administered dose; 88% was present as disulphide 1374 metabolites (74% of the administered dose). The metabolites are eliminated more 1375 slowly than the parent compound, with an elimination half-life of 23 hours (range 19.8-1376

29

37.5 hours). The elimination half-life in urine is 20 hours (Hurlbut et al., 1994). 1377 1378 The apparent difference in blood and urine half-lives in the Maiorino et al. (1991) oral 1379 and Hurlbut et al. (1994) intravenous study may be due to different metabolites 1380 produced following administration by different routes. In addition the total unithiol 1381 concentration was determined at different time points (Hurlbut et al., 1994). 1382 1383 9.4 Metabolism 1384 1385 Unithiol is extensively metabolised and unchanged drug is present as only a small 1386 concentration in blood and urine. In volunteers given unithiol 3 mg/kg intravenously 1387 over 5 minutes only 12% of unchanged unithiol was detected in the blood after 15 1388 minutes (Hurlbut et al., 1994). In volunteers given 3 x 100 mg unithiol capsules 3.7% 1389 was excreted as unchanged unithiol and 38.7% as metabolites by 15 hours. Of the 1390 total unithiol found in the urine by 15 hours, unchanged unithiol and metabolites 1391 represented 9% and 91%, respectively. The metabolites are thought to be 1392 disulphide compounds (Maiorino et al., 1991). In a later study (Maiorino et al., 1996) 1393 the unithiol disulphide metabolites were determined to be cyclic polymeric unithiol 1394 disulphides (97%), unithiol-cysteine mixed disulphide (2.5%) and acyclic unithiol 1395 disulphide (0.5%). 1396 1397 9.5 Effect of DMPS on the excretion of metals 1398 1399 Few volunteer studies on the effect of unithiol on metal excretion are available. 1400 1401 9.5.1 Arsenic elimination 1402 1403 In addition to increasing urinary elimination of arsenic, unithiol has also been shown 1404 to alter the relative urinary concentrations of organoarsenic metabolites. 1405 1406 The arsenic-antidote complex was determined in the urine of Romanian subjects 1407 exposed to high concentrations of arsenic in drinking water (up to 16 µg/L). Samples 1408 were collected before and after oral administration of 300 mg of unithiol. Subjects 1409 had been asked to avoid seafood for three days prior to and during the collection 1410 period. The presence of a unithiol-monomethylarsenous acid (As3) complex was 1411 demonstrated in the urine samples of subjects given unithiol. Administration of 1412 unithiol resulted in a decrease in dimethylarsenic acid (As5) and an increase in 1413 monomethylarsonic acid (As5) concentrations. Monomethylarsenous acid (As3) is a 1414 substrate for the biomethylation of arsenic from monomethylarsonic acid (As5) to 1415 dimethylarsenic acid (As5); the formation of the unithiol-monomethylarsenous acid 1416 complex reduces the availability of monomethylarsenous acid (As3) for 1417 biomethylation and inhibits further methylation (Gong et al., 2002). 1418 1419 A similar change in urine concentrations of dimethylarsinic acid and 1420 monomethylarsonic acid was also found in the study by Aposhian et al. (1997) 1421 investigating arsenic excretion in two populations in Chile. In one town the drinking 1422 water contained 593 µg/L of arsenic, and in the other 21 µg/L (controls). Subjects 1423 were asked to exclude seafood from their diet for 3 days prior to the study and 1424 bottled water (arsenic <0.5 mg/L) was drunk throughout. Subjects were excluded if 1425 they had a history of previous antidotal therapy, hypersensitivity to similar metal-1426

30

binding agents or administration of other investigational drugs, serious renal or 1427 psychiatric disease, abnormalities in blood biochemistry or urine analysis that could 1428 interfere with evaluation, pregnancy, lactation or abuse of alcohol or recreational 1429 drugs. There were 13 subjects in the high arsenic exposure group and 11 controls. 1430 Urine samples were collected before and after oral administration of 300 mg unithiol. 1431 In the 2 hour period after unithiol administration the urinary concentration of the 1432 metabolites monomethylarsonic acid and dimethylarsinic acid represented 42% and 1433 37-38%, respectively, with an inorganic arsenic concentration representing 20-22% 1434 of the total urinary arsenic. The normal range of monomethylarsonic acid is 10-20% 1435 and the percentage increase was almost the same for the two groups. The rise in 1436 monomethylarsonic acid was accompanied by a decrease in dimethylarsinic acid. 1437 The percentage of inorganic arsenic also increased with unithiol treatment. 1438 1439 9.5.2 Bismuth elimination 1440 1441 Two groups of 12 volunteers (age 26-65 years), who had been treated with colloidal 1442 bismuth subcitrate for 28 days because of Helicobacter pylori-associated gastritis, took 1443 part in a study of bismuth elimination with unithiol and succimer. Each subject 1444 received a single oral dose of succimer or unithiol 30 mg/kg in a randomised single 1445 blind study. The succimer or unithiol was given 7 to 14 days after the last dose of 1446 bismuth. Both antidotes produced a 50-fold increase in urinary bismuth excretion 1447 compared to control urine samples. The highest concentration was excreted within 1448 the first 4 hours after dosing. No significant difference was observed in bismuth 1449 elimination between succimer and unithiol and both were well tolerated (Slikkerveer et 1450 al., 1998). 1451 1452 9.5.3 Cadmium elimination 1453 1454 Of the 6 metal-binding agents investigated in an in vitro study using human cell 1455 cultures, unithiol, succimer and mercaptosuccinic acid were found to the most 1456 effective at increasing cadmium movement from cells. Unithiol produced the most 1457 rapid elimination from cells in the first two hours, but the other two agents mobilised 1458 more cadmium than unithiol (Bakka et al., 1981). 1459 1460 9.5.4 Mercury elimination 1461 1462 Significant increases in urinary mercury elimination have been demonstrated with 1463 unithiol administration. 1464 1465 The mercury elimination rate was determined in 5 healthy volunteers (4 male, 1 1466 female, aged 24 to 32 years, 49-93 kg) given unithiol 3 mg/kg intravenously over 5 1467 minutes. Unithiol increased mercury excretion by a factor of 24.2 in the 11 hours 1468 after administration. No relationship was observed between the dose of unithiol and 1469 the quantity of mercury excreted in the urine (Hurlbut et al., 1994). In the 4 male 1470 subjects who had taken part in a previous oral study (Maiorino et al., 1991) the 1471 quantity of mercury excreted in the 12 hours after intravenous administration was 1472 less than that excreted in the same period after oral unithiol (Hurlbut et al., 1994). 1473 1474 Mercury elimination was examined after a unithiol challenge test in 12 male (66-96 1475 kg), former chloralkali workers exposed to metallic mercury vapour for 2-18 years. 1476

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The investigation was undertaken 18-56 months after exposure has ceased. A 1477 single 300 mg dose of unithiol was given and this increased 24 urinary excretion of 1478 mercury by a factor of 7.6. A high proportion (62%) was excreted within the first 6 1479 hours and this probably reflects mercury stored in the kidney (Sallsten et al., 1994). 1480 1481 The clinical efficacy of unithiol was investigated in 10 male volunteers (aged 19-45 1482 years) with occupational mercury exposure (with a urine mercury concentration 1483 equal to or greater than 50 μg/g of creatinine). Each subject received unithiol 100 1484 mg orally three times a day for 5 days. They had been asked to omit seafood from 1485 the diet for one month and to take no iron-containing vitamin preparations or 1486 medications for 2 weeks prior to the study. One subject developed a macular rash 1487 which resolved in two days. Otherwise, all the subjects remained well with no 1488 changes in renal or liver function, blood biochemistry or vital signs. In 9 of the 10 1489 subjects mercury elimination was significantly enhanced during the first 24 hours 1490 after unithiol administration, and in all subjects the mean increase in mercury 1491 excretion was higher over the 5 day period compared to the baseline (Torres- Alanís 1492 et al., 1995). 1493 1494 Gonzalez-Ramirez et al. (1998) also studied the effect of unithiol on mercury 1495 elimination in volunteers with occupational exposure (5 males, 3 females, aged 21-1496 57 years). The unithiol was given in 3 cycles: 3 days after an initial challenge test, 1497 unithiol was given for 8 days with 5 subsequent days with no treatment. This was 1498 followed by second cycle of 7 days of unithiol, a 5 day period of no treatment and 1499 then 6 days of unithiol. The unithiol was given orally 1 hour before breakfast, lunch 1500 and dinner in doses of 100 mg, 100 mg and 200 mg, respectively, on each treatment 1501 day. One subject developed a maculopapular rash and raised liver enzymes after 1502 the first course and did not receive further doses. Prior to treatment the mean total 1503 urinary mercury excreted in 24 hours was 504 µg (range 140-1692 µg) and during 1504 the first course of unithiol this rose to 1754 µg (range 657-2880 µg). The figure for 1505 the two subsequent courses became progressively lower (314 µg [range 152-658 µg] 1506 and 173 µg [range 74-443 µg]) but in both cases was higher than the period of no 1507 treatment which preceded it (106 µg [range 66-212 µg] and 48 µg [range 30-97 µg]). 1508 Unithiol was effective in lowering the body burden of mercury and increasing the 1509 urinary mercury concentration. 1510 1511 The elimination of mercury following unithiol administration was examined in 75 1512 mercury-exposed volunteers from a gold mining area in the Philippines. Urine 1513 samples were collected before and 2 to 3 hours after unithiol administration (200 mg 1514 orally). In the first urine the mean concentration of inorganic mercury was 15.7 µg/g 1515 of creatinine and for organic mercury it was 2.2 µg/g of creatinine. In the samples 1516 after unithiol dosing the concentrations were 262 µg/g of creatinine and 14.5 µg/g of 1517 creatinine, respectively. Unithiol increased the inorganic urinary concentration by a 1518 factor of 16 and the organic mercury concentration by a factor of 5.1 (Drasch et al., 1519 2007). 1520 1521 Mercury clearance during dialysis was investigated in an in vitro experiment using 1522 pooled plasma samples to which mercury and metal-binding agents had been added. 1523 Of the agents investigated acetylcysteine was the most effective in clearing mercury 1524 after 90 minutes of in vitro dialysis, reducing the mercury concentration in the 1525 perfusate by 73%, whereas unithiol removed almost 70% of mercury in the perfusate. 1526

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Unithiol was more effective than succimer but the results were not statistically 1527 different. In contrast the quantity of mercury removed from plasma without the 1528 presence of a metal-binding agent was very low at 5% (Ferguson & Cantilena, 1992). 1529 1530 9.5.5 Palladium elimination 1531 1532 There is limited information on palladium elimination with unithiol. In a study of 50 1533 volunteers the elimination of palladium increased from 0.3 to 38 µg/g of creatinine 1534 and there was no difference between oral and intravenous administration of unithiol 1535 (Runow, 1996). 1536 1537 9.5.6 Trace element elimination 1538 1539 Unithiol has been shown to increase elimination of some trace elements in volunteer 1540 studies. 1541 1542 Trace element elimination was examined after a unithiol challenge test in 12 male 1543 (66-96 kg), former chloralkali workers exposed to metallic mercury vapour for 2-18 1544 years. The investigation was undertaken 18-56 months after exposure has ceased. 1545 A single 300 mg dose of unithiol was given and this increased 24 hour urinary 1546 excretion of copper by a factor of 12 and zinc by 1.5 (Sallsten et al., 1994). 1547 1548 Eleven patients presenting with concerns over exposure to mercury in dental amalgam 1549 were given a unithiol challenge test, and the urinary elimination of trace elements 1550 examined. The patients (8 female, 3 male) had no known occupational exposure to 1551 mercury. The dose of unithiol was 3 mg/kg intravenously and urine samples were 1552 taken 1 hour before and 1 hour after. There was a significant increase in mercury 1553 excretion (3-107 fold) in all subjects. The elimination of chromium and manganese 1554 was unchanged and the urinary concentrations of cobalt, aluminium and molybdenum 1555 were too low for reliable measurement. The urine copper (2-119 fold), selenium (3-1556 43.8 fold), zinc (1.6-44 fold) and magnesium (1.75-42.7 fold) concentrations were 1557 increased in most patients (Torres- Alanís et al., 2000). 1558 1559 Trace element blood concentrations were examined in 80 volunteers (51 females, 29 1560 males) given 2 mg/kg unithiol intravenously. An increase in urinary copper and zinc 1561 concentrations were observed 30 and 120 minutes after injection. The selenium 1562 concentration was unaffected. There was also a decrease in blood concentrations of 1563 copper, zinc and selenium. However, this may have been due to a dilution effect since 1564 the concentrations returned to normal within 120 minutes of the injection and a similar 1565 effect was observed within iron concentrations (Høl et al., 2003). 1566 1567 1568 10. Clinical studies – clinical trials 1569 1570 Few controlled clinical trials on unithiol are available, and most concern mercury. 1571 1572 10.1 Arsenic and unithiol clinical trials 1573 1574 Unithiol was investigated in a randomised placebo-controlled trial in the management 1575 of chronic arsenicosis due to contaminated drinking water (arsenic >50 μg/L) in 1576

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India. All the patients weighed 40-56 kg and had been drinking the water for more 1577 than 3 years. Subjects were excluded if they had ceased drinking arsenic-1578 contaminated water for more than 3 months, had been treated with other antidotes, 1579 had a history of smoking, alcoholism, were taking hepatotoxic drugs or were serum 1580 positive for hepatitis B virus surface antigen. Pregnant or lactating women were also 1581 excluded. 1582 1583 A total of 21 patients were randomly assigned to 2 treatment groups: 11 patients (9 1584 males, 2 females, mean age 30.63 years) received unithiol and 10 patients (5 males, 1585 5 females, mean age 34.4 years) received the placebo. The unithiol dosage regimen 1586 was 100 mg 4 times a day for 1 week, repeated in the third, fifth and seventh week. 1587 Further ingestion of arsenic-contaminated drinking water was also stopped. Therapy 1588 with unithiol resulted in significant clinical improvement as evaluated by an objective 1589 clinical scoring system. Cessation of exposure and placebo also reduced clinical 1590 scores but the post-treatment scores were significantly lower for unithiol-treated 1591 subjects. Improvement in clinical scores for the placebo group was attributed to 1592 cessation of exposure, rest and provision of an adequate hospital diet. The most 1593 significant improvement was seen in the clinical score of weakness, pigmentation 1594 and lung disease. There were also significant increases in total urinary arsenic 1595 excretion with unithiol treatment, compared to no increase in the placebo group. 1596 Unithiol was well tolerated with no adverse effects reported (Guha Mazumder et al., 1597 2001). 1598 1599 10.2 Copper (Wilson’s disease) and unithiol clinical trials 1600 1601 Wang et al. (2003) studied 28 patients with Wilson’s disease (18 males, 10 females 1602 aged 14-20 years). Patients were included on the basis of presence of 1603 extrapyramidal symptoms and signs, presence of characteristic corneal Kayser-1604 Fleischer ring observed with a slit lamp, a serum ceruloplasmin <200 mg/L and 1605 copper oxidase concentration <0.21 units and urinary copper >100 μg (1.56 µmol)/24 1606 hours. Group A received captopril, 1 mg/kg orally daily in 3 divided doses, Group B 1607 unithiol 20 mg/day intravenously and Group C both. Group D was the control group 1608 and did not receive either drug. Serum sulphydryl concentrations and 24 hour 1609 urinary copper concentrations were the markers of anticopper efficacy. Unithiol had 1610 a more potent anticopper effect than captopril and increased urinary copper 1611 concentrations and serum sulphydryl concentrations. Only 1 patient developed an 1612 adverse effect with transient elevation of alanine aminotransferase. 1613 1614 10.3 Lead and unithiol clinical trials 1615 1616 There are few clinical trials on the use of unithiol in lead poisoning. 1617 1618 One of the earliest reports of unithiol use in the treatment of lead poisoning was by 1619 Anatovskaya (1962). Sixty men with chronic lead toxicity were given 250 mg 1620 intramuscularly for 20 days. Blood concentrations of lead fell and urinary excretion 1621 increased. The clinical signs and symptoms improved subjectively and objectively. 1622 Haematological parameters and liver function improved in more patients in the 1623 unithiol-treatment group compared to controls given only supportive care. In addition 1624 patients treated with unithiol could be discharged from hospital 6 weeks earlier than 1625 controls. 1626

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1627 A more recent study involved children attending a lead poisoning treatment clinic in 1628 Baltimore, USA, where lead paint was the main source of poisoning. The criteria for 1629 inclusion were a blood lead concentration of 400-600 µg/L, normal hepatic, renal and 1630 haematological function, no history of antidotal therapy within the previous 3 months, 1631 no concurrent disease and an age of 30 to 72 months. Six children (5 males, 1 1632 female, aged 31 to 53 months) received a 5 day course of unithiol, 200 mg/m2 daily. 1633 Another 6 children (1 male, 5 females, aged 38-69 months), received 400 mg/m2 daily 1634 for 5 days. The drug was well tolerated in all cases. Administration of either dose 1635 reduced the blood lead concentration to approximately 78% of its pre-treatment 1636 concentration by 48 hours. After 96 hours the blood lead concentration was 76% and 1637 68% of its pre-treatment concentration in the low and high dose group, respectively. 1638 Blood lead concentrations did not increase until 48 hours after cessation of therapy. 1639 Urinary excretion of both copper and zinc were increased, 2-30 fold and 1.2-9.1 fold, 1640 respectively with higher concentrations of both in the higher treatment group. There 1641 were no significant changes in plasma copper or zinc concentrations. The urinary 1642 concentration of lead increased 1.3-15 fold between the last pre-treatment day and the 1643 first treatment day (Chisolm & Thomas, 1985). This trial was later terminated following 1644 the occurrence of at least one case of Stevens-Johnson syndrome and succimer was 1645 used instead (Chisolm, 1990). 1646 1647 10.4 Mercury and unithiol clinical trials 1648 1649 There are a small number of clinical trials investigating the effect of unithiol on 1650 mercury toxicity. An early study undertaken in Iraq, with preliminary results reported 1651 by Bakir et al. (1976) was limited in scope due to various circumstances (Clarkson et 1652 al., 1981). A more recent study examined patients with chronic mercury exposure in 1653 the Philippines (Böse-O’Reilly et al., 2003). Another study in China examined the 1654 effects of antidotal treatment in acutely poisoned patients treated 5 months after 1655 exposure (Zhang, 1984). A Mexican study examined the effect of unithiol on 1656 mercury excretion in patients with high mercury concentrations due to use of 1657 mercury-containing facial cream (Garza-Ocañas et al., 1997). 1658 1659 The early study investigated antidotal therapy in patients with methyl mercury 1660 poisoning. The source was homemade bread made from wheat contaminated with a 1661 methyl mercury fungicide over the winter of 1971-1972. Antidotal agents where in 1662 limited supply and were not available until February 1972 after exposure had ceased. 1663 The conditions of the time did not allow for the implementation of a clinically controlled 1664 study but it was possible to obtain data on the effects of antidotes on blood mercury 1665 concentrations. D-penicillamine (12 patients), N-acetyl-DL-penicillamine (17), unithiol 1666 (10) and a thiolated resin (8) were used. There were 27 females and 20 males, aged 1667 from 18 months to 55 years. Ten other patients received a placebo and 6 received no 1668 specific treatment. The duration of treatment was variable but for unithiol was 4 to 15 1669 days. All agents reduced blood mercury concentrations but unithiol was the most 1670 effective. However, the patients did not show any immediate clinical improvement, 1671 presumably because the duration of therapy was too short (Clarkson et al., 1981). 1672 1673 Unithiol was evaluated in the management of chronic mercury exposure, including 1674 exposure from mercury vapour, inorganic mercury and organic methyl mercury, in the 1675 gold mining area of Mount Diwata, in the Philippines. A total of 95 patients (no details 1676

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given) were included in the study. There was no control group. Unithiol was given 1677 orally at a dose of 200 mg twice daily for 14 days for adults and 5 mg/kg/day for 1678 children. Patients were assessed by questionnaire, medical, including neurological, 1679 examination and neuropsychological testing before and after therapy. Blood, urine 1680 and hair samples were analysed for mercury concentrations before therapy and blood 1681 and urine after therapy. Two study populations were identified: those living near 1682 Mount Diwata and exposed to metallic and/or inorganic mercury (60 patients), and 1683 those living downstream in Monkayo who were exposed to methyl mercury (35 1684 patients). Exposure to mercury continued during therapy with unithiol. The mercury 1685 concentrations in hair and blood did not differ between the two populations before 1686 unithiol therapy. The urine concentration was significantly higher in the Mount 1687 Diwata population (due to the differing pharmacokinetics of organic and inorganic 1688 mercury). There was therefore a higher renal excretion of mercury in the Mount 1689 Diwata population when treated with unithiol. However, the Monkayo population 1690 demonstrated a relative increase in renal excretion of mercury almost as high as the 1691 Mount Diwata population when given unithiol. Some patients (number not specified) 1692 did not respond to unithiol administration and showed no increase in mercury 1693 excretion. In the Monkayo group there was only a modest decrease in blood 1694 mercury concentrations, indicating the duration of treatment was too short to have a 1695 long-term effect on mercury stored in tissues. More than two-thirds of patients 1696 reported an improvement in subjective complaints after therapy and objective 1697 neurological parameters also showed significant improvement. Significant 1698 improvement was also demonstrated in two neuropsychological tests. The authors 1699 concluded that unithiol could increase mercury excretion but that a 14 day regimen 1700 was too short to have a permanent effect on mercury concentrations (Böse-O’Reilly 1701 et al., 2003). This study has a number of limitations including the absence of a control 1702 group, lack of details of patients (age, weight) and continued exposure to mercury 1703 during therapy. 1704 1705 Forty-one patients (aged 2 to 65 years) from 8 families were poisoned with mercury 1706 following ingestion of rice contaminated with an ethyl mercury seed dressing. One 1707 patient died soon after onset of symptoms and 8 were admitted in the initial acute 1708 phase of intoxication. The remaining 40 patients demonstrated a variety of clinical 1709 features 5 months after ingestion and 27 were treated with unithiol (250 mg daily by 1710 intramuscular injection) and/or succimer (500 mg twice daily by intravenous injection). 1711 The drugs were given for 3 days followed by a 4 day break and then another 3 day 1712 course, if required. Patients were given 1 to 8 courses until the urinary mercury 1713 concentration was normal. The 13 untreated patients showed little improvement in 1714 clinical features of toxicity but all those on therapy had some relief and 19 became 1715 asymptomatic. In 2 cases there was only slight improvement. In patients where the 1716 urinary concentration was measured before therapy, all but one had increased 1717 mercury excretion during antidotal administration. Side effects were mild and 1718 generally resolved within 30 minutes to 4 hours. Unithiol was found to be more 1719 effective, although no data distinguishing between the two drugs is given. In addition, 1720 the two antidotes were used interchangeably in some patients (Zhang, 1984). 1721 1722 Unithiol was also used in 12 females (aged 19-45 years) with mercury toxicity 1723 following use of a facial cream containing 5.9% mercurous chloride (calomel) for 2 to 1724 10 years. Two patients were symptomatic (exanthema and tremor) and all had 1725 elevated urinary mercury concentrations. Subjects were given a 5 day course of 1726

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unithiol (200 mg/day) as outpatients. Only 8 subjects took the unithiol as prescribed 1727 and supplied 24 hours urine samples for analysis. In all cases there was a significant 1728 increase in urinary mercury concentrations after 24 hours of unithiol. One 1729 symptomatic patient has complete resolution of effects and the other had persistent 1730 tremor. Unithiol was well tolerated in all patients (Garza-Ocañas et al., 1997). 1731 1732 1733 11. Case reports - clinical studies 1734 1735 In many clinical case reports of unithiol use in poisoning the authors do not determine 1736 a balance between the quantity of metal absorbed and excreted; this can be difficult 1737 where the dose ingested, injected or inhaled cannot be quantified. Clinical efficacy of 1738 unithiol is often only stated in terms of enhancing excretion and/or decreasing blood 1739 concentrations in the absence of severe adverse effects. In many cases, the authors 1740 are unable to distinguish between the effect of unithiol administration and the effect of 1741 supportive therapy (including removal from the source). 1742 1743 The efficacy of a metal-binding agent may be difficult to determine. After 1744 discontinuation of metal exposure (and absorption) a decrease in the blood 1745 concentration will occur without any therapy. Clinical efficacy should not be judged 1746 only by the amount of metal excretion or the decrease of blood concentrations. The 1747 reduction of the tissue content in the target organ and the restoration of pathological 1748 alterations also need to be considered. It is important to note that enhancement of the 1749 metal excretion by mobilisation may increase the metal burden of the target organ by 1750 redistribution, and conversely the body burden may be reduced without a striking 1751 decrease of the blood concentrations. However, in some case reports severe toxicity 1752 usually associated with a demonstrated high blood or urine concentration does not 1753 occur, and this may reasonably be assumed to be due to antidotal therapy. With 1754 these reservations in mind, it is clear that administration of unithiol can prevent 1755 development of toxicity and in symptomatic patients it can reduce recovery time and 1756 improve clinical signs and symptoms of toxicity. 1757 1758 Reference values for the metal and metalloids (Walker, 1998) are given as a guide at 1759 the start of each section to aid interpretation of the concentrations given in the case 1760 reports. 1761 1762 11.1 Use in antimony poisoning 1763 1764 Reference values for antimony (Walker, 1998): 1765 Children Serum/plasma 0.18 µg/L 1766 Blood 0.26 µg/L 1767 Urine 0.05 µg/L 1768 Adults (unexposed) Urine 0.8 µg/L 1769 1770 There is limited information on the use of unithiol in antimony poisoning, but it appears 1771 to be of benefit in the management of poisoning with trivalent antimony compounds. 1772 There is no information on its use in the management of poisoning with the less toxic 1773 pentavalent antimony compounds. 1774 1775 A 2 year 11 month old child was treated with unithiol after ingestion of an unknown 1776

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quantity of tartar emetic (antimony potassium tartrate). The clinical features, 1777 particularly massive fluid loss and dehydration, were consistent with antimony 1778 poisoning. She was given unithiol 65 mg intravenously then 3 x 100 mg daily for 10 1779 days followed by 3 x 50 mg daily for another 10 days. She was also treated with 1780 exchange transfusion at 39 hours post-ingestion. The serum antimony concentration 1781 at 4.5 hours post-ingestion was 0.6 mg/L and the urine concentration at 7 hours 31 1782 mg/L. Unithiol was considered effective in reducing the antimony concentration in the 1783 early stages of poisoning (Iffland & Bösche, 1987). 1784 1785 Unithiol has also been used with apparent success in other paediatric cases of 1786 antimony potassium tartrate poisoning (Kemper et al., 1989; Jekat & Kemper, 1990) 1787 and is recommended by others for the management of antimony poisoning (Chzhi-1788 Tysan, 1959; Lauwers et al., 1990). 1789 1790 11.2 Use in arsenic poisoning 1791 1792 Reference values for arsenic (Walker, 1998): 1793

Blood <0.13 µmol/L (<10 µg/L) 1794 Urine <0.13 µmol/24 hours (<10 µg/24 hours) of inorganic arsenic 1795

Urine <40 nmol/mmol creatinine (unexposed) 1796 <173 nmol/mmol creatinine (occupational exposure) 1797 1798 Unithiol has been used successfully in a number of cases of acute and chronic 1799 arsenic poisoning and is considered the antidote of choice for arsenic toxicity (Adam 1800 et al., 2003). 1801 1802 A 33-year-old female (62 kg) was treated with unithiol after accidental ingestion of 1803 arsenic paste (approximately 1.85 g of arsenic trioxide). She presented 100 hours 1804 after ingestion and was started on unithiol (250 mg intravenously every 6 hours for 1805 48 hours, then 250 mg orally 3 times a day for 48 hours, followed by 250 mg every 1806 12 hours for 23 days). Her condition improved after 2 days and she made a full 1807 recovery. The highest urinary arsenic concentrations occurred on hospital days 1-3 1808 (Kruszewska et al., 1996). 1809 1810 Two brothers, aged 21 and 19 years, were treated with unithiol after ingestion of 4 g 1811 and 1 g, respectively, of a powder which was later identified as arsenic trioxide. 1812 Treatment with unithiol was started 32 and 48 hours after ingestion. In the older 1813 brother the blood arsenic concentration at 26 hours was 400 µg/L. He suffered a 1814 cardiac arrest just before unithiol was started but was successfully resuscitated. At 1815 36 hours the blood arsenic concentration of the younger brother was 98 µg/L. Both 1816 men made a full recovery with no clinical or electrophysiological signs of arsenic 1817 neuropathy (Moore et al., 1994). 1818 1819 A 21-year-old male was treated with unithiol after intentional ingestion of 0.6 g of 1820 arsenic trioxide. He presented to hospital seven hours after ingestion and was 1821 dehydrated following diarrhoea, intestinal colic and vomiting. He was given a gastric 1822 lavage, rehydrated (on average 5.5 L of fluid/daily over 5 days) and given high dose 1823 unithiol. The parenteral regimen was 250 mg/hour on day 1, 125 mg/hour on day 2 1824 and then 62.5 mg/hour on days 3-5. From days 6-12 he was given oral doses of 600-1825 700 mg/day. The total dose given was 15.225 g. The serum arsenic concentration 1826

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on day 1 was 143 mg/L (urine 210,000 µg/L), on day 2, 29 mg/L (3800 µg/L), on day 1827 3, 10 mg/L (1675 µg/L) and by day 8 was <1 mg/L (115 µg/L). He developed mild 1828 increases in transaminase concentrations but remained otherwise well (Horn et al., 1829 2002). Heinrich-Ramm et al. (2003) examined the arsenic compounds excreted in this 1830 patient. In urine sampled on days 2-8 he excreted about 50 mg of arsenic of an 1831 estimated total ingested dose of 230 mg of arsenic. In the first urine sampled after 1832 unithiol therapy (11 hours post-ingestion) the arsenic concentration was 215 mg/L. 1833 This fell by a 1000-fold to 169 µg/L after 8 days of unithiol administration. As reported 1834 in other studies (see section 9.5.1), administration of unithiol resulted in a decrease 1835 in the urinary concentration of dimethylarsinic acid. In this patient the urinary 1836 concentration of dimethylarsinic acid did not exceed that observed in a reference 1837 population where it had accounted for 95% of the total arsenic excreted. In contrast, 1838 the urinary dimethylarsinic acid concentration in this patient accounted for <5% of 1839 the total arsenic excreted. This supports the theory that unithiol interferes with 1840 methylation of arsenic. 1841 1842 A 27-year-old female developed gastrointestinal effects, ECG changes and liver 1843 damage after ingestion of 9 g of arsenic trioxide. She was treated with intravenous 1844 fluids, activated charcoal and continous alkaline irrigation of the stomach over 36 1845 hours. She was given succimer (15 mg/kg every 8 hours for 26 days) and 1846 intramuscular dimercaprol (4 mg/kg every 4 hours) for 24 hours. A second course of 1847 dimercaprol was administered on day 5 due to continued deterioration. She was 1848 also started on a regimen designed to enhance methylation of arsenic derivatives 1849 which are less toxic and more readily excreted. She was given hydroxocobalamin, 1850 methionine, folinic acid, sodium bicarbonate, glutathione and intravenous unithiol 1851 (250 mg every 4 hours for 5 days). She began to improve within 48 hours with 1852 improved pulmonary function and ECG. Liver function tests began to resolve but 1853 were elevated for more than a month. She also received unithiol from days 15 to 18. 1854 Urinary arsenic concentrations fell rapidly in the first 10 days. At follow up one year 1855 later she had mild polyneuropathy. The role of succimer in this patient’s recovery 1856 was not evaluated. Unithiol, with and without the methylating regimen, increased the 1857 proportion of methylated arsenic metabolites (monomethylarsonic acid and 1858 dimethylarsenic acid) in the urine (Vantroyen et al., 2004). 1859 1860 A 33-year-old female with chronic arsenic poisoning from an unknown source was 1861 treated with unithiol. She presented with a 1.5 year history of episodes of peripheral 1862 neuropathy, pancytopenia, ventricular tachycardia, gastrointestinal symptoms, skin 1863 rash and nail changes. Her blood arsenic concentration was 56 µg/L and the 24 1864 hour urine concentration 130 µg/L. Analysis of well water demonstrated an arsenic 1865 concentration of 78 µg/L. Electromyography revealed moderate demyelinating 1866 neuropathy with axonal involvement. She was started on succimer 10 mg 3 times a 1867 day but serial urinary arsenic determination did not show any elimination. Her 1868 neuropathy continued to progress and she required ventilation. She was then 1869 started on unithiol at 250 mg/kg intravenously every 4 hours. During the first 24 1870 hours of treatment the urinary arsenic concentration rose from 101 to 300 µg/L. 1871 There was also improvement in her neuropathy and she was continued on unithiol 1872 for 12 days. She was much improved, extubated and discharged in a wheelchair 10 1873 days later. By 3 months she was walking on her own with some residual 1874 paraesthesiae and weakness in distal lower extremities. At follow up one year later 1875 she had only mild weakness and residual paraesthesiae controlled with amitriptyline 1876

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(Wax & Thornton, 2000). 1877 1878 Adam et al. (2003) reports 6 cases of arsenic poisoning, with retrospective 1879 comparison of the use of dimercaprol and unithiol. Three patients were treated with 1880 dimercaprol. Two of these patients with blood arsenic concentrations of 540 µg/L 1881 and 620 µg/L died within 34 hours and 6 days of exposure, respectively. One patient 1882 had a maximal blood arsenic concentration of 558 µg/L and developed paraplegia as 1883 a result of arsenic poisoning. The three patients treated with unithiol recovered fully. 1884 Two of these patients (including one with anuric renal failure) had very high blood 1885 arsenic concentrations of 2240 µg/L and 4469 µg/L. The third patient had a 1886 relatively mild course, after the early use of unithiol, despite a blood arsenic 1887 concentration of 245 µg/L. The authors concluded that unithiol is the treatment of 1888 choice for arsenic poisoning and that dimercaprol is obsolete. 1889 1890 11.3 Use in beryllium poisoning 1891 1892 No well documented case reports of unithiol use in beryllium poisoning could be 1893 found. 1894 1895 11.4 Use in bismuth poisoning 1896 1897 Reference values for bismuth (Walker, 1998): 1898

Blood <0.5 nmol/L (<0.1 µg/L) basal 1899 Up to 240 nmol/L (up to 50 µg/L) acceptable therapeutic 1900 >480 nmol/L (>100 µg/L) risk of toxicity 1901 Urine <0.5 nmol/L (<0.1 µg/L) 1902

1903 Unithiol has been used in both acute (Stevens et al., 1995; Bogle et al., 2000; 1904 Dargan et al., 2001; Dargan et al., 2003b; Ovaska et al., 2008) and chronic (Playford 1905 et al., 1990) bismuth poisoning, although clinical deterioration has been reported in 1906 one chronic case due to redistribution of tissue stores of bismuth (Teepker et al., 1907 2002). Unithiol is considered an effective antidote for acute bismuth poisoning 1908 (Andersen, 1999). 1909 1910 Unithiol was started 24 hours after intentional ingestion of 2.88 g of bismuth 1911 subcitrate in a 13-year-old girl. She was given 30 mg/kg orally for 10 days and 10 1912 mg/kg for 9 days. The serum bismuth concentration at 4 hours was 300 µg/L, 14 1913 μg/L at 48 hours and 8 µg/L at 72 hours. At 10 days post-ingestion the serum 1914 bismuth concentration was 1.8 µg/L. She remained well throughout (Bogle et al., 1915 2000). 1916 1917 In a similar case a 30-year-old male was started on unithiol after ingestion of 4.8 g of 1918 tripotassium dicitratobismuthate. He was admitted 2 hours post-ingestion with 1919 vomiting, but was otherwise well. The initial bismuth blood concentration was 424 1920 µg/L and urine 10,000 µg/L. Oral unithiol was started on day 2 (200 mg four times 1921 daily for 10 days, then 200 mg twice daily for 10 days). He remained well and after 1922 antidotal therapy the blood concentration was 17 µg/L and urine 37 µg/L (Dargan et 1923 al., 2001). 1924 1925 A 21-year-old male developed bismuth toxicity after ingestion of 50 to 60 tablets of 1926

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tripotassium dicitratobismuthate. The blood bismuth concentration was 590 µg/L 1927 and he was initially given dimercaprol 150 mg intramuscularly. Four hours later he 1928 was started on haemodialysis. He was subsequently given unithiol 250 mg 1929 intravenously 4 hourly for 48 hours and then 250 mg 6 hourly for 48 hours, then 500 1930 mg orally in two divided doses for 14 days (days 6 to 19 after ingestion). He 1931 received haemodialysis for the first 6 days and on days 8, 9 and 12. Each session 1932 lasted 4 hours and started 1 hour after unithiol administration. No significant 1933 clearance of bismuth occurred with dimercaprol. In contrast significant bismuth 1934 clearance was obtained with unithiol and despite improving renal function cessation 1935 of unithiol on day 20 resulted in a decrease in urinary bismuth clearance (Stevens et 1936 al., 1995). 1937 1938 Unithiol has been used in poisoning resulting from the use of bismuth iodoform 1939 paraffin paste used to pack a large wound of the sacrum following tumour excision. 1940 The patient began to develop neurological effects 5 days later and blood and urine 1941 bismuth concentrations were raised (340 µg/L and 2800 µg/L, respectively). The 1942 packing was removed and he was started on unithiol (intravenously 5 mg/kg four 1943 times daily for 5 days, 5 mg/kg 3 times daily for 5 days, and 5 mg/kg twice daily for 1944 17 days, then orally 200 mg 3 times daily for 10 days, 200 mg twice daily for 14 1945 days; 61 days in total). His neurological effects improved over the next month and 1946 the bismuth concentrations were normal after 55 days (Dargan et al., 2003b; Ovaska 1947 et al., 2008). 1948 1949 A 68-year-old male with renal impairment developed encephalopathy from ingestion 1950 of double the dose of tripotassium dicitratobismuthate (864 mg of bismuth daily) for 2 1951 years. He was given unithiol 100 mg 3 times daily for 10 days. Renal clearance of 1952 bismuth increased 10-fold from 0.24 mL/minute to 2.4 mL/minute during this time. 1953 The whole blood bismuth concentration decreased from 880 µg/L to 46 µg/L over a 1954 50 day period and he had marked improvement in cerebral function. There were no 1955 adverse effects (Playford et al., 1990). 1956 1957 In a 49-year-old female with encephalopathy from 5 years of chronic oral abuse of 1958 bismuth, unithiol had to be discontinued due to deterioration in her clinical condition. 1959 She was given 100 mg daily and there was a marked decrease in plasma bismuth 1960 concentrations with an increase in urinary bismuth concentrations. However, she 1961 deteriorated with cluster-like myoclonic jerks and stupor and the unithiol was stopped 1962 after 3 days. Her clinical condition improved over the next few weeks and lagged 1963 behind the plasma bismuth concentrations. They deceased from 550 µg/L to 30.4 1964 µg/L by day 21 when she demonstrated marked improvement. The unithiol may 1965 have caused redistribution of bismuth from tissue stores and caused the clinical 1966 deterioration in this patient (Teepker et al., 2002). 1967 1968 11.5 Use in cadmium poisoning 1969 1970 Reference values for cadmium (Walker, 1998): 1971

Blood <27 nmol/L (<3 µg/L) non smokers 1972 <54 nmol/L (<6 µg/L) smokers 1973 Urine 44.5 nmol/L (5 g/L) 1974

0.4-1.3 nmol/mmol creatinine (unexposed) 1975 1976

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There is limited information on the use of unithiol in cadmium poisoning. In a woman 1977 with chronic occupational exposure to cadmium and signs and symptoms of toxicity, 1978 administration of unithiol increased the urinary concentration of cadmium from 1.8 to 1979 4.2 μg/L (Daunderer, 1995). 1980 1981 11.6 Use in chromium poisoning 1982 1983 Reference values for chromium (Walker, 1998): 1984

Blood <10 nmol/L (<0.5 µg/L) 1985 Urine <5 nmol/L (<0.25 µg/L) non-occupational exposure 1986 Up to 98 nmol/mmol creatinine, depending on type of occupation 1987 1988

There is limited information on the use of unithiol in chromium poisoning. An adult 1989 who fell in a pool of chromic acid was started on unithiol within an hour of exposure. 1990 He became anuric and chromium was detected in the dialysate. Urine excretion 1991 returned 2 days after exposure and the chromium concentration was 5859 μg/L. The 1992 maximum urinary chromium concentration, 13,614 μg/L, was obtained 12 hours after 1993 the start of unithiol therapy. The serum chromium concentration was 1,983 mg/L 24 1994 hours after exposure. The patient recovered but no further details are given (Donner 1995 et al., 1986). 1996 1997 A 20-year-old male died after ingestion of 10-30 g of potassium dichromate. He was 1998 treated with haemodialysis (commenced at 2.5 hours) and continuous arterio-venous 1999 haemofiltration (at 16 hours). Unithiol (250 mg intravenously every 4 hours for 24 2000 hours then 6 hourly) was started 13 hours after admission. The initial plasma 2001 concentration of chromium was 5.8 mg/L and urine 159 mg/L. He had a cardiac 2002 arrest and died 48 hours after admission (Pudill et al., 1989). 2003 2004 11.7 Use in cobalt poisoning 2005 2006 There is limited information on the use of unithiol in cobalt poisoning. Unithiol was 2007 used in 2 paediatric patients with ingestion of an unspecified cobalt compound from 2008 a chemistry set. The children were initially treated with penicillamine and then on the 2009 fifth day started on the less toxic unithiol (3 x 50 mg orally). There were slight 2010 increases in serum cobalt concentrations during unithiol therapy and it was stopped 2011 once the urine cobalt concentrations were normal. No further details are given 2012 (Müller et al., 1989). 2013 2014 11.8 Use in copper poisoning 2015 2016 There is limited information on the use of unithiol in acute copper poisoning. A 3-2017 year-old child who ingested more than 3 g of copper sulphate received a gastric 2018 lavage within 30 minutes of ingestion and was commenced on unithiol therapy within 2019 an hour. The serum copper concentration did not reach a toxic concentration and 2020 urinary copper excretion was more than 10 times normal; no further details are given 2021 (Donner et al., 1986). 2022 2023 A 33-year-old female ingested an unknown quantity of copper sulphate and developed 2024 severe haemorrhagic gastroenteritis, dehydration, metabolic acidosis, renal failure, liver 2025 damage, intravascular haemolysis and methaemoglobinaemia. She was given unithiol 2026

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(250 mg intravenously every 4 hours) within 24 hours of ingestion until she developed 2027 anuria (more than 48 hours later). This patient survived with 10 days of intensive 2028 therapy and 5 weeks of hospitalisation. The serum copper concentration was normal 2029 on admission (5 hours post-ingestion) and as there were no measurements of copper 2030 excretion, the effectiveness of unithiol administration cannot be determined (Sinković et 2031 al., 2008). 2032 2033 11.8.1 Use in Wilson’s disease 2034 2035 Unithiol has been used with some success in patients with Wilson’s disease, usually 2036 those who fail to respond to penicillamine. 2037 2038 Walshe (1985) reported the use of unithiol (200 mg twice daily) in an adult patient with 2039 Wilson’s disease. He had initially been managed with penicillamine and trientine but 2040 had become intolerant to them. He developed proteinuria but remained well with 2041 unithiol. His copper excretion was maintained at 31.5-47.2 µmol (2000-3000 µg) daily. 2042 Two other patients were also tried on unithiol. One developed fever and a 2043 decreased leucocyte count which also occurred with a second test dose and further 2044 treatment was not given. The other patient took it for 10 days but then refused 2045 because of intense nausea. Other patients (number not specified) were given test 2046 doses and the resulting cupruresis was comparable with that obtained with 2047 penicillamine and trientine in most cases. 2048 2049 11.9 Use in gold poisoning 2050 2051 Unithiol has been used in iatrogenic gold poisoning and although the patient died 2052 from heart failure the unithiol was thought to be effective in removing gold from the 2053 body. No further details are given (Ashton et al., 1992a). 2054 2055 11.10 Use in lead poisoning 2056 2057 Reference values for lead (Walker, 1998): 2058

Blood <0.5 µmol/L (<10 µg/L) environmental exposure 2059 Urine <100 nmol/24 hours (<10 µg/24 hours) normal adults 2060 2061

Unithiol was first used in the management of chronic lead poisoning in Russia 2062 (Anatovskaya, 1962), and although unithiol has been used in subsequent cases of 2063 lead poisoning and has been shown to be of benefit in animal studies, succimer is 2064 usually the preferred antidote for lead poisoning, particularly in children (Angle, 2065 1993) as it is the less toxic of the two antidotes (Andersen, 1999). 2066 2067 An adult with lead poisoning due to the use of a lead-containing ointment was treated 2068 with oral unithiol (400 mg then 200 mg 2 hourly). She had a blood lead concentration 2069 of 1150 µg/L with gastrointestinal effects and a paraesthesia. Within 36 hours the 2070 concentration has decreased to 570 µg/L and the maximum urinary lead concentration 2071 in 24 hours was 73000 µg/L. The blood concentration rose to 890 µg/L during an 2072 interruption of therapy due to lack of drug supply, but decreased rapidly once unithiol 2073 was recommenced. Therapy was continued over 2 months without adverse effects 2074 (Donner et al., 1987; Hruby & Donner, 1987). 2075 2076

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A 24-year-old female developed lead poisoning after ingestion of instant lemon tea 2077 from a lead-glazed cup, over a period of 2.5 months. The reason for her illness was 2078 difficult to establish at first due to the unusual source, but once lead toxicity was 2079 confirmed she was treated with oral unithiol (5-10 mg/kg 3 times a day for 2 days, then 2080 2.5 mg/kg twice daily) until the blood and urine lead concentrations were normal. Her 2081 initial whole blood and urine lead concentrations were 600 µg/L and 1700 µg/L, 2082 respectively. She recovered over a 4 month period (Autenrieth et al., 1998). 2083 2084 Unithiol treatment (parenteral and then two 5 day cycles) improved optic neuropathy 2085 caused by lead deposition in the eye. There was improvement in visual acuity, visual 2086 field and dark adaptation (Dambite, 1966). 2087 2088 11.11 Use in mercury poisoning 2089 2090 Unithiol is the antidote of choice in patients with mercury poisoning. It has been used 2091 successfully in the treatment of acute and chronic poisoning, involving metallic 2092 mercury, inorganic mercury salts and organic compounds. Andersen (1999) suggests 2093 that unithiol is the optimal antidote for inorganic mercury poisoning and that succimer 2094 is more effective in organic mercury intoxication. 2095 2096 In acute poisoning with mercuric salts the initial doses usually have to be given by the 2097 parenteral route because of damage to the gastrointestinal tract. Unithiol should be 2098 used in combination with haemofiltration in patients with mercury-induced renal failure. 2099 2100 Reference values for mercury (Walker, 1998): 2101

Blood <20 nmol/L (<4 µg/L) 2102 Urine <50 nmol/24 hours (<10 µg/24 hours) 2103

2104 11.11.1 Inorganic mercury compounds 2105 2106 Campbell et al. (1986) reported two patients with high mercury concentrations, due to 2107 occupational exposure, treated with unithiol. One patient aged 22 years was 2108 asymptomatic despite a urinary mercury concentration of 832 µg/24 hours. The other, 2109 aged 23 years, had clinical features of mercury toxicity (weight loss, muscle twitching, 2110 excessive salivation, night sweats). His urinary mercury concentration was 429 µg/24 2111 hours. Both were given unithiol, 100 mg 3 times a day then 400 mg daily, for 2 2112 months. The drug was well tolerated and during therapy the elimination half-life of 2113 mercury deceased from 33 days to 11 days. After 2 months the symptomatic patient 2114 had improved and the urinary mercury concentration was within acceptable limits. 2115 2116 Ashton & House (1989) describe two patients treated with unithiol after ingestion of 2117 inorganic mercury. The first patient, aged 19 years, ingested approximately 29 g of 2118 mercuric nitrate and presented to hospital within 1.5 hours. He was treated with 2119 dimercaprol initially and he developed acute renal tubular necrosis and hypotension. 2120 He was then given high dose intravenous unithiol, haemodialysis, haemofiltration and 2121 plasma exchange. The initial blood mercury concentration was very high but renal 2122 function returned at 10 days post-ingestion and he made a full recovery. 2123 Haemodialysis, haemofiltration and plasma exchange were ineffective as promoting 2124 mercury removal. The second patient, aged 42, ingested approximately 1 g of 2125 mercuric chloride. He was treated with high dose intravenous unithiol and then oral 2126

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administration. He recovered without developing renal impairment, despite an initial 2127 mercury blood concentration of 600 µg/L. 2128 2129 Prompt administration of unithiol (at 8 hours post-ingestion) with intravenous fluid 2130 therapy was thought to be responsible for the lack of renal impairment in a 53-year-old 2131 man who had ingested approximately 50 g of mercuric iodide. He had vomited 2132 repeatedly and this may also have decreased absorption, however, the blood and 2133 urine mercury concentrations were high, at 1197 nmol/L and 159 nmol/L, respectively. 2134 He was given unithiol intravenously 250 mg every 4 hours for 60 hours and then orally 2135 twice daily for 18 days (Anderson et al., 1996). 2136 2137 A 19-year-old female vomited 30 minutes after ingestion of 3g of mercuric chloride and 2138 was given a gastric lavage. She developed anuria one hour later and was started on 2139 peritoneal dialysis and haemodialysis. She was given unithiol and dimercaprol and 2140 urine excretion returned after 10 days with a polyuric phase at 20 days. Creatinine 2141 clearance was normal by 100 days after ingestion (Nadig et al., 1985). 2142 2143 A 38-year-old male intentionally ingested 100 ml of mercuric chloride solution (of 2144 unknown concentration) and developed vomiting, haematemesis and bloody diarrhoea 2145 shortly afterwards. He was given a gastric lavage and once the history of ingestion 2146 had been determined was stated on dimercaprol. However, he rapidly developed 2147 oliguria and acute tubular necrosis. By 8 hours post-ingestion the urine output was 2148 less than 10 mL/hour. The blood mercury concentration was 14,300 µg/L. The urine 2149 mercury concentration before onset of anuria was 36,000 µg/L. He was started on 2150 intravenous unithiol 10 hours after ingestion: 250 mg every 4 hours for 48 hours, then 2151 250 mg every 6 hours for 48 hours and 250 mg 8 hourly. He required intravenous 2152 fluids for hypovolaemic shock and haemodialysis for renal failure. The blood mercury 2153 concentration remained high (at least 2 mg/L for the first 10 days) but kidney function 2154 returned within 10 days and haemodialysis was no longer required. Administration of 2155 unithiol was continued by the parenteral route because of ulceration of the 2156 oesophagus and stomach. After about 4 weeks oral unithiol was given (300 mg 3 2157 times a day), and unithiol was given for a total of 7 weeks until blood and urine 2158 concentrations were considered to be non-toxic. The average mercury half-life is 40-2159 60 days and in this patient it was 2.5 days in the initial distribution phase and 8.1 days 2160 in the terminal phase of metabolism and elimination. The patient made a full recovery 2161 (Toet et al., 1994). 2162 2163 Haemodialysis is ineffective in enhancing mercury elimination (Toet et al., 1994; Pai 2164 et al., 2000) even in patients treated with unithiol (Toet et al., 1994). However, 2165 continuous venovenous haemofiltration was successful in enhancing elimination of 2166 the mercury-unithiol complex in a patient with elevated blood mercury concentrations 2167 (initially 5200 µg/L) following ingestion of an inorganic mercury salt (Pai et al., 2000). 2168 2169 Continuous venovenous haemofiltration in combination with unithiol was also 2170 effective in the patient reported by Dargan et al. (2003a). A 40-year-old male 2171 ingested approximately 1 g of mercuric sulphate and soon after presentation required 2172 intubation and ventilation due to respiratory distress (his pharynx and epiglottis were 2173 oedematous and haemorrhagic). He was started on intravenous unithiol (250 mg 2174 every 4 hours for 4 days) 4.5 hours after ingestion. However he developed an 2175 erythematous maculopapular rash with blistering on the lower legs and the dose of 2176

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unithiol was reduced to 250 mg every 8 hours. After another 6 days he was started 2177 on oral unithiol (200 mg/day for 9 days). Anuria developed by 12 hours post-ingestion 2178 and he was commenced in continuous venovenous haemofiltration 7 hours after 2179 ingestion. He was anuric for 11 days with oliguria for days 12 to 43. Continuous 2180 venovenous haemofiltration was continued for 14 days with 8 sessions of 2181 haemodialysis for renal support between days 16 and 37. He was discharged on 2182 day 50 asymptomatic with no neurological signs or symptoms. Continuous 2183 venovenous haemofiltration removed 12.7% of the ingested dose, mostly over the 2184 first 72 hours. 2185 2186 11.10.2 Organic mercury compounds 2187 2188 A 20-year-old male was given a gastric lavage and activated charcoal within 2 hours of 2189 ingesting haloperidol, benztropine and 2-3 mouthfuls of a fungicide containing 0.69% 2190 methyl mercury and ethanol. The estimated dose of methyl mercury was 800 mg. He 2191 was started on oral D-penicillamine within 4 to 5 hours of ingestion. The whole blood 2192 mercury concentration 2 hours after ingestion was 1930 μg/L and at 24 hours was 2193 1007 μg/L. Approximately 36 hours after ingestion he was started on acetylcysteine 2194 and haemodialysis. D-penicillamine was discontinued 3 days after ingestion and oral 2195 unithiol (200 mg every 6 hours) was started. He received unithiol for 14 days but due 2196 to mild anorexia and nausea elected not to continue taking it as an outpatient. The 2197 whole blood mercury concentration at this time was 355 μg/L and he remained well. 2198 Serum zinc and copper concentrations remained normal during unithiol therapy. 2199 Neurological examination was normal at 6 weeks and 1 year post-ingestion. 2200 Haemodialysis and D-penicillamine were relatively ineffective in clearing the mercury. 2201 The unithiol was also relatively ineffective but this may have been due to 2202 administration of a multivitamin and mineral preparation containing copper and zinc at 2203 the same time, which may have reduced efficacy (Lund et al., 1984). 2204 2205 In a 49-year-old female who ingested 125 g of fungicide containing 3.5% mercury in 2206 the form of methoxyethyl mercury treated with penicillamine and unithiol alternating 2207 every 2 weeks, unithiol reduced the protein binding of methoxyethyl mercury from 93% 2208 to 83%. Antidote therapy was continued for 12 weeks and she developed no renal or 2209 neurological effects (Köppel et al., 1982). 2210 2211 A 44-year-old man was treated with unithiol and succimer after ingestion of a solution 2212 of thiomersal. The ingested dose was 83 mg/kg although he vomited about 15 2213 minutes later. He was given a gastric lavage just over 1 hour after ingestion and 300 2214 mg of unithiol was instilled into the stomach via a nasogastric tube. This dose was 2215 repeated on days 2, 3, 9 and 10, and he was given 250 mg of intravenous unithiol on 2216 days 3, 8 and 17, with 750 mg on days 4, 5 and 11, and 1000 mg on days 12-16 and 2217 23-29. He was also given oral succimer on days 17-23, 33-46 and 51-70. He 2218 received no antidotal therapy on days 6, 30-32 and 47-50. He developed renal failure 2219 on day 1 which persisted until day 40. He also developed gastritis, dermatitis, 2220 gingivitis, polyneuropathy and coma. He made a full recovery but the decline in 2221 mercury concentrations in the blood, urinary mercury excretion and renal mercury 2222 clearance were not influenced by antidotal therapy to a great extent. There was 2223 minimal or no increase in renal and blood clearance and no effect was detected after 2224 day 30 (Pfab et al., 1996). 2225 2226

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11.11.3 Metallic mercury 2227 2228 In a patient with intrabronchial aspiration of metallic mercury the urine and plasma 2229 mercury concentrations remained high despite intermittent therapy with unithiol and 2230 penicillamine over a 14 month follow up period. However, the patient remained well 2231 apart from vomiting and faintness on admission and intermittent mild arthralgia 2232 thereafter (Batora et al., 2001). In a similar case, a 35-year-old male remained 2233 asymptomatic after rupture of a Miller-Abbot tube and subsequent aspiration. The 24 2234 hour blood mercury concentration was 940 µg/L. He was treated with unithiol and the 2235 blood concentration decreased rapidly (Kummer & Michot, 1984). 2236 2237 There are a number of case reports of unithiol use in children with mercury toxicity 2238 (Bertram et al., 1989; Jekat & Kemper, 1990; Ruprecht, 1997). Unithiol was used in 2239 three children, aged 33 and 20 months and 6 years 10 months, with mercury toxicity 2240 from a broken thermometer. The thermometer had been broken on the carpet of the 2241 children’s room, which had under floor heating, about 8 months previously. Unithiol 2242 (50 mg 3 times daily) was given for up to 4 months and all the children recovered (von 2243 Mühlendahl, 1990). 2244 2245 Unithiol was used in the management of mercury poisoning in a 14-year-old girl with 2246 acrodynia. For 3 months her clinical features were diagnosed as a neurotic anxiety 2247 disorder but once the diagnosis of mercury toxicity was made she was treated with 2248 unithiol, 100 mg every other day and made a slow recovery. The source was metallic 2249 mercury spilt on a carpet that had been vacuumed up (Böckers et al., 1983). 2250 2251 11.11.4 Dermal mercury exposure 2252 2253 Unithiol was used in a 21-year-old diabetic male who developed mercury intoxication 2254 following use of a mercury-containing ointment for eczema for about 3 weeks. He 2255 developed classic signs of mercury poisoning with tiredness, sweating, mild 2256 fasciculation of extremities, ataxia, hand tremors, weight loss, proteinuria, anxiety 2257 and behavioural changes. The urinary mercury concentration was 0.252 mg/L and 2258 he was given oral unithiol for 12 days. The highest urine mercury concentration 2259 during therapy was 2.1 mg/L. He improved rapidly over a 2 week period, however, 2260 signs of neurological and renal damage, not typical of diabetes persisted (Pelclová et 2261 al., 2001). 2262 2263 Use of a mercury-containing cosmetic bleaching cream for 4 years caused nail 2264 dyschromia in a 56 year female. The nails were discoloured greenish-black and she 2265 had features of mercury toxicity with night sweats, insomnia and nervousness. 2266 Treatment with unithiol reduced the serum mercury concentration from 64 to15 µg/L; 2267 the urinary mercury concentration rose and peaked at 1660 µg/L on the 10th day. 2268 After the initial course of unithiol the serum mercury concentration increased and she 2269 was given a second course, lasting 3 weeks. The unithiol was well tolerated (Böckers 2270 et al., 1985). 2271 2272 11.11.5 Parenteral mercury exposure 2273 2274 Long-term unithiol administration has been used in the management of parenteral 2275 mercury poisoning. A 23-year-old male injected about 20 ml of metallic mercury 2276

47

intravenously and mercury was visible on x-ray in the right ventricle with multiple 2277 emboli in the lung. Mercury was also seen in the abdomen and right forearm. Only 2278 0.2 ml was removed from the heart by cardiac catheterisation and the blood mercury 2279 concentration rose to 294 µg/L. He was started on oral unithiol 300-800 mg a day and 2280 this continued for at least 4.5 years. The blood mercury and urine concentrations 2281 peaked at 1608 µg/L and 73,500 µg/L, respectively. He remained well except for 2282 intermittent chest pain. He developed no adverse effects to unithiol, and no reduction 2283 in plasma zinc, copper or selenium concentrations, although the urine copper 2284 concentrations were elevated (Ashton et al., 1992b). Batora et al. (2000) also used 2285 unithiol in a patient with intravenous mercury injection with elevated blood and urine 2286 concentrations (21.4 µg/L and 183.3 μg/L, respectively). They used only two courses 2287 of treatment over 17 days, and 6 weeks after therapy the blood concentration had 2288 fallen to 8.1 µg/L and the urine concentration increased to 397.6 µg/L. Mercury 2289 deposits were visible on both lung fields but by 1 year had almost completely 2290 disappeared. He remained well with only a temporary enzymuria (N-acetyl beta 2291 glucosaminidase) indicating subtle renal tubular damage. 2292 2293 A 35-year-old male presented with a history of gingivitis, weakness, pyrexia, anorexia 2294 and weight loss 6 weeks after intravenous injection of metallic mercury. Mercury was 2295 visible in the lungs, abdomen and injection site on X-ray and the urinary concentration 2296 was 500 µg/L. He was started on unithiol 2 days after admission on a 6 day course of 2297 600 mg/day in 3 divided doses. After the first 24 hours of treatment the urinary 2298 mercury concentration rose to 7750 µg/L and he developed a moderate 2299 hypersensivity-type skin reaction with resolved within 2 days. He was discharged 2300 one week after completion of unithiol therapy and had urine concentration 1100 µg/L 2301 with irritability and weakness. He did not return for a year when he presented with 2302 irritability, weakness, insomnia and tremor. The urinary mercury concentration was 2303 2206 µg/L. Mercury was still visible on X-ray and he was given another course of 2304 unithiol. Over the next 4 years he was reviewed every 6 months and the urinary 2305 mercury concentration was 800-1000 µg/L. Two years after the original presentation 2306 he was given a third course of unithiol. At 5 years the concentration was 807 µg/L 2307 and a fourth course of untihiol produced a less dramatic increase in urinary 2308 elimination. Tremor and weakness persisted. Although unithiol increased 2309 elimination there appeared to be no change in the radiographic deposits mercury in 2310 the lungs and abdomen. It was not clear that the infrequent administration of unithiol 2311 was of any benefit to this patient (Torres-Alanís et al., 1997). 2312 2313 In another case of elemental mercury injection, a 27-year-old male, 65 kg, injected 2314 1.5 mL (20 g) into his left cubital vein. Within 12 hours he developed pyrexia, 2315 tachycardia and dyspnoea and mercury was visible on chest X-ray. The serum 2316 mercury concentration was 172 µg/L on admission and peaked on day 6 at 274 µg/L. 2317 At 37 hours he was started on unithiol (200 mg orally every 8 hours) for 5 days, 2318 during which time he eliminated 8 mg of mercury. Three days later he was started 2319 on succimer (500 mg daily) for another 5 days. This resulted in the excretion of 2320 another 3 mg of mercury. Neither unithiol nor succimer were effective in enhancing 2321 elimination of mercury after intravenous injection (Eyer et al., 2006). 2322 2323 Unithiol was shown to be effective in enhancing mercury elimination and reducing 2324 renal irradiation in patients given radioactive chlormerodrin, an obsolete mercurial 2325 diuretic, for renal scintigraphy (Ogiński & Kloczkowski, 1973; Kloczkowski & Ogiński, 2326

48

1973). 2327 2328 11.12 Use in nickel poisoning 2329 2330 There are no case reports of unithiol administration in nickel poisoning but it has been 2331 recommended (Daunderer, 1982). 2332 2333 11.13 Use in palladium poisoning 2334 2335 No well documented case reports of unithiol use in platinum poisoning could be 2336 found. 2337 2338 11.14 Use in platinum poisoning 2339 2340 No well documented case reports of unithiol use in platinum poisoning could be 2341 found. 2342 2343 11.15 Use in polonium poisoning 2344 2345 Experience with use of unithiol in polonium exposure is limited. Shantyr et al. (1969) 2346 reported 10 children who were contaminated with polonium-210 from a damaged 2347 polonium-beryllium neutron source. They had body burdens of 0.2-7.0 µCi, far 2348 above the maximal permissible burden. Some were treated with unithiol (number 2349 unknown) and all remained well with no changes in general health, blood or renal 2350 function over the 46 month period of monitoring. However, most of the children 2351 developed impairment of protein formation in the liver, manifested as an increase in 2352 albumin and a decrease in globulin. This was observed from 21 months and 2353 persisted through the remaining period of observation. 2354 2355 11.16 Use in selenium poisoning 2356 2357 No well documented case reports of unithiol use in selenium poisoning could be 2358 found. 2359 2360 11.17 Use in silver poisoning 2361 2362 There is limited information on the use of unithiol in silver poisoning. Unithiol was 2363 used in a patient with argyria after penicillamine had failed to increase the urinary 2364 excretion of silver. The patient was 60 years old and had developed argyria after 15 2365 years use of silver nitrate to treat gingivitis due to ill-fitting dentures. Unithiol was 2366 given at a dose of 1-5 x 500 mg for 5 days then 2.5 g/day for another 5 days with a 5 2367 day period in between. Even though the renal excretion of silver was increased by 2368 unithiol administration the total amount of excreted silver was low, only 1 µmol of silver 2369 in total. This was estimated to be approximately 1% or less of the total body burden of 2370 silver and it was concluded that unithiol was of no benefit in argyria (Aaseth et al., 2371 1986). 2372 2373 In a 55-year-old patient with argyria, use of unithiol (3 x 100 mg/day) increased the 2374 urinary excretion of silver by approximately 100 fold. However, the total quantity of 2375 silver excreted was low. Administration of penicillamine did not affect silver excretion 2376

49

(Kemper et al., 1989; Jekat and Kemper, 1990). 2377 2378 11.18 Use in strontium poisoning 2379 2380 No well documented case reports of unithiol use in strontium poisoning could be 2381 found. 2382 2383 11.19 Use in tin poisoning 2384 2385 There is limited information on the use of unithiol in tin poisoning. Unithiol was used 2386 in dental assistant with tin exposure due to kneading of amalgam in the unprotected 2387 palm of the hand. The urinary concentration of tin was increased to 1094.4 µg/L with 2388 unithiol administration and clinical features (tiredness, dizziness and tremor) improved 2389 (Hruschka, 1990). 2390 2391 2392 12. Summary of evaluation 2393 2394 12.1 Indications 2395 2396 Unithiol appears to be effective (in terms of accelerating metal excretion without 2397 causing severe adverse effects) in most cases of: 2398

• acute and chronic intoxication by organic and inorganic mercury 2399

• acute and chronic intoxication by bismuth 2400

• chronic lead poisoning 2401

• acute and chronic arsenic poisoning. 2402 2403

It has also been used with some success in cases of human poisoning with the 2404 following, but data are limited: 2405

• trivalent antimony (there is no information on the less toxic pentavalent 2406 antimony compounds) 2407

• chromium 2408

• cobalt 2409

• copper, including patients with Wilson’s disease 2410

• gold 2411 2412 Animal studies have demonstrated apparent benefit but experience of unithiol use in 2413 human poisoning is lacking for the following: 2414

• beryllium 2415

• cadmium 2416

• nickel 2417

• tin 2418

• zinc 2419 2420 On the basis of animal or human case reports unithiol does not appear to be useful for 2421 the following: 2422

• palladium 2423

• platinum 2424

• polonium 2425

50

• silver (argyria) 2426

• strontium 2427

• thallium 2428

• vanadium 2429 2430 See Table 5 (section 16.2) for a summary of the available information on unithiol use 2431 in metal and metalloid poisoning. 2432 2433 12.2 Advised routes and dose 2434 2435 Unithiol may be administered both orally and parenterally. In cases of acute heavy 2436 metal ingestion the parenteral route is strongly recommended, because given orally 2437 the drug may bind residues of the metal in the gut, promote absorption and decrease 2438 body burden. 2439 2440 High dose therapy should be avoided in patients with chronic poisoning without severe 2441 clinical symptoms because of the risk of sudden mobilisation of the metal from tissue 2442 stores with resultant clinical deterioration. This occurred, for example, in a patient with 2443 chronic bismuth toxicity, necessitating cessation of unithiol therapy (Teepker et al., 2444 2002). 2445 2446 12.2.1 Oral administration 2447 2448 There is no standard regimen for unithiol administration; dosing and duration depends 2449 on clinical condition and blood and urine concentrations of the metal. High doses 2450 have been used and are well tolerated, but are not advised in chronically poisoned 2451 patients without severe clinical effects (see section 12.2). Unithiol should be taken on 2452 an empty stomach. 2453

• Adults: The usual initial dose is 100-200 mg every 6-8 hours (3-4 times/day); this is 2454 then tapered over the following days or weeks. 2455

• Children: The usual initial dose 50-100 mg every 6-8 hours (3-4 times/day); this is 2456 then tapered over the following days or weeks. Alternatively 5 mg/kg daily in 2 to 4 2457 divided doses has been used (Willig et al., 1984; Chisholm and Thomas, 1985; 2458 Karpinski & Markoff, 1997; Böse-O’Reilly et al., 2003). 2459

2460 12.2.2 Parenteral administration 2461 2462 Unithiol may be administered parenterally by the intramuscular or slow intravenous 2463 injection over 5 minutes (1 mL/minute). The parental doses of unithiol for adults and 2464 children are given in Table 1. From day 4 frequency of dosing should be based on the 2465 patient’s clinical condition; parental unithiol may be continued or oral treatment may be 2466 started. Unithiol solution must be administered immediately after opening the vials, 2467 and any remaining after dosing must be discarded. Parenteral unithiol must not be 2468 mixed with other infusion solutions (as this may reduce antidotal efficacy). 2469 2470

51

Table 1: Parenteral unithiol dosage 2471 2472

Adults Children Day of treatment Dose IM or

slow IV Total daily

dose Dose IM or slow IV Total daily

dose 1 250 mg every 3-

4 hours 1.5-2.0 g 5 mg/kg every 3-4

hours 30-40 mg/kg

2 250 mg every 4-6 hours

1.0-1.5 g 5 mg/kg every 4-6 hours

20-30 mg/kg

3 250 mg every 6-8 hours

0.75-1.0 g 5 mg/kg every 6-8 hours

15-20 mg/kg

4,5… 250 mg every 8-12 hours

0.50- 0.75 g 5 mg/kg every 8-12 hours

10-15 mg/kg

2473 12.3 Supportive therapy 2474 2475 Other supportive therapy, and gut decontamination, rehydration or cardiovascular 2476 support may be required. Haemofiltration in conjunction with unithiol administration is 2477 the renal replacement method of choice in patients with renal failure because it may 2478 enhance elimination of heavy metals (particularly mercury). 2479 2480 In cases of chronic poisoning identification and removal from source should also be 2481 undertaken. 2482 2483 12.4 Controversial issues 2484 2485 There are indications from animal studies that unithiol may be useful in the treatment of 2486 acute poisoning by beryllium, cadmium, nickel and tin and from a small number of 2487 human case reports for trivalent antimony, chromium, cobalt, copper and gold. 2488 However, there is a lack of good quality clinical data. 2489 2490 Diagnostic use of unithiol in cases of asymptomatic or suspected poisoning cannot be 2491 recommended. The metal-binding agent mobilises the metal resulting in redistribution 2492 which could increase the concentration at the target organ, despite an increased 2493 excretion. 2494 2495 It is still controversial, whether concurrent administration of more than one metal-2496 binding agent is harmful or beneficial. If adequate doses of unithiol are given, are well 2497 tolerated and the duration of therapy long enough, then use of more than one metal-2498 binding agent is not necessary. 2499 2500 12.5 Proposals for further studies 2501 2502 The mode of action of unithiol has not been fully elucidated and recent work has 2503 tended to focus on arsenic and mercury. Further study is required to determine the 2504 interaction of unithiol with individual metals and metalloids but also with the target 2505 organs. Modern tools, such as computer molecule modelling for complex formation, 2506 should be used to provide a better understanding of the biological processes involved. 2507

52

There is also a clear need for more clinical trials on the role of unithiol in the treatment 2508 of poisoned patients, in particular to define the optimum dose, duration of therapy and 2509 route of administration of unithiol and compare it to other metal-binding agents such as 2510 succimer and sodium calcium edetate. There are large populations of individuals 2511 poisoned with metals and metalloids, particularly arsenic and mercury, from 2512 environmental sources. For the metals which are less commonly involved in poisoning 2513 and where clinical trials are unlikely to be practical unless a mass incident occurs, there 2514 is a need for well documented case reports with biochemical and chemical analyses 2515 used to determine the efficacy of the antidote used. 2516 2517 Evaluation of the risks and benefits of combined antidotal therapy is also an area that 2518 warrants father investigation. 2519 2520 The suggestion that unithiol is the optimal antidote for inorganic mercury poisoning 2521 and that succimer is more effective in organic mercury intoxication requires verification 2522 (Andersen, 1999). 2523 2524 12.6 Adverse effects 2525 2526 Unithiol is generally well tolerated and the incidence of adverse effects is low. 2527 2528 Administration of unithiol also increases elimination of some trace elements, particularly 2529 zinc and copper (Bertram, 1977; Mant, 1985; Aaseth et al., 1986; Sallsten et al., 1994; 2530 Torres- Alanís et al., 2000; Høl et al., 2003), but also selenium and magnesium 2531 (Torres- Alanís et al., 2000). This effect is only likely to be of clinical significance in 2532 patients on chronic unithiol therapy. 2533 2534 Skin reactions including rashes, pruritis and blistering have been reported (Dubinsky & 2535 Guida, 1979; Mant, 1985; Ashton et al., 1992a; Hla et al., 1992; Toet et al., 1994; 2536 Torres- Alanís et al., 1995; Torres-Alanís et al., 1997; Gonzalez-Ramirez et al., 1998; 2537 Böse-O’Reilly et al., 2003; Dargan et al., 2003a). Erytheme multiforme with buccal 2538 ulceration (Ashton et al., 1992a) and depigmentation (Pagliuca et al., 1990) has been 2539 reported. Stevens-Johnson syndrome has been reported in a small number of cases 2540 (Chisholm, 1990; Chisholm, 1992; Van der Linde et al., 2008). Anaphylactic shock has 2541 not been reported (Ruprecht, 1997). In most cases allergic reactions have resolved 2542 within 3-5 days and generally no treatment is required. However, antihistamines and/or 2543 corticosteroids may be given if necessary. 2544 2545 Nausea may occur from oral administration (Lund et al., 1984; Walshe, 1985; 2546 Stevens et al., 1995; Gonzalez-Ramirez et al., 1998), and body fluids usually have a 2547 sulphur odour for 6-8 hours after unithiol administration. Mild elevation of liver 2548 enzymes (Chisolm & Thomas, 1985; Gonzalez-Ramirez et al., 1998; Wang et al., 2549 2003), diuresis (Glukharev, 1965), fever and leucocytosis (Walshe, 1985) have 2550 been reported. 2551 2552 With the parenteral preparation cardiovascular reactions may occur, particularly if 2553 injected too rapidly. These effects are hypotension (Hurlbut et al., 1994), nausea 2554 (Stevens et al., 1995), dizziness and weakness (Dubinsky & Guida, 1979; Zhang, 2555 1984). Necrosis and ulceration may occur at the injection site, but this is associated 2556 with high doses e.g. 100 mg/kg (Sanotsky et al., 1967). 2557

53

12.7 Restrictions of use 2558 2559 Unithiol should not be administered in acute arsine poisoning (AsH3), because it is 2560 ineffective and can increase arsine toxicity (Mizyukova & Petrunkin, 1974). 2561 2562 The administration of unithiol in cases of asymptomatic or suspected poisoning (a 2563 challenge or mobilisation test) cannot be recommended, because the metal-binding 2564 agent mobilises the metal from tissue stores resulting in redistribution and potentially 2565 increasing the concentration in the target organ, despite increasing excretion. 2566 2567 Renal impairment is not a restriction of use; haemofiltration is the renal replacement 2568 method of choice in patients with renal failure and in conjunction with unithiol 2569 administration may enhance elimination of heavy metals (particularly mercury). 2570 2571 2572 13. Model information sheet 2573 2574 13.1 Uses 2575 2576 Unithiol is a derivative of dimercaprol (2,3-dimercapto-1-propanol, British Anti-Lewisite, 2577 BAL), and is replacing dimercaprol as one of the main antidotes used in the 2578 management of heavy metal poisoning. Unithiol has several advantages over 2579 dimercaprol including lower toxicity, increased solubility in water and lower lipid 2580 solubility. It is due to these properties that it is effective by oral administration. 2581 2582 Unithiol has been used in the management of acute and chronic poisoning with a 2583 number of different metals and metalloids, and is particularly useful for arsenic, 2584 bismuth and mercury. It has been used for other metals and metalloids. Unithiol can 2585 be given parenterally or orally depending on the clinical situation and severity of 2586 poisoning. 2587 2588 13.2 Dosage and route 2589 2590 Unithiol may be administered both orally and parenterally. In severe cases and/or 2591 acute poisoning parenteral administration is recommended. In cases of acute heavy 2592 metal ingestion the parenteral route is strongly recommended, because given orally 2593 the drugs may bind residues of the metal in the gut, promote the absorption and 2594 increase the body burden by this way. 2595 2596 High dose therapy should be avoided in patients with chronic poisoning without severe 2597 clinical symptoms because of the risk of sudden mobilisation of the metal from tissue 2598 stores with resultant clinical deterioration. 2599 2600 13.2.1 Oral administration 2601 2602 There is no standard regimen for unithiol administration; dosing and duration depends 2603 on clinical condition and blood and urine concentrations of the metal. High doses 2604 have been used and are well tolerated, but are not advised in chronically poisoned 2605 patients without severity clinical effects (see section 12.2). Unithiol should be taken on 2606 an empty stomach. 2607

54

• Adults: The usual initial dose is 100-200 mg every 6-8 hours (3-4 times/day); this is 2608 then tapered over the following days or weeks. 2609

• Children: The usual initial dose 50-100 mg every 6-8 hours (3-4 times/day); this is 2610 then tapered over the following days or weeks. Alternatively 5 mg/kg daily in 2 to 4 2611 divided doses has been used. 2612

2613 13.2.2 Parenteral administration 2614 2615 Unithiol is given by IM or slow IV injection over 5 minutes (1 mL/minute). See table for 2616 dosing regimen. From day 4 frequency of dosing should be based on the patient’s 2617 clinical condition; parental unithiol may be continued or oral treatment may be started. 2618 Unithiol must not be mixed with other infusion solutions, as this may reduce its 2619 efficacy. 2620 2621 Table: Parenteral unithiol dosage 2622 2623

Adults Children Day of treatment Dose IM or slow

IV Total daily

dose Dose IM or slow IV Total daily

dose 1 250 mg every 3-4

hours 1.5-2.0 g 5 mg/kg every 3-4

hours 30-40 mg/kg

2 250 mg every 4-6 hours

1.0-1.5 g 5 mg/kg every 4-6 hours

20-30 mg/kg

3 250 mg every 6-8 hours

0.75-1.0 g 5 mg/kg every 6-8 hours

15-20 mg/kg

4,5… 250 mg every 8-12 hours

0.50-0.75 g 5 mg/kg every 8-12 hours

10-15 mg/kg

2624

13.3 Precautions and contraindications 2625 2626 Unithiol should not be administered in acute arsine poisoning (AsH3), because it is 2627 ineffective and can increase arsine toxicity. 2628 2629 The administration of unithiol in cases of asymptomatic or suspected poisoning (a 2630 challenge or mobilisation test) cannot be recommended, because the metal-binding 2631 agent mobilises the metal from tissue stores resulting in redistribution and potentially 2632 increasing the concentration in the target organ, despite increasing excretion. 2633 2634 The administration of more than one metal-binding agent at the same time cannot be 2635 recommended, as the risks and/or benefits of such therapy have not been evaluated. 2636 2637 The efficacy of a metal-binding agent may be difficult to determine. After 2638 discontinuation of metal exposure (and absorption) a decrease in the blood 2639 concentration will occur without any therapy. Clinical efficacy should not be judged 2640 only by the quantity of metal excretion or the decrease of blood concentrations. The 2641 reduction of the tissue content in the target organ and the restoration of pathological 2642 alterations also need to be considered. It is important to note that enhancement of the 2643

55

metal excretion by mobilisation may increase the metal burden of the target organ by 2644 redistribution, and conversely the body burden may be reduced without a striking 2645 decrease of the blood concentrations. 2646 2647 During long-term therapy the blood concentrations and excretion of trace elements 2648 should be monitored carefully, because depletion of trace metals may play a role in the 2649 toxicity of metal-binding agent agents. 2650 2651 13.4 Pharmaceutical incompatibilities and drug interactions 2652 2653 Unithiol must not be mixed with other infusion solutions, as this may reduce antidotal 2654 efficacy. 2655 2656 Unithiol solution should be administered immediately after opening of the vials and all 2657 remainder must be discarded, because the compound is oxidised rapidly in contact 2658 with air. 2659 2660 Unithiol should not be given orally with mineral preparations or activated charcoal 2661 because unithiol may be inactivated. For the same reason the unithiol capsules 2662 should be taken at least one hour before a meal. 2663 2664 13.5 Side effects 2665 2666 Unithiol is generally well tolerated and the incidence of adverse effects is low. 2667 2668 Administration of unithiol also increases elimination of some trace elements, particularly 2669 zinc and copper, but also selenium and magnesium. This effect is only likely to be of 2670 clinical significance in patients on chronic unithiol therapy. 2671 2672 Skin reactions including rashes, pruritis and blistering have been reported. Erytheme 2673 multiforme with buccal ulceration and depigmentation has been reported. Stevens-2674 Johnson syndrome has been reported in a small number of cases. Anaphylactic shock 2675 has not been reported. In most cases allergic reactions have resolved within 3-5 days 2676 and generally no treatment is required. However, antihistamines and/or corticosteroids 2677 may be given if necessary. 2678 2679 Nausea may occur from oral administration, and body fluids usually have a sulphur 2680 odour for 6-8 hours after unithiol administration. Mild elevations of liver enzymes, 2681 diuresis, fever and leucocytosis have also been reported. 2682 2683 With the parenteral preparation cardiovascular reactions may occur, particularly if 2684 injected too rapidly. These effects are hypotension, nausea, dizziness and weakness. 2685 2686 Necrosis and ulceration may occur at the injection site, but this is associated with high 2687 doses. 2688 2689 13.6 Pregnancy and lactation 2690 2691 Teratogenic effects have not been demonstrated in animal studies, indeed studies 2692 have demonstrated that unithiol can protect against the developmental toxicity of 2693

56

arsenic and mercury. Even though safety in human has not been established, 2694 pregnancy is not regarded as a contraindication. If unithiol is administered to 2695 pregnant women, the essential minerals should be monitored carefully, because 2696 metal-binding may cause a depletion of trace elements and it has been shown that 2697 zinc deficiency can cause teratogenic effects. 2698 2699 In general lactation should be avoided in metal poisoning. 2700 2701 13.7 Storage 2702 2703 The capsules should be stored in a dry place. 2704 2705 The shelf-life for the commercial available pharmaceutical preparations Dimaval® is 2706 claimed to be 5 years for the capsules 4 years for the ampoules. The expiry date is 2707 stated on each package. 2708 2709 2710 14. References 2711

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Tandon SK, Singh S, Jain VK & Prasad S (1996) Chelation in metal intoxication. 3544 XXXVIII: Effect of structurally different chelating agents in treatment of nickel 3545 intoxication in rat. Fundam Appl Toxicol, 31: 141-148. 3546 3547 Teepker M, Hamer HM, Knake S, Bandmann O, Oertel WH & Rosenow F (2002) 3548 Myoclonic encephalopathy caused by chronic bismuth abuse. Epileptic Disord, 4: 3549 229-233. 3550 3551 Toet AE, van Dijk A, Savelkoul TJ & Meulenbelt J (1994) Mercury kinetics in a case 3552 of severe mercuric chloride poisoning treated with dimercapto-1-propane sulphonate 3553 (DMPS). Hum Exp Toxicol, 13: 11-16. 3554 3555 Torres-Alanís O, Garza-Ocañas L & Piñeyro-López A (1995) Evaluation of urinary 3556 mercury excretion after administration of 2,3-dimercapto-1-propane sulfonic acid to 3557 occupationally exposed men. J Toxcol Clin Toxicol, 33: 717-720. 3558 3559 Torres-Alanís O, Garza-Ocañas L & Piñeyro-López A (1997) Intravenous self-3560 administration of metallic mercury: report of a case with 5-year follow-up. J Toxicol Clin 3561 Toxicol, 35: 83-87. 3562 3563 Torres-Alanís O, Garza-Ocañas L, Bernal MA & Piñeyro-López A (2000) Urinary 3564 excretion of trace elements in humans after sodium 2,3-dimercaptopropane-1- 3565 sulfonate challenge test. J Toxcol Clin Toxicol, 38: 697-700. 3566 3567 Twarog T & Cherian MG (1983) Chelation of lead with DMPS and BAL in rats injected 3568 with lead. Bull Environ Contaim Toxicol, 30: 165-169. 3569 3570 Twarog T & Cherian MG (1984) Chelation of lead by dimercaptopropane sulfonate and 3571 a possible diagnostic use. Toxicol Appl Pharmacol, 72: 550-556. 3572 3573 Van der Linde AAA, Pillen S, Gerrits GPJM & Bouwes Bavinck JN (2008) Stevens-3574 Johnson syndrome in a child with chronic mercury exposure and 2,3-3575 dimercaptopropane-1-sulfonate (DMPS) therapy. Clin Toxicol, 46: 479-481. 3576 3577 Vantroyen B, Heilier JF, Meulemans A, Michels A, Buchet JP, Vanderschueren S, 3578 Haufroid V & Sabbe M (2004) Survival after a lethal dose of arsenic trioxide. J 3579 Toxicol Clin Toxicol, 42: 889-895. 3580 3581 Volf V (1973) The effect of chelating agents on the distribution of 210 Po in rats. 3582 Experientia, 29: 307-308. 3583 3584 Volf V, Rencová J, Jones MM & Singh PK (1995) Combined chelation treatment for 3585 polonium after simulated wound contamination in rat. Int J Radiat Biol, 68: 395-404. 3586 3587 von Mühlendahl KE (1990) Intoxication from mercury spilled on carpets. Lancet, 3588 336:1578. 3589 3590 Walker AW (1998) SAS Trace Element Laboratories. Clinical and analytical 3591 handbook, 3rd edition. Guildford, Royal Surrey Hospital. 3592 3593

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Walshe JM (1985) Unithiol in Wilson's disease. Br Med J, 290: 673-674. 3594 3595 Wang XP, Yang RM, Ren MS & Sun BM (2003) Anticopper efficacy of captopril and 3596 sodium dimercaptosulphonate in patients with Wilson's disease. Funct Neurol, 18: 3597 149-53. 3598 3599 Wannag A & Aaseth J (1980) The effect of immediate and delayed treatment with 2,3-3600 dimercapto-propane-1-sulphonate on the distribution and toxicity of inorganic mercury 3601 in mice and in foetal and adult rats. Acta Pharmacol Toxicol, 46: 81-88. 3602 3603 Wax PM & Thornton CA (2000) Recovery from severe arsenic-induced peripheral 3604 neuropathy with 2,3-dimercapto-1-propanesulphonic acid. J Toxicol Clin Toxicol, 38: 3605 777-780. 3606 3607 Wiedemann P, Fichtl B & Szinicz L (1982) Pharmacokinetics of 14C-DMPS (14C-2,3-3608 dimercaptopropane-1-sulphonate) in beagle dogs. Biopharm Drug Dispos, 3: 267-74. 3609 3610 Wildenauer DB, Reuther H & Weger N (1982) Interactions of the chelating agent 2,3-3611 dimercaptopropane-1-sulfonate with red blood cells in vitro. I. Evidence for carrier 3612 mediated transport. Chem Biol Interactions, 42: 165-177. 3613 3614 Williams PAM & Baran EJ (2008) Vanadium detoxification: On the interaction of 3615 oxovanadium(IV) and other vanadium species with 2,3-dimercapto-1-3616 propanesulfonate. J Inorg Biochem, 102: 1195-1198. 3617 3618 Willig RP, Drohn W & Stegner H (1984) [Mercury poisoning: successful treatment with 3619 DMPS] (German). Monatschr Kinderheilkd, 132: 701. Cited in: Ruprecht J (1997) 3620 Scientific monograph Dimaval® (DMPS), 6th edition. Berlin, Heyl Company. 3621 3622 Xu ZF & Jones MM (1988) Comparative mobilization of lead by chelating agents. 3623 Toxicology, 53: 277-288. 3624 3625 Zalups RK, Parks LD, Cannon VT & Barfuss DW (1998) Mechanisms of action of 3626 2,3-dimercaptopropane-1-sulfonate and the transport, disposition, and toxicity of 3627 inorganic mercury in isolated perfused segments of rabbit proximal tubules. Mol 3628 Pharmacol, 54: 353-363. 3629 3630 Zalups RK & Bridges CC (2009) MRP2 involvement in renal proximal tubular 3631 elimination of methylmercury mediated by DMPS or DMSA. Toxicol Appl Pharmacol, 3632 235: 10-17. 3633 3634 Zhang J (1984) Clinical observations in ethyl mercury chloride poisoning. Am J Ind 3635 Med, 5: 251-258. 3636 3637 Zheng W, Maiorino RM, Brendel K & Aposhian HV (1990) Determination and 3638 metabolism of dithiol chelating agents. VII. Biliary excretion of dithiols and their 3639 interactions with cadmium and metallothionein. Fundam Appl Toxicol, 14: 598-607. 3640 3641

3642

15. Author names, address 3643

75

3644 Initial draft by FW Jekat & FH Kemper 3645 3646 Updated by Nicola Bates MSc, MA: Guy's & St Thomas' Poisons Unit, London, UK 3647 3648 3649 16. Additional information 3650

3651 16.1 Description of search strategy 3652

3653

Table 2: Results of EMBASE search, 5 April 2009. 3654

3655

Number Keywords Results 1 Unithiol [any field] 569 2 DMPS [any field] 446 3 (dimercaptopropanol OR dimercaptopropane AND sulfonate)

[any field] 100

4 poisoning OR toxicity OR overdose [any field] 186824 5 1 and 4 277 6 2 and 4 145 7 3 and 4 51 8 5 and 6 and 7 remove duplicates 295

3656

Table 3: Results of Pubmed search, using the EMBASE interface, 5 April 2009. 3657 3658 Number Keywords Results 1 Unithiol [any field] 541 2 DMPS [any field] 437 3 dimercaptopropanol OR dimercaptopropane AND sulfonate

[any field] 103

4 poisoning OR toxicity OR overdose [any field] 265512 5 1 and 4 208 6 2 and 4 158 7 3 and 4 62 8 5 and 6 and 7 remove duplicates 276

3659

Table 4: Results of Cochrane Library, 5 April 2009 3660

3661

Number Keywords Results 1 Unithiol [MeSH term] in Central Register of Controlled Trials 11 2 Unithiol [MeSH term] in Cochrane reviews 0 3 Unithiol [MeSH term] in other reviews 0 4 Unithiol [MeSH term] in economic evaluations 1

3662

16.2 Description of published evidence 3663

3664

Table 5: Summary of evidence of use of unithiol in metal and metalloid poisoning. 3665

76

3666

Metal Clinical trial data Case reports Animal studies Antimony No data. Used with apparent benefit

in a small number of paediatric trivalent antimony compounds (Iffland & Bösche, 1987; Kemper et al., 1989; Jekat & Kemper, 1990). There is no information on its use in pentavalent antimony compounds.

Unithiol has been shown to increase survival (Basinger & Jones, 1981a) and reduce the LD50 (Chih-Chang, 1958) in antimony-poisoned experimental animals.

Arsenic One randomized, single blinded, placebo-controlled study of 21 patients with chronic arsenicosis. Unithiol resulted in significant clinical improvement. Cessation of exposure and placebo also reduced clinical scores but these were significantly lower for unithiol-treated subjects. In the placebo group improvement was attributed to cessation of exposure and hospitalisation. There were significant increases in urinary arsenic excretion with unithiol treatment, compared to no increase in the placebo group. No adverse effects were reported (Guha Mazumder et al., 2001).

5 reports of acute arsenic poisoning involving 7 adult patients treated with unithiol as the only antidote (Moore et al., 1994; Kruszewska et al., 1996; Horn et al., 2002; Adam et al., 2003; Heinrich-Ramm et al. 2003). All patients showed increased urinary excretion and decreased plasma concentrations of arsenic. All patients recovered. 1 report where dimercaprol was used followed by succimer and unithiol together. Patient showed clinical improvement but contribution of unithiol difficult to determine (Vantroyen et al., 2004) 1 report of chronic arsenic poisoning. Administration of unithiol resulted in increased urinary excretion of arsenic and clinical improvement (Wax & Thornton, 2000).

Unithiol increases survival (Tadlock & Aposhian, 1980; Aposhian et al., 1982; Inns et al., 1990), increases the LD50 (Aposhian et al., 1981), increases urinary (Maiorino & Aposhian, 1985; Flora et al., 1995a) and faecal (Maehashi & Murata, 1986; Reichl et al., 1995) elimination, reduces tissue concentrations (Kreppel et al., 1989; Kreppel et al., 1990; Schäfer et al., 1991) and reduces the severity of toxicity (Kreppel et al., 1989, Inns & Rice, 1993; Flora et al., 1995a; Flora et al., 2005) in arsenic poisoned experimental animals.

Beryllium No data. No data. Unithiol increases beryllium excretion and reduces beryllium-induced toxic effects in experimental animals (Mathur et al., 1994; Flora et al., 1995b; Johri et al., 2002; Johri et al., 2004).

Bismuth No data. 3 cases of acute bismuth poisoning, involving a 13-year-old (Bogle et al., 2000) and 2 adults (Stevens et al., 1995; Dargan et al., 2001;

Unithiol increases survival (Basinger et al., 1983) and reduces tissue concentrations (Slikkerveer et al., 1992; Jones et al.,

77

Metal Clinical trial data Case reports Animal studies Ovaska et al., 2008). All showed improved clearance of bismuth, though one patient was also haemodialysed (Stevens et al., 1995). 2 cases of chronic bismuth poisoning. In 1 case unithiol resulted in improvement of neurological toxicity (Playford et al., 1990) and in the other the patient deteriorated and unithiol was stopped (Teepker et al., 2002).

1996) in bismuth-poisoned experimental animals.

Cadmium No data. 1 case report. Unithiol in a patient with chronic occupational exposure increased urinary cadmium concentrations. No further information available (Daunderer, 1995).

Unithiol increases the LD50 (Pethran et al., 1990), increases survival (Aposhian (1982; Andersen & Nielsen, 1988; Basinger et al., 1988; Srivastava et al., 1996), reduces tissue concentrations (Planas-Bohne & Lehman, 1983; Eybl et al., 1984; Srivastava et al., 1996) in cadmium poisoned experimental animals. However, in some studies there was no effect on survival (Eybl et al., 1984), excretion (Eybl et al., 1984, Rau et al., 1987; Zheng et al., 1990) or tissue distribution of cadmium (Cherian, 1980) and an increase in tissue concentrations was reported in some studies (Shinobu et al., 1983; Basinger et al., 1988). Although unithiol may be effective, other metal-binding agents appear to be more efficacious (Eybl et al., 1984; Eybl et al., 1985; Andersen & Nielsen, 1988; Srivastava et al., 1996).

Chromium No data. 2 case reports in adults; the effect of unithiol is unclear in both. 1 patient recovered (Donner et al., 1986); the other also received haemodialysis and haemofiltration but died 48 hours after admission (Pudill et al., 1989).

Apparent benefit demonstrated, but data are limited. Unithiol reduces chromate-induced cytotoxicity (in some circumstances) (Susa et al, 1994), reduces lethality and increases renal excretion of (Susa et al, 1994) in chromium-poisoned experimental animals.

78

Metal Clinical trial data Case reports Animal studies Cobalt No data. 2 paediatric cases of acute

exposure. Unithiol was used after penicillamine and was associated within increased urinary cobalt concentrations (Müller et al., 1989).

Apparent benefit demonstrated, but data are limited. Unithiol has been shown to reduce the lethality of cobalt (Cherkes & Braver-Chernobulskaya, 1958; Eybl et al., 1985) but to increase cobalt concentrations in some tissues (Eybl et al., 1985).

Copper No data. One paediatric case of acute ingestion; unithiol was associated with increased urinary copper excretion (Donner et al., 1986). In an adult case there was no measurement of copper excretion; the patient survived (Sinković et al., 2008).

Apparent benefit demonstrated, but data are limited. Unithiol increased the LD50 (Pethran et al., 1990) and reduced toxicity (Mitchell et al., 1982) in copper-poisoned experimental animals.

Gold Wilson’s disease In a randomised trial of 28 patients comparing unithiol and captopril, unithiol had a more potent anticopper effect. 1 patient on unithiol developed a transient, adverse effect (Wang et al., 2003).

Wilson’s disease In an unspecified number of patients copper excretion with unithiol was comparable to that of penillicamine. Unithiol was discontinued in 2 patients due to adverse effects (Walshe, 1985) Copper poisoning 1 case of iatrogenic poisoning where unithiol was thought to be effective in removing gold although no details are given (Ashton et al., 1992a).

Unithiol increases survival (Basinger et al., 1985), reduces renal concentrations (Gabard, 1980; Kojima et al., 1991; Takahashi et al., 1994), increases urinary excretion (Kojima et al., 1991) and reduces renal toxicity (Kojima et al., 1991; Takahashi et al., 1994) in gold-poisoned in experimental animals

Lead One controlled study of 60 males with chronic lead toxicity. Unithiol-treated patients had increased elimination and biochemical and clinical improvement, and were discharged 6 weeks earlier than controls (Anatovskaya, 1962). 12 children (aged 31 to 69 months) with chronic lead toxicity received one of two dose regimens of unithiol. All had reduced blood lead concentrations and

In 2 adult patients with chronic lead exposure unithiol decreased blood lead concentrations and increased urinary lead excretion (Donner et al., 1987; Hruby & Donner, 1987; Autenrieth et al., 1998). Succimer is more commonly used in the management of lead poisoning.

Unithiol increases survival (Llobet et al., 1990), increases excretion (Hofmann & Segewitz, 1975; Llobet et al., 1990), reduces tissue concentrations (Twarog & Cherian, 1983; Twarog & Cherian, 1984; Xu & Jones, 1988; Llobet et al., 1990), except in the brain (Sharma et al., 1987; Aposhian et al., 1996) and reduces toxicity (Twarog & Cherian, 1983; Sharma et al., 1987; Tandon et al., 1994) in lead-poisoned experimental animals.

79

Metal Clinical trial data Case reports Animal studies increased urinary excretion (Chisolm & Thomas, 1985).

Mercury In patients with chronic exposure penicillamine (12 patients), N-acetyl-DL-penicillamine (17), unithiol (10) and a thiolated resin (8) were compared. The study was not clinically controlled. Although all agents reduced blood mercury concentrations and unithiol was the most effective there was no immediate clinical improvement, presumably because the duration of therapy was too short and therapy was started months after exposure (Clarkson et al., 1981). 27 patients were treated with unithiol and/or succimer. All had some relief (19 became asymptomatic). Most had increased mercury excretion. Unithiol was found to be more effective (data not provided and the two antidotes were used interchangeably in some patients) (Zhang, 1984). In a study of 95 patients with chronic exposure (vapour, inorganic mercury and methyl mercury) unithiol increased urinary mercury excretion in some but others (number not specified) showed no increase in mercury excretion. In more

Inorganic mercury 6 acute cases reported; 3 patients initially received dimercaprol (Nadig et al., 1985; Ashton & House, 1989; Toet et al., 1994) and 4 developed renal failure (Nadig et al., 1985; Ashton & House, 1989; Toet et al., 1994; Dargan et al., 2003a). Unithiol was shown to reduce the mercury elimination half-life in two cases (Toet et al., 1994; Dargan et al., 2003a). All patients recovered. In 2 chronic exposure cases unithiol decreased the elimination half-life of mercury (Campbell et al., 1986). Organic mercury 3 acute cases in adults. In 1 case unithiol was determined to be relatively ineffective possibly due to coadministration of a copper and zinc supplement (Lund et al., 1984). In another case where unithiol was alternated with penicillamine, the unithiol reduced mercury protein binding (Köppel et al., 1982). The 3rd patient was also treated with succimer; neither significantly increased mercury clearance (Pfab et al., 1996). Metallic mercury 2 cases of aspiration; both patients remained well but in 1 the mercury blood and urine concentrations remained high (Batora et al., 2001) and in the other the blood concentration decreased rapidly (Kummer & Michot, 1984).

Unithiol increases excretion (Gabard, 1976a; Gabard, 1976b; Wannag & Aaseth, 1980; Planas-Bohne, 1981; Aaseth et al., 1982; Buchet & Lauwerys, 1989), decreases tissue concentrations (Gabard, 1976a; Gabard, 1976b Cikrt & Lenger, 1980; Wannag & Aaseth, 1980; Aaseth et al., 1982; Aaseth, 1983; Kachru & Tandon, 1986) and reduces toxicity (Planas-Bohne, 1977; Jones et al., 1980; Nielson & Andersen, 1991) in mercury-poisoned experimental animals.

80

Metal Clinical trial data Case reports Animal studies than two-thirds there was improvement in subjective complaints and objective neurological parameters. The 14 day regimen was too short to have a permanent effect on mercury concentrations (Böse-O’Reilly et al., 2003). Study limitations: absence of a control group, lack of details of patients (age, weight) and continued exposure to mercury during therapy. In 8 patients with chronic exposure from facial mercurous chloride cream there was a significant increase in urinary mercury concentrations after 24 hours of unithiol. One symptomatic patient recovered and the other had persistent tremor (Garza-Ocañas et al., 1997).

In 3 children (<7 years) unithiol was associated with improved clinical signs and enhanced mercury excretion (von Mühlendahl, 1990). In a 14-year-old with acrodynia there was slow recovery with unithiol (Böckers et al., 1983). Dermal mercury In 2 cases of chronic exposure, unithiol increased urinary mercury concentrations (Böckers et al., 1985l Pelclová et al., 2001). Parenteral mercury 4 cases of mercury injection (Ashton et al., 1992b; Torres-Alanís et al., 1997; Batora et al., 2000; Eyer et al., 2006). Although unithiol increases urinary mercury concentrations, the effect may be small (Eyer et al., 2006) and there may be no change in radiographic deposits of mercury (Torres-Alanís et al., 1997) or clinical signs.

Nickel No data. No data. Unithiol increases survival (Basinger et al., 1980), increases excretion (Sharma et al., 1987) and reduces nickel-induced toxic effects (Sharma et al., 1987; Tandon et al., 1996).

Palladium No data. No data. No benefit demonstrated, but data are limited. Unithiol did not influence toxicity or reduce lethality in palladium-poisoned animals (Mráz et al., 1985).

Platinum No data. No data. No benefit demonstrated, but data are limited. A single dose of unithiol had no significant effect on renal platinum concentrations. After 4 treatments there was significant increase in urinary excretion of platinum, but this was low (Planas-Bohne et al., 1982).

Polonium No data. Children (number not Not recommended.

81

Metal Clinical trial data Case reports Animal studies known, but <10) treated with unithiol after contamination with poloniumn-210 remained well over the 46 month period of monitoring with only impairment of protein formation in the liver (Shantyr et al., 1969).

Although unithiol can remove polonium-210 from most tissues (Aposhian et al., 1987) it results in concentration of polonium in the kidneys (Volf, 1973; Rencová et al., 1993; Volf et al., 1995).

Selenium No data. No data. No benefit demonstrated, but data are limited. Unithiol had no effect in selenium-poisoned animals; the concentration of selenium in urine and faeces was unchanged (Paul et al., 1989).

Silver No data. In 2 cases of chronic silver toxicity unithiol increased urinary silver excretion but the quantity excreted was low (Aaseth et al., 1986; Kemper et al., 1989; Jekat and Kemper, 1990).

Unithiol increased the LD50 of silver chloride in mice (Pethran et al., 1990), prevented the development of toxic pulmonary oedema and death in dogs (Romanov, 1967) and in vitro completely reversed silver inhibition of Na,K-ATPase (Hussain et al., 1994).

Strontium No data. No data No benefit demonstrated, but data are limited. Unithiol did not affect survival rate in strontium-poisoned experimental animals (Domingo et al., 1990; Pethran et al., 1990).

Thallium No data. No data. Unithiol is ineffective in thallium-poisoned experimental animals (Pethran et al., 1990; Mulkey & Oehme, 2000).

Tin No data. In 1 case unithiol increased urinary tin concentrations and clinical signs improved (Hruschka, 1990).

Apparent benefit demonstrated with reduced tin-induced lesions in rats (Merkord et al., 2000), but data are limited.

Vanadium No data. No data. Unithiol had no effect on lethality in vanadium-poisoned mice (Jones & Basinger, 1983) and no significant effect on the death rate, body weight reductions, or reduction in weights of legs and toes in chick eggs incubated with vanadium (Hamada, 1994). An in vitro study has shown that oxovanadium forms a complex with unithiol (Williams & Baran, 2008).

82

Metal Clinical trial data Case reports Animal studies The significance of this in vivo remains to be elucidated.

Zinc No data. No data. Unithiol increases excretion (Domingo et al., 1988) and reduced lethality in zinc-poisoned experimental animals (Basinger & Jones, 1981b), but more effective antidotes are available (Basinger & Jones, 1981b; Domingo et al., 1988; Llobet et al., 1988).

3667 Abbreviations 3668 3669 AAS Atomic Absorption Spectroscopy 3670 ABCC2 ATP-binding cassette, sub-family C 3671 AES Atomic Emission Spectroscopy 3672 ALAD δ-aminolevulinate dehydratase 3673 BAL 2,3-dimercaptopropanol; British Anti-Lewisite; dimercaprol (rINN) 3674 BAPSA 2,3-bis-(acetylthio)-propanesulphonamide 3675 CAS Chemical Abstracts Service 3676 CDTA cyclohexanediamintetraacetic acid 3677 DDC sodium diethyldithiocarbamate 3678 DMPA N-(2,3-dimercaptopropyl) phthalamidic acid 3679 DMSA Dimercaptosuccinic acid; succimer (rINN) 3680 DTPA Diethylenetriaminepentaacetic acid; pentetic acid (rINN) 3681 EDTA Ethylenediaminetetraacetic acid; sodium calcium edetate (rINN) 3682 g gram 3683 HOEtTTC N,N’-di-(2-hydroxyethyl)-ethylenediamine-N’N’-biscarbodithioate 3684 HPLC High Performance Liquid Chromatography 3685 ICP Inductively Coupled Plasma 3686 i.m. intramuscular 3687 i.p. intraperitoneal 3688 IPCS International Programme on Chemical Safety 3689 IR infra-red 3690 i.v. intravenous 3691 k kilo (103) 3692 L litre 3693 LD Lethal Dose (subscript indicates percent mortality) 3694 m milli (10-3) 3695 µ micro (10-6) 3696 µCi microCurie 3697 min minute 3698 MRP2 multidrug resistance protein 2 3699 NOEL no observed effect level 3700 OAT1 organic anion transporter 1 3701 OAT3 organic anion transporter 3 3702 PAH p-aminohippurate 3703 ppm parts per million (10-6) 3704

83

rINN recognised international non-proprietary name 3705 3706 3707