Measurement of WR-2721, WR-1065, and WR-33278 in plasma

Alkaline phosphatase was used in developing two new assays for WR-2721; one involved simple spectrophotometric titration with Ellman's reagent and the other was based upon HPLC analysis of a monobromobimane derivative. Both methods gave acceptable results when applied to whole plasma. Assays for WR-1065 and disulfide forms of the drug were also developed but were found to give unreliable results with whole plasma owing to apparent rapid reaction of these drug forms with plasma constituents. The methods were applied to the analysis of blood samples in rat 0-70 min after i.v. injection of WR-2721.     

Dephosphorylation of WR-2721 with mouse tissue homogenates

Mouse liver homogenate had an optimum pH of 8.6 to 8.8 for dephosphorylation of WR-2721 in the analyzed pH range from 5.2 to 10.0. At this optimum pH condition, the dephosphorylation activities of six mouse tissue homogenates were analyzed. Kidney, liver and small intestine homogenates showed higher dephosphorylation activities (935, 336 and 314 nmoles/mg protein/hr, respectively) than spleen and lung homogenates (86, 49 nmoles/mg protein/hr, respectively). Furthermore, serum did not show any dephosphorylation activity. The high activity found in liver homogenate agrees well with our previous data with mouse L cells. However, optimum pH from 8.6 to 8.8 in liver homogenate is quite different from the data reported by using Ehrlich ascites tumor cells (optimum pH was 5.6). Therefore, it is suggested that WR-2721 administered into mouse is efficiently dephosphorylated in certain tissues such as liver to its active form with the enzyme(s) different from that found in Ehrlich ascites tumor cells.     

WR-2721 (amifostine) infusion in patients with Ewing's sarcoma receiving ifosfamide and cyclophosphamide with mesna: drug and thiol levels in plasma and blood cells, a Pediatric Oncology Group study

Purpose: Previous WR-2721 human pharmacokinetic studies were limited to plasma levels in patients receiving platinum-based compounds, and none includes the effects of WR-2721 on endogenous thiols. In the present study (Pediatric Oncology Group study no. 9457), we measured the levels of WR-2721, its active metabolites, as well as cysteine and glutathione in whole blood, plasma, and blood cells in patients receiving high-dose alkylating agents with mesna. Methods: WR-2721 was administered (15 min intravenous infusion of 825 mg/m² per dose ×2) to five patients with metastatic Ewing's sarcoma receiving ifosfamide and cyclophosphamide with mesna. Intracellular and extracellular blood thiols were labeled with monobromobimane (mBBr) at the time of collection, and the low molecular weight (LMW) thiols were subsequently separated by HPLC and detected by fluorescence. Results: The active metabolite of the drug, WR-1065, peaked at 100 μM in plasma and blood cells at the end of WR-2721 infusion and decayed with a rapid initial half-life. Detectable levels of WR-1065 and its LMW disulfides were present in plasma and blood cells at ∼1 h after the WR-2721 infusion. By the end of the first WR-2721 infusion (prior to mesna infusion), the mean cysteine level more than doubled and the mean Cys-SS-LMW (cystine and the mixed LMW disulfides) level decreased by ∼50% in both plasma and blood cells. In four of five patients, reduced glutathione levels in blood cells increased by the end of the first WR-2721 infusions, the average increment being ∼36%. Conclusions: (1) WR-1065 is rapidly formed from WR-2721 and equilibrates between plasma and blood cells; (2) WR-1065 decays in plasma and blood cells with similar rapid initial half-lives of ∼16 min; (3) WR-2721 treatment increases cysteine in plasma and blood cells, an effect similar to that of mesna; (4) WR-2721 treatment appears to increase glutathione levels in blood cells; (5) Mesna does not have a substantial effect on the fate of WR-2721 in patients. Received: 14 December 1998 / Accepted: 10 May 1999     

Determination of the cytoprotective agent WR-2721 (Amifostine, Ethyol®) and its metabolites in human blood using monobromobimane fluorescent labeling and high-performance liquid chromatography

Purpose: WR-2721 [S-2-(3-aminopropylamino)ethylphosphorothioic acid] is a chemoprotective agent that is currently in pediatric clinical trials. It is a prodrug that is dephosphorylated by alkaline phosphatase to the active free thiol form, WR-1065 [S-2-(3-aminopropylamino)ethanethiol]. It is likely that adequate and sustained cellular levels of the drug are necessary for optimum cytoprotection. To date, a method to measure both plasma and cellular levels of WR-2721 and its metabolites in clinical samples has not been available. Methods: In the study reported here the monobromobimane (mBBr) fluorescent labeling method was used to measure these levels when drug was added in vitro to blood samples from normal volunteers. In addition, we present pharmacokinetic data from a pediatric patient receiving WR-2721 (825 mg/m² × 2). Results: The results can be summarized as follows: (1) WR-2721 was detected in the patient's plasma with a half-life of about 10 min; (2) the WR-1065 concentration in the blood cellular fraction was similar to that of plasma; (3) both WR-1065 and WR-SS-low molecular weight (WR-SS-LMW) metabolites disappeared from plasma and the cellular fraction by 3.6 h after WR-2721 infusion; (4) a large proportion of WR-1065 was oxidized in plasma to WR-SS protein and WR-SS-LMW; (5) a large proportion of WR-1065 in the cellular fraction was oxidized to WR-SS-protein; (6) the WR-SS-LMW concentration in the cellular fraction was low; and (7) saturation of plasma and cellular protein binding sites was possible. Conclusions: The pharmacokinetic data that were generated with this technique could guide clinical trials using WR-2721. Received: 18 September 1997 / Accepted: 16 January 1998     

Cytotoxic effects of WR-2721 on mouse testicular cells

WR-2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid) has been demonstrated to be cytotoxic to stem spermatogonia in the mouse. Five and 10 injections of 300 mg/kg killed sufficient numbers of stem cells to reduce sperm production 56 days after treatment by 16 and 43%, respectively. Single injections of 300 or 400 mg/kg of WR-2721 given 15 min after irradiation produced negligible toxicity to stem cells as measured by counts of repopulated tubules; 600 mg/kg reduced stem cell survival by 47%. Four daily injections of 300 mg/kg given 4, 3, 2, and 1 days prior to irradiation (with or without a fifth injection 15 min after irradiation) reduced stem cell survival by about 60%. The cytotoxic effects of WR-2721 on testicular stem cells at least partially explains the reduced protection factors observed in the testis with low doses of radiation and during fractionated treatments involving multiple injections of drug.     

Human pharmacokinetics of WR-2721

The pharmacokinetic properties of WR-2721 were investigated in 13 cancer patients given a 150 mg/M2 intravenous bolus dose of the drug. An average plasma clearance value of 2.17 L/min was obtained. Very little of the drug or the two metabolites, WR-1065 and WR-33278, were excreted in urine obtained after the blood collection schedule. Plasma concentrations of WR-2721 decreased by 94% within 6 minutes of drug administration. The mean value of 6.44 L obtained for the steady-state volume of distribution indicates that the extravascular space occupied by the drug is small. These observations suggest that in human cancer patients, WR-2721 is rapidly taken up by tissues and converted to metabolites.     

Lens epithelial cell protection by aminothiol WR-1065 and anetholedithiolethione from ionizing radiation

Lens epithelium disorganization, glutathione (GSH) depletion, and epithelial cell death have been incriminated in the cytopathogenic mechanisms that lead to cataract formation following UVB and x-ray exposures. The objective of this study was to determine the in vitro capacity of the aminothiol WR-1065, the active metabolite of amifostine, and anetholedithiolethione (ADT or Sulfarlem®) to protect bovine lens epithelial cells against x-ray irradiation. WR-1065 and ADT were used at a concentration of 20 μM. A single dose of 10 Gy was delivered at a rate of 2 Gy/min. Fluorimetric assays were then performed using a neutral red probe to evaluate cell viability, a Hoechst 33342 probe (HO) to evaluate nuclear condensation and apoptosis, and a monobromobimane probe to estimate the intracellular GSH pool. Twenty-four hours after x-ray exposure, cells pretreated with WR-1065 showed increased GSH levels, improved cell viability, and decreased HO fluorescence in addition to a lesser proportion of cells with apoptotic nuclear modifications. Between 72 and 120 hr postirradiation, ADT-pretreated cells also showed increased intracellular GSH levels and cell viability and decreased HO fluorescence and apoptotic cell morphology. This in vitro study demonstrates that WR-1065 and ADT protects lens epithelial cells from x-ray injury; thus, ADT and amifostine are appropriate candidates for clinical trials in humans. They are currently used in preventing radiation-induced xerostomia and should be further tested in the prevention of late radiation-induced ocular complications such as sicca syndrome and cataract. © 2002 Wiley-Liss, Inc.     

Modification of WR-2721 toxicity and radioprotection by an inhibitor of alkaline phosphatase

We used levamisole, an inhibitor of alkaline phosphatase, to study the role of that enzyme in mediating the metabolic activation, toxicity, and radioprotection of WR-2721 in intact mice. We found the toxicity of WR-2721 was slightly decreased by prior subcutaneous (SQ) injection of 40 mg/kg of levamisole. In studying the effect of levamisole on WR-2721 radioprotection, we found that intraperitoneal (i.p.) injection of levamisole had little or no effect on radioprotection of the gastrointestinal and the hematopoietic systems. Even this small reduction of protection was due in part to the toxicity of levamisole as demonstrated when levamisole was injected following, rather than before, WR-2721-radiation treatment. To determine whether levamisole inhibited the activation (i.e., dephosphorylation) of WR-2721 to WR-1065, we assayed WR-1065 in the jejunum using an HPLC electrochemical assay. SQ injection of 75 mg/kg levamisole 10 min prior to WR-2721 reduced the WR-1065 observed 10 min after WR-2721 administration by 37%. In conclusion, levamisole appears to be too toxic and non-specific to be useful in studying and regulating the metabolism, toxicity and radioprotection of WR-2721.     

HPLC assay for 2-(3-aminopropylamino)ethanethiol (WR-1065) in plasma

A high pressure liquid chromatography (HPLC) plasma assay for WR-1065 is described which is both precise (coefficient of variation less than 5%) and accurate (% average deviation less than or equal to 6.1) throughout the concentration range from 1 to 500 micrograms/mL of plasma. The analyte is separated by HPLC and detected with a thiol specific electrochemical transducer cell. The detector response is linear over the ranges 1 to 10 micrograms/mL (R2 = 0.995), 10 to 100 micrograms/mL (R2 = 0.995), and 100 to 500 micrograms/mL (R2 = 0.974). The absolute retention times for WR-1065 and WR-1729 are 9 and 12 minutes, respectively. The assay uses 100 microL of plasma and requires a total chromatography cycle time of 40 minutes. The method has been found suitable for the determination of WR-1065 in plasma from a beagle dog after i.v. administration of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721).     

Comparative biodistribution and radioprotection studies with three radioprotective drugs in mouse tumors

The organ level biodistribution and tumor radioprotective properties of three drugs have been compared: WR-2721 (NSC 296961), WR-3689 (NSC 327729), and WR-77913 (NSC 318809). The three drugs have similar distribution patterns in normal mouse tissues. At 30 minutes after intraperitoneal injection, highest levels of 35S from radiolabeled protector are found in kidney and submandibular salivary gland, with lowest levels in brain and moderately low values in tumor and skin. Three of four tumors examined take up less WR-3689 than the other two protectors. For the three protectors, the dose modifying factors for the RIF-1 tumor irradiated in vivo and assayed in vitro are 1.5-1.7, but do not vary as predicted by differential uptake of drug into this neoplasm. In RIF-1, WR-3689 is taken up most avidly, but the three drugs tend to be equally protective.     

WR-2721 protection of pneumonitis and fibrosis in mouse lung after single doses of x rays

The radioprotective effect of WR-2721 has been studied in mouse lung after single doses of radiation. Using the breathing rate assay and lethality, radioprotection was assessed at monthly intervals between 3 and 18 months after irradiation during both pneumonitis and chronic fibrosis. The degree of radioprotection was greater for fibrosis than for pneumonitis using both assays. In replicate experiments, dose modifying factors (DMF's) ranging from 1.2 to 1.4 were obtained for pneumonitis and 1.5 and 1.6 for fibrosis. The differences in DMF's for the two phases of lung damage were significant. A difference in the time course of expression of damage was seen in both the breathing rate and lethality assays between mice irradiated with and without WR-2721: the damage ended sooner in the drug-treated mice. This difference is best explained by protection of all damage after 5 months by WR-2721. No evidence of drug toxicity was found. We conclude that WR-2721 protects against chronic lung fibrosis caused by radiation at least as well as against the earlier appearing pneumonitis after single doses of radiation. Thus, if WR-2721 is dose modifying and if late tissue complications are dose limiting in clinical radiotherapy, then a therapeutic benefit would be obtained by the use of this drug in clinical radiotherapy, provided that the radioprotection of tumors did not exceed a factor of 1.5-1.6.     

Protection of cultured mammalian cells by S-2-(3-aminopropylamino)ethylphosphorothioic acid(WR-2721)

The ability of WR-2721 to protect cultured mammalian cells against radiation-induced killing was nearly the same as that of cysteamine when WR-2721 was activated by mouse liver extract. Without the liver extract, protection by WR-2721 required long incubations with the cells prior to irradiation. The protective activity increased in proportion to the cell concentration. The dose reduction factor at a concentration of 4 mM WR-2721 was 1.11 and 1.41 for 1.5 X 10(5) cells/ml and 15 X 10(5) cells/ml of cultured cells, respectively. A non-protein bound sulfhydryl group was detected in cell suspensions after incubation with WR-2721, but it was not a dephosphorylated product of WR-2721.     

Effect of tumor type, size, and endpoint on tumor radioprotection by WR-2721

Experiments are reported showing that the degree of tumor radioprotection afforded by WR-2721 varies with the type of tumor and assay endpoint, and that for a given tumor system, microaggregates are protected better than larger cell masses. The tumors used were a methylcholanthrene-induced fibrosarcoma (FSa), and two tumors of spontaneous origin, another fibrosarcoma (NFSa), and a mammary carcinoma (MCa-4), all syngeneic to C3Hf/Kam mice. WR-2721 was given in a dose of 400 mg/kg 30 minutes before irradiation in all experiments. In TCD50 assays, WR-2721 protected 5 mm diameter and impalpable 3 day-old transplants of 5 X 10(5) FSa cells growing in the leg by factors of 1.11 and 1.13, respectively. Using the tumor latency endpoint, 3 day-old s.c. transplants of 10(3) FSa in the abdominal wall were protected by a factor of 1.27, a degree of protection similar to that reported earlier for sterilization of lung micrometastases of the same tumor. MCa-4 tumors growing in the leg were protected better than FSa in TCD50 assays with protection factors of 1.3 for 4 day-old transplants, 1.24 for 5 mm tumors, and 1.23 for 8 mm tumors. MCa-4 tumors recurrent after irradiation as 4 day-old transplants grew more rapidly in mice that had received WR-2721, and this was shown to be most likely due to protection by the drug against expression of the tumor bed effect. Using the lung micrometastases assay, NFSa was protected by a factor of 1.22. This variability in protection with different tumor types, sizes, and assay endpoints is discussed in terms of drug delivery and uptake, and also in relation to the influence of tumor hypoxia on the radioprotective ability of WR-2721.     

Modification of the radiation response of the mouse kidney by misonidazole and WR-2721

The radiation response of the mouse kidney has been assayed after a range of X ray doses given with or without the nitroimidazole misonidazole or the aminothiol WR-2721. Sensitization and protection of the kidney were investigated by comparing the X ray dose needed to achieve a particular level of injury in the presence or absence of the drug. Two functional assays and kidney weight at sacrifice were used to obtain dose response curves. Urine output and 51Chromium EDTA excretion were used as functional assays at 25 and 49 weeks after irradiation. They demonstrated no radio-sensitization by misonidazole with 1, 2 or 5 fractions of X rays. Significant radioprotection was seen when 400 mg kg 1 WR-2721 was given before single X ray doses (PF = 1.34). Similar radioprotection was observed when renal weight at 1 year after irradiation was used as the third assay of damage. These results confirm that the kidney responds as a well-oxygenated normal tissue with only a small protection being afforded against radiation injury by WR-2721.     

Protection of spermatogonial survival and testicular function by WR-2721 against high and low doses of radiation

The radioprotection of normal cells with WR-2721 at doses of radiation extending down to less than 1 Gy was investigated using testicular cells. Survival of stem spermatogonia after single doses of radiation was measured by counts of repopulating tubules and by sperm head counts, with consistent results obtained for both endpoints. Protection factors (PF) obtained by injection of 400 mg/kg WR-2721 at 15 min prior to irradiation decreased from about 1.4 at radiation doses above 10 Gy to 1.0 at 2 Gy. Similarly, the radioprotection by 300 mg/kg WR-2721 was reduced from a PF of about 1.35 when the drug was given prior to a single high dose of radiation to 1.0-1.1 when the drug was given prior to each of 5 daily fractions of 2 Gy. Thus, less protection of testicular stem cells by WR-2721 was observed at lower doses of radiation. This lowered protection may be explained, at least in part, by a direct cytotoxic effect of WR-2721 on testicular stem cells. Protection of differentiated spermatogonia was observed with 400 mg/kg WR-2721; the PF was 1.4 at 1 Gy and decreased at lower doses. The protection of testicular function by WR-2721, as assayed by the return of fertility and the maximum recovered level of sperm production, was compared to the protection of stem cell survival. At about 8 Gy the PF with 400 mg/kg WR-2721 for both functional endpoints was about 1.5, which was not significantly different from the value of 1.3 obtained using the stem cell assays.     

Pulse radiolysis studies of WR-1065

WR-1065, the dephosphorylated sulfhydryl form of WR-2721 has been suggested as the metabolite responsible for the radioprotective abilities of the latter and as being active during the radiation chemical stage of damage production. Hence this study was performed to determine some of the radiation chemical parameters of the compound. Pulse radiolysis techniques, developed for use with SH compounds, were employed in the current work: Three systems were used to attempt to measure the rate constant for reaction of OH. radicals with WR-1065; (a) Competition with phenylalanine, in which the decrease in the amount of RSSR- caused by the addition of phenylalanine is monitored; this failed because of the absence of an absorbance attributable to RSSR-. (b) Competition with 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS), monitoring the decrease in OH. induced ABTS absorbance caused by WR-1065 addition. This was unsuccessful due to reaction of RS. with ABTS. (c) Competition with CNS-, observing the decrease in (CNS)-2 caused by WR-1065 addition indicates that the second order rate constant for reaction of OH. with WR-1065 is 9.2 +/- 0.3 X 10(9) M-1 s-1. To investigate the ability of WR-1065 to react with DNA radicals the yield of ABTS radicals was monitored in a situation where DNA scavenges 50% of the OH. radicals in the presence of ABTS (20%) and WR-1065 (30%). DNA radicals were shown not to react with ABTS but WR-1065 radicals do, the absence of additional ABTS absorbing species indicates that DNA radicals do not react with WR-1065 under the experimental conditions used. An attempt was made to investigate the possibility that WR-1065 is concentrated close to DNA macromolecules in solution: The rate constant for reaction Br2- with WR-1065 was measured in the presence and in the absence of excess DNA (with which Br2- was not observed to react). The observed rates of reaction were independent of the presence of DNA indicating that the latter does not affect the availability of WR-1065 for reaction with Br2- radical. Reaction of WR-1065 with Br2- was used as a probe for determining the pK of the SH group. The rate of reaction increases from 1.8 X 10(8) M-1 s-1 at pH 6.3 to 1.6 X 10(9) M-1 s-1 at pH 8.4, the mid-point of the increase indicates a pK of 7.3.     

Dose and interval relationship for the interaction of WR-2721 and nitrogen mustard with normal and malignant cells

The protective effect of WR-2721 on nitrogen mustard (HN2) cytotoxicity against hematopoietic stem cells as well as the potentiation effect previously noted against leukemia cells was further examined for dose and interval dependency for these opposite effects. For AKR leukemia cells, a dose dependent potentiation of HN2 cytotoxicity was noted with WR-2721. The magnitude of the potentiation was about 10-fold with 5 mg/mouse WR-2721 and nearly 100-fold at 10 mg/mouse. Interval studies showed maximum potentiation occurred when WR-2721 was given close in time to HN2 and decreased in magnitude as the interval between the agents increased. However, a pronounced potentiation of cytotoxicity was also found when HN2 followed WR-2721 by 4 to 12 hr. For hematopoietic stem cells, a protective effect of WR-2721 on HN2 cytotoxicity was observed. This effect decreased rapidly with interval between the agents with no protection noted for an interval of 6 hr. These results indicate that agents like WR-2721 may have an important therapeutic role in chemotherapy.     

Radioprotection of cells in culture by WR-2721 and derivatives: form of the drug responsible for protection

Studies of V79-171 cells were undertaken to determine what extracellular or intracellular derivative of the drug WR-2721 is associated with radioprotection. The effect of preincubation at 23 +/- 1 degree C with WR-2721, and with derivatives of WR-2721 produced in medium containing alkaline phosphatase, upon survival of cells following subsequent gamma-irradiation was examined. It was established that WR-2721, WR-1065, WR-33278, WRSSCys, and other disulfide forms produced by reactions of WR-1065 with the medium do not provide significant protection when present only extracellularly. Protection was found to correlate with cellular levels of the thiol form of the drug (WR-1065) but not with the cellular level of the disulfide forms of WR-1065. Similar results were obtained with HeLa, Me-180, Ovary 2008, HT-29/SP-1d, and Colo 395 cell lines showing that human tumor cell lines behave in the same fashion as the V79-171 nontumorigenic hamster diploid cell line. None of the drug forms produced significant cytotoxicity under the conditions used. It was concluded that it is the cellular level of WR-1065 at the time of irradiation which determines protection. The results are consistent with protection mechanisms involving scavenging of hydroxyl radicals, hydrogen atom transfer to DNA radicals, depletion of oxygen near DNA, enhancement of rapid biochemical repair processes, or some combination of these mechanisms.     

Promotion of cystine uptake, increase of glutathione biosynthesis, and modulation of glutathione status by S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) in Chinese hamster cells

We recently found that exposure of cells to different aminothiols promotes cystine uptake and leads to an increase of cellular glutathione by new biosynthesis (Issels et al., Biochem. Pharmacol., 37: 881-888, 1988). Therefore, we further investigated whether the known radioprotective and chemoprotective aminothiol derivative S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) or its dephosphorylated form (WR-1065) will lead to similar effects. In order to convert WR-2721 to the free thiol compound (WR-1065) in vitro, the medium also contained 20 U/ml alkaline phosphatase (AP). For uptake studies a modified McCoy's 5A medium supplemented with 0.1 mM [35S]cystine was used. In Chinese hamster ovary (CHO) and Chinese hamster ovarian carcinoma (OvCa) cells, WR-2721 exposure alone did not increase the cystine uptake relative to that of control (untreated) cells, while WR-2721 + AP enhanced the uptake of cystine more than twofold in both cell lines. The increase of cystine uptake was dependent on the time of exposure (0-60 min) and the concentrations of WR-2721 (0-8 mM) + AP. Half-maximal uptake of cystine was observed at concentrations of 0.69 and 0.57 mM WR-2721 in CHO and OvCa cells, respectively. Determination of both reduced (GSH) and oxidized (GSSG) cellular glutathione levels after the exposure (0-300 min) to WR-2721 + AP in CHO cells showed a depletion of GSH to less than 10% of the pretreatment value and a 4-fold reduction of the GSH/GSSG ratio. In contrast, in OvCa cells the amount of total glutathione rather increased with no significant change of the GSH/GSSG ratio by the exposure to WR-2721 + AP. Further analysis using high-performance liquid chromatography of cell extracts revealed that the relative amount of incorporated [35S]-cystine into glutathione was increased similarly in both cell lines. The data show that precursor availability and new biosynthesis of glutathione is enhanced by the exposure to WR-2721 + AP in vitro despite the differential modulation of the cellular glutathione status in the two cell lines. These findings may have important implications for the use of aminothiols like WR-2721 in various cells and tissues in regard of their response to chemotherapeutic agents, ionizing radiation and/or hyperthermia.     

The sulfhydryl containing compounds WR-2721 and glutathione as radio- and chemoprotective agents. A review, indications for use and prospects.

Radio- and chemotherapy for the treatment of malignancies are often associated with significant toxicity. One approach to reduce the toxicity is the concomitant treatment with chemoprotective agents. This article reviews two sulfhydryl compounds, namely the agent WR-2721 (amifostine), a compound recently registered for use in human in many countries, and the natural occurring compound glutathione (GSH). GSH is not registered as a chemoprotective agent. WR-2721 is an aminothiol prodrug and has to be converted to the active compound WR-1065 by membrane-bound alkaline phosphatase. WR-1065 and GSH both act as naturally occurring thiols. No protective effect on the tumour has been found when these compounds are administered intravenously. There is even in vitro evidence for an increased anti-tumour effect with mafosfamide after pretreatment with WR-2721, and in vivo after treatment with carboplatin and paclitaxel. Randomized clinical studies have shown that WR-2721 and GSH decrease cisplatin-induced nephrotoxicity and that WR-2721 reduces radiation radiotherapy-induced toxicity. Side-effects associated with WR-2721 are nausea, vomiting and hypotension, GSH has no side-effects. An exact role of WR-2721 and GSH as chemoprotectors is not yet completely clear. Future studies should examine the protective effect of these drugs on mucositis, cardiac toxicity, neuro- and ototoxicity, the development of secondary neoplasms and their effect on quality of life.