Introduction

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The toxicity of chemotherapy remains significant and, in theory, can be ameliorated by optimal utilization of protective drugs. Mesna (sodium 2-mercaptoethanesulfonate, HS-CH₂-CH₂SO₃Na), a successful example of a chemoprotector, is primarily used to reduce hemorrhagic cystitis induced by oxazaphosphorine (e.g., cyclophosphamide and ifosfamide) (Brock et al., 1982). Mesna presumably binds to the oxazaphosphorine toxic product acrolein, forming the inert thioether conjugate OHC-CH₂-H₂C-S-CH₂-CH₂SO₃Na (Ormstad et al., 1983; Manz et al., 1985). In contrast, binding of mesna to the (4-hydroxylated) oxazaphosphorine (the active metabolite) occurs mainly at the 4-hydroxy position, preserving the chloroethyl reactive moieties (Seitz et al., 1989; Kawabata et al., 1990). Thus, mesna is thought to alter neither the kinetics nor the alkylating properties of oxazaphosphorine. The popular mechanistic model for mesna involves its rapid oxidation (plasma mesna half-life, <17 min) in the blood to dimesna (Burkert, 1983). Dimesna in turn is reduced in the liver and renal tubular epithelium by glutathione; mesna is excreted in the bladder to produce the desirable uroprotection (Ormstad and Uehara, 1982; Ormstad and Ohno, 1984; Goren et al., 1998a). Other protective functions of mesna include reducing the teratogenicity and genotoxicity of oxazaphosphorine and reducing the toxicity and lethality of platinum-based compounds (reviewed in Kempf and Ivankovic, 1987).

Amifostine [WR-2721; Ethyol, S-2-(3-aminopropylamino)ethyl phosphorothioic acid, ⁺H₃N-(CH₂)₃-NH₂⁺-(CH₂)₂-S-PO₃H], on the other hand, is primarily used to decrease platinum nephropathy (Treskes and van der Vijgh, 1993). Amifostine is a prodrug that is activated in the blood by alkaline phosphatase to produce the free thiol metabolite WR-1065 [WR-SH; S-2-(3 aminopropylamino)ethanethiol, ⁺H₃N-(CH₂)₃-NH₂⁺-(CH₂)₂-SH]; the latter enters cells by passive diffusion in the millimolar concentration range (Calabro-Jones et al., 1988; Mitchell et al., 1995; Newton et al., 1996). Uptake of WR-1065 and WR-33278 (the symmetric disulfide) by the polyamine transport system may be important in the micromolar concentration range (Mitchell et al., 1995; Newton et al., 1996). The protective mechanisms of WR-1065 involve its thiol group, which reacts with free radicals and readily ionizes to a thiolate ion that can neutralize epoxides and other electrophilic agents (Treskes and van der Vijgh, 1993). In addition, the cationic character of WR-1065 and WR-33278 allows them to concentrate near DNA, which enhances their protective capacity (Smoluk et al., 1988; Zheng et al., 1992). Both amifostine and mesna are highly concentrated in the kidney (Utley et al., 1984; Shaw et al., 1986, 1994).

Measurement of mesna levels during combined administration of amifostine and mesna has not been previously reported. To investigate the rationale of using the two thiol drugs, we determined plasma, RBC¹, and highly-purified PBMC thiol levels after in vitro treatment of blood from a healthy volunteer, four patients with metastatic Ewing's sarcoma who received amifostine and mesna before cyclophosphamide and ifosfamide infusions (Pediatric Oncology Group study no. 9457), and two local patients who received mesna alone. We also sought to investigate a report that mesna does not undergo a thiol-disulfide exchange reaction with oxidized glutathione under physiological conditions because such restricted reactivity might be important to its fate in vivo (Pendyala et al., 2000).

	Materials and Methods

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Reagents. WR-1065 (mol. wt., 207.16) was obtained from U.S. Bioscience Corporation (West Conshohocken, PA); mesna (mol. wt., 164.18; 100 mg/ml solution) from Bristol-Myers Squibb Co. (Stamford, CT); 4-hydroperoxycyclophosphamide (4-OOH-CP, D-18864; mol. wt., 293.1) was obtained from Dr. Ulf Niemeyer (ASTA Medica AG, Frankfurt am Main, Germany); monobromobimane (mBBr) from Molecular Probes (Eugene, OR); 5,5'-dithio-bis(2-nitrobenzoic acid), cysteine (Cys-SH), GSH, GSSG (containing 0.2% GSH), Tris, and HPLC grades methanol, acetonitrile (AcCN), dimethyl sulfoxide (DMSO), and dichloromethane from Sigma (St. Louis, MO); trifluoroacetic acid and methanesulfonic acid (MSA) from Fluka Chemical Corp. (Ronkonkoma, NY); highest purity DTT from Calbiochem (San Diego, CA); perchloric acid from Aldrich (Milwaukee, WI); Ficoll-Paque from Amersham Pharmacia Biotech AB (Uppsala, Sweden); Hanks' balanced salt solution from Invitrogen (Carlsbad, CA); RPMI 1640 medium (pH 7.15, without L-glutamine) from Mediatech, Inc. (Herndon, VA); phosphate-buffered saline (PBS without calcium or magnesium) from Bio Whittaker (Walkersville, MD); and CD45 MicroBeads, OctoMACS magnet, and MiniMACS high-gradient magnetic separation columns with maximum capacity of ~10₈ cells from Miltenyi Biotec (Auburn, CA).

Patients. Patient 1 was an 18-year-old male; patient 2, an 18-year-old male; patient 3, a 9-year-old male; patient 4, a 15-year-old male; patient 5, a 19-year-old female; and patient 6, a 7-year-old female. The study was approved by the Institutional Review Board for the Protection of Human Subjects at each of the participating institutions. Informed consent was obtained from each patient.

Drug Administration and Pharmacokinetic Sampling. The chemotherapy and pharmacokinetic sampling for patients 1 to 4 were as previously described (Souid et al., 1999). Briefly, they received amifostine (825 mg/m² = ~3.1 mmol/m²) i.v. over 15 min, beginning 30 min before ifosfamide (mol. wt., 261.1) and cyclophosphamide (mol. wt., 279.1) infusions. Amifostine was repeated 3 h after the first dose. Mesna (400 mg/m² = ~2.4 mmol/m²/dose i.v.) was given as well, with the first dose just after the first amifostine dose. The cyclophosphamide course started 3 weeks after the ifosfamide course.

Volunteer's Blood. Blood was freshly drawn from a normal volunteer into a purple-top Vacutainer tube. WR-1065 and/or mesna were added, and the samples were mixed by inversions at RT for 2 min and incubated at 37°C for 15 min. mBBr was then added (from a 1.0 M stock in DMSO) to a final concentration of 30 mM, and the samples were mixed by inversions at RT for 3 min and incubated in the dark at RT for 30 min. The PBMC acid-soluble supernatants were prepared and analyzed as described below. The plasma and RBC acid-soluble supernatants were prepared, stored, and analyzed as described (Souid et al., 1998, 1999).

PBMC Preparation and Processing. Blood samples from patients and a normal volunteer were diluted with an equal volume of Hanks' balanced salt solution and gently layered on the top of an equal volume of Ficoll-Paque. The solutions were centrifuged (RT, 3000g) for 1 h. The PBMC were collected, washed with 2.0 ml of PBS (supplemented with 2 mM EDTA and 1.0% bovine serum albumin, final pH 7.4), and resuspended in 80 µl of PBS. Twenty microliters of CD45 MicroBeads were added, and the mixtures were incubated at 4°C for 20 min. The antibody-coated PBMC were diluted with PBS and centrifuged (RT, 2000g) for 5 min (to remove unbound MicroBeads). The cells were resuspended in 0.5 ml of PBS and loaded on a MiniMACS column (attached to the magnet and prewashed with 3.0 ml of PBS). The cells were then washed with 3.0 ml of PBS, eluted (away from the magnet) with 1.0 ml of PBS, and counted on the Coulter Z2 model (Beckman Coulter, Inc., Fullerton, CA). The final yield was ~10⁶ PBMC/1.0 ml blood; the mean ± S.D. of the cell volume was 190 ± 50 fl. The preparations appeared homogenous on cytospin smears prepared with the Wright stain virtually free of RBC, platelets, and granulocytes. The acid-soluble supernatants were prepared by the addition of 150 µl of 2.5% perchloric acid/2 M sodium methanesulfonate to the PBMC pellets, followed by vigorous vortexing. The supernatants were collected by centrifugation and analyzed as described below.

In Vitro Incubation of PBMC with Thiol Drugs. Purified PBMC (8 × 10⁶ cells/incubation, with a mean ± S.D. cell volume of 189 ± 53 fl) from a healthy volunteer were incubated (15 min at 37°C) with 2 mM WR-1065 or mesna. At the end of incubation, mBBr was added to a final concentration of 30 mM, and the derivatization was allowed to continue in the dark at RT for 20 min. The PBMC were then washed with PBS, and their acid supernatants were prepared and analyzed as described below.

In Vitro Incubation of PBMC with 4-OOH-CP. In all experiments, ~10⁷ cells/condition were incubated in RPMI medium in a final volume of 1.0 ml at 37°C. A control sample with no addition was incubated along with the experimental conditions. PBMC were incubated with WR-1065, mesna, and 4-OOH-CP at 37°C for 60 min. The alkylating agents were added last and rapid mixing followed each addition. At the end of the incubation period, the cells were collected by centrifugation and suspended in 20 mM Tris-MSA and 5 mM mBBr (final volume, 0.5 ml). The mixtures were incubated at RT for 15 min. The concentrations of GSH in the cellular acid-soluble supernatants were determined on HPLC, as described (Souid et al., 1998, 1999). The final pH in all reaction mixtures was ~7.2 and did not change significantly throughout the incubation period. The free thiols in the mesna reaction mixture decayed with a half-life of ~19 min and in the WR-1065 mixture with a half-life of ~16 min.

Sample Analysis. The acid-soluble supernatants were analyzed with (reaction 1) and without (reaction 2) reduction with DTT. Reaction 1 contained a 100-µl sample and 30 µl of 1.0 M Tris-base (final pH 8.0). DTT was added to a final concentration of 3.2 mM (from a freshly made 0.1 M ice-cold stock). The mixture was incubated at RT for 20 min. Additional DTT was added to achieve a total final concentration of 4.2 mM, and the incubation was continued for another 20 min. Then, 10.7 mM mBBr was added (from a 0.1 M stock solution in AcCN). The reaction was allowed to continue in the dark at RT for 20 min and repeated with 5.0 mM mBBr. The derivatization reaction was stopped by adding 5 µl of 5.0 M MSA. The sample was then diluted with an equal volume of 10 mM MSA and extracted with H₂O-saturated dichloromethane. Fifty microliters of the supernatant was injected onto the HPLC and analyzed as described (Souid et al., 1998, 1999). Reaction 2 was exactly as Reaction 1, except that the samples were incubated with distilled H₂O instead of DTT and AcCN instead of mBBr. The final concentrations were calculated using the measured volume of plasma, RBC, and PBMC. Values for the total reducible thiols [i.e., the free thiols plus their low-molecular weight (LMW) disulfides] were calculated as thiol equivalents.

HPLC Analysis. The analysis was performed on the HPLC, as described (Souid et al., 1998, 1999). The minimum quantifiable level was ~1 pmol, and the minimum quantifiable concentration in the blood samples was ~0.3 µM. Representative chromatograms are shown in Fig. 1.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Representative chromatograms of the PBMC of patient 4.  The patient received amifostine followed by mesna before cyclophosphamide. The dashed chromatogram is an analytical run of 10 µl of 2 µM standard. Peak 1 is the cysteine-bimane derivative, peak 2 the WR-S-bimane derivative, peak 3 the reagent, peak 4 the mesna-bimane derivative, and peak 5 the glutathione-bimane derivative. The dotted and solid chromatograms are analytical runs of 50 µl of the PBMC acid-soluble supernatants of patient 4 at 3.25 h (i.e., immediately after the second amifostine infusion) and 3.75 h, respectively (the schedule and sampling time are the same as those described in the legend to Table 9).

	Results

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Thiol-Disulfide Exchange Reactions. To ascertain whether the thiol-disulfide exchange reactions of mesna are unusual, we compared the ability of mesna to reduce GSSG with that of WR-1065, cysteine, and DTT (Table 1). The reactions were carried out with 1 mM GSSG in the presence of 5 mM thiol at pH 8.0, and the production of GSH was measured as a function of time. WR-1065 produced ~30% reduction very rapidly, but no further reduction occurred with continued incubation, suggesting that equilibrium had been achieved. GSH production with cysteine and DTT was similar, but with these thiols, the GSH level continued to increase, with DTT producing the highest overall conversion at 30 min. Reaction with mesna was slower but resulted in an overall reduction of GSSG intermediate between that of cysteine and that of DTT at 30 min.

In Vitro. We next examined the fate of mesna added to volunteer's blood at 500 µM in the absence of and together with an equal quantity of WR-1065 (Table 2). The RBC uptake of mesna was significantly lower than that of WR-1065 (10% versus 60% of plasma level) when each was tested alone. When tested in combination the results were very similar, indicating that the presence of one thiol does not markedly influence the uptake of the other. The presence of WR-1065 appeared to have little effect on or to actually decrease the fraction of mesna found in the thiol form in the plasma and RBC. Similar results were observed at 50 µM the drug thiols.

In Vivo. We expanded our previous study of WR-1065 (Souid et al., 1999), to include measurements of mesna in four Pediatric Oncology Group patients who received WR-2721 and mesna (patients 1-4) and two local patients who received mesna alone (patients 5 and 6). The concentration-time profiles of mesna for patients 1, 2, and 6 are shown in Fig. 2, with the time course of chemotherapy indicated on the top of the panels.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Concentration-time profiles of mesna and total reducible mesna in the plasma and RBC. The plasma levels are displayed in the left panels and the RBC levels in the right panels. The time course of drug delivery is shown on the top of each panel (A, amifostine; M, mesna; and C, cyclophosphamide). The concentration of mesna is displayed with open circles and total reducible mesna (i.e., mesna plus dimesna plus mixed LMW disulfides) with closed circles.

	Discussion

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We first addressed the question of whether mesna is unusual in its ability to reduce GSSG. The results in Table 1 indicate that the reduction of GSSG by mesna is only modestly slower than that by WR-1065, cysteine, or DTT at pH 8.0 and 37°C. This corresponds with the previous findings of Bast et al. (1987), who measured the reaction in Tris-Cl at pH 7.4 and 37°C, but not those of Pendyala et al. (2000), who failed to detect a reaction in PBS at 37°C and an unspecified pH. Thiol-disulfide exchange reactions take place by nucleophilic attack of a thiolate anion on a disulfide bond, so the pK_a of the thiol can be an important factor in determining the observed rate at a given pH (Szajewski and Whitesides, 1980). However, the pK_a of mesna (9.2) is the same as the pK_a1 of DTT, so ionization differences should not produce rate differences at pH 8.0 (Whitesides et al., 1977). However, the second step of the overall reduction by DTT involves a rapid intramolecular reaction, whereas that for reduction by mesna involves a slow bimolecular reaction; this predicts a 2-fold faster reduction by DTT than by mesna, in general accord with the present results. WR-1065 appeared to be the most reactive toward GSSG of the four thiols studied, probably reflecting the fact that its first pK_a is 7.7, so the thiol group is largely ionized at pH 8.0 and available to promote thiol-disulfide exchange (Newton et al., 1992).

In vitro studies of uptake by RBC of mesna and WR-1065 from blood demonstrated that significant uptake of mesna does occur but at a level much lower than that of WR-1065 (Table 2). For both agents, the predominant intracellular form is the thiol, whereas substantial disulfide levels were found in plasma. The ratio of RBC to plasma thiol, calculated from Table 2, was 0.19 for treatment with mesna alone compared with a corresponding ratio of 1.05 for treatment with WR-1065 alone. For treatment with both thiols in combination, the corresponding ratios were 0.21 (mesna) and 0.90 (WR-1065). These values indicate extensive equilibration of WR-1065 and modest uptake of mesna. Both thiols bind to plasma and blood cell proteins, forming drug-protein disulfides (Table 3).

Similar results were found when purified PBMC were incubated with 2 mM thiol. Equilibration of intracellular with extracellular levels was found in the experiment with WR-1065, but with mesna uptake over 15 min at 37°C proceeded to only 7% of the equilibrium value. Treatment of whole blood with 500 µM each of mesna and WR-1065 followed by labeling, isolation of PBMC, lysis, and analysis of the thiol content (Table 4) showed that incorporation of mesna was about 5-fold lower than that of WR-1065.

Mesna uptake was similar in PBMC and RBC, so the absence of nuclei in RBC does not make them unusual in this respect. All of the results with PBMC indicate that labeling of cellular thiols with mBBr in whole blood, isolation of the intact cells, extraction, and analysis of the bimane derivatives provide a viable method for determination of cellular thiol levels.

The cytoprotective capacities of WR-1065 and mesna were compared in PBMC (Table 5). Both thiols protected GSH depletion by 4-OOH-CP. However, WR-1065 was more effective. A 5-fold molar excess of WR-1065 was required for full GSH protection. In contrast, 5-fold molar excess of mesna produced at most 50% protection. The more effective cytoprotective capacity of WR-1065 in comparison with that of mesna is consistent with WR-1065 efficient cellular uptake (Tables 2, 4, 6, 8, and 9).

The results for analysis of blood samples from patients 1 and 2 were qualitatively similar to those from the in vitro studies. Whereas patient plasma and RBC levels of WR-1065 were largely equilibrated, as previously reported (Souid et al., 1999), the RBC levels of mesna were only 18 and 31% of the plasma values, even though much longer time intervals were involved in the patient studies (Fig. 2) than the 15-min intervals used in the in vitro studies. Uptake of mesna and dimesna by blood cells has not been previously reported. However, uptake by isolated animal cells from small intestine and kidney is very efficient, whereas uptake by hepatocytes and pulmonary cells is slow and uptake by myocardial cells was not detectable (Ormstad and Uehara, 1982; Ormstad et al., 1983; Ormstad and Ohno, 1984).

In patients 1 and 2, the peak levels of WR-1065 measured in the plasma (80 and 67 µM, respectively) and RBC (100 and 80 µM, respectively) are in the range of those reported previously, and the concentration-time profiles are very similar (Souid et al., 1999). Mesna, on the other hand, distributes mostly in the plasma (Fig. 2).

The peak plasma and RBC levels of mesna were used to calculate whole blood values (assuming 60% plasma volume), yielding values of 174 µM for patient 1 and 225 µM for patient 2, with corresponding values for total reducible mesna of 370 and 260 µM, respectively. These are higher than those found in whole blood following i.v. infusion of 240 mg/m² over 10 to 40 min (79 ± 19 µM for mesna and 129 ± 23 µM for mesna plus 2 times dimesna) (Goren et al., 1998b), which presumably reflect the higher dose and different administration time employed in our protocol (400 mg/m² over 15 min). Also, our values for total reducible mesna represent not only dimesna but also its mixed LMW-disulfides.

Our data show less rapid oxidation of mesna to disulfide forms in the plasma of patients 1 and 2 than previously reported, possibly reflecting the concomitant administration of amifostine (Fig. 2). This may represent one aspect in which the combined use of mesna and amifostine deviates from use of mesna alone. Moreover, the transient increase in plasma and RBC levels of mesna following the second amifostine infusion in (Fig. 2; patients 1 and 2) suggests that WR-1065 is releasing disulfide bound mesna via thiol-disulfide exchange reactions. In contrast, rapid oxidation of mesna to disulfide forms was observed in the plasma of patient 6, who received only mesna (Fig. 2; patient 6).

Both WR-1065 and mesna reduce cysteine disulfides, an effect that increases cellular cysteine influx and renal cysteine excretion (Pendyala et al., 2000). Furthermore, high-dose chemotherapy may deplete cellular glutathione, an effect that can be prevented by thiol administration (Whitesides et al., 1977; Dorr and Lagel, 1994; Meier et al., 1994). Our findings support these observations.

We lack a useful therapeutic model for monitoring drug thiol administration. Our methods for drug thiol determinations in RBC and PBMC may fulfill this purpose. For example, exceeding the capacity of a protective agent may account for the lack of protection with dose-intensive combination chemotherapy (Shapiro et al., 1998). In our trial, the bioavailable phosphoramide mustard is ~1.5 mmol/m² (~20% of the cyclophosphamide dose) (Bakke et al., 1972), amifostine 6.2 mmol/m², and mesna 12.0 mmol/m². Thus, the total thiol is ~12-fold higher than the phosphoramide mustard. However, a significant proportion of the thiol dose is used to reduce endogenous disulfides, and the remaining portion may decay faster than the alkylating agent.

In summary, the results demonstrate similarities between mesna and WR-1065 with respect to their participation in thiol-disulfide exchange reactions. Mesna distributes mostly in the extracellular compartment, whereas WR-1065 distributes more efficiently in the intracellular compartment. Combined treatment with amifostine and mesna does not cause major modification in their distribution; hence, their protective properties may be additive. However, the data should be considered preliminary because they represent only four patients. A more comprehensive study is needed to provide conclusive evidence on any potential interaction between the two agents when used in combination.