Amifostine Prior to Lethal Irradiation Prevents Allogeneic Bone Marrow Engraftment in Mice

ORIGINAL ARTICLE Amifostine prior to lethal irradiation prevents allogeneic bone marrow engraftment in mice

JS Thompson1,2, R Asmis3, Y Chu1,2, J Glass1,2, B Nelson1,2 and SA Brown1,2

1Veterans Affairs Medical Center, Lexington, KY, USA; 2Department of Internal Medicine, University of Kentucky, Lexington, KY, USA and 3Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA

We and others have demonstrated that the milieu created reactive oxygen species that may account for much of their byionizing radiation (IR) used for conditioning playsa lytic properties. Thus, the desired effects of these therapies major role in the development of acute graft-versus-host may be offset by undesirable inflammatory reactions that disease (aGVHD). We reasoned that antioxidants that limit therapy in humans. The majority of these agents could inhibit IR induction of inflammatorycytokines adversely affect host hematopoietic and lymphoid tissues and/or apoptosis might reduce the incidence or severity resulting in anemia, thrombocytopenia, leukopenia and of aGVHD. Therefore, BALB/c mice were treated suppression of the immune response. To offset some of the with amifostine, n-acetyl cysteine (NAC) or pyrrolidine undesirable effects, dietary supplements and antioxidants dithiocarbamate (PDTC) prior to transplantation with have been increasingly utilized.1–6 They are presumed to allogeneic C57Bl/6 bone marrow and spleen cells. None of reduce the toxic effects of the therapy, reduce oxidative 30 amifostine-pretreated mice developed weight loss or stress and enhance the host immune response to some other signs of aGVHD and theyrejected their allogeneic cancers. Some have antitumor activity and others may transplants. However, pretreatment to groups of five mice enhance the effect of other anticancer treatments.3,5,6 each with molar equivalent doses of NAC or PDTC Despite a great number of reported studies, an excellent accelerated death, and lower doses did not prevent review by Malmberg5 and a meta-analysis by Block et al.6 aGVHD. In vitro tests demonstrated that PDTC and point out that their ability to enhance tumor killing is NAC acted as pro-oxidants when incubated with isolated variable and that the loss of host immune response to normal mouse lymphocytes, whereas amifostine and its tumor antigens is a potential drawback. In this regard, active metabolite WR-1065 did not. The conclusion that there are very few agents that have proven clinically amifostine protected immune function from IR in vivo was effective in reducing normal tissue injury induced by IR further supported bythe fact that amifostine and WR- and cytoreductive chemotherapy.7,8 1065 preserved the response of radiated normal lympho- Of these, the aminothiol pro-drug amifostine is a free cytes to respond to PHA and both stimulated growth radical scavenger and is clinically effective in reducing IR of non-radiated, non-PHA-treated normal lymphocytes and chemotherapy-induced injury to normal tissues within vitro. Taken together, these data caution the use of out apparently compromising their antitumor properties.2–4 amifostine in allogeneic transplantation. In autologous transplantation, it has allowed higher doses Bone Marrow Transplantation (2008) 41, 927–934; of radiation by reducing toxicities such as mucositis. doi:10.1038/sj.bmt.1705995; published online 4 February 2008 Because destruction of the oral mucosa reduces the barrier Keywords: amifostine; engraftment; acute graft-versus- to infection, reduction in mucositis is an important host disease; immune response advantage. Our own experimental work and data from the literature9–12 led us to propose that the milieu created by conditioning played a very major role in the development Introduction of acute graft-versus-host disease (aGVHD). We reasoned that the administration of antioxidants that could inhibit Ionizing radiation (IR)and cytoreductive drugs, such as IR induction of inflammatory cytokines and/or inhibit cyclophosphamide, adriamycin, etoposide and paclitaxil, IR-induced apoptosis could have a beneficial effect on the are widely used to destroy neoplastic tissues. They initiate initiation of aGVHD. In addition to amifostine, our choices of other likely candidates were the thiol-containing antioxidants n-acetyl cysteine (NAC)and pyrrolidine dithiocarbamate (PDTC), both of which inhibit nuclear Correspondence: Dr JS Thompson, Department of Internal Medicine, factor-kB activation and nuclear translocation and thereby University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA. the transcription of many cytokines, chemokines and E-mail: [email protected] 13,14 9,10 11,12 Received 21 May 2007; revised 13 December 2007; accepted adhesion molecules. We and others have demon- 13 December 2007; published online 4 February 2008 strated the critical role of tumor necrosis factor a and Amifostine prevents allogeneic engraftment JS Thompson et al 928 interleukin-1b in pathogenesis of aGVHD. We tested their animal laboratory care (AAALAC)and local guidelines, administration prior to lethal total body irradiation (TBI) animals that lost more than 2 g of body weight in 48 h, and allogeneic bone marrow (BM)/spleen transplantation developed severe diarrhea or became too lethargic to eat or in our standard protocol that typically leads to fatal drink were euthanized and counted as a death. aGVHD. In this study, we report that treatment with 400 mg/kg amifostine 30 min before 8.5 Gy lethal TBI of Thiol compounds BALB/c H2d mice and transplantation with fully allogeneic b Amifostine, NAC and PDTC were purchased from Sigma- H2 C57Bl/6 BM and spleen cells (SCs)preserved the Aldrich Inc.; WR-1065 was purchased from Ash Stevens ability of the host immune system to reject the allo- Inc., Detroit, MI, USA. transplant within 7 days. On the contrary, neither PDTC nor NAC prevented allogeneic engraftment and the animals died from toxicity and/or aGVHD. Phenotypic analysis The viability, H2d (host)and H2 b (donor)phenotypes and absolute numbers of CD3 þ T cells were compared in the amifostine-treated and transplanted versus the non-amifos- Methods tine-treated and transplanted recipients. The spleens were obtained from groups of animals killed on day 7 (before the Cell preparation signs of aGVHD usually begin), from the survivors on day To obtain the necessary SCs, 8- to 12-week-old C57Bl/6 14 and from the long-term amifostine-treated survivors on (B6)mice were euthanized with CO asphyxiation followed 2 days 36 and 61. After isolating and washing the cells at by cervical dislocation. Whole spleens were washed with 10 1C, they were resuspended in 5 ml cold flow cytometry phosphate-buffered saline (PBS), placed in RPMI (Gibco, medium containing 0.5% bovine serum albumin þ 0.02% Grand Island, NY, USA)containing 10% fetal calf serum azide in PBS, pH 7.4, counted with crystal violet (Hyclone, Logan, UT, USA), penicillin–streptomycin and (5%) þ acetic acid (0.0125%)and resuspended in flow L-glutamine (Invitrogen, Carlsbad, CA, USA)and pro- cytometry medium at 20 Â 106 cells/ml. To prevent Fc cessed in a Stomacher Biomaster (Sampling Solutions Ltd., binding, the cells were blocked with CD16/32 (clone Rugby, Warwickshire, UK). The cell suspension was MFCR00)for 5 min, following which they were incubated removed from the processing bag and passed through a with antibodies purchased from Caltag (Carlsbad, CA, nylon mesh filter. BM was obtained by aspiration from the USA)unless otherwise noted: anti-H2K b (AFG-885)and femur and tibia of the same animals, dispersed though a -H2Kd (34-2-12); and isotype controls: hamster immuno- steel sieve and centrifuged to remove fat globules. Mono- globulin (Ig) G (HM01); rat Ig (R2a01, R2a04); rat IgG2b nuclear cells were separated on a Ficoll-Hypaque gradient (R2b20, R2b04). After incubation for 15 min on ice, the (Sigma-Aldrich Inc., St Louis, MO, USA). Interface cells cells were washed twice in cold wash buffer (0.01% azide in were removed and washed twice with complete media. The PBS), following which 0.2–0.3 ml 1% paraformaldehyde (in cells were resuspended at the desired concentration and PBS pH 7.4)was added and submitted for flow cytometry. aliquoted into tubes for treatment. Two-color flow cytometry measured the percentage of the total and CD3 þ donor (H2Kb)versus host (H2K d) Radiation cells of each phenotype. Radiation was performed with a Mark IV 137Cesium irradiator (JL Shepard, Glendale, CA, USA)at a dose À1 Glutathione disulfide ratio as a measure of cellular redox rate of 2.0 Gy min . TBI (8.5 Gy)was administered in vivo Normal mouse SCs (NMSCs)were harvested in PBS 1 h and the doses are listed in the in vitro experiments. after the indicated treatments. Each sample was split into two separate 500 ml aliquots, one for the determination of Transplant model total non-protein-bound glutathione and glutathione dis- Transplantation of fully allogeneic BM þ SCs from C57Bl/6 ulfide (GSH þ GSSG)and the second for the determination (B6)H2 b donors into lethally radiated BALB/c H2d hosts of oxidized GSSG.15,16 For the determination of GSSG, consistently results in aGVHD.9,10 Depending on the the samples were supplemented with N-ethylmaleimide number of T-cell-containing SCs added to the standard (3.8 mM)to alkylate-free thiol groups. Proteins were 10 Â 106 BM inoculum, we can manipulate the outcome precipitated with 100 ml cold 18% perchloric acid. Super-

from 100% fatal in 7–10 days to a more lingering yet fatal natants (150 ml)were neutralized with 100 ml1MKP1 outcome or to a milder form from which most of the (pH ¼ 7.0)and reduced with 10.4 m M dithiotreitol for animals will recover. The addition of 2.5 Â 106 or more T- 60 min at room temperature. GSH was derivatized in the cell-containing donor SCs results in generally fatal aGVHD presence of 11.2 mM o-phthalaldehyde and separated by within 9–17 days. Unless otherwise speciﬁcally stated reverse-phase high-performance liquid chromatography set groups of 8- to 10-week-old BALB/c mice received 8.5 Gy to an excitation wavelength of 340 nm and an emission TBI followed within 1 h by the intravenous (i.v.)injection wavelength of 420 nm. The sample was separated isocrati- 6 6 of 10 Â 10 allogeneic B6 BM cells þ 2.5 Â 10 spleen SCs cally on Brownlee columns with propionate buffer (in 35 nM

from the same donors. The animals were observed daily for NaPi pH ¼ 6.5)/acetonitrite at a ﬂow rate of 1.2 ml/min. signs of diarrhea, hunched posture, rufﬂed fur, skin lesions Levels of reduced GSH were calculated as the difference and decreased activity, and weighed three times weekly. between GSH þ GSSG and GSSG. The cellular GSH redox According to association for assessment and accredition of state is expressed as the GSH/GSSG ratio. Three samples

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 929 were taken from each culture, each performed in triplicate intraperitoneally (i.p.)with the molar concentrations of and subjected to the above methods. The mean and s.d. NAC and PDTC equal to that of amifostine, that is, of the three samples was calculated for GSH, GSSG and 400 mg/kg amifostine (1.8 mM/kg) ¼ 304 mg/kg NAC and for the GSH/GSSG ratio for each of the experimental 307 mg/kg PDTC. Four of the five PDTC-treated mice died treatments. within a few hours after TBI (Figure 1a). None of the amifostine-pretreated mice developed weight loss or other Quantitation of hydrogen peroxide signs of aGVHD and all survived for 21 days. The NAC After incubation with the various compounds, the cells mice all rapidly lost weight and died probably as a result of were pelleted via centrifugation and supernatants were the high toxic 304 mg/kg dose. Although two of the control assayed by the Amplex Red Hydrogen Peroxide/Peroxidase PBS animals survived 21 days, they lost weight and Assay Kit (Molecular Probes, Eugene, OR, USA). The exhibited other signs of aGVHD. Amifostine survival assay was performed according to the manufacturer’s versus NAC (P ¼ 0.0018); versus PDTC (P ¼ 0.0025); instructions. The results were determined from standards versus PBS (P ¼ 0.0126). of known concentration assayed alongside the experimental Since the doses of PDTC and perhaps NAC were toxic, samples. we repeated the experiment comparing survival of 400 mg/ kg amifostine with 100 mg/kg PDTC, 100 mg/kg NAC or PBS i.p. After 30 min, they received 8.5 Gy TBI followed by Mitogen response the i.v. injection of 10 Â 106 BM þ 2.5 Â 106 SCs from Lymphocytes were isolated from BALB/c mice dispersed B6 donors (Figure 1b). Again, none of the amifostine- SCs isolated by centrifugation over Ficoll-Hypaque lym- pretreated animals lost significant weight, developed phocyte separation medium (LSM-Mediatec 25-072-CV). diarrhea or hunched posture characteristic of aGVHD The lymphocyte layer was carefully removed and the cells were washed in PBS. The cells were adjusted to 1 Â 106 per well and added in triplicate to 96-well trays. Some wells Survival of BALB/c mice transplanted with C57Bl/6 BM and SCs were left otherwise untreated. Others were pretreated with Experiment 1 (each group n = 5) either 100 mM amifostine or 100 mM WR-1065. Phytohema- 100 glutinin (PHA)(5 mg/ml; Sigma-Aldrich)was added to each AMI 400 mg/kg (1.8 mM/kg) well and followed by 0, 4, 8 or 10 Gy IR. The cells were 75 PBS cultured for 1–6 days. At 18 h prior to harvest, 10 mlof NAC 304 mg/kg (1.8 mM/kg) 3 PDTC 307 mg/kg (1.8 mM/kg) 0.2 mCi/ml H-thymidine was added. The cells were har- 50 vested into a Titertek Multiple Cell Harvester, the filters Survival % were dried, added to scintillation vials containing 1 ml 25 OPTI-FLUOR (PerkinElmer Inc., Waltham, MA, USA) and the disintegrations per minute counted. 0 0 2 4 6 8 10 12 14 16 18 20 Statistics Days For the survival data, the data were analyzed by log rank statistics. The P-values reflect the difference between the Experiment 2 (each group n = 5) survival curves. For the other data, two-tailed t-test 100 statistics assuming unequal samples were performed. The AMI 400 mg/kg (1.8 mM/kg) data are expressed as means±s.d. that is reflected as error 75 PBS bars above the data bars. Po0.05 was taken as statistically significant. 50 Survival %

25 PDTC 100 mg/kg (0.6 mM/kg) Results NAC 100 mg/kg (0.6 mM/kg) 0 Amifostine prevents engraftment of allogeneic BM SCs 0 2 4 6 8 10 12 14 16 18 20 avoiding aGVHD Days At 30 min following 8.5 Gy TBI, 8- to 10-week-old BALB/c 6 6 Figure 1 Mice pretreated with amifostine prior to fully allogeneic bone mice were transplanted i.v. with 10 Â 10 BM þ 2.5 Â 10 marrow (BM) þ spleen cell (SC)transplantation did not develop acute graft- SCs isolated from 8- to 10-week-old C57Bl/6 (B6)mice. versus-host disease (aGVHD)and survived the 21 days of the experiment. The mice were observed daily and weighed three times Groups of normal BALB/c 8- to 10-week-old mice were injected weekly. Since there was no change in the body weight or intraperitoneally (i.p.)with either pyrrolidine dithiocarbamate (PDTC), amifostine (AMI), n-acetyl cysteine (NAC)or phosphate-buffered saline appearance of the amifostine-treated animals while most of (PBS). After 30 min, they received 8.5 Gy total body irradiation (TBI) and the NAC, PDTC or PBS animals had died, the experiments were transplanted intravenously (i.v.)with 10 Â 106 BM þ 2.5 Â 106 SCs were terminated at 21 days. Previous experiments had isolated from 8- to 10-week-old H2-incompatible C57Bl/6 (B6)mice. established that BALB/c mice that received 400 mg/kg Experiment 1 compares the survival of transplanted mice that had been pre-treated with 400 mg/kg amifostine pre-treatment with that of molar amifostine followed by 8.5 Gy TBI alone survived for the equivalent doses of NAC and PDTC. Experiment 2 compares the survival of duration of the 30-day experiment. Therefore, for compar- another group of transplanted mice pre-treated with 400 mg/kg amifostine ison, in the ﬁrst experiment the mice were pretreated with that of non-toxic doses of NAC and PDTC.

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 930 and all lived for the 21 days of this experiment. The NAC- Table 1 Amifostine prevents engraftment and aGVHD following treated mice rapidly lost weight and developed signs of BM/SC transplantation aGVHD and all died by day 16. Although less pronounced Days post-transplant PBSAmifostine early in the experiment, the PDTC- and PBS-pretreated animals also developed aGVHD. One control PBS pre- Day 7 (n ¼ 11)( n ¼ 9) treated animal survived for 21 days but none of the PDTC- Body weight (g)19.2 22.6 Spleen weight (mg)94.6 43.7 and NAC-treated mice survived. Amifostine survival versus Simonsen Indexa 4.9 1.9 NAC (P ¼ 0.0018); versus PDTC (P ¼ 0.0016); versus PBS Phenotype (%)97.6 ±2.2 95.8±8.7 (P ¼ 0.0495). total H2b cellsb total H2d cellsc There were several possible explanations for this rather 41.8±19.9 45.9±21.3 b b d c surprising result. One was that amifostine had inhibited CD3 H2 cells CD3 H2 cells donor T-cell antihost activation. Another was that allo- Day 14 (n ¼ 2)d (n ¼ 9) recognition had occurred but the inflammatory reaction was Body weight (g)20.9 22.8 prevented. However, based on the observation that the Spleen weight (mg)92.6 61.2 amifostine-treated mice exhibited no signs of aGVHD, the Simonsen Indexa 4.3 2.7 Phenotype (%)91.5 ±5.0 93.9±6.9 possibility also existed that the amifostine had so altered total H2b cellsb total H2d cellsc the effect of TBI that host BALB/c-immunocompetent cells 11.7±2.1 27.1±7.6 survived the radiation and rejected the donor cells. Therefore, CD3 H2b cellsb CD3 H2d cellsc two additional experiments were performed. A total of 47 BALB/c mice received 8.5 Gy TBI and were transplanted Day 36 (n ¼ 0)( n ¼ 5) 6 6 Body weight (g)26.7 with 10 Â 10 BM þ 2.5 Â 10 SCs. Twenty-nine of the mice Spleen weight (mg)46.9 were pretreated with 400 mg/kg amifostine and the other 18 Simonsen Indexa 1.7 with a similar volume of PBS i.p. SCs were obtained and Phenotype (%)97.3 ±0.7 examined for the percentage of dually labeled donor H2b total H2d cellsc d 30.8±2.2 versus host H2 -positive–CD3-positive donor/host cells CD3 H2d cellsc determined by flow cytometry (Table 1). Additionally, the ratio of spleen weight to body weight has been shown to be a Day 61 (n ¼ 0)( n ¼ 6) good indication of aGVHD (Simonsen Index); higher ratios Body weight (g)29.9 ±1.8 correlate with aGVHD. The mice were weighed daily and Spleen weight (mg)81.9 ±13.1 Simonsen Indexa 2.8 inspected for signs of aGVHD. If an animal lost more than Phenotype (%)98.1 ±0.9 2.0 g body weight within 48 h, the animal was euthanized and total H2d cellsc counted as an experimental death. None of the amifostine- 30.7±2.8 treated mice lost significant weight and all appeared healthy CD3 H2d cellsc at the time of euthanasia for cytometric analysis. Of the 29 Abbreviations: aGVHD, acute graft-versus-host disease; BM, bone amifostine-treated mice, 9 were euthanized on day 7, 9 on marrow; PBS, phosphate-buffered saline; SC, spleen cell. day 14, 5 on day 36 and 6 on day 61. Whereas more than aRatio of spleen weight divided by body weight Â 10À3. 98% of the SCs in 8/9 of the amifostine-treated mice bThe percentage of CD3-positive H2b cells. examined on day 7 were recipient H2d positive, one animal cThe percentage of CD3-positive H2d cells. d was partially engrafted with 27.3% donor H2b cells. On day Only two PBS-treated mice survived to this date for testing. 14, more than 97% of the SCs in 7/9 mice were recipient H2d, but 4.6 and 5.7% H2b donor cells were present in 2 mice. By and developed aGVHD. Although it was anticipated that days 36 and 61, only amifostine-treated animals survived. amifostine would prevent some of the effects of lethal IR, The percentage of H2d host cells in their spleens was 97.3 and the degree was unexpected. Not only did the mice survive, 98.1%, respectively, indicating that the allogeneic graft had suggesting multi-lineage recovery, but they also did not lose been completely rejected. significant weight, suggesting that the bowel was protected. In contrast, control mice that received a similar volume The rapidity and completeness of the rejection of the of PBS developed severe aGVHD. Daily weights confirmed allogeneic transplant also suggested that amifostine might marked weight loss in the PBS controls. Eleven of these actually have accelerated the host immune response to the animals were euthanized on day 7. The ratio of spleen allograft. In contrast, molar equivalent doses of NAC and weight divided by body weight (Simonsen Index)for this PDTC were toxic and lower doses did not prevent aGVHD. group was 4.9 as compared to 1.9 in the amifostine-treated mice, supporting the diagnosis of aGVHD. By day 14, only two of the PBS-treated mice were still alive for testing. The In vitro tests with NMSCs demonstrate that NAC and percentage of H2b cells was 91.5%, indicating that there PDTC act as pro-oxidants in contrast to amifostine and had not been complete donor engraftment in the two mice WR-1065 that survived to this day. However, the Simonsen Index was The obvious question was why did these three thiol- elevated at 4.3, again reflecting the occurrence of aGVHD. containing compounds have such profoundly different Taken together, this data demonstrate that the amifos- effects on engraftment of allogeneic cells and thus on the tine-treated hosts rejected the allogeneic transplant and did development of aGVHD? We reasoned that at least one not develop aGVHD. In contrast, the mice that received explanation might relate to their acting as pro- or only PBS were populated with allogeneic H2b donor cells antioxidants. Therefore, NMSCs were incubated in vitro

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 931 with amifostine, WR-1065, NAC and PDTC to determine 0.0, 0.1, 0.5, 1.0 and 10.0 mM NAC, amifostine and its active their effects on the GSH level, on the GSH/GSSG ratio and metabolite WR-1065 (Figure 3). Whereas 10.0 mM PDTC, on the release of H2O2 as indicators of oxidant stress. 1.0 and 10 mM NAC and 10.0 mM amifostine signiﬁcantly

increased H2O2 release above control, WR-1065 did not. PDTC acts as a pro-oxidant when added to NMSC The greatest increase was by 1.0 and 10.0 mM NAC as First, we determined the effect of these four drugs on the compared to the increases by PDTC and amifostine. Doses level of GSH and the GSH/reduced GSSG ratio. NMSCs less than 1.0 mM had no effect. were incubated for 1 h with doses of 0.0, 1.0 and 10.0 mM amifostine, WR-1065, PDTC and NAC. The data are Amifostine and WR-1065 prior to IR reduce/prevent presented as a percentage change compared to that in the radiation-induced loss of the response of NML to respond untreated but similarly cultured PBS control. PDTC to PHA lowered GSH and the GSH/GSSG ratio (P 0.001) o As documented in Table 1, there were few to zero (induced thiol oxidation). Although both NAC and WR- detectable H2b donor cells in the spleens of the amifos- 1065 tended to increase GSH above the PBS-treated tine-pretreated BALB/c mice transplanted with H2b C57Bl/ control, the increases did not quite reach statistical 6 BM and SC at 7 and 14 days and none on days 36 and 61. significance (Figure 2a). However, both 1.0 mM (P 0.01) o This suggested that amifostine had prevented TBI-induced and 10.0 mM (P 0.01)WR-1065 significantly increased the o destruction of BALB/c host immune function. To test this GSH/GSSG ratio, reflecting a protective effect on the hypothesis, we examined the effect of amifostine and its intracellular environment (Figure 2b). active metabolite WR-1065 to either prevent or reduce radiation-induced suppression of the response of isolated NAC to a greater degree than PDTC or amifostine normal mouse BALB/c lymphocytes (NMLs)to PHA stimulates H202 from NMSC (Figure 4). This is relevant because T-cell recognition and Using the Amplex Red assay, we directly measured the activation are essential for the rejection of allogeneic release of H2O2 from NMSC cultures treated for 2 h with tissues. If amifostine could prevent radiation-induced killing of normal lymphocytes in vitro at doses similar to Effect of PDTC, NAC, that used in transplantation, then this would strongly Amifostine and WR-1065 on GSH: support this hypothesis. Lymphocytes were isolated from means ± s.d. (n = 3) 10 mice; 5 Â 106/ml were placed in 96-well U-bottom plates following which 100 mM amifostine or WR-1065 or control PDTC Amifostine 125 NAC WR-1065 PBS was added. The cells were then radiated with 0, 4, 8 or 10 Gy IR followed by the addition of 5.0 mg/ml PHA in 100 30 min. The cells were cultured for 2, 3, 4, 5 or 6 days and ∗∗∗ 3 75 ∗∗∗ 16 h prior to harvest, 1.0 mCi H-thymidine (ICN Bio- medical, Costa Mesa, CA, USA)was added. The cultures were 50 harvested in triplicate and the b emissions were counted. 25 Percent untreated control 0 1.0 10.0 pmol/µg DNA H2O2 release by PDTC, NAC, Effect of PDTC, NAC, Amifostine and WR-1065: means ± s.d. (n = 3) Amifostine and WR-1065 on the 0.7 PDTC GSH/GSSG ratio: means ± s.d. (n = 2) aaa 0.6 NAC 200 PDTC NAC aa Amifostine 0.5 ∗∗ ∗ Amifostine xx 150 ∗ WR-1065 WR-1065 M)

µ ( 0.4 a bb 100 2 ∗ O bb 2 0.3 x b ∗∗∗ H 50 ∗∗∗ Percent untreated control 0.2

0 0.1 1.0 10.0 µM drug 0.0 0.00 1.00 10.00 Figure 2 Pyrrolidine dithiocarbamate (PDTC)but not n-acetyl cysteine Treatment (µM) (NAC), amifostine or WR-1065 decreases the level of glutathione (GSH) and the GSH/glutathione disulﬁde (GSSG)ratio when incubated with Figure 3 Pyrrolidine dithiocarbamate (PDTC)and n-acetyl cysteine normal mouse spleen cell (NMSC) in vitro. NMSCs were incubated for 1 h (NAC)signiﬁcantly increase the release of H 2O2 from normal mouse with doses of 0.0, 1.0 and 10.0 mM amifostine, WR-1065, PDTC and NAC. spleen cells (NMSCs) in vitro. The Amplex Red assay was used for

(a)GSH pmol/ mg DNA. The data are presented as a percentage change determining H2O2 levels. NMSC cultures were treated for 2 h with 0.0, 0.1, compared to the similarly cultured phosphate-buffered saline (PBS) 0.5, 1.0 and 10.0 PDTC, NAC, amifostine and its active metabolite control: (n ¼ 3); (***Po0.001). (b)GSH/GSSG ratio. The data are WR-1065. PDTC: (** versus *, Po0.05); NAC: (aa versus a, Po0.05); presented as a percentage change compared to the similarly cultured PBS (aaa versus a, Po0.01); amifostine: (xx versus x, Po0.05); WR-1065 bb: control: (n ¼ 2); (***Po0.001); (*Po0.05). (versus b, P40.05).

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 932 IR + PHA NML + 100 µM Amifostine or WR-1065 + 5 µg/ml 4000 No treatment ∗ day 4 PHA Amifostine (Days 1, 2, 4, 6) ∗ 3000 WR-1065 ∗ Untreated PHA 2000 8000 PHA + Amifostine ∗∗∗ 7000 PHA + WR-1065 H-Thymidine

3 1000 6000 aa ∗∗ aa incorporation:DPM 5000 0 4000 4.0 8.0 10.0 DPM Gy IR 3000 a H-Thymidine 3

incorporation: ∗ Figure 4 WR-1065 protects the PHA response of normal mouse BALB/c 2000 lymphocyte (NML)after 4, 8 and 10 Gy ionizing radiation (IR). 1000 Lymphocytes were incubated with 100 mM amifostine, WR-1065 or control 0 phosphate-buffered saline (PBS)followed by 0, 4, 8 or 10 Gy IR and by the 1246 addition of 5 mg phytohemaglutinin (PHA). The cells were cultured for 2, 3, Days 4, 5 or 6 days (n ¼ 3)and DNA synthesis was determined by 3H-thymidine incorporation. As compared to no treatment: (*Po0.05). Figure 5 Amifostine and WR-1065 prolong the response of normal mouse BALB/c lymphocyte (NML)to PHA. Lymphocytes were incubated with 100 mM amifostine, 100 mM WR-1065 or control phosphate-buffered Cell proliferation measured in disintegrations per minute saline (PBS). Phytohemaglutinin (PHA) (5 mg)was added to one-half of the was compared daily between the amifostine- and WR-1065- cultures and they were incubated for 1, 2, 4, 5 or 6 days (n ¼ 3). DNA treated and control PBS-treated cultures. Figure 4 demon- synthesis was determined by 3H-thymidine incorporation for the final 16 h strates that amifostine and WR-1065 protected the of culture. (aa versus a, Po0.05); (** versus *, Po0.05); (*** versus *, P 0.01). lymphocytes from 4, 8 and even 10 Gy IR, preserving their o response to PHA. NML + 100 µM Amifostine or WR-1065 (Days 1, 2, 4, 6) Amifostine and WR-1065 prolong the response to PHA and 5000 3 No treatment stimulate H incorporation de novo ∗ This protection raised the question whether amifostine and Amifostine 4000 ∗∗ WR-1065 only protect cells when they are administered WR-1065 before an oxidant stress or could they actually serve to promote lymphoid growth in the absence of agents causing 3000 ∗ oxidant stress and cell death? Could they actually heighten DPM the normal immune response? Amifostine (100 mM), WR- 2000 H-Thymidine 3 1965 (100 mM)or control PBS was added to NML cultures. incorporation: PHA (5 mg/ml)was added to one-half of the cultures. They 1000 were incubated for 1, 2, 4, 5 or 6 days. As above, 16 h prior to harvest 1 mCi 3H-thymidine (ICN Biomedical) 0 was added. They were harvested in triplicate and the b 1246 emissions were counted. Whereas the response to 5 mg/ml Days PHA alone peaked at day 4, it remained elevated to at least day 6 in the cultures treated with either amifostine or WR- Figure 6 Amifostine and WR-1065 alone stimulate normal mouse BALB/c lymphocyte (NML). Lymphocytes were incubated with 100 mM 1065 (Figure 5). Furthermore, WR-1065 alone significantly amifostine, 100 mM WR-1965 or control phosphate-buffered saline (PBS). 3 stimulated H incorporation into untreated NML culture They were incubated for 1, 2, 4, 5 or 6 days (n ¼ 3)and DNA synthesis was for 2 days and both amifostine and WR-1065 alone determined by 3H-thymidine incorporation. As compared to the untreated significantly stimulated incorporation into cells cultured cultures for that day: (*Po0.05; **Po0.01). for 4 days (Figure 6). This pattern is similar to the response of NML to PHA alone (Figure 5), suggesting that WR- that amifostine facilitates early recovery of hematological 1065 and perhaps amifostine were acting as mitogens to and lymphoid cells following autologous transplantation, induce DNA synthesis. some chemotherapy20 and more localized radiotherapy for cancer therapy,21,22 its effect on recovery of these tissues in allogeneic stem cell transplantation (allo-SCT)has not been Discussion as well documented.23 Of concern is the possibility that the very same ability to protect lymphocytes may have negative It is now well established that amifostine protects normal consequences when the intent of treatment is to cause cells from IR17 and chemotherapy-induced injury.18 In immunosuppression. For example, its use in transplant- contrast, it does not generally appear to protect tumor cells ation of either allogeneic stem cells or in association with from the effects of these agents.8,18 Thus, its potential immunosuppressive treatment for solid organ transplant- therapeutic index can be of benefit in the treatment of many ation could be to prevent or inhibit the destruction of cancers. This protection seems to include a variety of normal lymphocytes and thereby preserve the immune normal cells including lymphocytes.19 Whereas it is clear system in unwanted situations.

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 933 The effect of concomitant amifostine administration may The second test was to measure the effect of these agents be to reduce the effectiveness of these drugs. Not only did to stimulate the oxidant H2O2. NAC along with PDTC amifostine and WR-1065 prevent radiation-induced inhibi- signiﬁcantly stimulated the release of H2O2, indicating that tion of the NMSC’s response to PHA, it prolonged their it also could act as a pro-oxidant when incubated with response to PHA on day 5 (data not shown)and day 6 in NMSC. In contrast, neither amifostine nor WR-1065 the absence of IR. Furthermore, they actually enhanced the decreased GSH and the GSH/GSSG ratio or signiﬁcantly

NML growth in culture in the absence of either PHA or IR stimulated H2O2, providing at least one explanation for (Figure 6). The capacity of amifostine to stimulate BM their observed in vivo effects. colony forming units granulocyte, erythrocyte, monocyte, However, their use in allogeneic stem cell or solid organ megakaryocyte (CFU-GEMM)and burst forming units- transplantation to reduce toxicity should take into account erythroid (BFU-E)cell growth has been observed in murine that preservation of the immune system could prevent cells after radiation18 and normal non-irradiated human engraftment and/or allow solid organ rejection. Alterna- marrow,24 but to our knowledge has not heretofore been tively, there remains considerable controversy as to whether noted with lymphoid cells. antioxidants may also reduce the tumoricidal effects of Does this apply to other agents that could reduce the chemotherapy that act as strong oxidants.5,6 In this regard, toxicity of IR or chemotherapy? The data presented herein Weiss et al.36 have recently reported that NAC adminis- document that another clinically used antioxidant NAC did tered for 10 days after allo-SCT to BALB/c mice led to a not prevent allogeneic stem cell engraftment nor did it mild reduction in graft-versus-leukemia. prevent aGVHD. What distinguishes amifostine from NAC Our data provide a clear example of the ability of and PDTC? We hypothesize that the observed benefit of amifostine to preserve T-lymphocyte function following amifostine on the immune system could be the result of lethal TBI and thereby cause the rejection of normal several factors: first, its free radical scavenging function allogeneic BM supplemented with lymphocyte-containing could eliminate the majority of the initial TBI-induced SCs. The ability of pretreatment with amifostine and WR- burst of free radicals; second, amifostine induced genera- 1065 to preserve lymphocyte function in vivo was supported tion of MnSOD to protect against second-generation free by the observation that amifostine preserved the response radicals and third, arrest of the cell cycle until DNA repair of isolated radiated normal lymphocytes to respond to and replication can occur. As noted above, both NAC and PHA. Furthermore, they stimulated growth of non- PDTC are excellent free radical scavengers but they did not radiated non-PHA-treated normal lymphocytes over the 6 preserve the host’s ability to reject the allogeneic graft. days of culture. NAC has been used to treat steroid resistant aGVHD Taken together, this in vivo and in vitro data herald a following BM transplantation7 and acetaminophen toxi- cautionary note when the use of amifostine is considered to city.25 Yet under other experimental conditions, NAC and reduce the toxicities of radiation or chemotherapy em- PDTC stimulated apoptosis in vascular smooth muscle ployed in conditioning allogeneic stem cell transplants or to cells.26 PDTC is another effective free radical scavenger. prevent solid organ allogeneic graft rejection. To our In vivo, PDTC protected mice from endotoxin and tumor knowledge, only one randomized trial of 30 patients has necrosis factor a-induced lethal shock,27,28 rabbits from been reported for its use in allogeneic transplantation.23 staphylococcal enterotoxin A-induced fever29 and cold Amifostine was given on each day of conditioning or twice perfusion-induced injury to orthotopic rat liver trans- daily if the chemotherapy or TBI was given twice a day. plants.30 In vitro, it has been reported to reduce the effect The patients receiving amifostine experienced less frequent of ultraviolet radiation in experiments with cultured cell mucositis and statistically less infections but the details of lines.31,32 On the other hand, under other experimental the time course of multilineage engraftment were not conditions PDTC has acted as a pro-oxidant.33,34 provided. This is relevant since the use of fully ablative Given the clear distinction in the in vivo transplant conditioning is now frequently replaced by lower intensity experiments and conflicting in vitro reports in the literature, conditioning for allo-SCT that relies to a greater degree on we chose two in vitro tests to compare the effects of amifostine the use of immunosuppressive drugs. and its active metabolite WR-1065 with NAC and PDTC. The first was to determine the level of GSH and the GSH/GSSG ratio as a measure of intracellular redox because these assays have been reported to distinguish NAC from PDTC. Whereas References NAC protected mice from endotoxemia by regulating 35 1 Erol FS, Topsakai C, Ozveren MF, Kaplan M, Ilhan N, intracellular redox, the pro-oxidant effect of PDTC was Ozerman IH et al. Protective effects of melatonin and vitamin 16 demonstrated by increasing the generation of GSSG. Our E in brain damage due to gamma radiation: an experimental data again document that PDTC acts as a pro-oxidant when study. Neurosurg Rev 2004; 27: 65–69. incubated in vitro with NMSC by decreasing GSH and the 2 Hospers GAP, Eisenhauer EA, de Vries EGE. The sulfhydryl GSSG ratio in contrast to NAC, amifostine and WR-1065. In containing compounds WR-2721 and glutathione as radio- another study, we have demonstrated that when PDTC and chemoprotective agents. A review: indications for use and precedes IR in vitro, it enhanced IR-induced cell death of prospects. Br J Can 1999; 80: 629–638. NMSC (submitted for publication). This may help to explain 3 Movsas B, Scott C, Langer C, Werner-Wasik M, Nicolaou N, why the higher dose of PDTC accelerated death after TBI and Komaki R et al. Randomized trial of amifostine in locally transplantation prior to the time when engraftment could advanced non-small cell lung cancer patients receiving occur and aGVHD develop. chemotherapy and hyperfractionated radiation: Radiation

Bone Marrow Transplantation Amifostine prevents allogeneic engraftment JS Thompson et al 934 Therapy Oncology Group Trial 98–01. J Clin Oncol 2005; 23: chemotherapy-induced apoptosis of peripheral blood lympho- 2145–2154. cytes from cancer patients. Life Sci 1999; 64: 1525–1532. 4 Cassatt DR, Fazenbaker CA, Kifle G, Bachy CM. Preclinical 21 Rick O, Beyer J, Scgwella N, Siegert W. Influence of studies of the radioprotective efficacy and pharmacokinetics of amifostine on reconstruction of lymphocyte subpopulations subcutaneously administered amifostine. Semin Oncol 2002; after conventional- and high-dose chemotherapy in patients 29: 2–8. with germ cell tumors. Ann Hemat 2002; 81: 717–722. 5 Malmberg K-J. Effective immunotherapy against cancer. 22 Koukourakis MJ, Ktenidou-Kartel S, Bourikas G, Kartalis G, Cancer Immunol Immunother 2004; 53: 879–892. Tsatalas C. Amifostine protects lymphocytes during radio- 6 Block KI, Koch AC, Mead MN, Tothy PK, Newman RA, therapy and stimulates expansion of the CD95/Fas and CD31 Gyllenhaal C. Impact of antioxidant supplementation on chemo- expressing T-cells, in breast cancer patients. Cancer Immunol therapeutic efficacy: a systematic review of the evidence from Immunother 2003; 52: 127–131. randomized controlled trials. Cancer Treat Rev 2007; 33: 407–418. 23 Hwang WYK, Koh L-P, Ng HJ, Tan PHC, Chuah CTH, 7 Colombo AA, Alessandrina EP, Bernasconi P, Arcese GW, Fook SC et al. A randomized trial of amifostine as a Rabusin M, Bacigalupa A et al. N-Acetyl cysteine in the cytoprotectant for patients receiving myeloablative therapy treatment of steroid-resistant acute graft-versus-host disease. for allogeneic hematopoietic stem cell transplantation. Bone Transplantation 1999; 68: 1414–1416. Marrow Transplant 2004; 34: 51–56. 8 Grdina DJ, Murley JS, Kataoka Y. Radioprotectants: current 24 List AF, Heaton R, Glinsmann-Gibson B, Capizzi RL. status and new directions. Oncology 2002; 63 (Suppl 2): 2–10. Amifostine stimulates formation of multipotent and erythroid 9 Xun CQ, Thompson JS, Jennings CD, Brown SA, Widmer bone marrow progenitors. Leukemia 1998; 12: 1596–1602. MB. Effect of total body irradiation, busulfan–cyclophos- 25 Kozer E, McGuigan M. Treatment strategies for early phamide or cyclophosphamide conditioning on inflammatory presenting acetaminophen overdose: a survey of medical cytokine release and development of acute and chronic graft- directors of poison centers in North America and Europe. versus-host disease in H-2 incompatible transplanted SCID Hum Exp Toxicol 2002; 21: 123–127. mice. Blood 1994; 83: 2360–2366. 26 Tsai JC, Jain M, Hsieh CM, Lee WS, Yoshizumi M, Patterson 10 Xun CQ, Tsuchida M, Thompson JS. Delaying transplantation C et al. Induction of apoptosis by pyrrolidine dithiocarbamate after total body irradiation is a simple and effective way to and N-acetylcysteine in vascular smooth muscle cells. J Biol reduce acute graft-versus-host disease mortality after major H2 Chem 1996; 271: 3667–3670. incompatible transplantation. Transplantation 1997; 64: 297–302. 27 Lauzurica P, Martinez-Martinez S, Marazeula M, Gomez del 11 Ferrara JLM, Abhyankar S, Gilliland DG. Cytokine storm of Arco P, Martinez C, Sanchez-Madrid F et al. Pyrrolidine graft-versus-host disease: a critical role of interleukin-1. dithiocarbamate protects mice from lethal shock induced by Transplant Proc 1993; 25: 1216–1217. LPS or TNF-alpha. Eur J Immunol 1999; 29: 1890–1900. 12 Holler E, Kolb HJ, Moller A, Kempini A, Liesenfeld S, 28 Nemeth SH, Hasko G, Vizi ES. Pyrrolidine dithiocarbamate Pechumer H et al. Increased serum levels of tumor necrosis augments IL-10, inhibits TNF-alpha, MIP-1alpha, IL-12, and factor alpha precede major complications of bone marrow nitric oxide production and protects from the lethal effect of transplantation. Blood 1990; 75: 1011–1016. endotoxin. Shock 1998; 10: 49–53. 13 Araki S, Dobashi K, Kubo K, Yamamoto Y, Asayama K, 29 Shao D-Z, Lee J-J, Huang W-T, Liao J-F, Lin M-T. Inhibition Shirahata A. N-acetylcysteine attenuates TNF-alpha induced of nuclear factor-kappaB prevents staphylococcal enterotoxin changes in secretion of interleukin-6, plasminogen activator A-induced fever. Mol Cell Biochem 2004; 262: 177–185. inhibitor-1 and adiponectin from 3T3-L1 adipocytes. Life Sci 30 Gu X-P, Xu F-T, Jiang Y, Qui Y-D, Ding Y-T. Pyrrolidine 2006; 79: 2405–2412. dithiocarbamate added to University of Wisconsin solution 14 Parodi FE, Mao D, Terri L, Ennis BS, Bartoli MA, Thompson inhibits reperfusion injury after orthotopic liver transplant- RW. Suppression of experimental abdominal aortic aneurysms ation in rats. Ann Clin Lab Sci 2004; 34: 187–194. in mice by treatment with pyrrolidine dithiocarbamate, an 31 Kulms D, Zeise E, Poppelmann B, Schwartz T. DNA damage, antioxidant inhibitor of nuclear factor-kB. J Vasc Surg 2005; death receptor activation and reactive oxygen species con- 41: 479–489. tribute to ultraviolet radiation-induced apoptosis in an 15 Asmis R, Wang Y, Xu L, Kisgati M, Begly JG, Mieyal JJ. essential and independent way. Oncogene 2002; 21: 5844–5851. A novel thiol oxidation-based mechanism for adriamycin- 32 Qin J-Z, Bacon PA, Panella J, Sitalilo LA, Denning MF, induced cell injury in human macrophages. FASEB J 2005; 19: Nickoloff BJ. Low-dose UV-radiation sensitizes keratinocytes 1866–1875. to TRAIL-induced apoptosis. J Cell Physiol 2004; 200: 16 Thompson JS, Amis R, Glass J, Liu H, Wilson C, Nelson B 155–166. et al. p53 status influences regulation of HSPs and ribosomal 33 Brennan P, O’Neill LA. 2-Mercaptoethanol restores the ability proteins by PDTC and radiation. Biochem Biophys Res of nuclear factor kB (NFkB)to bind DNA in nuclear extracts Commun 2006; 343: 435–442. from interleukin 1-treated cells incubated with pyrrolidine 17 Sasse AD, de Oliveira G, Clark L, Sasse EC, Clark OAC. dithiocarbamate (PDTC): evidence for oxidation of gluta- Amifostine reduces side effects and improves complete thione in the mechanism of inhibition of NFkB by PDTC. response rate during radiotherapy: results of a meta-analysis. Biochem J 1996; 320: 975–981. Int J Radiat Oncol Biol Phys 2006; 64: 784–791. 34 Malaguarnera L, Pilastro MR, DiMarco R, Scitto C, 18 Taylor CW, Wang LM, List AF, Fernandes D, Paine-Murrieta Massarino MC, Messina A. Cell death in human acute GD, Johnson CS et al. Amifostine protects normal tissues myelogenous leukemic cells induced by pyrrolidine dithio- from paclitaxel toxicity while cytotoxicity against tumour cells carbamate. Apoptosis 2003; 8: 539–545. is maintained. Eur J Can 1997; 33: 1693–1698. 35 Victor VM, Rocha M, De la Fuente M. N-Acetylcysteine 19 Clark LS, Albertini RJ, Nicklas JA. The aminothiol WR-1065 protects mice from lethal endotoxemia by regulating the redox protects T lymphocytes from ionizing radiation-induced state of immune cells. Free Radic Res 2003; 37: 919–929. deletions of the HPRT gene. Cancer Epidemiol Biomarkers 36 Weiss L, Reich S, Zeira M, Or R, Resnick IG, Slavin S et al. Prev 1997; 6: 1033–1037. N-Acetylcysteine mildly inhibits the graft-versus-leukemia 20 Provinciali M, Ciavattini A, Di Stefano G, Argentati K, effect but not the lymphokine activated cells (LAK)activity. Garzette GG. In vivo amifostine (WR-2721)prevents Transplant Immunol 2007; 17: 198–202.

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