Subcutaneous administration of amifostine (ethyol) is equivalent to intravenous administration in a rat mucositis model

PURPOSE: Amifostine (Ethyol) is currently approved for intravenous (IV) administration to prevent xerostomia in patients receiving radiotherapy for head-and-neck cancer. Recently, subcutaneous (SC) administration has been explored as an alternative route. To determine whether SC administration was equivalent to IV administration, we used models to follow pharmacokinetics and oral mucosal protection in rats. METHODS: Amifostine was administered to rats at doses of 200, 100, or 50 mg/kg (1300, 650, or 325 mg/m(2)) IV or SC at various times before radiation at 15.3 Gy (protection studies) or harvest of blood and tissues for analysis by HPLC (pharmacokinetic studies). RESULTS: Amifostine administered IV or SC 1 h before radiation protected rats from mucositis, but the protective effect was more prolonged when amifostine was administered SC. Tissue levels of the active metabolite (WR-1065) were equivalent after SC administration. The correlation between tissue levels of WR-1065 and protection was strong, but that between blood levels of WR-1065 and protection was only weak. CONCLUSION: These data demonstrate that, in a rat model, SC administration of amifostine was at least as effective as that by IV.     

Subcutaneous administration of amifostine during fractionated radiotherapy: a randomized phase II study.

PURPOSE: Amifostine (WR-2721) is an important cytoprotective agent. Although intravenous administration is the standard route, pharmacokinetic studies have shown acceptable plasma levels of the active metabolite of amifostine (WR-1605) after subcutaneous administration. The subcutaneous route, due to its simplicity, presents multiple advantages over the intravenous route when amifostine is used during fractionated radiotherapy. PATIENTS AND METHODS: Sixty patients with thoracic, 40 with head and neck, and 40 with pelvic tumors who were undergoing radical radiotherapy were enrolled onto a randomized phase II trial to assess the feasibility, tolerance, and cytoprotective efficacy of amifostine administered subcutaneously. A flat dose of amifostine 500 mg, diluted in 2.5 mL of normal saline, was injected subcutaneously 20 minutes before each radiotherapy fraction. RESULTS: The subcutaneous amifostine regimen was well tolerated by 85% of patients. In approximately 5% of patients, amifostine therapy was interrupted due to cumulative asthenia, and in 10%, due to a fever/rash reaction. Hypotension was never noted, whereas nausea was frequent. A significant reduction of pharyngeal, esophageal, and rectal mucositis was noted in the amifostine arm (P <.04). The delays in radiotherapy because of grade 3 mucositis were significantly longer in the group of patients treated with radiotherapy alone (P <.04). Amifostine significantly reduced the incidence of acute perineal skin and bladder toxicity (P <.0006). CONCLUSION: Subcutaneous administration of amifostine is well tolerated, effectively reduces radiotherapy's early toxicity, and prevents delays in radiotherapy. The subcutaneous route is much simpler and saves time compared with the intravenous route of administration and can be safely and effectively applied in the daily, busy radiotherapy practice.     

Amifostine (ETHYOL) protects rats from mucositis resulting from fractionated or hyperfractionated radiation exposure

PURPOSE: The cytoprotective drug amifostine (Ethyol) protects rats from oral mucositis resulting from a single dose of gamma-irradiation. We expanded earlier studies to determine whether multiple doses of amifostine protect against fractionated or hyperfractionated radiation and whether the active metabolite of amifostine (WR-1065) accumulates in tissues upon repeated administration. METHODS AND MATERIALS: Rats received amifostine daily for 5 days in conjunction with a 1-week fractionated radiation schedule and were evaluated for oral mucositis. Rats also received amifostine before the am or pm exposure or b.i.d. in conjunction with hyperfractionated radiation. To determine the pharmacokinetics of WR-1065 after repeated dosing, amifostine was given 5 days a week for 1 or 3 weeks, and rat tissue and plasma were collected at intervals during and after treatment and analyzed for WR-1065. RESULTS: Amifostine protected rats from mucositis resulting from fractionated or hyperfractionated radiation. When the number of days of amifostine administration was reduced, protection was diminished. A dose of 100 mg/kg given in the morning or 2 doses at 50 mg/kg provided the best protection against hyperfractionated radiation. WR-1065 did not accumulate in tissues or tumor upon repeated administration. CONCLUSIONS: Amifostine prevented radiation-induced mucositis in a rat model; protection was dose and schedule dependent.     

Phase I clinical and pharmacologic study of weekly cisplatin and irinotecan combined with amifostine for refractory solid tumors.

PURPOSE: This Phase I study was designed primarily to determine the maximum tolerated dose (MTD) and dose-limiting toxicities (DLTs) of irinotecan and cisplatin with and without amifostine in children with refractory solid tumors. PATIENTS AND METHODS: Cisplatin, at a fixed dose of 30 mg/m(2), and escalating doses of irinotecan (starting dose, 40 mg/m(2)) were administered weekly for four consecutive weeks, every 6 weeks. After the MTD of irinotecan plus cisplatin was determined, additional cohorts of patients were enrolled with amifostine (825 mg/m(2)) support. Leukocyte DNA-platinum adducts and pharmacokinetics of cisplatin and WR-1065 (amifostine-active metabolite) were also determined. RESULTS: Twenty-four patients received 43 courses of therapy. The MTD for irinotecan administered in combination with cisplatin (30 mg/m(2)) was 50 mg/m(2). The DLTs of this combination were neutropenia and thrombocytopenia. With the addition of amifostine, at an irinotecan dose of 65 mg/m(2) and cisplatin dose of 30 mg/m(2), the DLT was hypocalcemia. Although no objective responses were observed, six patients received at least three courses of therapy. The amounts of platinum adducts (mean +/- SD) were 10 +/- 20 molecules/10(6) nucleotides. The maximum plasma concentrations (C(max)) for free cisplatin and WR-1065 were 4.5 +/- 1.6 micro M and approximately 89 +/- 10 micro M, respectively. The half-life (t(1/2)) for free plasma cisplatin was 25.4 +/- 5.4 min. The initial t(1/2) for plasma WR-1065 was approximately 7 min and terminal t(1/2) approximately 24 min. CONCLUSION: The combination of cisplatin and irinotecan administered weekly for 4 weeks in children with refractory cancer is well tolerated. Amifostine offers some myeloprotection, likely permitting >/=30% dose escalation for irinotecan, when administered in a combination regimen with cisplatin. However, effective antiemetics and calcium supplementation are necessary with the use of amifostine. Further escalation of irinotecan dosing, using these precautions for amifostine administration, may be possible.     

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.     

Selective radioprotection of hepatocytes by systemic and portal vein infusions of amifostine in a rat liver tumor model

PURPOSE: The tolerance of the liver to radiation is too low to permit an effective dose to be delivered to patients who have diffuse intrahepatic cancer. In this study we evaluated whether systemic or portal venous administration of the aminothiol compound, amifostine, could protect the normal liver from the effects of ionizing radiation without compromising tumor cell kill in a rat liver tumor model. METHODS AND MATERIALS: Rats implanted with liver tumors were infused with 200 mg/kg amifostine over 15 min via the femoral or portal vein. The livers were irradiated with a single 6-Gy fraction 15-20 min after the termination of amifostine infusion. Protection of the liver was assessed by an in vitro hepatocyte micronucleus assay and tumor protection by an in vivo-in vitro clonogenic survival assay. Tissue levels of the active metabolite, free WR-1065, were determined in the tumor and in the normal liver using a specific HPLC assay with electrochemical detection. RESULTS: After a 6-Gy fraction, the frequency of hepatocyte micronuclei after administration of saline, systemic amifostine, and portal venous amifostine was 18.7+/-1%, 6.8+/-1%, and 9.9+/-2%, respectively, corresponding to a radiation equivalent effect of 6 Gy +/- 0.5, 1.8 Gy +/- 0.3, and 2.5 Gy +/- 1.3, respectively. Both amifostine conditions showed considerably less radiation effect than saline-treated controls (p < 0.01); the two amifostine conditions did not differ (p = 0.3). The surviving fraction of tumor cells was not affected by amifostine treatment and was 0.03+/-0.02 and 0.05+/-0.03 for systemic and portal venous delivery and 0.06+/-0.02 for control animals (ANOVA analysis showed no significant difference of the means p = 0.34). Portal venous delivery produced significantly less WR-1065 in the tumor compared to systemic administration (54 microM +/- 36 vs. 343 microM +/- 88, respectively, p = 0.03). CONCLUSIONS: Both systemic and portal venous administration of amifostine effectively protect hepatocytes from ionizing radiation without compromising tumor cell kill in a clinically relevant animal model. These findings suggest that amifostine may be a selective normal tissue radioprotectant in liver cancer and that regional/portal infusions may be preferable to systemic dosing.     

WR-1065, the active form of amifostine, protects HL-60 cells but not peripheral blood mononuclear cells from radiation and etoposide-induced apoptosis

PURPOSE: Developing myeloid cells are particularly sensitive to chemotherapy and ionizing radiation. Mature cells of the hematopoietic lineages, such as are found in the peripheral blood mononuclear cells (PBMCs), are much less sensitive for reasons that are not yet understood. Protecting the myeloid precursors from radiation or chemotherapy is an important goal. METHODS: We have used fluorescence microscopy to assess the ability of WR-1065, the active metabolite of amifostine (Ethyol), to protect cultured myeloid leukemic HL-60 cells or freshly isolated PBMCs from the induction of apoptosis by ionizing radiation or etoposide. RESULTS: WR-1065 greatly reduced the percentage of radiation-induced apoptosis in the p53 negative HL-60 cells 24 h after exposure to 8 Gy. WR-1065 also greatly reduced the percentage of HL-60 cells undergoing apoptosis 24 h after a 1-h exposure to 1 microM etoposide. The pan-caspase inhibitor ZVAD-fmk completely inhibited radiation-induced apoptosis in HL-60 cells when present for the first hour after exposure to radiation, but had no effect on cell survival. In contrast, neither WR-1065 nor ZVAD-fmk reduced the level of radiation-induced apoptosis in normal human PBMCs. CONCLUSION: These results suggest that pro-apoptotic pathways are present in immature myeloid cells that can be selectively protected from radiation or chemotherapy-induced apoptosis.     

Phase I and II study of azacitidine in Japanese patients with myelodysplastic syndromes

Azacitidine, an inhibitor of DNA methyltransferase, is reported to have antileukemic efficacy and is approved for the treatment of myelodysplastic syndromes in Western countries. We have conducted a Phase I/II study of azacitidine in Japanese patients with myelodysplastic syndromes to evaluate its pharmacokinetics, efficacy, and safety. In all, 53 patients received 75 mg/m(2) azacitidine subcutaneously or intravenously once daily for seven consecutive days on a 28-day cycle. The C(max) following intravenous administration was approximately 3.7-fold higher than that following subcutaneous administration, whereas the area under the plasma concentration-time curve from time zero to infinity was comparable for subcutaneous and intravenous administration. The bioavailability of azacitidine following subcutaneous administration was 91.1%, indicating that azacitidine is nearly completely absorbed after subcutaneous administration. The hematologic improvement and hematologic response rates were 54.9% (28/51) and 28.3% (15/53), respectively, and there were no differences between the two routes of administration. Azacitidine was generally well tolerated and clinically manageable in Japanese patients with myelodysplastic syndromes. Adverse events occurred in ≥ 20% of patients included hematologic toxicity, gastrointestinal events, and general disorders, such as malaise. Grade 3/4 adverse events that occurred in ≥ 50% of patients were all due to hematologic toxicity. The safety profile of azacitidine was generally similar for both routes of administration, with the exception of injection site reactions observed following subcutaneous administration. These results indicate that azacitidine can be expected to be a useful therapeutic agent in Japanese patients with myelodysplastic syndromes.     

A method for the combined measurement of ethiofos and WR-1065 in plasma: application to pharmacokinetic experiments with ethiofos and its metabolites

An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 micrograms/mL (0.75 to 186 mumol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120-150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 micrograms/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 micrograms/mL, 0.23 microM) 2-3 hours postinfusion. Clearances averaged 43.5 +/- 13.4 (SD) mL min-1 kg-1 and half-lives observed in the 20-60 minute postinfusion period were 8-15 min.     

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     

Inhibition of spontaneous metastases formation by amifostine.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo.     

Phase I trial of a twice-daily regimen of amifostine with ifosfamide, carboplatin, and etoposide chemotherapy in children with refractory carcinoma

BACKGROUND: Amifostine protects normal tissues against chemotherapy and radiation-induced toxicity without loss of antitumor effects. Evidence suggests that multiple daily doses of amifostine may improve its cytoprotective effects. The purpose of this study was to assess the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of twice-daily doses of amifostine with ifosfamide, carboplatin, and etoposide (ICE) chemotherapy for children with refractory malignancies and to determine the pharmacokinetic properties of amifostine, WR-1065, and the disulfide metabolites of amifostine. METHODS: Patients with refractory malignancies were treated with amifostine 15 minutes before and 2 hours after chemotherapy with ifosfamide (3 g/m(2) per dose on Days 1 and 2) and carboplatin (635 mg/m(2) on Day 3). Etoposide was administered on Days 1 and 2 (150 mg/m(2)). The starting dose of amifostine was 740 mg/m(2). Pharmacokinetic studies were performed after the first dose of amifostine. RESULTS: Twelve patients received 23 courses of ICE and amifostine. Dose-limiting toxicities for amifostine at 740 mg/m(2) were somnolence and anxiety. The other Grade 3 and 4 toxicities included asymptomatic, reversible hypocalcemia, vomiting, and reversible hypotension. At a dose of 600 mg/m(2), amifostine was well tolerated. Hypocalcemia, due to rapid, transient suppression of parathyroid hormone production, required close monitoring and aggressive intravenous calcium supplementation. Pharmacokinetic studies revealed high interpatient variability with rapid plasma clearance of amifostine and WR-1065. The median elimination half-life of amifostine (9.3 minutes) and WR-1065 (15 minutes) was much shorter than the disulfide metabolites (74.4 minutes). CONCLUSIONS: The recommended pediatric dose of amifostine for a twice-daily regimen is 600 mg/m(2) per dose (1200 mg/m(2)/day) with DLTs of anxiety and somnolence, lower than the previously recommended single dose of 1650 mg/m(2).     

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.     

Pharmacokinetics of WR-1065 in mouse tissue following treatment with WR-2721

Levels of reduced glutathione (GSH) and N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were measured in tissues of Balb/c mouse bearing EMT₆ tumors at time intervals ranging from 5 min to 48 hr after i.v. injection of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) at 500 mg per kg. In all tissues examined (liver, kidney, lung, heart, muscle, brain, tumor, spleen, and salivary gland), maximal WR-1065 levels occurred 5–15 min after injection, with levels in liver, kidney, lung, and salivary gland exceeding one μmole per gm. The post-maximum decline in WR-1065 varied markedly with tissue, lung exhibiting a 6-fold drop by 30 min and salivary gland falling only 1596 after 3 hr. In a mouse treated with carbon-14 labeled WR-2721 it was found after 15 min that WR-1065 accounted for over half of the total drug in all tissues except tumor, where it accounted for a third of the total drug. There was no evidence that GSH levels were substantially altered by WR-2721 treatment. The results provide the first direct evidence supporting the widely held view that WR-2721 treatment results in intracellular WR-1065 and they demonstrate that high levels of WR-1065 occur very soon after i.v. injection.     

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.     

Repression of c-myc gene expression by the thiol and disulfide forms of the cytoprotector amifostine

The clinically approved cytoprotector amifostine, designated WR-2721, [S-2-(3-aminopropylamino)ethylphosphorothioic acid], protects against both radiation and drug-induced mutagenesis in animal systems. These effects extend over a wide concentration range making amifostine a strong candidate for evaluation as a possible cancer chemopreventive agent. To better identify and develop potential intermediate biomarkers for chemoprevention at the molecular level we applied the technique of differential display RT-PCR to assess the effects of both the thiol (SH), i.e. WR1065 and the disulfide (SS), i.e. WR-33278, metabolites of amifostine on gene expression in CHO-AA8 cells. Cells were exposed to either 40 microM or 4 mM of each agent for 30 min, and subsequent changes in gene expression were identified and contrasted to that found in corresponding untreated control cells. One band that showed a differential response was sequenced and was found to have 78% homology with a segment of the human pHL-1 cDNA clone contained in GenBank. This clone contains a COX III mitochondrial DNA insert and two exons of human c-myc. Northern blot analyses were performed by using the cloned human c-myc exon 1 probe to confirm whether c-myc gene expression was affected. Repression of c-myc expression was observed under all of the conditions evaluated. An exposure of cells to 40 microM of the disulfide form of amifostine was the most effective in repressing c- myc, i.e. 27% of control level. A concentration of 4 mM of the disulfide form reduced gene expression to 45% of the control level, while the thiol form was less effective, with 4 mM and 40 microM concentrations reducing c-myc gene expression to 65% and 46% of control levels, respectively.      

The effects of cycloheximide and WR-1065 on radiation-induced repair processes: a mechanism for chemoprevention

The effects of cycloheximide (CHX) and 2-[(aminopropyl)-amino]ethanethiol(WR-1065), each alone or in combination, on radiation-inducedmutation induction at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus and cell killing were investigatedusing a Chinese hamster ovary (CHO) AA8 cell system. Treatmentwith CHX, a potent inhibitor of protein synthesis, at a concentrationof 10 µg/ml administered 30 min prior to irradiation with7.5 Gy had no effect on cell survival but did reduce the radiation-inducedmutation frequency (per 10⁶ survivors) from 106.5±8.8(SEM) to 36.2±5.6 (SEM). Exposure of cells to 4 mM WR-1065reduced the mutation frequency to 44.8±4.2 (SEM), butthe combination of agents afforded no additional protection,that is 41.1±3.3 (SEM). The mechanism of action attributedto CHX in reducing mutation frequency is its ability to preventthe induction of an error-prone repair system. Split-dose radiationexperiments, that is 8 Gy versus 4 Gy + 4 Gy separated by 3h, were performed to evaluate and contrast the relative abilitiesof CHX and WR-1065, each alone or in combination, in affectingcell survival. Cycloheximide administered to cells 30 min beforethe first radiation dose and present throughout the 3 h incubationtime prior to the second dose inhibited split-dose repair asevidenced by a reduction in surviving fraction by 60% as comparedwith the value obtained for non-CHX-treated cells that wereexposed to two equal doses of 4 Gy. Cells exposed to 4 mM WR-1065immediately following the first 4 Gy radiation dose and thenwashed free 2.5 h before exposure to a second 4 Gy dose, whichwas also followed by a 30 min exposure to WR-1065, increasedthe surviving fraction by 80% over the value obtained for cellsnot exposed to WR-1065 during their split-dose radiation treatment.When CHX treatment was combined with WR-1065 exposure, the protectiveeffect of WR-1065 was abolished, that is surviving cell fractionwas again reduced by 60% as compared with untreated controlgroups. These results indicate that protein synthesis is requiredfor WR-1065 to affect split-dose related repair processes. Presumably,the inhibition of the induction of an error-prone repair systemby CHX would account for its effects on both resultant decreasesin mutation frequency and cell survival. In contrast, WR-1065and/or its disulfide metabolite appear to facilitate the efficacyand fidelity of such a repair system once it is induced. Thisis most probably the result of their polyamine-like structuresand properties, which can influence the stability of DNA damagedsites as well as inhibit cell cycle progression, thus leadingto longer times available in which to complete repair beforedamage is fixed at cell division.