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.     

Tissue levels of WR-1065, the active metabolite of amifostine (Ethyol), are equivalent following intravenous or subcutaneous administration in cynomolgus monkeys

Amifostine (Ethyol) is a cytoprotective drug approved for the reduction of xerostomia in head and neck cancer when administered to patients receiving postoperative radiation therapy. Although amifostine is approved for intravenous infusion, the off-label subcutaneous route of administration has become more prevalent. Although human patient data indicate higher plasma bioavailability of the active metabolite (WR-1065) following intravenous compared to subcutaneous administration, there are no corresponding data showing human tissue levels of WR-1065 following either route of administration due to the difficulty in obtaining human specimens. In our study we compared plasma and tissue pharmacokinetics of WR-1065 in primates following both routes of administration. Monkeys received amifostine at a dose of 260 mg/m2 either intravenously or subcutaneously. Plasma samples were analyzed for total WR-1065 by reverse-phase high-pressure liquid chromatography (HPLC) and fluorescence detection up to 4 h after amifostine administration. Tissues were analyzed for free WR-1065 by reverse-phase HPLC and electrochemical detection 30 and 60 min after administration. Following intravenous administration, plasma WR-1065 levels peaked rapidly and showed a bi-exponential decline, while following subcutaneous administration WR-1065 levels rose slowly and declined exponentially. The relative plasma bioavailability of WR-1065 given subcutaneously was lower at 30 and 60 min. Interestingly, after 30 min, tissues showed equal or slightly greater concentrations of WR-1065 following subcutaneous administration. Levels following 60 min were comparable following both routes. The plasma bioavailability studies performed in primates confirm human plasma data. Expanding the study to evaluate primate tissue levels of WR-1065 revealed that despite lower plasma bioavailability following subcutaneous administration, tissue levels of the active metabolite were surprisingly greater than or equal to those measured in animals that received the drug intravenously. These studies strengthen the argument for subcutaneous administration of amifostine in radiation oncology.     

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.     

Selective exclusion by the polyamine transporter as a mechanism for differential radioprotection of amifostine derivatives.

Amifostine metabolites WR-1065 and the disulfide WR-33278 are thiol-containing polyamine analogues with potent radio- and chemoprotective properties. Some studies suggest that amifostine exerts differential cytoprotection in normal versus neoplastic tissues, but this finding remains controversial. To assess the role of the polyamine transport system in radioprotection by amifostine derivatives, human DU-145 prostate cancer cells were transfected with a cDNA that encodes antizyme (OAZ), a polyamine-inducible protein that suppresses polyamine transport under control of a minimal heat shock promoter. Selected clones expressing OAZ displayed heat shock-dependent suppression of polyamine uptake. When added to culture medium under nonreducing conditions, both WR-1065 and WR-33278 were detected as the disulfide form. Each derivative protected both parental and OAZ-transfected DU-145 cells from X-ray-induced cell killing at 37 degrees C. When cultures were heat shocked at 42 degrees C, both derivatives protected parental, but not OAZ-transfected cells from radiation-induced cell killing. Treatment of DU-145 cells with difluoromethylornithine (DFMO) suppressed intracellular putrescine and spermidine content, but increased the uptake of WR-33278-derived aminothiols. The concentration-dependent radioprotection of DU-145 cells by WR-33278 was enhanced by DFMO. Addition of exogenous putrescine reduced WR-33278-mediated radioprotection in both DFMO-treated and untreated DU-145 cells. These data demonstrate that negative regulation of the polyamine transporter, mediated by polyamines or antizyme, suppresses the uptake and radioprotective activity of amifostine derivatives. Selective exclusion of amifostine derivatives by the polyamine transporter could account for differential radio- or chemoprotection in normal versus neoplastic tissues in specific situations.     

Further evidence that the radioprotective aminothiol, WR-1065, catalytically inactivates mammalian topoisomerase II

It has recently been proposed that the thiol form of the cytoprotective drug amifostine that is designated WR-1065 [2-((aminopropyl)amino)ethanethiol] exerts its cytoprotective effects in part via a catalytic inhibition of DNA topoisomerase II (topo II)alpha. This in turn leads to the subsequent accumulation of cells in G2 phase and a prolongation of the cell cycle. We have used a Chinese hamster V79 cell-based micronucleus assay to further evaluate this hypothesis. It is demonstrated that WR-1065 strongly inhibits the clastogenesis of the topo II poisons etoposide and clinafloxacin at clinically attained exposure levels while having no effect on clastogenesis induced by topo II-noninteractive chemicals. These findings are consistent with the hypothesis that WR-1065 is a catalytic inhibitor of topo II in mammalian cells. These studies also suggest that WR-1065 might be expected to reduce the toxicity and clastogenicity in clinical applications of etoposide or quinolone antibiotics in dose-limiting normal tissues.     

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).     

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.     

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.      

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.     

Phase III randomized trial of amifostine as a radioprotector in head and neck cancer.

PURPOSE: Radiotherapy for head and neck cancer causes acute and chronic xerostomia and acute mucositis. Amifositine and its active metabolite, WR-1065, accumulate with high concentrations in the salivary glands. This randomized trial evaluated whether amifostine could ameliorate these side effects without compromising the effectiveness of radiotherapy in these patients. PATIENTS AND METHODS: Patients with previously untreated head and neck squamous cell carcinoma were eligible. Primary end points included the incidence of grade > or =2 acute xerostomia, grade > or =3 acute mucositis, and grade > or =2 late xerostomia and were based on the worst toxicity reported. Amifostine was administered (200 mg/m(2) intravenous) daily 15 to 30 minutes before irradiation. Radiotherapy was given once daily (1.8 to 2.0 Gy) to doses of 50 to 70 Gy. Whole saliva production was quantitated preradiotherapy and regularly during follow-up. Patients evaluated their symptoms through a questionnaire during and after treatment. Local-regional control was the primary antitumor efficacy end point. RESULTS: Nausea, vomiting, hypotension, and allergic reactions were the most common side effects. Fifty-three percent of the patients receiving amifostine had at least one episode of nausea and/or vomiting, but it only occurred with 233 (5%) of 4,314 doses. Amifostine reduced grade > or =2 acute xerostomia from 78% to 51% (P<.0001) and chronic xerostomia grade > or = 2 from 57% to 34% (P=.002). Median saliva production was greater with amifostine (0.26 g v 0.10 g, P=.04). Amifostine did not reduce mucositis. With and without amifostine, 2-year local-regional control, disease-free survival, and overall survival were 58% versus 63%, 53% versus 57%, and 71% versus 66%, respectively. CONCLUSION: Amifostine reduced acute and chronic xerostomia. Antitumor treatment efficacy was preserved.     

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.     

Carboplatin combined with amifostine, a bone marrow protectant, in the treatment of non-small-cell lung cancer: a randomised phase II study.

Amifostine (WR-2721), a thiol compound, has been shown to protect normal tissue from alkylating agents and cisplatin-induced toxicity without loss of anti-tumour effects. To confirm this result, we conducted a phase II randomised trial to determine if the addition of amifostine reduces the toxicity of carboplatin without loss of anti-tumour activity in patients with inoperable non-small-cell lung cancer (NSCLC). After the first course of carboplatin (600 mg m-2 i.v. infusion), 21 patients were randomised to receive three cycles of carboplatin alone (C arm) or three infusions of amifostine at 910 mg m-2 (CA arm) at 28 day intervals. The amifostine was given 20 min before and at 2 and 4 h after carboplatin. Since the 910 mg m-2 amifostine infusion led to hypotension in six patients, the dosage was reduced by 25%, to 683 mg m-2 t.i.d., in the other four patients. Amifostine was well tolerated at this dose level. Five patients in the CA arm and three in the C arm had their planned treatment discontinued owing to progressive disease (n = 3), amifostine side-effects (hypotension, sneezing and sickness, n = 4), and carboplatin-induced thrombocytopenia (n = 1). Bone marrow and renal function at study entry and after the first course of carboplatin before randomisation were similar in both treatment arms. Twenty courses of carboplatin+amifostine have been compared with 25 courses of carboplatin alone. Although there was no statistically significant difference with respect to haematological values comparing both arms, the median time to platelet recovery (> 100 x 10(9) l-1) (13.5 days vs 21 days; P = 0.04) and the need for hospitalisation for i.v. antibiotic and other supportive treatment tended to be reduced in the CA arm (0/20 vs 6/25 patient courses; P = 0.06). Response rates and median survival (14 vs 9 months) were no different, excluding tumour protection activity by amifostine. These results with a small number of patients suggest that amifostine given with carboplatin may reduce the duration of thrombocytopenia and hospitalisation.     

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.     

In Vitro Protection from Cisplatin-induced Neurotoxicity by Amifostine and its Metabolite WR1065

Cisplatin-induced neuropathy is a major dose-limiting toxicity. Counteraction by amifostine and its metabolite WR1065 may reduce peripheral neurotoxicity in a number of patients. Using the nerve growth factor (NGF)-dependent neurite outgrowth from the PC12 pheochromocytoma cell line as an in vitro assay for neurotoxicity, the protective effects of amifostine and WR1065 against cisplatin action on neurite formation by PC12 cells were studied.Cisplatin in a concentration of 10 µg/ml significantly decreased the percentage of neurite forming cells from 84% to 40%. Amifostine in doses of 0.4 and 0.8 mM proved to protect significantly against the cisplatin-induced decrease in neurite formation, when co-incubated with cisplatin. Also the metabolite WR1065 protected significantly against cisplatin neurotoxicity in a dose of 0.12 mM. Our results show a significant protection by amifostine and its main metabolite WR1065 against cisplatin-induced neurotoxicity using an in vitro model.     

Uptake of WR-2721 derivatives by cells in culture: identification of the transported form of the drug

When V79-171 cells are incubated in medium to which WR-1065 has been added the cells accumulate WR-1065 and disulfides of WR-1065 (WRSS) in a ratio of about 10:1. Analysis of the culture medium showed that it contained primarily WR-1065 but that significant levels of the symmetrical disulfide WR-33278 and of the mixed disulfide of WR-1065 with cysteine were also present. Since incubation of cells with either of the latter disulfides did not lead to uptake it was concluded that WR-1065 is the form of the drug taken up. The uptake rate on a per cell basis was shown to be independent of cell density, to be first order in the WR-1065 concentration in the incubation medium, to increase as [H+]-1.2 at medium pH values from pH 6.8 to 8.0, and to have a Q10 value (rate increase per 10 degrees C temperature increase) of 2.9 +/- 0.3 between 2 and 37 degrees C. Rates of WR-1065 uptake measured for HeLa, HT29/SP-1d, Me-180-VCII, Ovary 2008, and WI-38 cell lines were found to be similar to that measured for V79-171 cells. The results are consistent with uptake by nonmediated, passive diffusion of the uncharged form of WR-1065 across the plasma membrane but uptake mediated by a membrane transport system could not be rigorously excluded. Based upon these results and earlier findings it is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of (a) slower conversion of WR-2721 to WR-1065 in tumors as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and (b) a decreased uptake rate of the WR-1065 present in tumors as a consequence of their lower pH.     

Amifostine Does Not Inhibit the Toxic Effects of Anthracycline Derivates or Mitoxantrone on MDR Tumor Cell Lines

In this work three hum cell lines with multidrug resistance (MDR) caused by a P-glycoprotein (PGP) overexpression, CEM VLB, HL60 DNR, LOVO DX and two cell lines with MDR associated with a multidrug related protein (MRP) or a lung resistance-related protein (LRP) overexpression named GLC4 ADR and SW1573/2R120 were tested for Amifostine protection against Daunorubicin, Doxorubicin, Idarubicin and Mitoxantrone toxicity. This class of anticancer agents was chosen because they are commonly used in the first line treatments of acute leukemias where a PGP, an LRP or an MRP overexpression often occurs even at onset. A 7-day incubation with escalating doses of anticancer agents with or without a 15 minute preincubation in Amifostine or its active metabolite WR-1065 were used. In conclusion, in none of the MDR positive and negative cell lines did Amifostine modify the toxicity of the anticancer drugs. The observation that even the WR-1065 metabolite gave no protection against Anthracyclines toxicity strengthened the data and provided confirmation for the further in vivo testing of the safety and efficacy of Amifostine in leukemias.     

Extracellular: intracellular and subcellular concentration gradients of thiols.

Chinese hamster V79 cells in Eagle's minimum essential medium in vitro at room temperature were incubated with the aminothiol, WR-1065, or glutathione (GSH) at extracellular concentrations of approximately 1 mmol dm-3. Average intracellular concentrations of GSH, cysteine, and WR-1065 were measured by high performance liquid chromatography, and the effective reducing environment near DNA probed by staining the cells with acridine orange (AO) and measuring the delayed fluorescence. Exposure to either thiol resulted in a rapid, 10-fold increase in average intracellular cysteine concentrations (to about 1 mmol dm-3). Adding extracellular GSH after prior depletion of GSH by treatment with L-buthionine sulfoximine (BSO) did not restore intracellular GSH, but intracellular cysteine was elevated 10-fold. These results are ascribed to thiol/disulfide exchange with cystine in the medium. WR-1065 slowly concentrated intracellularly to approximately 160% of the extracellular concentration. Chemical conjugation of GSH in cells decreased the reducing environment near DNA, but BSO treatment altered the uptake of AO. The electrostatic attraction of WR-1065 toward isolated DNA was markedly affected by ionic strength.