Ginkgo biloba extract attenuates cisplatin-induced renal interstitial fibrosis by inhibiting the activation of renal fibroblasts through down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells.

BACKGROUND: Activation of renal fibroblasts into myofibroblasts plays an important role in promoting renal interstitial fibrosis (RIF). Ginkgo biloba extract (EGb) can alleviate RIF induced by cisplatin (CDDP). PURPOSE: To elucidate the effect of EGb treatment on cisplatin-induced RIF and reveal its potential mechanism. METHODS: The two main active components in EGb were determined by high-performance liquid chromatography (HPLC) analysis. Rats were induced by CDDP and then treated with EGb, 2ME2 (HIF-1α inhibitor) or amifostine. After HK-2 cells and HIF-1α siRNA HK-2 cells were treated with CDDP, EGb or amifostine, the conditioned medium from each group was cultured with NRK-49F cells. The renal function of rats was detected. The renal damage and fibrosis were evaluated by H&E and Masson trichrome staining. The IL-6 content in the cell medium was detected by ELISA. The expression levels of indicators related to renal fibrosis and signaling pathway were examined by western blotting and qRT-PCR. RESULTS: HPLC analysis showed that the contents of quercetin and kaempferol in EGb were 36.0 µg/ml and 45.7 µg/ml, respectively. In vivo, EGb and 2ME2 alleviated renal damage and fibrosis, as well as significantly decreased the levels of α-SMA, HIF-1α, STAT3 and IL-6 in rat tissues induced by CDDP. In vitro, the levels of HIF-1α, STAT3 and IL-6 were significantly increased in HK-2 cells and HIF-1α siRNA HK-2 cells induced by CDDP. Notably, HIF-1α siRNA significantly decreased the levels of HIF-1α, STAT3 and IL-6 in HK-2 cells, as well as the IL-6 level in medium from HK-2 cells. Additionally, the α-SMA level in NRK-49F cells was significantly increased after being cultured with conditioned medium from HK-2 cells or HIF-1α siRNA HK-2 cells exposed to CDDP. Furthermore, exogenous IL-6 increased the α-SMA level in NRK-49F cells. Importantly, the expression levels of the above-mentioned indicators were significantly decreased after the HK-2 cells and HIF-1α siRNA HK-2 cells were treated with EGb. CONCLUSION: This study revealed that EGb improves CDDP-induced RIF, and the mechanism may be related to its inhibition of the renal fibroblast activation by down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells.

Enteral Activation of WR-2721 Mediates Radioprotection and Improved Survival from Lethal Fractionated Radiation.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Bioprinting cell-laden matrigel for radioprotection study of liver by pro-drug conversion in a dual-tissue microfluidic chip.

The objective of this paper is to introduce a novel cell printing and microfluidic system to serve as a portable ground model for the study of drug conversion and radiation protection of living liver tissue analogs. The system is applied to study behavior in ground models of space stress, particularly radiation. A microfluidic environment is engineered by two cell types to prepare an improved higher fidelity in vitro micro-liver tissue analog. Cell-laden Matrigel printing and microfluidic chips were used to test radiation shielding to liver cells by the pro-drug amifostine. In this work, the sealed microfluidic chip regulates three variables of interest: radiation exposure, anti-radiation drug treatment and single- or dual-tissue culture environments. This application is intended to obtain a scientific understanding of the response of the multi-cellular biological system for long-term manned space exploration, disease models and biosensors.

Radioprotection of craniofacial bone growth.

In this review, the potential of pharmacologic therapy for prevention of radiation-induced bone growth inhibition is discussed. Significant radioprotection using the radioprotector Amifostine has been achieved in animal models of radiation-induced retardation of long and craniofacial bone growth. Moreover, radioprotection in vitro has been achieved in a number of cell lines, including osteoblast-like, endothelial, and fibroblastic. This evidence may support future clinical investigations of radioprotector Amifostine or similar substances for radioprotection of the growing craniofacial skeleton.

Amifostine protects rat kidneys during peptide receptor radionuclide therapy with [177Lu-DOTA0,Tyr3]octreotate.

PURPOSE: In peptide receptor radionuclide therapy (PRRT) using radiolabelled somatostatin analogues, the kidneys are the major dose-limiting organs, because of tubular reabsorption and retention of radioactivity. Preventing renal uptake or toxicity will allow for higher tumour radiation doses. We tested the cytoprotective drug amifostine, which selectively protects healthy tissue during chemo- and radiotherapy, for its renoprotective capacities after PRRT with high-dose [(177)Lu-DOTA(0),Tyr(3)]octreotate. METHODS: Male Lewis rats were injected with 278 or 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate to create renal damage and were followed up for 130 days. For renoprotection, rats received either amifostine or co-injection with lysine. Kidneys, blood and urine were collected for toxicity measurements. At 130 days after PRRT, a single-photon emission computed tomography (SPECT) scan was performed to quantify tubular uptake of (99m)Tc-dimercaptosuccinic acid (DMSA), a measure of tubular function. RESULTS: Treatment with 555 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate resulted in body weight loss, elevated creatinine and proteinuria. Amifostine and lysine treatment significantly prevented this rise in creatinine and the level of proteinuria, but did not improve the histological damage. In contrast, after 278 MBq [(177)Lu-DOTA(0),Tyr(3)]octreotate, creatinine values were slightly, but not significantly, elevated compared with the control rats. Proteinuria and histological damage were different from controls and were significantly improved by amifostine treatment. Quantification of (99m)Tc-DMSA SPECT scintigrams at 130 days after [(177)Lu-DOTA(0),Tyr(3)]octreotate therapy correlated well with 1/creatinine (r(2)=0.772, p<0.001). CONCLUSION: Amifostine and lysine effectively decreased functional renal damage caused by high-dose [(177)Lu-DOTA(0),Tyr(3)]octreotate. Besides lysine, amifostine might be used in clinical PRRT as well as to maximise anti-tumour efficacy.

Determination of WR-1065 in human blood by high-performance liquid chromatography following fluorescent derivatization by a maleimide reagent ThioGlo3.

In order to improve the sensitivity and stability of human blood samples containing WR-1065 (i.e., active metabolite of the cytoprotective agent amifostine), a high-performance liquid chromatographic method was developed and validated using fluorescent derivatization with ThioGlo3. Using a sample volume of only 100 microl, the method was specific, sensitive (limit of quantitation=10 nM in deproteinized blood or 20 nM in whole blood), accurate (error < or = 3.2%) and reproducible (CV < or = 8.7%). In addition, the stability of WR-1065 in deproteinized and derivatized blood samples was assured for at least four weeks at -20 degrees C. This method should be particularly valuable in translating the kinetic-dynamic relationship of WR-1065 in preclinical models to that in cancer patients.

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.

Amifostine protection against doxorubicin cardiotoxicity in rats.

Aminothiol amifostine (AMI) protects against toxic effects of both ionizing radiation and numerous anticancer drugs. The aim of this study was to investigate the potential protective effects of AMI against doxorubicin (DOX)-induced cardiotoxicity in rats. Male Wistar rats were treated with AMI (75 mg/kg i.p.) and/or DOX (1.25 mg/kg i.p.), 4 times per week, for 4 weeks. Mortality, general condition and body weight of the animals were observed during the whole treatment, and for a further 4 weeks, until the end of experiment. Evaluation of cardioprotective efficacy of AMI was performed by analyzing the electrocardiographic parameters and response to the pro-arrhythmic agent aconitine, as well as activity registration of the in situ rat heart preparations. Necropsy was also performed at the end of the experiment, and heart excision, weight and macroscopic examination were done before histological evaluation. Doxorubicin caused rat heart disturbances manifested by prominent electrocardiographic changes (Salpha-T prolongation and T-wave flattening), significantly enhanced response to aconitine, decrease of the heart rate and contractility, as well as histopathologically verified myocardial lesions. The heart changes were accompanied by 40% mortality rate, significant decline in body mass and severe effusion intensity score in 66.6% of the animals. Application of AMI before each dose of DOX significantly reduced or completely prevented its toxic effects. Therefore, since AMI had very good protective effects against a high dose of DOX given as a multiple, low, unitary dose regimen, not only on the heart but on the whole rat as well, it could be recommended for further investigation in this potentially new indication for clinical application.

Disposition of WR-1065 in the liver of tumor-bearing rats following regional vs systemic administration of amifostine.

PURPOSE: Amifostine is a prodrug in which selectivity is largely determined by the preferential formation and uptake of its cytoprotective metabolite, WR-1065, in normal tissues as a result of differences in membrane-bound alkaline phosphatase activity. It was hypothesized that amifostine may be a good candidate for regional drug delivery to the liver because of its large hepatic extraction and total body clearance. METHODS: Rat livers were implanted with Walker-256 tumors. The tumor-bearing rats received 15 min infusions of amifostine (200 mg/kg) via the portal vein or the femoral vein. WR-1065 concentrations in the blood, liver and tumor were measured at various times. RESULTS: The WR-1065 tumor portal dosing AUC15-60 was 40% of systemic dosing, and tumor concentrations following portal dosing were one-fifth of that following systemic dosing. The portal dosing WR-1065 liver AUC15-60 was 60% higher than the values for systemic dosing. The liver/tumor concentration ratios of WR-1065 following portal dosing were up to 8-fold higher than the ratio following systemic administration. Unfortunately, systemic exposure to WR-1065 was greater following portal vs systemic amifostine. CONCLUSIONS: Amifostine may provide increased liver protection and decreased tumor protection from radio- or chemotherapy when administered by the portal vein. However, portal dosing also increases systemic exposure to WR-1065, which is associated with hypotension.

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.

Amifostine has antiangiogenic properties in vitro by changing the redox status of human endothelial cells.

Amifostine is a broad-spectrum cytoprotective agent, selective for normal tissues. It is a pro-drug metabolised to the free thiol WR-1065 that may act as a scavenger of free radicals, generated in tissues exposed to chemotherapeutic agents or radiation. WR-1065 can be further oxidized to its symmetric disulfide WR-33278 or degraded to hydrogen peroxide (H2O2). Both WR-1065 and WR-33278 resemble endogenous polyamines. Although amifostine is used in some cases in the clinic, there are only few studies concerning its actions at the cellular level. We have previously shown that amifostine inhibits angiogenesis in vivo, affecting the expression of several angiogenic genes. In the present work, we studied the effect of amifostine on human umbilical vein endothelial cell (HUVEC) functions in vitro, in order to further clarify its mechanism(s) of action. Amifostine increased HUVEC proliferation, an effect that was reversed by the intracellular H2O2 scavenger sodium pyruvate, agents that increase intracellular cAMP levels and L-valine. On the other hand, amifostine decreased HUVEC migration, an effect that was reversed by L-valine or L-arginine but not sodium pyrouvate. The decrease in migration was in line with decreased tube formation on matrigel and decreased amounts of metalloproteinase-2 released into the culture medium of HUVEC. Finally, amifostine reduced tyrosine nitration of the cytoskeletal proteins actin and alpha-tubulin in a time dependent manner. This last action could be due to the reduced production of nitric oxide (NO) or to other not yet identified mechanisms. Collectively, our results suggest that amifostine acts on endothelial cells through pathways that affect the redox status of the cells, either by producing H2O2 or by modulating NO production.

New liquid chromatographic assay with electrochemical detection for the measurement of amifostine and WR1065.

A high-performance liquid chromatographic method (HPLC) was developed for the analysis of the radio- and chemo-protectant, amifostine and its active metabolite-WR1065 in deproteinized human whole blood and plasma. The two compounds were quantified by measuring WR1065 after two different sample pretreatment procedures. During these procedures, amifostine was quantitatively converted into WR1065, by incubating the sample at 37 degrees C for 4 h at pH<1.0. The resulting amounts of WR1065 were determined by HPLC with coulometric detection (analytical cell: E(1)=200 mV and E(2)=600 mV; guard cell: E(G)=650 mV). The WR1065 standard curve ranged from 0.37 to 50.37 microM. The lower limit of quantitation of WR1065 was 0.25 microM. The within- and between-day precisions were < or = 4.3% and < or = 6.0% for amifostine, < or = 4.4% and < or = 3.8% for WR1065, respectively. The within- and between-day accuracy ranged from 95.4 to 97.7% and 95.4 to 97.8% for amifostine, and from 97.1 to 101.7% and 97.2 to 99.7% for WR1065, respectively. This method minimizes WR1065 loss during sample preparation, and allows for rapid analysis of both compounds on one system. Furthermore, the application of a coulometric electrode is more efficient and requires less maintenance than previously published methods for the two compounds.

Preclinical studies on the radioprotective efficacy and pharmacokinetics of subcutaneously administered amifostine.

The radioprotective effects and pharmacokinetics of subcutaneously (SC) administered amifostine have been investigated in animal studies. Studies in rats using a single dose of amifostine showed that SC administration gave protection from radiation-induced mucositis that is at least equivalent to that achieved by intravenous administration of the drug. These studies also indicate that tissue levels of the active metabolite WR-1065 correlated better with the radioprotective effects of amifostine than do plasma WR-1065 levels. Multiple-dose studies in rats show radioprotective effects equal to or greater than those obtained with intravenous dosing in the setting of fractionated irradiation. In addition, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. Preliminary data from studies of SC amifostine in monkeys indicate a plasma pharmacokinetic profile similar to that reported earlier in humans. Tissue WR-1065 levels were higher at 30 minutes after SC dosing than they were after intravenous dosing and were comparable for the two routes at 60 minutes.

Down-regulation of intestinal-type alkaline phosphatase in the tumor vasculature and stroma provides a strong basis for explaining amifostine selectivity.

Strong clinical and experimental evidence indicates that amifostine confers cytoprotection in normal, but not in tumor, tissues. However, the mechanism of such selective action is poorly understood. Intestinal-type alkaline phosphatase (IAP) is a major isoenzyme involved in the hydrolysis of amifostine (WR-2721) to its active thiol form WR-I065. Could differences in IAP expression between normal and malignant tissues account for amifostine's selectivity? Paraffin-embedded material from normal breast, lung, colon, and head and neck tissues, together with their malignant counterparts, were retrieved and stained immunohistochemically for human IAP (antibody 7324, Abcam, Cambridge, UK) and endothelial cell CD31 antigen. Normal tissues (epithelium, fibroblasts, and vessels) consistently displayed strong nuclear and cytoplasmic IAP reactivity. The vascular density (number of positive vessels per x 200 optical field), whether assessed in anti-IAP or anti-CD31 stained sections, was very similar, indicating a strong IAP content for the entire normal vasculature. Therefore, amifostine hydrolysis is ensured in normal tissues and may occur at both vascular and interstitial levels. By contrast, 60% of the tumors analyzed showed a loss of IAP expression in both epithelial cells and stroma, and only 10% to 15% of them demonstrated nuclear/cytoplasmic reactivity, which was confined to the epithelial cells. Similarly, the percentage of tumor vessels exhibiting some IAP reactivity was very low (6% to 17%). This dramatic loss of IAP expression from tumor stroma/vasculature may form a strong basis for explaining amifostine selectivity. In contrast, the abundance of IAP expression in normal tissues, stromal and vascular, ensures an intense hydrolysis of WR-2721 and rapid intracellular accumulation of WR-1065.

Blood thiols following amifostine and mesna infusions, a pediatric oncology group study.

The Pediatric Oncology Group study for metastatic Ewing's sarcoma used amifostine and mesna with the alkylating agents. To determine the fate of combined drug thiols, we measured thiol levels in plasma, red blood cells (RBC), and peripheral blood mononuclear cells (PBMC) of four patients. We also conducted analogous measurements on two patients who received mesna alone and a volunteer's blood following in vitro treatment. Thiols were labeled with monobromobimane, separated on high-pressure liquid chromatography, and detected by fluorescence. Incubation of a volunteer's blood with mesna, WR-1065, or both revealed that cellular uptake of total reducible drug was approximately 10% of plasma level for mesna but approximately 60% for WR-1065. Cellular drugs were mainly the thiol form, whereas half of the plasma drugs were disulfides. Combined incubation with both thiols did not change the extent or form of uptake. WR-1065 and mesna prevented glutathione depletion by 4-hydroperoxycyclophosphamide. Results from patients were similar. WR-1065 and mesna appeared in the cells by the end of the drug infusions, although WR-1065 uptake was more efficient than mesna. The concentration-time profiles of mesna in RBC paralleled those in plasma. Amifostine administration during mesna infusion caused transient increase in mesna levels. Both agents increased blood cysteine and decreased total reducible cysteine. Mesna alone and mesna plus amifostine prevented cellular glutathione depletion. In conclusion, mesna is imported by RBC and PBMC, but less efficiently than WR-1065. When present at equal levels, these thiols do not influence each other's uptake. Adequate dosing of either drug is necessary for protecting the cells from toxic effects of alkylating agents.

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).

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography.

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 micromol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).

A risk-benefit assessment of amifostine in cytoprotection.

Recent advances in chemotherapy have focused on the benefit of high dose regimens, increasing the dose intensity of conventional chemotherapy and using intensified chemotherapy with or without autologous bone marrow rescue. Dose intensity usually increases objective response rates of antineoplastic drugs and might, in some circumstances, improves survival. However, unacceptable acute and/or cumulative toxicity often impairs the proper management of patients, leading to dose reduction or treatment delay, thus reducing the efficacy and potentially the quality of life of patients. Therefore, considerable efforts have been made to manage, to prevent, and to delay many acute and cumulative treatment-related toxicities. Amifostine (WR-2721 ) is a multiorgan cytoprotector which has demonstrated cytoprotective effects, in vitro and in vivo, against the most common cytotoxic drug-related toxicities and against radiation-induced adverse effects in healthy tissues. In vitro and in vivo, cytoprotection was observed in several organs including kidney, haematopoietic stem cells, myocardial cells, neural cells, and mucosa, without detectable protection of malignant cells. In addition, in preclinical studies, amifostine appeared to be able to reduce the risk of radiation-induced secondary neoplasms. Phase I studies showed that nausea/vomiting and hypotension are the dose-limiting toxicities of amifostine and these may be controlled by reducing the duration of injection of amifostine. Phase II and randomised studies have confirmed the efficacy of amifostine in protecting against radiotherapy-induced mucositis, cisplatin-induced nephrotoxicity, cyclophosphamide-induced neutropenia and carboplatin-induced thrombocytopenia. Importantly, the cytoprotection of healthy tissues occurred without any significant deleterious effect on response rate, time to progression, and survival of patients receiving amifostine. However, in addition to the potential quality of life benefit, the most important question of whether the use of a cytoprotective agent might translate into the possibility of maintaining the dose intensity of anticancer therapies has still to be answered. The real benefit of amifostine in the overall management of patients with cancer requires additional studies to determine whether this chemoprotective approach can be of benefit to patients by increasing response rate, time to progression, and long term survival in patients receiving the more recent combination therapies involving new drugs such as the taxanes and oxaliplatin.

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

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