Determination of alkaline phosphatase activity based on enzyme-triggered generation of a thiol and the fluorescence quenching of silver nanoclusters.

A fluorimetric method is described for the determination of alkaline phosphatase (ALP) activity. It is based on the use of polyethyleneimine-coated silver nanoclusters (AgNCs), which display an intense blue fluorescence peaking at 450 nm (under 375 nm excitation). ALP catalyzes the dephosphorylation of the thiophosphate amifostine to generate a thiol that binds to the AgNCs and causes its fluorescence to be quenched. Under the optimal experimental conditions, fluorescence linearly drops in the 0.08-2.0 U L-1 ALP activity range, and the limit of detection is 0.02 U L-1. The method was successfully applied to the determination of ALP activity in spiked human serum samples. Graphical abstract Alkaline phosphatase (ALP) catalyzes the degradation of amifostine with a generation a thiol product. The thiol quenches the fluorescence of silver nanoclusters, and a method for the detection of ALP down to 0.02 U L-1 was developed.

Monoterpene's multiple free radical scavenging capacity as compared with the radioprotective agent cysteamine and amifostine.

Monoterpenes are major active components of lavender, thyme, and mint. The X-ray radioprotective activity of pure monoterpenes is attributed to their scavenging ability against active species, but so far no firm evidence has been demonstrated. The objective of this study is to quantitatively determine antioxidant abilities of monoterpenes and collate it with radioprotective activity. Using multiple free-radical scavenging (MULTIS) method, we have determined the scavenging abilities of monoterpenes (linalool, thymol, and menthol) against six active species. A previous study has shown that the monoterpene linalool is a radioprotector for cellular systems, therefore, its scavenging ability was compared with known radioprotective agents such as cysteamine and amifostine. Results indicated that the monoterpene menthol but not linalool is a potent scavenger of reactive oxygen species and its scavenging magnitude is comparable to cysteamine and amifostine. This paper is first to show a correlation between ROS scavenging ability and radioprotective action.

Binding of amifostine to human serum albumin: a biophysical study.

The aim of this present work is to investigate the interaction between amifostine and human serum albumin (HSA) in simulated physiological conditions by spectroscopic methods to reveal potential toxic effects of the drug. The results reflected that amifostine caused fluorescence quenching of HSA through a static quenching process, which was further confirmed by the electrochemical experiments. The binding constants at 290, 297 and 304 K were obtained as 2.53 × 10(5) /M, 8.13 × 10(4) /M and 3.59 × 10(4) /M, respectively. There may be one binding site of amifostine on HSA. The thermodynamic parameters indicated that the interaction between amifostine and HSA was driven mainly by hydrogen bonding and electrostatic forces. Synchronous fluorescence spectra, circular dichroism and Fourier transform infrared spectroscopy results showed amifostine binding slightly changed the conformation of HSA with secondary structural content changes. Förster resonance energy transfer study revealed high possibility of energy transfer with amifostine-Trp-214 distance of 3.48 nm. The results of the present study may provide valuable information for studying the distribution, toxicological and pharmacological mechanisms of amifostine in vivo.

Radioprotectors - the evergreen topic.

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation-induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical-scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR-2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical-scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.

Preparation of amifostine polylactide-co-glycolide microspheres and its irradiation protective to mouse through oral administration.

The objectives of this study were to prepare the amifostine polylactide-co-glycolide (PLGA) microsphere and investigate its irradiation protective to mouse through oral administration. Amifostine-loaded PLGA microsphere was formulated using a modified double emulsion-solvent evaporation technique. The microsphere particle was spherical with a mean diameter of 2.8 ± 0.1 µm. Release data of amifostine PLGA microsphere was tested in phosphate-buffered saline at 37°C using a dialysis method and its release profiles was biphasic, showing a relatively large burst effect (50%) over the first 6 h, followed by a slower release phase, which sustained with 80% amifostine released in 48 h and almost 100% release till 6 days (144 h). A diffusion-controlled release model (Higuchi equation, R² = 0.9725) was obtained for amifostine releasing from PLGA microsphere. The radiation experiment was performed by applied cobalt-60 γ-radiation source. One hour before γ-radiation exposure, the mouse was orally given free amifostine and PLGA microsphere, respectively. The irradiation effects, such as blood cell concentration, superoxidase dismutase (SOD) activity and malondialdehyde (MDA) level were monitored. The results indicated that amifostine PLGA microsphere was more irradiation protective to mouse than that of free amifostine under the same oral administration route.

Drde-07: a possible antidote for sulphur mustard toxicity.

Chemical Warfare Agents are classified in various categories and vesicating agents are one among them. Vesicating agents are mostly mustard agents. Sulphur mustard which is chemically known as bis(2-chloro ethyl) sulphide (SM), was first used in World War-I and in recent past in Iran-Iraq war. Its possible use by the terrorist groups can't be overlooked in the present scenario. As the mode of its action is still lacking, no specific treatment is so far known against SM induced systemic toxicity. The major drawback with the development of antidote against sulphur mustard is low efficacy of the potential compounds in vivo models. This review summarizes the current update about the work done so far and the future strategies.

Comparative toxic effect of nitrogen mustards (HN-1, HN-2, and HN-3) and sulfur mustard on hematological and biochemical variables and their protection by DRDE-07 and its analogues.

The chemical warfare agents sulfur mustard (SM) and nitrogen mustards (HN-1, HN-2, and HN-3) are highly reactive vesicants. The study was planned to investigate the protective efficacy of amifostine, DRDE-07 and their analogues, and few conventional antidotes (30 minutes pretreatment) against dermally applied SM and nitrogen mustards in preventing hematological and biochemical changes in mice. Mustard agents (1.0 median lethal dose [LD(50)]) induced a significant decrease in the body weight and spleen weight. A significant decrease in the white blood cell (WBC) count and an increase in serum transaminases and alkaline phosphatases (ALPs) were observed. A significant decrease in reduced (GSH) and oxidized glutathione (GSSG) and an increase in thiobarbituric acid reactive substances were also observed. All the mustard agents increased DNA fragmentation. The effects of SM were significantly ameliorated by DRDE-07 analogues, and with nitrogen mustards the protection was partial. Overall, DRDE-30 (propyl analogue) followed by DRDE-35 (butyl analogue) are favored as safer and better compounds.

Prophylactic efficacy of combination of DRDE-07 and its analogues with amifostine against sulphur mustard induced systemic toxicity.

Sulphur mustard, [bis (2-chloroethyl)] sulphide (SM), is a bifunctional alkylating agent. SM forms sulphonium ion in the body which alkylates DNA and several other macromolecules, and induces oxidative stress. Although several antidotes have been screened for the treatment of systemic toxicity of SM in experimental animals none of them are recommended so far. In the search for more effective and less toxic antidotes, various combinations were tried against SM induced toxicity and skin lesions. SM exposed through percutaneous route was used to evaluate the prophylactic efficacy of various combinations. Low dose of DRDE-07 (S-2(2-aminoethylamino) ethyl phenyl sulphide), DRDE-30 [S-2(2-aminoethyl amino) ethyl propyl sulphide], DRDE-35 [S-2(2-aminoethyl amino) ethyl butyl sulphide] with amifostine combinations, were given orally 30 min prior to SM exposure. Significant depletion was observed in body weight, organ body weight index and hepatic GSH and GSSG content in mice after SM exposure. Pretreatment with low dose of different combinations of DRDE-07, DRDE-30 and DRDE-35 with amifostine could recover biochemical alterations and histopathological changes caused by SM exposures.

Preparation of polylactide-co-glycolide and chitosan hybrid microcapsules of amifostine using coaxial ultrasonic atomizer with solvent evaporation.

The objective of this study was to evaluate the effect of various processing and formulation factors on the characteristics of amifostine hybrid microcapsules. Amifostine-loaded hybrid microcapsules were prepared using PLGA and chitosan. In short, amifostine powder was dissolved in de-aerated water with or without chitosan. The amifostine solution was later emulsified into PLGA solution in dichloromethane containing phosphatidylcholine. The resultant emulsion was fed through the inner capillary of a coaxial ultrasonic atomizer. The liquid fed through the coaxial outer capillary was either water or chitosan solution. The atomized droplets were collected into PVA solution and the droplets formed microcapsules immediately. The hybrid microcapsules prepared with chitosan solution only as an outer layer liquid showed the maximum efficiency of encapsulation (30%). The median sizes of all three formulations were 33-44 microm. These formulations with chitosan showed positive zeta-potential and sustained drug release with 13-45% amifostine released in 24 h. When chitosan was incorporated into inner as well as outer liquid layers, the drug release increased significantly, 45% (compared with other formulations) released in 24 h and almost 100% released in 11 days. Hybrid microcapsules of amifostine showed moderately high efficiency of encapsulation. The cationic charge (due to the presence of chitosan) of these particles is expected to favour oral absorption and thus overall bioavailability of orally administered amifostine.

Application of Multifunctional Nanomaterials in Radioprotection of Healthy Tissues.

Radiotherapy has been extensively used in clinic for malignant tumors treatment. However, a severe challenge of it is that the ionizing radiation needed to kill tumors inevitably causes damage to surrounding normal tissues. Although some of the molecular radioprotective drugs, such as amifostine, have been used as clinical adjuvants to radio-protect healthy tissues, their shortcomings such as short systemic circulation time and fast biological clearing from the body largely hinder the sustained bioactivity. Recently, with the rapid development of nanotechnology in the biological field, the multifunctional nanomaterials not only establish powerful drug delivery systems to improve the molecular radioprotective drugs' biological availability, but also open a new route to develop neozoic radioprotective agents because some nanoparticles possess intrinsic radioprotective abilities. Therefore, considering these overwhelming superiorities, this review systematically summarizes the advances in healthy tissue radioprotection applications of multifunctional nanomaterials. Furthermore, this review also points out a perspective of nanomaterial designs for radioprotection applications and discusses the challenges and future outlooks of the nanomaterial-mediated radioprotection.

Examination of the effect of ovarian radiation injury induced by hysterosalpingography on ovarian proliferating cell nuclear antigen and the radioprotective effect of amifostine: an experimental study.

AIM: The aim of this study was to examine the effect of amifostine on cellular injury in the ovarian tissue induced by hysterosalpingography (HSG). METHODS: In total, forty 4-month old female Wistar Albino rats were assigned into 8 groups. Each group contained 5 rats. Group 1 (G1): rats were decapitated without any procedure. Group 2 (G2): rats were decapitated after 3 hours of total body irradiation. Group 3 (G3): rats were decapitated 3 hours after HSG procedure. Group 4 (G4): rats were decapitated 3 hours after HSG procedure performed 30 min after receiving amifostine 200 mg/kg intraperitoneally. Group 5 (G5): rats were decapitated after 1 month without any procedure. Group 6 (G6): rats were decapitated after 1 month of total body irradiation. Group 7 (G7): rats were decapitated 1 month after HSG procedure. Group 8 (G8): rats were decapitated 1 month after HSG procedure performed 30 min after receiving amifostine 200 mg/kg intraperitoneally. After rats were decapitated under general anesthesia in all groups, blood samples were obtained and bilateral ovaries were removed. One of the ovaries was placed in 10% formaldehyde solution for histological germinal epithelial degeneration, apoptosis and proliferating cell nuclear antigen scoring. The other ovary and blood sera were stored at -80°C. TNF-α, total antioxidant status, total oxidant status, and malondialdehyde levels were studied in tissue samples and anti-mullerian hormone levels in blood samples. RESULTS: At the end of the first month, there was significant ovarian germinal epithelium degeneration. Proliferating cell nuclear antigen immunoreactivity was significantly reduced in all other groups when compared with G1 and G5. CONCLUSION: In conclusion, amifostine could significantly reduce the ovarian cellular injury induced by HSG.

Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs.

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.

CCND1-BCL2 Gene Network: A direct target of Amifostine in human acute megakaryocytic leukemia cells.

Amifostine, 2-(3-aminopropyl) aminoethyl phosphorothioate, is a broad-spectrum cytoprotective agent used to treat nuclear radiation and chemical weapon injuries. Recently, amifostine has been shown to have a profound biological influence on tumor cells. To examine the effects and mechanisms underlying the effects of amifostine on human acute megakaryocytic leukemia, we evaluated the efficacy of amifostine against Dami cells and observed a cell cycle arrest in G2 /M phase. Amifostine treatment also induced cell apoptosis of Dami cells which corresponds to formal studies. Through whole-genome microarray and bioinformatics analyses, we found that amifostine affected the gene expression of CCND1, BCL2, and CASP3 which revealed the mechanism amifostine acted on Dami cells. Thus, CCND1-BCL2 Gene Network is predicted to be a direct target of amifostine treating human acute megakaryocytic leukemia, which may provide a novel potential target for the therapy of several subtypes of human AML.

Improvement of the in vitro safety profile and cytoprotective efficacy of amifostine against chemotherapy by PEGylation strategy.

Amifostine, an organic thiophosphate prodrug, has been clinically utilized for selective protection of normal tissues with high expression of alkaline phosphatase from oxidative damage elicited by chemotherapy or radiotherapy. However, the patients receiving amifostine suffer from severe dose-dependent adverse effects. Strategies for improvement of the protective efficacy and toxicity profile of amifostine are urgently required. Here we constructed a PEGylated amifostine (PEG-amifostine) through conjugation of amifostine to the 4-arm PEG (5000 Da) by a mild one-step reaction. The relatively large PEG-amifostine molecules clustered into spherical nanoparticles, resulting in distinct hydrolysis properties, cell uptake profile and antioxidative activity compared with the free small molecules. PEGylation prolonged the hydrolysis time of amifostine, providing sustained transformation to its functional metabolites. PEG-amifostine could be internalized into cells and translocated to acidic organelles in a time-dependent manner. The intrinsic cytotoxicity of amifostine, which is related to the reductive reactivity of its metabolites and their ability to diffuse readily, was attenuated after PEGylation. This modification impeded the interaction between free sulfhydryls and functional biomolecules, providing PEG-amifostine with an improved safety profile in vitro. Moreover, PEG-amifostine showed higher efficiency in the elimination of reactive oxygen species and prevention of cisplatin-induced cytotoxicity compared with free amifostine. Overall, our study for the first time developed a PEGylated form of amifostine which significantly improved the efficacy and decreased the adverse effects of this antioxidant in vitro with great promise for clinical translation. In vivo study is urgently needed to confirm and redeem the cytoprotective effects of the PEG-amifostine in chemotherapy.

Sustained-release microspheres of amifostine for improved radio-protection, patient compliance, and reduced side effects.

A biweekly administration of sustained-release microsphere dosage form of amifostine, a radioprotective drug used in radiotherapy, was performed to examine the feasibility to minimize injection frequency and blood concentration-associated side effects. Model animal trials indicated that this subcutaneously injecting microspheres, 50-100 µm in diameter, achieved bi-weekly prolonged radio-protective efficacy and, at the same time, significantly reduced skin irritation than the solution form of amifostine given by the same administration route. In addition, the hypertension associated with blood concentration of amifostine was not observed in the drug-treated rats. The animals given the amifostine microspheres and amifostine showed significantly differences in white blood cell, red blood cell, hematocrit, hemoglobin and spleen tissue histopathology after exposed under a cobalt-60 γ-radiation at a dose rate of 1.0 Gy/min for 6 min. The in vitro release profile of amifostine from the micropsheres showed a minor initial burst (less than 20% of total drug loading in the first day of administration), consisting with the side effects observations. The results suggest that amifostine encapsulated in sustained-release microspheres may be an ideal dosage form for prolonged radio-protective efficacy and improved patient compliance.

Amifostine-conjugated pH-sensitive calcium phosphate-covered magnetic-amphiphilic gelatin nanoparticles for controlled intracellular dual drug release for dual-targeting in HER-2-overexpressing breast cancer.

We developed a surfactant-free method utilizing amifostine to stably link a targeting ligand (Herceptin) to amphiphilic gelatin (AG)-iron oxide@calcium phosphate (CaP) nanoparticles with hydrophobic curcumin (CUR) and hydrophilic doxorubicin (DOX) encapsulated in the AG core and CaP shell (AGIO@CaP-CD), respectively. This multi-functional nanoparticle system has a pH-sensitive CaP shell and degradable amphiphilic gelatin (AG) core, which enables controllable sequential release of the two drugs. The dual-targeting system of AGIO@CaP-CD (HER-AGIO@CaP-CD) with a bioligand and magnetic targeting resulted in significantly elevated cellular uptake in HER2-overexpressing SKBr3 cells and more efficacious therapy than delivery of targeting ligand alone due to the synergistic cell multi-drug resistance/apoptosis-inducing effect of the CUR and DOX combination. This nanoparticle combined with Herceptin and iron oxide nanoparticles not only provided a dual-targeting functionality, but also encapsulated CUR and DOX as a dual-drug delivery system for the combination therapy. This study further demonstrated that the therapeutic efficacy of this dual-targeting co-delivery system can be improved by modifying the application duration of magnetic targeting, which makes this combination therapy system a powerful new tool for in vitro/in vivo cancer therapy, especially for HER2-positive cancers.

Ultrasensitive detection of amifostine and alkaline phosphatase based on the growth of CdS quantum dots.

In this study, we reported a simple and sensitive fluorescence nanosensor for rapid detection of amifostine and alkaline phosphatase (ALP). The novel nanosensor was based on the fluorescence "turn on-off" of CdS quantum dots (QDs). Firstly, Cd(2+) cation could react with S(2-) anion to generate fluorescent CdS QDs in the presence of amifostine. The fluorescence (FL) intensity of amifostine-capped CdS QDs (Amifostine-CdS QDs) was increased with the increasing amounts of amifostine, and could be used for amifostine detection. However, amifostine could be converted to 2-(3-aminopropylamino) ethanethiol (WR1065) in the presence of ALP based on the dephosphorylation of ALP. Under the optimum conditions, the affinity of WR1065 to CdS QDs was weaker than that of amifostine. Therefore the new generation of WR1065-CdS QDs would reduce the FL intensity with the increase of ALP concentration, and the fluorescence of CdS QDs was turn off. The metabolic process of amifostine in the presence of alkaline phosphatase could be also studied via the change of FL intensity of CdS QDs. The present method was cost-effective, convenient, and does not require any complicated synthetic procedures.

Ranking the Binding Energies of p53 Mutant Activators and Their ADMET Properties.

The guardian of the genome, p53, is the most mutated protein found in all cancer cells. Restoration of wild-type activity to mutant p53 offers promise to eradicate cancer cells using novel pharmacological agents. Several molecules have already been found to activate mutant p53. While the exact mechanism of action of these compounds has not been fully understood, a transiently open pocket has been identified in some mutants. In our study, we docked twelve known activators to p53 into the open pocket to further understand their mechanism of action and rank the best binders. In addition, we predicted the absorption, distribution, metabolism, excretion and toxicity properties of these compounds to assess their pharmaceutical usefulness. Our studies showed that alkylating ligands do not all bind at the same position, probably due to their varying sizes. In addition, we found that non-alkylating ligands are capable of binding at the same pocket and directly interacting with Cys124. The comparison of the different ligands demonstrates that stictic acid has a great potential as a p53 activator in terms of less adverse effects although it has poorer pharmacokinetic properties.

Radioprotectants: pharmacology and clinical applications of amifostine.

Amifostine (Ethyol, ALZA Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) is a phosphorylated cysteamine derivative that was originally developed by the US Army Walter Reed Institute (Washington, DC) as a radioprotectant. Amifostine, a prodrug, is metabolized by the enzyme alkaline phosphatase to an active sulfhydryl compound (WR-1065) capable of scavenging radiation-generated free radicals and preventing cell damage. The disulfides of WR-1065 are structurally analogous to endogenous polyamines, which can bind to DNA molecules and stabilize them in a compact form less vulnerable to damage by cytotoxic agents. Preclinical and clinical studies show that amifostine is a selective radioprotectant that reduces both early and late radiation-induced toxicities to normal tissues while leaving tumor cells exposed to the cytotoxic effects of radiation. Preclinical data indicate that amifostine could reduce the risk of secondary cancers caused by radiation and certain forms of chemotherapy.

Pharmacokinetic profile of amifostine.

This article reviews the chemistry, measurement, metabolism, and pharmacokinetics of the cytoprotective agent amifostine. Validated analytic methodology to measure parent drug and pharmacologically active metabolites and pharmacokinetic studies are essential to the efficient performance and analysis of clinical studies. Well-validated analytic methods developed in the authors' laboratory were used to characterize this agent. Amifostine [s-2-(3-aminopropylamino)ethyl-phosphorothioate] is the phosphorylated pro-drug form of the active free thiol drug WR-1065 [2-(3-aminopropylamino)ethanethiol]. Observations described here support the hypothesis that amifostine is dephosphorylated rapidly by alkaline phosphatase present on the plasma membranes of the arteriolar endothelium of various normal tissues and on the plasma membranes of the kidneys' proximal tubular epithelium to its active thiol metabolite WR-1065, which in turn immediately enters normal tissues. Other metabolites that have been identified are WR-33278, the symmetrical disulfide of WR-1065; the mixed disulfides WR-1065-cysteine and WR-1065-glutathione; and cysteamine. Amifostine was recently approved by the US Food and Drug Administration for use as a cytoprotector in cancer patients receiving chemotherapy. Current pharmacokinetic studies in cancer patients are focusing on establishing a population model as a basis for developing limited sampling strategies for future investigations of the pharmacokinetic-pharmacodynamic behavior of amifostine.