Fluorometric determination of amifostine and alkaline phosphatase on amphiprotic molecularly imprinted silica crosslinked with binary functional silanes and carbon dots.

A silica-based molecularly imprinted polymer (MIP) formed by functional silanes (basic 3-aminopropyltriethoxysilane (APTES) and acidic 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane (CSPTMS)) was crosslinked with carbon dots (CDs) to develop a fluorescent sensor toward an amphiprotic template, amifostine (AMF). The CDs were synthesized by hydrothermal carbonization of succinic acid and an ionic liquid and possessed hydroxyl and pyrrolic functional groups, which enabled the CDs to be derivatized with silanes for subsequent sol-gel polymerization. Except for the CDs derivatized with tetraethoxysilane, CD-APTES, CD-CSPTMS, and CD-APTES/CSPTMS (molar ratio = 1/1) all presented distinct fluorescence dynamic quenching when interacted with AMF. However, APTES/CSPTMS was selected as the sol-gel monomer for the formation of MIP because its quenching ratio and imprinted factor were the highest among the CD-silane-MIPs. Moreover, 0.5 mg/mL of CD-APTES/CSPTMS -MIP in pH 7.5 buffer was used to quantify AMF (0.5-200 nM, LOD = 0.15 nM) and alkaline phosphatase (ALP) (2-150 µU/mL, LOD = 0.5 µU/mL), which activates the metabolism of AMF, and the calibration curves of AMF and ALP were determined via fluorescence quenching and restoration, respectively. The recoveries of 1, 10, and 60 nM AMF from 360-fold-diluted human serum solutions were 95, 104, and 103%, respectively, with RSD values that were lower than 4.2%. The average ALP activity of the original human serum was determined to be 32.1 U/L (RSD = 5.41%).

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.

Isotachophoretic determination of phosphate splitting from Amifostine and p-nitrophenyl phosphate in serum and neuroblastoma cells.

Amifostine [WR-2721; H2N-(CH2)3-NH-(CH2)2-S-PO3H2] is used as a protecting agent in the chemotherapy of neuroblastoma. It is supposed that Amifostine will be transformed into its active form, the free thiol (WR-1065), easier by normal cells than by tumour cells. Analytical capillary isotachophoresis was used to determine the dephosphorylation of Amifostine in serum and on neuroblastoma cells and peripheral blood cells. Furthermore, the biological effects of Amifostine and its free thiol, on cell proliferation of neuroblastoma cells were measured in combination with Carboplatin. It was found that neuroblastoma cells did not split phosphate less efficiently than normal peripheral blood cells. Furthermore, neither Amifostine (as expected) nor the free thiol (not expected according to the theory) were able to inhibit the effects of Carboplatin. Therefore, the current hypothesis concerning the mode of action of Amifostine must be questioned.

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.

High-performance liquid chromatographic assay using electrochemical detection for the combined measurement of amifostine, WR 1065 and the disulfides in plasma.

A high-performance liquid chromatographic (HPLC) method was developed for the combined analysis of the chemoprotective agent, amifostine, its active metabolite, WR 1065, and the (symmetrical and mixed) disulfides of WR 1065 in plasma. These three compounds were quantified by measuring WR 1065 after three different sample pretreatment procedures. During these procedures, amifostine and the disulfides were quantitatively converted into WR 1065, by incubating the sample either at a low pH or in the presence of dithiothreitol, respectively. The resulting amounts of WR 1065 were determined by HPLC with electrochemical detection (Au electrode, + 1.00 V). The lower limit of quantitation of WR 1065 was 0.15 microM. The within-day and between-day precision were < or =4.4 and < or =8.2% for WR 1065, < or =4.9 and < or =13.1% for amifostine and < or =8.5 and < or =5.5% for the disulfides, respectively. The within-day and between-day accuracy ranged from 97.2 to 109.8% and from 97.6 to 101.5% for WR 1065, from 88.3 to 110.7% and from 99.4 to 101.5% for amifostine and from 99.2 to 110.2% and from 103.3 to 104.9% for the disulfides, respectively. This method is superior to other described methods due to its simple and relatively rapid analysis of all three compounds in one system. Furthermore, it is at least as sensitive as earlier reported methods for one of the compounds and the application of the gold electrode requires only minor maintenance. Therefore, this method is very suitable for pharmacokinetic studies of amifostine and its metabolites. As an example, the plasma concentrations of amifostine, WR 1065 and the disulfides are shown in a patient after receiving an i.v. dose of 740 mg/m2 amifostine.

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.

Clearance of S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913) in rats.

Complications of radiotherapy in the treatment of retinal and choroidal neoplastic diseases include cataract formation, radiation retinopathy, neovascular glaucoma, cystoid macular edema, and subretinal neovascularization. These side effects may be minimized by the use of compounds known to have a protective effect on normal ocular tissues without impeding the benefits of therapy. Phosphorothioates, first developed under the Antiradiation Drug Development Program of the U.S. Army Medical Research and Development Command, have been reported to protect normal tissues during radiation therapy in a variety of animal models. One of the phosphorothioates, WR-77913 (S-[3-amino-2-hydroxylpropyl]phosphorothioate) was found to inhibit cataract formation in rats after radiation exposure. To test the efficacy of WR-77913 in the retina, we established a high-pressure liquid chromatography method to measure the levels of dephosphorylated WR-77913 and studied the drug's clearance from the lens, retina, blood, kidney, and liver in rats.

Analysis of S-35 labeled WR-2721 and its metabolites in biological fluids.

Studies with WR-2721 and related compounds have been hindered by the lack of a suitable assay for the drug and its major metabolites. We have developed a chromatographic method which requires no derivatization for the separation and detection of WR-2721, the free thiol, its symmetrical disulfide and other mixed disulfides. Our procedure involves ion-pairing for separation of ionizable compounds by causing polar molecules to become more lipophilic and hence separable using reverse phase HPLC. Detection is based upon liquid scintillation counting of S-35 incorporated during the synthesis of the parent compound. This method requires no pre-column preparation of samples and, by detecting the S-35 label, eliminates the chance that a coeluting species could interfere with detection, as might occur with post-column derivatization. Chromatography was done using a 10 micron C8RP column and 35% MeOH/65% 0.0113M NaH2PO4, 0.005 M hexanesulfonate, pH 5.9, flowing at 1 ml/min. Half-minute fractions were collected into scintillation vials for counting. Retention volumes for the various compounds were: column breakthrough (3.5 ml), WR-2721 (4.5 ml), WR-1065 (9 ml), and WR-33278 (24 ml). This analytical technique employing radiotracers can be used to study radioprotective mechanisms by time dependent measurements of the tissue distribution and chemical form of labeled drug. Such chemical information can then be correlated with biological measures of radiation protection.