WR 1065

WR-1065, a dephosphorylated metabolite of amifostine?(Ethyol), can protect against the immediate and delayed effects of radiation exposure.

WR-1065 can protect against zidovudine (AZT) – induced genetoxicity. The lymphoblastoid cell line MOLT-3 cells were exposed to 0/10μM AZT for 96h. In the first 24h 0/5?μM WR-1065 was added and Cyt B was added in 76h in the cells, the viability of AZT treated MOLT-3 cells did not altered.[1] Moreover, in RKO36 cell lines (derivative RKO human colorectal carcinoma carrying a GFP-pCMV-EGFP2Xho), 4 mM final concentration (EC50) WR-1065 treatment for 30min immediately before irradiation showed protective effects against cell chromosomal damage and death induced by ionizing radiation and delayed genomic instability. But 40??M WR-1065 did not show immediate radio-protective effects in irradiated RKO36 cells.[2] WR-1065 acts as radioprotective agents mainly through suppression of the homologous recombination pathway. In SPD8 Chinese hamster cell line, both 4 mM WR-1065 for 30min and 10 ?M for 24h significantly reduced the homologous recombination induced by 0.2 mM Hydroxyurea for 24h or 100 nM camptothecin for 1h. While WR-1065 did not show its radioprotective effects in irradiated homologous recombination-deficient irs 1SF cells compared with homologous recombination-proficient cells AA8/CXR3(P＜0.05).[3]

With spray drying technique using PLGA (polylactide co-glycolide) as the polymer matrix, WR-1065 were prepared into nanoparticles. 500mg/kg WR-1065/PLGA nanoparticles in which containing 21.7(w/w WR-1065) were administrated orally in mice to determined its radio-protective role. WR-1065PLGA nanoparticles treatment mice showed noteworthy higher 30-day survival, less bone marrow suppression and less intestinal injury compared non-treated control mice, indicated its significant radio-protective effects.[4]

References:
[1].  Ofelia A . Olivero, Michael O. Ong, Han nan M. Braun, Ariadna Marrogi, Kathyiani Divi, James B. Mitchel l, and Miriam C. Poirier. Selective protection of zidovadine-induced DNA-damage by antioxidants WR-1065 and tempol. Environmental and Molecular Mutagenesis (2014)55: 566-572
[2].  Jaroslaw Dziegielewski, Janet E. Baulch, Wilfried Goetz, Mitchell C. Coleman, Douglas R. Spitz, Jeffrey S. Murley, David J. Grdina, and William F. Morgan. WR-1065, the active metabolite of amifostine, mitigates radiation-induced delayed genomic instability. Free Radic Biol Med. (2008)45(12): 1674–1681
[3].  Jaroslaw Dziegielewski, Wilfried Goetz, Jeffrey S. Murley, David J. Grdina, William F. Morgan, and Janet E. Baulch. Amifostine Metabolite WR-1065 Disrupts Homologous Recombination in Mammalian Cells. Radiat Res. (2010) 173(2): 175–183
[4].  Sarala Pamujula,?Vimal Kishore,?Barbara Rider,?Krishna C. Agrawal, and?Tarun K. Mandal. Radioprotection in mice following oral administration of WR-1065/PLGA nanoparticles. (2008) 84(11): 900-908

Amifostine metabolite WR-1065 disrupts homologous recombination in mammalian cells.[Pubmed:20095849]

Radiat Res. 2010 Feb;173(2):175-83.

Repair of DNA damage through homologous recombination (HR) pathways plays a crucial role in maintaining genome stability. However, overstimulation of HR pathways in response to genotoxic stress may abnormally elevate recombination frequencies, leading to increased mutation rates and delayed genomic instability. Radiation-induced genomic instability has been detected after exposure to both low- and high-linear energy transfer (LET) radiations, but the mechanisms responsible for initiating or propagating genomic instability are not known. We have demonstrated that WR-1065, the active metabolite of amifostine, protects against radiation-induced cell killing and delayed genomic instability. We hypothesize that hyperstimulation of HR pathways plays a mechanistic role in radiation-induced genomic instability and that, in part, WR-1065 exerts it radioprotective effect through suppression of the HR pathway. Results of this study demonstrate that WR-1065 treatment selectively protected against radiation-induced cell killing in HR-proficient cell lines compared to an HR-deficient cell line. Further, WR-1065 treatment decreases HR in response to DNA damage using two different mammalian cell systems. This suppression of hyper-recombination is a previously unrecognized mechanism by which WR-1065 effects radioprotection in mammalian cells.

Selective protection of zidovudine-induced DNA-damage by the antioxidants WR-1065 and tempol.[Pubmed:24833597]

Environ Mol Mutagen. 2014 Aug;55(7):566-72.

The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 muM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 microM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 microM WR-1065 and/or 0 or 200 microM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage.

Development and Validation of an HPLC Method for Determination of Amifostine and/or Its Metabolite (WR-1065) In Human Plasma Using OPA Derivatization and UV Detection.[Pubmed:26664371]

Iran J Pharm Res. 2015 Fall;14(4):1051-7.

A rapid, sensitive and reproducible HPLC method was developed and validated for the analysis of amifostine (AMF) and/or its metabolite, WR-1065 in human plasma. The method involves the alkylation of free sulfydryl group with iodoacetic acid followed by derivatization of the drug and its metabolite with o-phthaldialdehyde (OPA) and UVdetection at 340 nm. The derivatized AMF and WR-1065 were eluted in less than 11 min, and in the case of the metabolite with no interferences from the endogenous plasma peaks. Cystein was used as the internal standard. Analysis was carried out on a Eurosphere Performance (RP-18e, 100 x 4.6 mm) analytical column. The mobile phase was a mixture of methanol and phosphate buffer 0.03 M pH = 2.7 at a ratio of 40: 60v/v, respectively, with a flow rate of 1.5 mLmin(-1). Limit of detection was 0.5 microgmL(-1). The method involved a simple extraction procedure for AMF and/or its metabolite and analytical recovery was 90 +/- 0.9%.The calibration curve was linear over the concentration range of 1-200 microgmL(-1). The coefficients of variation for intra-day and inter-day assays were less than 10%.

Effect of WR-1065 on 6-hydroxydopamine-induced catalepsy and IL-6 level in rats.[Pubmed:27403255]

Iran J Basic Med Sci. 2016 May;19(5):490-6.

OBJECTIVES: Neuroinflammation and oxidative stress play a key role in pathogenesis of Parkinson's disease (PD). In the present study we investigated the effect of reactive oxygen species (ROS) scavenger WR-1065 on catalepsy and cerebrospinal fluid (CSF) level of interleukin 6(IL-6) and striatum superoxide dismutase (SOD) activity in 6-hydroxydopamine (6-OHDA) induced experimental model of PD. MATERIALS AND METHODS: Seventy two male Wistar rats were divided into 9 equal groups and 6-OHDA (8 mug/2 mul/rat) was infused unilaterally into substantia nigra pars copmacta (SNc) to induce PD. Catalepsy was measured by standard bar test, CSF level of IL-6 was assessed by enzyme-linked immunosorbent assay (ELISA) method and SOD activity measured by spectrophotometric method. In pre-treatment groups WR-1065 (20, 40 and 80 mug/2 mul/rat/day, for 3 days) was infused into the SNc before 6-OHDA administration and 21 days later, as a recovery period, behavioral and molecular assay tests were done. RESULTS: Our results showed that pre-treatment with WR-1065 improved (P<0.001) 6-OHDA-induced catalepsy in a dose dependent manner. In 6-OHDA-lesioned animals SOD activity in SNc and CSF level of IL-6 was decreased markedly (P<0.001) when compared with non-lesioned group, while pre-treatment with WR-1065(P<0.001) restored their levels up to the normal range. CONCLUSION: Our study indicated that pre-treatment with WR-1065 could modulate catalepsy and IL-6 level in 6-OHDA-lesioned rats. Also WR1065 could increase SOD activity up to normal range. It can be regarded as an anti-oxidative drug in prevention or adjunctive therapy of PD.

The cytoprotective aminothiol WR1065 activates p53 through a non-genotoxic signaling pathway involving c-Jun N-terminal kinase.[Pubmed:12531896]

J Biol Chem. 2003 Apr 4;278(14):11879-87.

WR1065 is an aminothiol with selective cytoprotective effects in normal cells compared with cancer cells. In a previous study (North, S., El-Ghissassi, F., Pluquet, O., Verhaegh, G., and Hainaut, P. (2000) Oncogene 19, 1206-1214), we have shown that WR1065 activates wild-type p53 in cultured cells. Here we show that WR1065 induces p53 to accumulate through escape from proteasome-dependent degradation. This accumulation is not prevented by inhibitors of phosphatidylinositol 3-kinases and is not accompanied by phosphorylation of Ser-15, -20, or -37, which are common targets of the kinases activated in response to DNA damage. Furthermore, WR1065 activates the JNK (c-Jun N-terminal kinase), decreases complex formation between p53 and inactive JNK, and phosphorylates p53 at Thr-81, a known site of phosphorylation by JNK. A dominant negative form of JNK (JNK-APF) reduces by 50% the activation of p53 by WR1065. Thus, WR1065 activates p53 through a JNK-dependent signaling pathway. This pathway may prove useful for pharmacological modulation of p53 activity through non-genotoxic mechanisms.

The cytoprotective aminothiol WR1065 activates p21waf-1 and down regulates cell cycle progression through a p53-dependent pathway.[Pubmed:10713709]

Oncogene. 2000 Feb 24;19(9):1206-14.

The phosphoaminothiol WR1065, the active metabolite of the pro-drug amifostine (WR2721), protects cultured cells and tissues against cytotoxic exposure to radiation or chemotherapeutic agents. We show here that WR1065 and the pro-drug WR2721 activate the p53 tumor suppressor protein and induce the expression of the cyclin-dependent kinase inhibitor p21waf-1 in the breast cancer cell line MCF-7, and in the mouse fibroblast cell line balb/c 3T3. Using two MCF-7 derived cell lines, MN1 and MDD2, we show that induction of p21waf-1 is detectable in MN1 (expressing a functional p53) but not in MDD2 (p53 disabled). These effects are observed at concentrations of WR1065 (0.5 to 1 mM) identical to those required to protect against cytotoxicity by hydrogen peroxide. Induction of p53 is not prevented by addition of aminoguanidine, an inhibitor of Cu-dependent amine-oxidases which blocks the extra-cellular degradation of WR1065 into toxic metabolites. Moreover, spermidine, a natural polyamine structurally related to amifostine, does not activate p53. Induction of p53 by WR1065 results in a delay in the G1/S transition in MCF-7 and MN-1 cells, but not in the p53 disabled cells MDD2. These data indicate that WR1065, a polyamine analog with thiol anti-oxidant properties, activates a cell cycle check-point involving p53.

WR-1065 dihydrochloride can protect normal tissues from the toxic effects of certain cancer drugs and activate p53 through a JNK-dependent signaling pathway.

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