WR-2721 Treatment Research Articles

Radioprotector WR-2721 and mitigating peptidoglycan synergistically promote mouse survival through the amelioration of intestinal and bone marrow damage.

The identification of an agent effective for the treatment of intestinal and bone marrow injury following radiation exposure remains a major issue in radiological medicine. In this study, we evaluated the therapeutic impact of single agent or combination treatments with 2-(3-aminopropylamino) ethylsulphanyl phosphonic acid (WR-2721) and peptidoglycan (PGN, a toll-like receptor 2 (TLR-2) agonist) on radiation-induced injury of the intestine and bone marrow in lethally irradiated male C57BL/6 mice. A dose of 3 mg of WR-2721 per mouse (167 mg/kg, intraperitoneally) was given 30 min before irradiation, and 30 μg of PGN per mouse (1.7 mg/kg) was injected intraperitoneally 24 h after 10 Gy irradiation. Bone marrow cluster of differentiation (CD)45+ and CD34+ markers of multiple haematopoietic lineages, number of granulocyte–erythroid–macrophage–megakaryocyte (GEMM) progenitor colonies, bone marrow histopathology, leucine-rich repeat-containing G-protein-coupled receptor 5 (Lgr5) expression in the intestines, xylose absorption and intestinal histopathology were all assessed at various time-points after irradiation. Furthermore, nuclear factor kappa B (NF-κB) p65 protein in the ileum was stained by immunofluorescent labelling. PGN-treated irradiated mice showed an increase in CD45+CD34+ cells compared with untreated mice 1.25 days after 10 Gy ionizing radiation (IR) (P < 0.05). Furthermore, combined PGN and WR-2721 treatment had an obviously synergistic radio-protective effect in nucleated cells in the bone marrow, including GEMM progenitors and CD45+CD34+ cells 4 days after 10 Gy IR. Single agent PGN or WR-2721 treatment after 10 Gy IR clearly increased Lgr5-positive pit cells (P < 0.05) and xylose absorption (P < 0.05). However only PGN and WR-2721 combination treatment markedly increased villus height (P < 0.05), number of crypts (P < 0.05) and whole-body weights after 10 Gy whole-body irradiation (WBI). The NF-κB p65 subunit was translocated to the nucleus, and phosphate-IκBα (Ser32/Ser36) was detected after stimulation with either PGN or WR-2721, which indicates that these two agents act synergistically through the activation of the NF-κB pathway. Administration of PGN in combination with WR-2721 was demonstrated to have a synergistic effect on the increase in haematopoietic cells and intestinal reconstitution, as well as improved survival in lethally irradiated mice, but resulted in some degree of an immune disorder.

Effects of Amifostine and Fengling Polysaccharide on Proliferation and Chemoprotection of Human Granulocyte-Macrophage Progenitor Cells.

Objective: This paper is to study the proliferation and chemoprotection effects of amifostine (WR-2721) and fengling polysaccharide (FLPS) on the human granulocyte-macrophage progenitor cells.Method: (1) Mononuclear cells (MNC) were deparated by Ficoll (1.077g/ml). The MNC of WR-2721 and FLPS treatment were cultured in methylcellulose senisolid medium, the colony forming unit-granulocyte macrophage(CFU-GM) was measured. The effects of WR-2721 and FLPS on CFU-GM were studied. (2) MNC of WR-2721 treatment or containing FLPS were cultured 14h at 37°C with VP16. The cytoprotective activity against VP16 toxic effects of WR-2721 and FLPS on CFU-GM were observed. (3)To study the effects of WR-2721 on proliferation inhibition and apoptosis of HL-60 human leukemia cell line, the cell apoptosis rate of HL-60 was determined by annexinV/PI double staining method, cell proliferation and chemotherapy sensitivity were analyzed with XTT assay, and the changes of cell cycle were observed through flow cytometry.(4) HL-60 cells of containing FLPS were cultured 14h at 37°C with VP16. The cytoprotective activity against VP16 toxic effects of FLPS on HL-60 cells were observed.Results: (1) The number of CFU-GM was significantly increased in 10 groups by addition of 0.5–25μg/ml FLPS and 12 groups treated with 0.01–5mmol/L WR-2721( 30 min, 37°C), p<0.05. The mean value of CFU-GM in groups of negative control, WR-2721 (1mmol/L), FLPS(5μg/ml) and WR-2721+FLPS were 91.4±50.4,119.8±62.9,143.2±76.4 and 179.2±97.6 per 1×105, respectively, Compared with control, singnificant differences were seen between each group, p<0.05. WR-2721+FLPS group compared with WR-2721 and FLPS group, p<0.01 and p<0.05. (2) The number of CFU-GM was significantly increased in MNC of adding FLPS or WR-2721 treatment. The mean value of CFU-GM in groups of VP-16 control, negative control, WR-2721 + VP16, FLPS + VP-16 and WR-2721 + FLPS + VP16 were 30.9±22.5, 83.2±43.8, 64.6±41.2, 55.3±33.5 and 78.3±48.2 per 1×105, respectively. Compared with VP16 control group, singnificant differences were seen between each group, p < 0.01. (3) After treatment (30min,37°C)with WR-2721, the sensitivity of HL-60 cells to VP-16 was enhanced, and the IC50 descended from 52.5μg/ml to 40.5μg/ml. After 72hours trentment of HL-60 cells with WR-2721, the early apoptotic cells (annexinV-FITC positive/PI negative) were increased from(5.5±1.9)% to (48.5±8.4)%(p<0.001), late apoptotic cells(annexinV-FITC positive/PI positive)were increased from(1.2±0.5)% to (39.0±4.0)% (p<0.001), and HL-60cells were arrested in G2-M phase. (4) HL60 cells were cultured 14h at 37°C with 20μg/ml VP16 and 50μg/ml FLPS, the cell survival rate was 88.0%±2.3%, negetive control was 90.5%±2.9%, no singnificant difference was seen between two group, n =21, p >0.05.Conclusion :(1) WR-2721 and FLPS can increase the prolification of CFU-GM. Combination of WR-2721 and FLPS show stronger effect than the WR-2721 or FLPS alone. (2) WR-2721 and FLPS selectiveiy protect human peripheral blood CFU-GM from the cytotoxicity of VP16 without decreasing its cytotoxic effect on HL60 cells. (3) WR-2721 treatment can enhance HL-60 cells chemotherapy sensitivity to VP-16, inhibit proliferation, induce HL-60 cells apoptosis and accumulation of cells in G2-M phase.

Adipocyte differentiation in Sod2−/− and Sod2+/+ murine bone marrow stromal cells is associated with low antioxidant pools

Adipocytogenesis in bone marrow stromal cells (BMSCs) from manganese-superoxide dismutase-deficient (Sod2(-/-)) and wild-type (Sod2(+/+)) mice and the effect of antioxidant pool size were determined. BMSCs from Sod2(-/-) or Sod2(+/+) mice were cultured with and without adipocytogenic supplements including: 10 mug/mL insulin, 1 muM dexamethasone, and 100 muM indomethacin. Oil Red-O-positive cells and reverse-transcriptase polymerase chain reaction measurement of peroxisome proliferator-activated receptor-gamma (PPARgamma) and lipoprotein lipase (LPL) were measured. Antioxidant glutathione levels (GSH) and glutathione peroxidase activity (GPX) were determined. Sod2(-/-) cells demonstrated constitutive adipocytogenesis in basal medium and generated 34% more adipocytes in adipocytogenic media. Growth of cells in the free radical scavenger antioxidant, amifostine (WR2721; 4 mM) decreased numbers of adipocytes in Sod2(-/-) BMSCs in both basal (38.0%, p = 0.037) and adipocytogenic (37.5%, p = 0.021) media and reduced to undetectable the levels of expression of PPARgamma and LPL. In contrast, Sod2(+/+) cells showed no detectable constitutive adipocytogenesis but formed adipocytes in adipocytogenic medium, with a decrease (43.7%, p = 0.001) by addition of WR2721. In basal conditions, Sod2(-/-) cells had lower GSH (78.6%; p = 0.0089) and GPX (52.7%; p < 0.001) levels than did Sod2(+/+) cells, which were increased in either medium by WR2721 treatment of Sod2(-/-) or Sod2(+/+) cells (all p < 0.001). Differentiation of BMSCs to adipocytes was inversely correlated with the level of GSH (r = -0.9427, p = 0.0167). Sod2(-/-) long-term bone marrow cultures had decreased hematopoiesis compared to those from Sod2(+/-) or Sod2(+/+) mice. The cellular redox pathway has a role in adipocyte differentiation of cells of the hematopoietic microenvironment.

WR-2721 reduces bone loss after hindlimb tenotomy in rats

WR-2721 is a thiophosphate analog of cysteamine that produces hypocalcemia in vivo. Previous studies suggest that WR-2721 produces hypocalcemia by independent inhibitory effects on parathyroid hormone (PTH) secretion, osteoclastic bone resorption, and tubular reabsorption of calcium. We sought to determine if WR-2721 would decrease bone loss in an animal model of disuse osteoporosis produced by unilateral knee tenotomy in the rat. Tenotomy significantly increased osteoclast number in tibias on the side of the procedure compared with tibias on the opposite side which had not undergone the procedure at 3 and 14 days. Femoral weight of tenotomized limbs were also reduced significantly compared with the contralateral limb at 3 and 14 days. WR-2721 treatment (240 mg/kg daily) prevented 26% of the loss of femoral dry weight and 29% of the loss of femoral ashed weight produced 14 days after tenotomy. In addition, WR-2721 treated (240 mg/kg daily) animals had fewer osteoclasts in tenotomized tibias than control animals at 3 days (6.6 +/- 0.7/mm versus 10.3 +/- 0.9/mm, p less than 0.02) and at 14 days (5.8 +/- 0.3/mm versus 8.7 +/- 0.4/mm, p less than 0.02). These data suggest that WR-2721 decreases bone loss in this model by decreasing osteoclastic bone resorption.

Effect of WR-2721 on Radiation-induced Lipid Peroxidation and Enzyme Release in Erythrocytes and Microsomes

The effect of WR-2721 on radiation-induced lipid peroxidation and enzyme release was studied after gamma-irradiation of microsomes and erythrocytes. WR-2721 rendered protection to microsomes by reducing lipid peroxidation whereas it did not exert significant protection against radiation-induced lipid peroxidation in erythrocytes. The non-protective effect of WR-2721 was also observed in radiation-induced enzyme (AChE) release of erythrocytes. It is proposed that erythrocytes do not dephosphorylate WR-2721 into the free thiol derivative (WR-1065). This was confirmed by the microsomal treatment of erythrocytes that had shown the protective effect of WR-2721. Further, a significant increase in sulphydryl content in microsomes, and no such increase in erythrocytes, after the WR-2721 treatment indicated that dephosphorylation of WR-2721 is necessary for its protective effect.

Prevention of parathyroid hormone-dependent nephrocalcinosis by chronic administration of the organic phosphorothioate WR-2721.

Pharmacological inhibition of parathyroid hormone (PTH) secretion has been the object of several experimental and clinical trials in the past. It is only recently that a drug, WR-2721 [S-,2-(3-aminopropylamino)-,ethylphosphorothioic acid], has been shown to effectively inhibit PTH secretion in euparathyroid subjects and in parathyroid cancer patients within a few hours after its administration. In the present study we tested its long term efficacy as a PTH inhibitor in a model of nephrocalcinosis resulting from secondary hyperparathyroidism. Intact and thyroparathyroidectomized (TPTX) rats were pair-fed on a low Ca (0.2%)-high phosphorus (1.6%) diet (nephrocalcinotic diet) or on a normal Ca (1.1%)-normal phosphorus (0.8%) diet for 7 days. Simultaneously, either 0.2 mmol/kg of WR-2721 or its solvent was injected subcutaneously twice daily. The intact animals on the nephrocalcinotic diet had an increased urinary cyclic AMP excretion and important renal Ca accumulation. This nephrocalcinosis was markedly reduced by WR-2721 treatment. The kidney Ca content of the WR-2721-treated rats was 58 +/- 6% lower than that of the nontreated animals. The low Ca-high phosphorus diet did not cause nephrocalcinosis in the TPTX rats. WR-2721 failed to reduce the nephrocalcinosis induced by 1,25 dihydroxyvitamin D3 intoxication in TPTX rats fed the normal Ca-normal phosphorus diet. In conclusion, the present study suggests that chronic treatment with WR-2721, a potent inhibitor of PTH secretion, may be effective for preventing the deleterious consequences of hyperparathyroidism, such as nephrocalcinosis.

Normalization of hypercalcemia associated with a decrease in renal calcium reabsorption in Leydig cell tumor-bearing rats treated with WR-2721.

The Rice-500 Leydig cell tumor (LCT) in Fischer rats is a model of humoral hypercalcemia of malignancy (HHM). In this model, the elevation of plasma calcium (Ca) does not result merely from an increased bone resorption, but also from an enhanced tubular Ca reabsorption (TRCa). We investigated the hypocalcemic response to WR-2721 [S-2,2-(3-aminopropylamino)-, ethylphosphorothioic acid] in LCT-bearing Fischer rats. WR-2721 is a potent inhibitor of normal and aberrant parathyroid hormone (PTH) secretion. Moreover, it exerts a PTH-independent inhibitory effect on TRCa. In hypercalcemic LCT-bearing rats WR-2721 induced a fall in plasma Ca from 3.24 +/- 0.12 to 2.66 +/- 0.23 mmol/liter within 2 h after one single injection of 0.7 mmol/kg body wt. The decrement in plasma Ca was associated with a marked increase in urinary Ca excretion, indicating an inhibition of TRCa. The elevated urine cyclic AMP of LCT-bearing rats, however, was not altered by WR-2721 treatment. These results suggest that in this HHM model, WR-2721 can normalize calcemia through its PTH-independent inhibitory effect on TRCa. WR-2721 could therefore be an effective drug to treat human hypercalcemia of malignancy, particularly in those tumors wherein a markedly enhanced renal Ca reabsorption contributes to the elevation of the plasma Ca level.

Behavioral Toxicity and Efficacy of WR-2721 as a Radioprotectant

S-2-(3-Aminopropylamino)ethylphosphorothioic acid (WR-2721) is a promising protectant for radiation-induced lethality. However, treatment with WR-2721 also produces nausea, vomiting, diarrhea, and hypotension, which implies severe functional consequences. Three studies were conducted to assess the effects of WR-2721 on rat motor performance and weight and to assess the ability of WR-2721 to mitigate the early performance decrement (PD) produced by ionizing radiation. In the first study, rats trained on the accelerod motor performance task were give 200, 300, or 400 mg/kg WR-2721 intraperitoneally (ip). The highest dose used referenced the maximum tolerated dose in the rat, which is two-thirds the median lethal dose (590 mg/kg). The subjects were tested immediately after treatment, at 30-min intervals for 3 h, and again at 24 h. All groups (N = 6/group) demonstrated a significant decrease in accelerod performance compared to control levels across the eight test trials, which ranged from 24 to 44% in the 200 and 400 mg/kg dose groups, respectively. Performance returned to baseline levels at 24 h. Some deaths occurred at all dose levels. In the second study, motor performance was measured after exposure to radiation alone or a drug/radiation combination (N = 8/group). WR-2721 was administered 30 min before exposure to 130 Gy of gamma radiation from a 60Co source at a dose rate of 20 Gy/min. Rats were tested on the accelerod immediately after WR-2721 treatment and at 10, 15, 30, 60, and 120 min and 24 h following radiation. Performance was significantly depressed compared to control throughout the 24 h following radiation exposure, with and without WR-2721. The decrement produced by WR-2721 and radiation alone appeared to add up to the combined drug/radiation decrement found over the 15- to 120-min test periods. In the third study assessing the effects of WR-2721 on weight, untrained rats treated with 200 or 400 mg/kg WR-2721 exhibited significant weight loss that lasted up to 3 days. Weight returned to pretreatment levels in 15 days, and no deaths occurred. In summary, the data suggest that in the rat (1) WR-2721 is behaviorally toxic at doses relevant to radioprotection, (2) WR-2721 treatment along with the stress of motor performance may combine to lower the level at which lethalities occur, (3) WR-2721 does not protect for radiation-induced PD, and (4) WR-2721 combined with radiation disrupts performance more severely than either radiation or WR-2721 alone.

Pharmacokinetics of WR-1065 in mouse tissue following treatment with WR-2721

Levels of reduced glutathione (GSH) and N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were measured in tissues of Balb/c mouse bearing EMT 6 tumors at time intervals ranging from 5 min to 48 hr after i.v. injection of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) at 500 mg per kg. In all tissues examined (liver, kidney, lung, heart, muscle, brain, tumor, spleen, and salivary gland), maximal WR-1065 levels occurred 5–15 min after injection, with levels in liver, kidney, lung, and salivary gland exceeding one μmole per gm. The post-maximum decline in WR-1065 varied markedly with tissue, lung exhibiting a 6-fold drop by 30 min and salivary gland falling only 1596 after 3 hr. In a mouse treated with carbon-14 labeled WR-2721 it was found after 15 min that WR-1065 accounted for over half of the total drug in all tissues except tumor, where it accounted for a third of the total drug. There was no evidence that GSH levels were substantially altered by WR-2721 treatment. The results provide the first direct evidence supporting the widely held view that WR-2721 treatment results in intracellular WR-1065 and they demonstrate that high levels of WR-1065 occur very soon after i.v. injection.