Comparison of the protective effects of three phosphorothioate radioprotectors in the RIF-1 tumor

Three aminoalkyl phosphorothioates, WR-2721, WR-3689, and WR-77913, were compared as radioprotectors of RIF-1 tumors irradiated in vivo and assayed for cell survival in vitro. The protector doses were 50% of the acute drug LD50. The radiation dose modifying factors for the three drugs were nearly equal, ranging from 1.5 to 1.7 at surviving fractions of 0.1 and 0.05. Using biodistribution data obtained with 35S labeled drugs, the uptake in tumors was calculated as micromoles drug per gram of tumor. On this basis, tumor levels of WR-77913 were 4.5-fold those of WR-2721, and WR-3689 uptake was 2.7-fold greater than uptake of WR-2721. Thus, on a molar basis, WR-2721 appears to be the most effective protector, but all three phosphorothioates protect this tumor moderately well. In diffusible substance autoradiographs of 3H WR-3689 labeled tumors, label was generally distributed over cells with no evidence of preferential localization over nuclei.     

Protection of mice against fission neutron irradiation by WR-2721 or WR-151327

Two phosphorothioate compounds, WR-2721 and WR-151327, were examined for their radioprotective efficacies against the effects of fission neutron irradiation in male and female mice. Within sex groups no significant difference in lethality at 30 or 100 days postirradiation was found between WR-2721 or WR-151327 pretreatment. The dose modification factors (DMFs) for male mice treated with either compound were 1.29 (LD50/30) and 1.24 (LD50/100), and those for drug-treated female mice were 1.21 (LD50/30) and 1.19 (LD50/100). Both WR-2721 and WR-151327 were found to be equally radioprotective when compared using DMFs as the end point. WR-151327 (500 mg/kg, ip) was found to be significantly more toxic to both male and female B6D2F1 mice than equimolar amounts of WR-2721. Small but significant sex differences in radioprotection were found: the DMFs for female mice pretreated with either compound were lower than those for similarly treated male mice; the incidence of mortality 31-100 days postexposure in male mice pretreated with WR-151327 was greater than for female mice. In addition, sex differences were noted in drug toxicity. Toxic death in female mice given WR-151327 (500 mg/kg, ip) is 2.6 times more probable than in males.     

Radioprotective effects of the thiols GSH and WR-2721 against X-ray-induction of micronuclei in erythroblasts

The frequency of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow and peripheral blood of adult male Swiss mice treated with reduced glutathione (GSH) and S-2-/3-aminopropylamino/ethyl phosphorothioic acid (WR-2721), at a dose of 400 mg/kg body weight, and exposed to 6 Gy X-rays. GSH or WR-2721 was applied alone, or 60 and 30 min, respectively, prior to X-ray-exposure. The number of MNPCEs was determined at 24 h after the thiol treatment and X-irradiation. The radioprotection and toxicity caused in the mouse erythroblasts by GSH and WR-2721, as indicated by the number of MNPCEs were dependent on the thiol applied. The stronger radioprotective effect is obtained following WR-2721 administration than after GSH application. WR-2721 showed greater toxicity than GSH. The combination of GSH and WR-2721 given before X-ray-exposure resulted in the most radioprotective effect as compared to the respective single-drug treatment of mice. Application of the both thiols, without subsequent X-irradiation appeared to be the most toxic, compared with administration of WR-2721 or GSH alone. The effective radioprotection by the combined action of GSH and WR-2721 against genomic instability induced in the mouse erythroblasts by X-rays was shown.     

Opposite effects of WR-2721 and WR-1065 on radiation-induced hypothermia: possible correlation with oxygen uptake

Ionizing radiation induces hypothermia in guinea pigs. While systemic injection of the radioprotectant S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) did not block hyperthermia induced by exposure to 10 Gy of gamma radiation, central administration did attenuate it. The dephosphorylated metabolite of WR-2721, N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065), accentuated radiation-induced hypothermia by both routes of administration. In brain homogenates, oxygen uptake was inhibited by WR-2721 but elevated by WR-1065. These results suggest that the antagonism of radiation-induced hypothermia found only after central administration of WR-2721 is due to its direct actions and not to its dephosphorylated metabolite and that this effect may be correlated with the inhibition by WR-2721 of oxygen uptake.     

Radioprotective effect of combinations of WR-2721 and mercaptopropionylglycine on mouse bone marrow chromosomes

The radioprotective and toxic effects of low to moderate doses of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721) and its combination with mercaptopropionylglycine (MPG, 20 mg/kg body wt) on the chromosomes of the bone marrow cells of Swiss albino mice were studied at 24 h and 14 days postirradiation. Significant protection against radiation-induced chromosome aberrations was observed with 50 mg/kg WR-2721. The protection increased with the dose of the drug administered, and the degree of protection per unit dose increment was more pronounced at lower than at higher doses. A combination of WR-2721 and MPG given before exposure resulted in a significantly greater number of normal metaphases at 24 h postirradiation compared to the respective single-drug treatment groups. On Day 14 postirradiation, when the presence of WR-2721 resulted in an increase in the frequency of aberrant cells, combination with MPG helped to reduce this value markedly, especially at WR-2721 doses below 200 mg/kg. On the basis of these results it is suggested that 150 mg/kg WR-2721 may be considered an optimum dose for combination with MPG for protection of chromosomes of bone marrow cells when repeated drug administrations are not needed. Changes in the level of glutathione (GSH) in the blood were studied at different times following the administration of 150 mg/kg WR-2721 and its combination with MPG (20 mg/kg) before sham irradiation or exposure to 4.5 Gy 60Co gamma rays. The results showed that WR-2721 elevated blood GSH levels significantly above normal values by the time radiation was delivered, while MPG did not. Glutathione appears to have an important role in the action of WR-2721, while protection by MPG may not be mediated through GSH. Injection of MPG after WR-2721 helps to maintain the higher GSH level for a longer duration compared to treatment with WR-2721 alone. It is possible that MPG delays the metabolism of GSH.     

Effect of WR-2721 on fetal development in the rat

The radioprotector WR-2721 has been shown to radioprotect all tissues studied except the central nervous system. However, it has not yet been used to radioprotect the fetus. In this study we determined that [14C]WR-2721 injected into pregnant rats quickly passed the placenta and was concentrated by the fetus. In addition, we evaluated the toxicity of WR-2721 (2.5-600 mg/kg) to pregnant rats and their fetuses during the period of major organogenesis at Days 9, 11, and 14 postconception. Pregnant animals were only slightly more (10%) sensitive (LD50, 580 mg/kg) to WR-2721 than nonpregnant cohort animals (LD50, 640 mg/kg). At concentrations of 50 mg/kg or less there was a small but statistically significant increase in fetal deaths, while at doses greater than 300 mg/kg a larger degree of fetal mortality occurred. Maximal fetal weight loss, to about 84% of control, was found at 500 mg/kg. No changes in head dimensions or gross malformations of the surviving fetuses were detected at any time or concentration. Of all the parameters measured in this study none demonstrated a predilection for any specific period of major organogenesis. The results of this study indicate that while WR-2721 demonstrates a dose-related embryotoxicity it is not teratogenic.     

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.     

Postirradiation glucan administration enhances the radioprotective effects of WR-2721

Based on murine survival studies, endogenous hemopoietic spleen colony formation (E-CFU), and recovery of bone marrow and splenic granulocyte-macrophage colony-forming cells (GM-CFC), it was demonstrated that the postirradiation administration of glucan, an immunomodulator and hemopoietic stimulant, enhances the radioprotective effects of WR-2721. LD50/30 dose reduction factors for mice treated with WR-2721 (200 mg/kg approximately 30 min before irradiation), glucan (250 mg/kg approximately 1 h after irradiation), or both agents were 1.37, 1.08, and 1.52, respectively. Enhanced survival in mice treated with both agents appeared to be due in part to glucan's ability to accelerate hemopoietic regeneration from stem cells initially protected from radiation-induced lethality by WR-2721. Following a 10-Gy radiation exposure, E-CFU numbers in mice treated with saline, WR-2721, glucan, or both WR-2721 and glucan were 0.05 +/- 0.03, 6.70 +/- 1.05, 0.95 +/- 0.24, and 33.90 +/- 2.96, respectively. Similarly, bone marrow and splenic GM-CFC numbers were greater in mice treated with both WR-2721 and glucan than in mice treated with either agent alone. These results demonstrated at least additive radioprotective effects when mice were given WR-2721 prior to irradiation and glucan following irradiation. These effects appeared to depend on the sequential cell protection mediated by WR-2721 and hemopoietic repopulation mediated by glucan.     

Histopathologic effects of soluble glucan and WR-2721, independently and combined in C3H/HeN mice.

Soluble glucan, an immunomodulator, and Walter Reed (WR)-2721, a radioprotectant, increase postirradiation survival when administered before and after exposure, respectively. Combined, these agents act synergistically through WR-2721's ability to spare hematopoietic stem/progenitor cells from radiation injury and glucan's ability to subsequently stimulate spared cells to proliferate. In this study, the histopathologic effects of WR-2721 (200 mg/kg, ip) and glucan (250 mg/kg, iv), at doses capable of increasing survival in lethally irradiated mice, were evaluated in unirradiated and irradiated female C3H/HeN mice. After treatment, whole body weights and wet organ weights of liver, spleen, and kidney, as well as gross and histologic changes in these and other tissues, were monitored on Days 1, 4, 7, 11, 15, 21, and 28. Morphometric studies of splenic white and red pulps were also performed. Soluble glucan, with or without WR-2721, in unirradiated groups, was associated with splenomegaly, transient morphometrically determined perturbations of white and red pulp areas, and histologic alterations of white pulp. In irradiated mice, splenic weight loss was initially dampened in glucan groups and accompanied by morphologic and histologic changes similar to those seen in unirradiated counterparts. The subsequent rebound of splenic parameters in irradiated mice was limited to WR-2721-treated mice and was associated with hematopoietic reconstitution. Glucan, with or without WR-2721, in unirradiated groups was associated with transient hepatomegaly and associated histologic changes. Similar changes in irradiated animals were seen only in the combined treatment group.     

Transdermal absorption of radioprotectors using permeation-enhancing vehicles

Radioprotectors are not currently used clinically due to concerns regarding toxicity and uncertainties regarding tumor protection. Topical radioprotection of skin might find clinical applications with protectors such as WR-2721, but laboratory studies in which protectors have been applied in water have not been promising. We have studied the absorption of 14C-WR-2721 and [14C]cysteine dissolved in skin permeation-enhancing vehicles through the skin of hairless mice and compared the absorption to that in water. Skin concentration of WR-2721 was increased most by dimethylformamide (DMF), but only propylene glycol increased absorption as far as the dermis, as measured by plasma concentration. Skin concentration of cysteine was improved by DMF, 2-pyrrolidone (2-P), and methyl-2-pyrrolidone (M-2-P); only dimethylsulfoxide (DMSO) resulted in increased plasma levels of the protector. Pretreating skin with DMSO before application of WR-2721, irrespective of the vehicle, improved its concentration within the skin. Plasma levels were improved (10 and 12 times) only with 2-P and DMF. Therefore, by choosing the appropriate vehicle, it is possible to breach the barrier of the stratum corneum and enhance the presence of the protector in all layers of the skin.     

WR-2721, its derivatives and their radioprotective effects on mammalian cells in culture

Although it is well known that WR-2721 is very efficient in protecting mice against lethal irradiation, we could not find any radioprotective effect of WR-2721 on mouse L cells in culture. But WR-1065 alone (free SH form of WR-2721), and WR-2721 pre-incubated with mouse liver homogenate, showed radioprotective ability. It was found that mouse liver homogenate dephosphorylated WR-2721 to WR-1065. The highest WR-2721 metabolizing activity was found in mouse liver homogenate and Chang liver cell homogenate. Homogenates of human liver and kidney were also shown to possess moderate activity for metabolizing WR-2721. These results suggest to us that WR-2721 must be dephosphorylated before exerting its radioprotective effect and that this dephosphorylating activity varies with tissues. It is demonstrated therefore that mouse L cell in culture is a novel system to assess the extent of dephosphorylation of WR-2721 in various tissues.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Selenium Pretreatment Enhances the Radioprotective Effect and Reduces the Lethal Toxicity of Wr-2721

Although WR-2721, S-2-(3-aminopropylamino)ethylphosphorothioic acid, is an effective radioprotector, its use is limited by its toxicity. Combining WR-2721 with other agents might decrease its toxicity and/or increase its effectiveness. The effect of selenium (Se) pretreatment on the acute toxicity and radioprotective effect of WR-2721 was studied in male CD2F1 mice. Injection of 1.6mg/kg Se 24 hr before WR-2721 (800-1200 mg/kg, IP) decreased the lethality of WR-2721 significantly. Lower doses of Se were also effective, but simultaneous administration was not effective. Se injection alone (1.6 mg/kg) 24 hr before cobalt-60 irradiation increased the survival (dose reduction factor, DRF = 1.1) significantly. A synergistic effect on post-irradiation survival was observed when Se was injected 24 hr before WR-2721 (200-600 mg/ kg IP before irradiation). For example, after exposure to 22 Gy (1 Gy/min), 30-day survival was 100% when mice were treated with both Se and 600mg/kg WR-2721, and was 13% with WR-2721 alone. The DRF after 400 mg/kg WR-2721 was 2.6 with Se compared to 2.2 without Se pretreatment. Alkaline phosphatase activity in bone marrow cells and serum was significantly depressed after treatment with 1.6 mg/kg Se, suggesting that a retardation of conversion of WR-2721 to its active free sulfhydryl form through the action of alkaline phosphatase might be partly responsible for the effects of Se. Other possible mechanisms related to the antioxidant properties of Se are under investigation.     

Induction of micronucleated erythrocytes by MEA,AET, WR-2721 and X-rays

The induction of micronucleated polychromatic erythrocytes (MNPCEs) was assessed in the bone marrow of adult male Swiss mice treated with MEA (cysteamine HCl), AET (2-aminoethylisothiouronium Br.HBr), or WR-2721 (S-2-(3-aminopropylamino)ethyl phosphorothioic acid), at a dose of 200 mg/kg body weight, and/or exposed to 6 Gy X-rays. MEA, AET, or WR-2721 was given alone or 15 min prior to X-ray exposure, and the frequency of MNPCEs was determined 24 h after the aminothiol treatment and X-irradiation of mice. A genotoxic effect was shown for MEA, AET, WR-2721, and X-rays, as well as a protective effect of the aminothiols against X-ray-induced genotoxicity in the mouse erythropoietic system. The aminothiol drugs given alone, without subsequent X-irradiation, elevated the frequency of MNPCEs, and WR-2721 appeared to be less toxic than AET and MEA. After exposure of mice to X-rays, the number of MNPCEs was distinctly increased. MEA, AET, or WR-2721 administration prior to X-irradiation resulted in a reduction of the X-ray-induced elevation of the frequency of micronuclei, but a stronger radioprotective effect was obtained following WR-2721 and AET treatment than after MEA application. So, the genotoxic and radioprotective effect of the aminothiols was dependent on the compound applied.     

Inhibitory effects of WR-2721 and cysteamine on tumor initiation in mammary glands of pregnant rats by radiation

We evaluated the effect of WR-2721 [S-2-(3-aminopropylamino)-ethylphosphorothioic acid] and cysteamine (2-mercaptoethylamine) on the development of radiation-induced mammary tumors in rats. Pregnant rats were treated with WR-2721 or cysteamine 30 min prior to whole-body irradiation with gamma rays from a (60)Co source at a dose of 1.5 or 2.6 Gy. Additional pregnant rats were given saline and then exposed to gamma rays at a dose of 0, 1.5 or 2.6 Gy as a control. All rats were implanted with pellets of diethylstilbestrol, a tumor promoter, 1 month after termination of nursing and were observed for 1 year to detect palpable mammary tumors. No mammary tumors developed in the saline-injected nonirradiated rats. However, when rats were irradiated with 1.5 or 2. 6 Gy after saline treatment, the incidence of mammary tumors was high (71.4 and 92.3%, respectively). Administration of WR-2721 or cysteamine prior to irradiation with 1.5 Gy significantly decreased the tumor incidence (23.8 and 20.8%, respectively). Tumor prevention by either agent was less effective at the higher dose. The appearance of the first mammary tumor occurred later in rats treated with WR-2721 or cysteamine than in the control rats. An increasing rate of adenocarcinoma in the control group was observed with increasing dose from 1.5 Gy up to 2.6 Gy. However, the development of adenocarcinoma did not increase after pretreatment with WR-2721 or cysteamine in rats irradiated with 2.6 Gy. Many of the mammary tumors that developed in the control rats were of the ER(+)PgR(+) type. Administration of WR-2721 produced no tumors of the ER(+)PgR(+) type. Cysteamine treatment increased the development of ER-negative tumors. The serum concentration of progesterone was significantly higher in rats treated with WR-2721 or cysteamine than in the control rats. On the other hand, the estradiol-17beta concentration was reduced by treatment with WR-2721, but not significantly compared to the control. WR-2721 and cysteamine had no effect on the prolactin concentration of the irradiated rats. The results suggest that administration of WR-2721 or cysteamine prior to the irradiation has a potent preventive effect on theinitiation phase during mammary tumorigenesis.     

Protection of mice against mixed fission neutron-gamma (n: gamma = 1:1) irradiation by WR-2721, 16,16-dimethyl PGE2, and the combination of both agents

The survival of mice after whole-body exposure to a modified fission neutron-gamma field (n: gamma = 1:1) was used to examine radiation protection by WR-2721, 16,16-dimethyl PGE2(DiPGE2), and the combination of both agents. Administration of WR-2721 (453 mg/kg) increased the LD50/30 from 5.24 to 7.17 Gy (DMF = 1.37), whereas pretreatment with DiPGE2 (1.6 mg/kg) increased the LD50/30 to 5.77 Gy (dose modification factor (DMF) = 1.10). The combination of 453 mg/kg WR-2721 and 0.4 mg/kg DiPGE2 resulted in an LD50/30 of 7.33 Gy, yielding a DMF of 1.39. However, no significant difference in protection was obtained with the combination of the two agents compared to that seen with WR-2721 alone.     

Radiation protection of murine intestine by WR-2721, 16,16-dimethyl prostaglandin E2, and the combination of both agents

The survival of murine intestinal clonogenic cells (ICC) and the survival of mice after whole-body exposure to 137Cs irradiation were used to measure radiation protection by ethiophos (WR-2721), 16,16-dimethyl prostaglandin E2, and the combination of the two. Doses from 2 to 12.5 mg/mouse of WR-2721 increased cell survival linearly from 3.2 +/- 0.3 in controls given 15.0 Gy to 93.1 +/- 5.2 per jejunal circumference. In contrast, 16,16-dm PGE2 increased ICC survival at 15.0 Gy rapidly from 1 to 10 micrograms/mouse, followed by a plateau up to 100 micrograms/mouse. Animal survival at 6 days (LD50/6) increased from 16.3 +/- 0.4 Gy (95% confidence limits) in controls to 20.3 +/- 0.6 Gy in the PG-treated animals. WR-2721 increased the LD50/6 to 26.1 +/- 1.4 Gy. The dose modification factors were 1.25 and 1.60, respectively. The combination of agents increased ICC survival above that seen with each agent alone up to 8 mg WR-2721, above which no additional protection was seen. Animals given 10 micrograms PG plus 10 mg WR-2721 survived longer than with either agent given alone. The LD50/6 was 36.3 +/- 1.8 Gy for a dose modification factor (DMF) of 2.23. In addition, the slope of the probit curve was reduced from those of each agent alone. PG-induced changes in villus epithelial cell morphology and survival may account, in part, for these observations. The results suggest that either the mechanisms for these two types of radiation protectors are different or they act on separate subcellular targets which are critical to survival from radiation injury.     

Transdermal absorption of radioprotectors in the rat using permeation-enhancing vehicles

Topical radioprotection of rat skin with WR-2721 has not been effective presumably because the drug does not cross the stratum corneum to reach the epidermis and dermis. Earlier, we showed in the mouse that WR-2721 and cysteine dissolved in permeation-enhancing vehicles passed through the skin more readily than when in water. However, the most effective vehicles in the mouse were not necessarily as effective in the rat. Here we report that the most effective transport vehicles in the rat were (1) water with WR-2721, (2) water and dimethylformamide (DMF) with cysteine, and (3) water and DMF with prostaglandin E2 (PGE2). Pretreatment of the skin with dimethylsulfoxide (DMSO) further improved the transfer of the radioprotectors across the skin in most cases. After pretreatment with DMSO, the most effective vehicles were (1) water for WR-2721, (2) water and methyl-2-pyrrolidone (M-2-P) for cysteine, and (3) DMF for PGE2.     

Immunocytochemical analysis of apoptotic bone marrow cells after treatment of mice with WR-2721 and chemotherapeutic drugs

The effects of the aminothiol WR-2721 (Amifostine) and the chemotherapeutic drugs, cyclophosphamide (CP) and cisplatin (CDDP) on induction of apoptosis in bone marrow cells of adult male Swiss mice were studied. The mice received intraperitoneal injections of WR-2721 (400 mg/kg), cyclophosphamide (200 mg/kg), and cisplatin (10 mg/kg). WR-2721 was administered alone, or 30 min before treatment of mice with CP or CDDP. The number of apoptotic bone marrow cells was determined at 7 h and 24 h after the agent(s) administration. The In Situ Cell Death Detection Kit, AP based on TUNEL technique, and Fast Red Substrate System were used for microscopic analysis of immunocytochemically stained apoptotic cells. Application of cyclophosphamide and cisplatin resulted in a distinct increase of the number of apoptotic cells in the mouse bone marrow. After treatment of mice with WR-2721 prior to administration of CP or CDDP, as compared to the chemotherapeutic treatment only, the tendency to a decrease--albeit statistically insignificant--in the number of apoptotic cells was observed. Application of WR-2721 alone, without subsequent administration of the chemotherapeutic agents caused an inconsiderable increase in the number of apoptotic cells. The degree of apoptotic DNA cleavage in cells of the mouse bone marrow varied depending on the agent(s) given and the time interval after the drug administration.     

A comparison of radioprotection from three neutron sources and 60Co by WR-2721 and WR-151327

Two thiophosphoroate radiation protectors (WR-2721 and WR-151327) were assessed for their ability to modify the effects of neutron or gamma irradiation on the gastrointestinal tract. Three neutron sources (DOSAR, JANUS, and FERMILAB) were compared to the response obtained after 60Co irradiation. The end points studied were intestinal stem cell survival and LD50(6). DOSAR and JANUS, both fission-spectrum neutrons, showed somewhat different gut sensitivities [LD50(6)] of about 240 and 400 cGy respectively. The intestinal LD50 obtained with FERMILAB neutrons (25 meV) was closer (875 cGy) to that obtained after 60Co (1068 cGy) irradiation. WR-151327 protected against the lethal effects of fission neutron (DOSAR and JANUS) to a greater degree (DMF = 2.2) than with lower LET sources such as FERMILAB neutrons (DMF = 1.7) or 60Co (DMF = 1.7). The results did not correlate with the intestinal stem cell assays where WR-2721 when compared to WR-151327 showed either similar (DOSAR; fission spectrum neutrons) or somewhat better (60Co and FERMILAB neutrons) protection. Possible explanations for the differing results are discussed.