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Purpose: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect., Methods and Materials: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined., Results: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes., Conclusions: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

Purpose: To determine the role of RLIP76 in providing protection from radiation and chemotherapy. In the present report, we used RLIP76 to refer to both the mouse (Ralbp1) and the human (RLIP76) 76-kDa splice variant proteins (RLIP76) for convenience and to avoid confusion. In other reports, Ralbp1 refers to the mouse enzyme (encoded by the Ralbp1 gene), which is structurally and functionally homologous to RLIP76, the human protein encoded by the human RALBP1 gene., Methods and Materials: Median lethal dose studies were performed in RLIP76(-/-) and RLIP76(+/+) C57B mice after treatment with a single dose of RLIP76 liposomes 14 h after whole body radiation. The radiosensitivity of the cultured mouse embryonic fibroblasts and the effects of buthionine sulfoximine (BSO), amifostine, c-jun N-terminal kinase (JNK), protein kinase B (Akt), and MAPK/ERK kinase (MEK) were determined by colony-forming assays. Glutathione-linked enzyme activities were measured by spectrophotometric assays, glutathione by dithiobis-2-nitrobenzoic acid (DTNB), lipid hydroperoxides by iodometric titration, and aldehydes and metabolites by thiobarbitauric acid reactive substances and liquid chromatography-mass spectrometry (LCMS)., Results: RLIP76(-/-) mice were significantly more sensitive to radiation than were the wild-type, and RLIP76 liposomes prolonged survival in a dose-dependent manner in both genotypes. The levels of 4-hydroxynonenal and glutathione-conjugate of 4-hydroxynonenal were significantly increased in RLIP76(-/-) tissues compared with RLIP76(+/+). RLIP76(-/-) mouse embryonic fibroblasts were markedly more radiosensitive than RLIP76(+/+) mouse embryonic fibroblasts, despite increased glutathione levels in the former. RLIP76 augmentation had a remarkably greater protective effect compared with amifostine. The magnitude of effects of RLIP76 loss on radiation sensitivity was greater than those caused by perturbations of JNK, MEK, or Akt, and the effects of RLIP76 loss could not be completely compensated for by modulating the levels of these signaling proteins., Conclusion: The results of our study have shown that RLIP76 plays a central role in radiation resistance.

Purpose: The purpose of this study was to determine by using (99m)Tc-diethylenetriaminepentaacetic acid (DTPA) lung scintigraphy whether amifostine given before irradiation protects alveolocapillary integrity in a rabbit model., Methods and Materials: Twenty white New Zealand rabbits were randomly divided into 4 groups: (1) control (CONT), (2) amifostine alone (AMF), (3) radiation (RAD), and (4) radiation plus amifostine (RAD+AMF). The AMF and RAD+AMF groups received amifostine. The RAD and RAD+AMF groups were irradiated to the right hemithorax with a single dose of 20 Gy using a (60)Co treatment unit. Amifostine (200 mg/kg) was given i.p. 30 min before irradiation. The (99m)Tc-DTPA radioaerosol study was performed 14 day after irradiation., Results: The mean clearance rate of (99m)Tc-DTPA in control subjects was 140 +/- 21 min. The highest t((1/2)) value was noted in the RAD group (603 +/- 105 min, p = 0.001). There were no significant differences between the (99m)Tc-DTPA lung clearance rates of the CONT, RAD+AMF (238 +/- 24 min), and AMF groups (227 +/- 54 min). The mean penetration index values of CONT, RAD, AMF, and RAD+AMF are 63% +/- 1.6%, 63% +/- 2.5%, 60% +/- 2.9%, and 63% +/- 2%, respectively., Conclusions: We concluded that amifostine treatment before the lung irradiation protects the lung alveolocapillary integrity. This study confirms the protective effect of amifostine in an acute phase of radiation lung injury.

Purpose: Amifostine is a prodrug that requires dephosphorylation by alkaline phosphatase to become activated. This process occurs rapidly within the bloodstream after its i.v. administration to patients undergoing cancer treatment with selected radiation and chemotherapies. Vascular endothelial cells will, therefore, represent a normal cell system that is among the first to experience the radioprotective effects of this agent. Amifostine's active free thiol WR-1065 was investigated to determine its effect on radiation-induced changes in transcriptional patterns and subsequent apoptosis in human microvascular endothelial cells (HMEC) growing in vitro., Methods and Materials: Human microvascular endothelial cells were grown to confluency and then exposed to WR-1065 at a concentration of 4 mM for 30 min, radiation doses that ranged from 0 to 6 Gy, and WR-1065 at a concentration of 4 mM for 30 min before exposure to ionizing radiation. Cell survival was assessed by clonogenic assay, cell cycle phase was analyzed by flow cytometry, apoptosis was also assessed by flow cytometry in which Anexin V staining and sub-G1 fraction analysis were applied, and gene expression was analyzed by the Clontech Atlas Human cDNA array to identify synergistic and antagonistic effects as a function of amifostine and radiation exposure conditions with a focus on apoptotic-related factors., Results: Exposure of HMEC to 4 mM WR-1065 30 min before irradiation resulted in a protection enhancement factor of 2.0; that is, D(O-IRR) of 1.25 Gy and D(O-IRR+WR) of 2.56 Gy. Expression profiling revealed 29 genes that were synergistically activated by the combined action of WR-1065 and ionizing radiation, and an additional 12 genes were synergistically or additively suppressed. In particular, a subset of apoptosis-related genes that included caspases 2, 4, and 9 and different members of the bcl family, along with apoptosis-related receptors, were identified as being significantly affected by the combined treatment of WR-1065 and radiation exposure. In addition, a number of cell cycle-related genes that express cyclins A, G1, G2, and D3 and DNA damage/check point proteins ATM, DNA-PK and RAD23B were also found to be significantly affected. Functional assays of apoptosis were also performed that demonstrated the ability of WR-1065 to protect against radiation-induced apoptosis., Conclusions: WR-1065, the active thiol form of amifostine, is an effective radioprotector of HMEC as determined by use of clonogenic and apoptotic assays for cell survival. Expression profiling successfully defined the transcriptional response of HMEC to both WR-1065 and ionizing radiation exposure, either alone or in combination, and demonstrated both synergistic and antagonistic effects on the expression of different cellular genes, along with corresponding functional responses. The radioprotective effects of amifostine are not limited to its well-characterized physiochemical properties, which include free-radical scavenging, auto-oxidation leading to intracellular hypoxia, and chemical repair by hydrogen atom donation, but include its ability to modulate the complex transcriptional regulation of genes that are involved in apoptosis, cell cycle, and DNA repair.

Purpose: To evaluate the protective learning and memory effect of Ethyol in irradiated young rats., Methods and Materials: One hundred twenty-eight 45-day-old Wistar rats received whole brain fractionated radiation (30 Gy), whereas 48 rats received sham irradiation. Four irradiated subgroups were defined: saline, 37.5 mg/kg, 75 mg/kg, and 150 mg/kg Ethyol. Sequential behavioral studies including one-way and two-way avoidance tasks were undertaken before and after radiation., Results: Before radiation, the performances of all groups were similar. For the one-way avoidance task, at 1, 3, and 6 months postradiation, saline-irradiated rats had a lower percentage of avoidance than sham- or Ethyol- (75 or 150 mg/kg) irradiated rats (p

Purpose: Clinical symptomatic late injury to the rectal wall occurs in about one-third of patients with prostate cancer treated with external beam irradiation. Reducing the physical dose to the anterior rectal wall without a similar reduction in the posterior peripheral zone is difficult because of the proximity of the prostate to the anterior rectal wall. On the basis of our previous observations in an animal model that intrarectal application of amifostine resulted in very high concentrations of amifostine and its active metabolite WR-1065 in the rectal wall, a Phase I dose-escalation clinical trial was undertaken., Methods and Materials: Twenty-nine patients with localized prostate cancer were accrued. Eligibility criteria included histologically confirmed adenocarcinoma, Karnofsky performance status >or=70, and no pelvic lymphadenopathy or distant metastases. The total dose to the prostate was 70.2 Gy in 20 patients and 73.8 Gy in 9 patients. Therapy was delivered using a 4-field technique with three-dimensional conformal planning. Amifostine was administered intrarectally as an aqueous solution 30 min before irradiation on the first 15 days of therapy. Amifostine was escalated in cohorts from 500 to 2500 mg. Proctoscopy was performed before therapy and at 9 months after completion. Most patients underwent repeat proctoscopy at 18 months. On Days 1 and 10 of radiotherapy, serum samples were collected for pharmacokinetic studies. The clinical symptoms (Radiation Therapy Oncology Group scale) and a proctoscopy score were assessed during follow-up., Results: All patients completed therapy with no amifostine-related toxicity at any dose level. The application was feasible and well tolerated. No substantial systemic absorption occurred. With a median follow-up of 26 months, 9 patients (33%) developed rectal bleeding (8 Grade 1, 1 Grade 2). At 9 months, 16 and 3 patients developed Grade 1 and Grade 2 telangiectasia, respectively. This was mostly confined to the anterior rectal wall. No visible mucosal edema, ulcerations, or strictures were noted. No significant differences were found between the proctoscopy findings at 9 and 18 months. Four patients (14%) developed symptoms suggestive of radiation damage that, on sigmoidoscopy, proved to be secondary to unrelated processes. These included preexisting nonspecific proctitis (n = 1), diverticular disease of the sigmoid colon (n = 1), rectal polyp (n = 1), and ulcerative colitis (n = 1). Symptoms developed significantly more often in patients receiving 500-1000 mg than in patients receiving 1500-2500 mg amifostine (7 [50%] of 14 vs. 2 [15%] of 13, p = 0.0325, one-sided chi-square test)., Conclusion: Intrarectal application of amifostine is feasible and well tolerated. Systemic absorption of amifostine and its metabolites is negligible, and close monitoring of patients is not required with rectal administration. Proctoscopy is superior to symptom score as a method of assessing radiation damage of the rectal wall. The preliminary efficacy data are encouraging, and further clinical studies are warranted.

Purpose: Amifostine has been approved as a therapy to decrease the incidence of moderate-to-severe xerostomia in patients undergoing postoperative radiation treatment for head-and-neck cancer. As a reducing agent capable of participating in intracellular reductive/oxidative processes, it has the potential to affect redox-sensitive transcription factors and gene expression. Amifostine's active free thiol WR-1065 was investigated to determine its effect on nuclear transcription factor kappaB (NFkappaB) activation and subsequent gene expression in U87 glioma cells., Methods and Materials: The human glioma cell line U87 was grown to confluency and then exposed to WR-1065 at a concentration of 40 microM for times ranging from 30 min to 24 h. Changes in cell cycle were monitored by flow cytometry. The effect of WR-1065 on NFkappaB activation was determined by a gel shift assay. Changes in gene expression as a function of time of exposure to WR-1065 were determined by Northern blot and the Atlas Human cDNA Expression Array (Clontech, Palo Alto, CA). Changes in gene expression using the Atlas Array were verified by reverse transcriptase-polymerase chain reaction (RT-PCR) with gene-specific primers., Results: Exposure of U87 cells to 40 microM WR-1065 resulted in a marked activation of NFkappaB between 30 min and 1 h after treatment. Expression of MnSOD, an NFkappaB-responsive gene, was enhanced by over 2-fold after 16 h of treatment and remained elevated at 24 h. During this period of time, no changes in cell cycle distribution were observed. To assess changes in the expression levels of NFkappaB-responsive genes as a function of WR-1065 exposure, cDNA arrays containing 49 genes identified as having DNA-binding motifs for NFkappaB were used. Only five genes were found to be significantly affected at 1, 4, and/or 16 h of treatment. GST-3 and c-myc were repressed up to 2- and 4-fold, respectively. The expression levels of IL-2Ra, RANTES, and c-myb, in contrast, were enhanced up to 14-, 3-, and 2-fold, respectively. The remaining genes having NFkappaB-responsive elements in their promoter regions were either not expressed (20 genes) or were not affected (24 genes) by exposure to WR-1065., Conclusions: The redox-sensitive transcription factor NFkappaB can be activated in U87 glioma cells by the active thiol form of the cytoprotector amifostine. Activation of NFkappaB by the antioxidant WR-1065 is accompanied by a reduced expression of the oncogene c-myc and an enhanced expression of the antioxidant gene MnSOD, a gene whose expression in tumor cells is relatively low, but when overexpressed has been correlated with a suppression of the malignant phenotype. Activation of NFkappaB by WR-1065, however, results in selective rather than global changes in the expression of genes containing NFkappaB-responsive elements.

The aim of this study was to determine the independent and combined effects of 100 mg/kg and 200 mg/kg doses of the radioprotectant amifostine and radiotherapy dose fractionation in preserving the integrity of or minimizing damage to the physis during high-dose radiation exposure in an animal model. Thirty-six weanling four-week-old male Sprague-Dawley rats were randomized into six study groups of six animals each. The distal femur and proximal tibia in the right leg of each animal was exposed to X-irradiation, with the contralateral left leg serving as the nonirradiated control. Three groups received a single 25 Gy radiotherapy dose: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Three groups received a total of 25 Gy in three equal fractions: one group alone, a second group preceded by 100 mg/kg amifostine, and a third preceded by 200 mg/kg amifostine. Fractionation of the 25 Gy radiation dose reduced the mean percent overall limb growth loss to 44.8%, a statistically significant reduction compared to a mean 58.8% reduced growth with the single 25 Gy dose. Addition of amifostine at 100 and 200 mg/kg before each of the three fractions of radiotherapy further decreased the mean percent overall limb growth loss to 35.2% and 28.5%, respectively, both statistically significant reductions beyond that achieved by fractionation alone.

Purpose: The purpose of this study was to determine the relative benefits of sparing longitudinal bone growth by fractionation alone compared to pretreatment with amifostine, a chemical that provides differential radioprotection of normal tissues., Methods and Materials: Twenty-four weanling 4-week-old male Sprague-Dawley rats were randomized into 2 overall treatment groups: fractionation alone (n = 12) and amifostine pretreatment (n = 12). The distal femur and proximal tibia in the right leg of each animal were exposed to a therapeutic X-irradiation dose (17.5 Gy total in 3 or 5 fractions) with the contralateral left leg as control. In 12 of the animals, amifostine (100 mg/kg) was administered intraperitoneally 20 min before radiation exposure. Six weeks later, growth was calculated based upon measurement of the bone lengths., Results: Fractionated radiation resulted in a mean percent overall limb growth loss of 21. 1 +/- 7.0%. The addition of amifostine brought the mean percent overall limb growth loss to 16.3% +/- 4.6%, which showed a strong trend toward significance compared to fractionation alone (p = 0. 061). The addition of radioprotection with amifostine to 5 fractions irradiation significantly reduced the femoral and overall percentage growth arrest and limb length discrepancy compared to 5 fractions alone., Conclusions: These results support further investigation of amifostine and other radioprotectants in combination with fractionation for use in growing children requiring radiotherapy to the extremity for malignant tumors.

The effects of the radiosensitizer misonidazole (MISO) and the radioprotector WR-2721 on radiation-induced carcinogenesis in C3Hf/Kam mice were investigated. The right hind legs were exposed to graded single doses of gamma-rays. MISO and WR-2721 were given i.p. 30 min before irradiation at a dose of 1 mg/g and 0.4 mg/g, respectively. The RCD50, or radiation dose inducing tumors in 50% of the irradiated legs, was determined 650 days after treatment. The same animals were also checked for the effect of these drugs on hair loss and radiation-induced leg contractures. MISO enhanced radiation carcinogenesis by a factor of 1.43, whereas WR-2721 reduced it by a factor of 1.75. These effects on carcinogenesis correlated well with the modifying effects of the two agents on radiation-induced hair loss (early damage) and leg contractures (late damage).

The radioprotectant WR 2721 administered intraperitoneally to rats at 30--40 min before exposure to 1000 rad of 60Co caused a reduction in spleen lysosomal enzyme activity and fluctuations in cyclic nucleotide levels. Liver and spleen cAMP levels from drug-treated animals were higher at 3--6 hr postirradiation whereas spleen cGMP levels were lower at 1--4 hr postirradiation than irradiated controls not given the radioprotectant. At 3 days after radiation exposure, spleen cyclic nucleotide levels and lysosomal enzyme activities in rats given WR 2721 were closer to the level of nonirradiated control rats than of irradiated rats receiving saline.

The radioprotective effect of WR-2721 has been studied in mouse lung after single doses of radiation. Using the breathing rate assay and lethality, radioprotection was assessed at monthly intervals between 3 and 18 months after irradiation during both pneumonitis and chronic fibrosis. The degree of radioprotection was greater for fibrosis than for pneumonitis using both assays. In replicate experiments, dose modifying factors (DMF's) ranging from 1.2 to 1.4 were obtained for pneumonitis and 1.5 and 1.6 for fibrosis. The differences in DMF's for the two phases of lung damage were significant. A difference in the time course of expression of damage was seen in both the breathing rate and lethality assays between mice irradiated with and without WR-2721: the damage ended sooner in the drug-treated mice. This difference is best explained by protection of all damage after 5 months by WR-2721. No evidence of drug toxicity was found. We conclude that WR-2721 protects against chronic lung fibrosis caused by radiation at least as well as against the earlier appearing pneumonitis after single doses of radiation. Thus, if WR-2721 is dose modifying and if late tissue complications are dose limiting in clinical radiotherapy, then a therapeutic benefit would be obtained by the use of this drug in clinical radiotherapy, provided that the radioprotection of tumors did not exceed a factor of 1.5-1.6.

The radioprotection of normal cells with WR-2721 at doses of radiation extending down to less than 1 Gy was investigated using testicular cells. Survival of stem spermatogonia after single doses of radiation was measured by counts of repopulating tubules and by sperm head counts, with consistent results obtained for both endpoints. Protection factors (PF) obtained by injection of 400 mg/kg WR-2721 at 15 min prior to irradiation decreased from about 1.4 at radiation doses above 10 Gy to 1.0 at 2 Gy. Similarly, the radioprotection by 300 mg/kg WR-2721 was reduced from a PF of about 1.35 when the drug was given prior to a single high dose of radiation to 1.0-1.1 when the drug was given prior to each of 5 daily fractions of 2 Gy. Thus, less protection of testicular stem cells by WR-2721 was observed at lower doses of radiation. This lowered protection may be explained, at least in part, by a direct cytotoxic effect of WR-2721 on testicular stem cells. Protection of differentiated spermatogonia was observed with 400 mg/kg WR-2721; the PF was 1.4 at 1 Gy and decreased at lower doses. The protection of testicular function by WR-2721, as assayed by the return of fertility and the maximum recovered level of sperm production, was compared to the protection of stem cell survival. At about 8 Gy the PF with 400 mg/kg WR-2721 for both functional endpoints was about 1.5, which was not significantly different from the value of 1.3 obtained using the stem cell assays.

The protective effect of WR-2721 on nitrogen mustard (HN2) cytotoxicity against hematopoietic stem cells as well as the potentiation effect previously noted against leukemia cells was further examined for dose and interval dependency for these opposite effects. For AKR leukemia cells, a dose dependent potentiation of HN2 cytotoxicity was noted with WR-2721. The magnitude of the potentiation was about 10-fold with 5 mg/mouse WR-2721 and nearly 100-fold at 10 mg/mouse. Interval studies showed maximum potentiation occurred when WR-2721 was given close in time to HN2 and decreased in magnitude as the interval between the agents increased. However, a pronounced potentiation of cytotoxicity was also found when HN2 followed WR-2721 by 4 to 12 hr. For hematopoietic stem cells, a protective effect of WR-2721 on HN2 cytotoxicity was observed. This effect decreased rapidly with interval between the agents with no protection noted for an interval of 6 hr. These results indicate that agents like WR-2721 may have an important therapeutic role in chemotherapy.

The radioprotective effect of WR-2721 has been studied in two murine tumors using single doses or five daily fractions. Single dose irradiations of the SA FA resulted in a highly variable radio-protective response. In one experiment large protection factors (1.2-2.5) were obtained, with the greatest protection at low X ray doses. In later experiments with the same tumor, there was little or no radioprotection. The Ca MT was significantly protected against single dose irradiation with both 250 mg/kg and 400 mg/kg of the drug. In a five fraction schedule the extent of radioprotection for CA MT was greater than with single X ray doses for the same drug dose per fraction. Tumor protection factors from the present work and from the literature are compared with published protection factors for normal tissues. Significant tumor radioprotection is seen in most studies. The data indicate more variability in the extent of tumor protection for a given drug dose than is seen in normal tissues. Tumor protection is often greatest at low X ray doses which may be a result of preferential protection of the better-oxygenated tumor cells.

WR-2721 (S-2-(3 aminopropylamino) ethylphosphorothioic acid) has been investigated for its ability to protect gut, lung, and testis, as well as fibrosarcoma (FSa) tumor nodules, in the lungs of mice from gamma-radiation injury. This compound greatly protected jejunum and testis epithelial cells. FSa micrometastases in the lung were protected to a lesser extent than jejunum and testis. Conversely, WR-2721 was not able to protect the lung against radiation-induced enhancement of tumor metastases formation generated by intravenously injected FSa cells.

The radiation response of the mouse kidney has been assayed after a range of X ray doses given with or without the nitroimidazole misonidazole or the aminothiol WR-2721. Sensitization and protection of the kidney were investigated by comparing the X ray dose needed to achieve a particular level of injury in the presence or absence of the drug. Two functional assays and kidney weight at sacrifice were used to obtain dose response curves. Urine output and 51Chromium EDTA excretion were used as functional assays at 25 and 49 weeks after irradiation. They demonstrated no radio-sensitization by misonidazole with 1, 2 or 5 fractions of X rays. Significant radioprotection was seen when 400 mg kg 1 WR-2721 was given before single X ray doses (PF = 1.34). Similar radioprotection was observed when renal weight at 1 year after irradiation was used as the third assay of damage. These results confirm that the kidney responds as a well-oxygenated normal tissue with only a small protection being afforded against radiation injury by WR-2721.

Experiments have been carried out to compare the toxicity and radioprotective effect of sodium hydrogen-S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913) with those of S-2-(3-aminopropylamino)ethyl phosphorothioic acid (WR-2721). The drugs were given intraperitoneally to 12 week-old female BALB/c mice 30 minutes before whole body irradiation. Lethality at 30 days was the endpoint used. The drug LD50/30 was 678 mg/kg for WR-2721 and 3574 mg/kg for WR-77913. The LD50/30 for WR-77913 combined with 500 mg/kg of WR-2721 was 3328 mg/kg. The LD50/30 for misonidazole was 380 mg/kg when given in combination with 500 mg/kg of WR-2721 and 801 mg/kg when combined with 2200 mg/kg of WR-77913. Protection of bone marrow by WR-77913 and WR-2721 was comparable at doses close to the maximum tolerable dose (MTD, drug dose lethal to 10% of the animals at 30 days), but WR-77913 gave better protection at 35% of the MTD. These characteristics of low toxicity, non-additive toxicity with WR-2721, less toxicity in combination with misonidazole and adequate bone marrow protection at 25% of the MTD, make WR-77913 a protector worthy of further investigation.

The degree of radioprotection obtained with WR-2721 is critically dependent upon the oxygen tension of the tissue concerned. It was therefore considered of interest to examine the response of vascular tissue, which would be supposedly well oxygenated, to treatment with WR-2721 plus X rays. The response of this tissue is of interest because of its role in late radiation damage. In addition, for therapeutic gain an agent must protect normal tissue without concomitant tumor protection. However, data on tumor radioprotection have been conflicting and therefore the effect of WR-2721 on an experimental tumor was also tested. Vascular damage was assessed using the fact that tumors grow more slowly in irradiated beds (Tumor Bed Effect). The response of the RIF-1 tumor treated in vivo was measured by in vitro assay of clonogenic survival. WR-2721 (400 mg kg-1) injected 30 minutes before 5 to 30 Gy X rays protected skin stroma by a factor of 1.6. However, WR-2721 given 10 to 60 minutes before 20 Gy X rays to the RIF-1 tumor had either no effect or was protective, according to the method of immobilizing the mice during irradiation. Differences between the ways animals are physically restrained for tumor irradiation could account for the conflicting data concerning tumor radioprotection with WR-2721.

Changes in rat parotid salivary gland weight and functional parameters were evaluated at 8 to 10 days post irradiation in WR-2721 protected and non-protected animals following exposure to a single 15.3 Gy dose of Cs-137 radiation to the head. Glandular fluid secretory capacity was assessed by maximum flow rate, total volume of saliva and duration of secretion following pilocarpine stimulation. Protection against radiomucositis was also evaluated indirectly by daily monitoring of food and water intake, body weight and paraoral symptomatology. WR-2721 provided a significant degree of protection for all glandular functional parameters as well as gland weight. Relative protective factors (RPF) were computed for irradiated protected and non-protected animals compared to their sham-irradiated, pair-fed controls. The calculated RPFs were: Gland weight 1.9, maximum flow rate 2.9, volume of saliva 2.1 and duration of secretion 2.1 for a mean "relative protection" of 2.25. Substantial protection against radiomucositis in protected animals was evident by a progressive gain in body weight and lack of oral signs and symptoms as compared to non-protected animals. Protection against radiomucositis and preservation of residual parotid gland secretory capacity as determined by functional parameters suggests that WR-2721 may be of significant benefit in alleviating oral symptoms and maintaining salivary gland function for patients receiving radiotherapy for head and neck tumors.

The ability of WR-2721 to protect jejunal crypt cells after single doses and multifractionated doses of radiation was studied. Effective dose survival curves for jejunal crypt cells were constructed over the dose range of 230 to 1600 cGy. WR-2721 was given 30 minutes before each fraction, in a regimen consisting of 200 mg/kg before the first radiation fraction, followed at 3 hr intervals by 100 mg/kg for a total of 12 drug doses for the largest number of fractions. Fractionation protocols were designed with common dose fractions in regimens with different fraction numbers, allowing a test of the hypothesis of equal effect per fraction and an estimate of the initial number of clonogens per crypt in both the drug treated and non-drug treated mice. The hypothesis of equal effect per fraction could not be rejected in either the drug or non-drug treated mice. An average number of 137 clonogens per crypt was estimated for the non-drug treated mice and 81 clonogens per crypt in the drug treated mice; the difference between these two values was not significant. The protection factor decreased with decreasing dose ranging from a high of 1.47 (95% C.L. = 1.44 to 1.50) after a single dose of 2000 cGy to a low of 1.21 (95% C.L. = 1.08 to 1.37) after 200 cGy. Analysis of the data using either the linear quadratic (LQ) or two-component (TC) model of cell survival showed that WR-2721 was not dose-modifying over the dose range tested. Analysis using the LQ model showed that both beta and alpha were modified by WR-2721, by 50% and 20% respectively. These data indicate that protection by WR-2721 can be expected to decrease with dose although there is some protection after clinically relevant doses.

The ability of a radioprotector-gammaphos to modify the development of late vascular changes in the rat brain after local irradiation with 40 and 60 Gy was investigated by means of an angiographic method. The relative number of animals with gross vascular abnormalities, the size of foci with vascular lesions, and the changes in size and number of vessels were less in the drug-treated groups. Thus, it may be possible to increase the tolerance of host tissues to curative radiation therapy by chemical radioprotectors such as gammaphos.

Cellular thiols appear to have two separable mechanisms for influencing cellular radiosensitivity: 1) a direct role, through radical scavenging and/or hydrogen donation processes, and 2) an indirect role, regulating the amount of oxygen (or other electron affinic sensitizer) able to reach the radiosensitive targets of the cell. The contribution of each is easily measured with multicell spheroids, using fluorescence activated cell sorting techniques for selective recovery of cells from any depth within spheroids (i.e., from areas with different oxygenation). We have found that the region in the spheroid over which the transition from aerobic to anoxic conditions occurs is highly dependent on cellular thiol levels. Combining thiol depletion by DL-buthionine-S, R-sulfoximine (BSO) and electron affinic radiosensitization using misonidazole resulted in a markedly potentiated response to radiation, which we interpret as being primarily a result of reoxygenation.

The toxicity of radiation and alkylating agent chemotherapy is in part related to free radical formation in DNA and associated proteins, which is enhanced by oxygen and other electron affinic compounds. During the Phase I clinical trials of WR-2721 and alkylating agent chemotherapy, venous blood samples were obtained prior to and immediately following the WR-2721 infusion. We have observed a marked increase in the oxygen saturation of venous blood following WR-2721 (430-910 mg/m2 at a rate of 15 mg/m2/minute). The mean venous PO2 (PvO2) rose from a baseline of 34.0 +/- 13.54 torr to a mean value of 54.60 +/- 12.47 torr (p less than 0.001). The percent venous oxygen saturation increased from 54.52 +/- 21.38% to 82.73 +/- 7.66%. The highest values generally occurred within the first 2 hours after the completion of the WR-2721 infusion. No significant differences were found between the pre and post WR-2721 values for either the venous pH, CO2, or bicarbonate levels. Despite increased PvO2, the patients' heart rates, blood pressures, or respiratory rates did not change pre and post WR-2721. No apparent differences were noted in the effects of WR-2721 or PvO2 when given at doses of 740 or 910 mg/m2. However, when WR-2721 (418-1100 mg/m2) was administered over 15 minutes, the PvO2 increased in only 8/13 courses. We postulated that the marked increase in venous blood oxygen concentration was secondary to one of the following mechanisms: 1) Increased affinity of hemoglobin for oxygen, 2) microcirculatory shunting, or 3) decreased tissue requirements for oxygen. In 12 patients oxygen-hemoglobin dissociation curves and intracellular red cell pH's were obtained before and after 740-910 mg/m2 of WR-2721. Despite the significant increase in oxygenation of venous blood after WR-2721, there were no changes in either oxygen-hemoglobin dissociation or intracellular red cell pH to account for this increase. The oxygen consumption of peripheral blood granulocytes was measured prior to and following 15 courses of WR-2721. A marked decrease in granulocyte oxygen consumption was observed in 6/8 patients who had a significant increase in PvO2 and only 1/7 patients who had either a decrease or no change in PvO2 after WR-2721. Our preliminary investigations suggest that the increased venous blood oxygen content may be secondary to a reduction in normal tissue oxygen consumption. WR-2721 may afford protection by both decreasing oxygen consumption and delivery to normal tissues and increasing intracellular sulfhydryls.

The epidermal clone assay has been used to study the radioprotective effect of WR-2721 on mouse skin under different conditions of oxygenation and under anoxia. The skin has shown a progressive decrease in sensitivity as the inspired gas was changed from 100% oxygen towards 0% oxygen. Compared with mice breathing 100% oxygen, those breathing air are partially protected. The inspired oxygen concentration to given half the full oxygen effect is 10--12%. The radioprotection observed with 400 mg/kg WR-2721 is markedly dependent on the ambient oxygen concentration. The protection factor is 1.1 or less in mice breathing 5%, 1% of 0% oxygen. Protection is maximal (1.95) in air and in 50% oxygen and diminishes to 1.6 at higher oxygen tensions.

Tumor regrowth delay is an assay which reflects tumor cell kill but can be modified by growth rate changes resulting from damage to the stroma (tumor bed effects). If the stromal damage is modified by radiosensitizers and radioprotectors to a different degree from the tumor cells, the overall measurement of dose modifying factors may be influenced by the choice of a regrowth size for the growth delay analysis. We have studied the response of two mouse tumors; a fibrosarcoma which showed only a small TBE and a carcinoma which showed a very large TBE. Sensitizer enhancement ratios and protection factors have been obtained by assessing regrowth to a variety of endpoint sizes. The dose modifying effects on the stroma have then been determined by analyzing the regrowth rates of tumors after irradiation. The choice of endpoint size modified both the sensitizer enhancement ratio and the protection factor for both tumors. It appears that stromal damage may be the cause of the radioprotection observed in the carcinoma, whereas direct tumor cell radioprotection is indicated in the fibrosarcoma. Both direct tumor cell killing and cell death secondary to stromal damage will play an important role in determining the local control of irradiated tumors.

This paper reviews the role of oxygen in the protection of both normal tissues and tumors in vivo by WR-2721. Although the presence of hypoxic cells in tumors is well accepted, data are presented that suggest there is a range of oxygen concentrations and thus OERs in normal tissues in vivo, too, and that some may, in fact, be radiobiologically hypoxic. Thus, the range of protection factors in normal tissues (which appears unrelated to tissue drug levels), as well as tumors can perhaps be explained by the range of OERs in these same tissues. The question of whether protection decreases with radiation dose per fraction is related to the distribution of oxygen in both normal tissues and tumors, i.e., whether oxygen is homogeneously or heterogeneously distributed among the cells. It is hypothesized that sulphydryl compounds may equalize the OER differential between tumors and normal tissues and thus remove the natural advantage (radioresistance) of the tumor when treated with radiation. Thus, no loss of therapeutic benefit would occur when sulphydryl compounds were given with radiation if the normal tissue were better oxygenated than the tumor.

Although WR-2721 preferentially protects normal tissues against irradiation, it seemed desirable to find other drugs presenting a lower toxicity and the same radioprotective properties. A new compound, I 102, was selected; it was characterized on one hand by a coupling between cysteamine and an amino-acid, and on the other hand by an acetyl-group, which protects the thiol function. The effects of WR-2721 and of I 102 were studied on EMT6 tumors grafted on BALB/c mice. Whatever the size of the tumor, the cell survival increased as a function of the time elapsed between the injection of I 102 and the end of the irradiation (TI). In contrast, the radioprotection afforded by WR-2721 was found to be independent of TI. The survival curves suggest that, like WR-2721, I 102 protects essentially oxygenated cells.