Ethylphosphorothioic Acid Research Articles

Sodium hydrogen-S-(3-amino-2-hydroxypropyl) phosphorothioate (WR-77913): Toxicity and bone marrow radioprotection

Experiments have been carried out to compare the toxicity and radioprotective effect of sodium hydrogen-S-(3-amino-2-hy(iroxypropyl) 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 LD 50 30 was 678 mg/kg for WR-2721 and 3574 mg/kg for WR-77913. The LD 50 30 for WR-77913 combined with 500 mg/kg of WR-2721 was 3328 mg/kg. The LD 50 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 W R-77913 gave better protection at 25% 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`,0 of the MTD, make WR-77913 a protector worthy of further investigation.

Clinical trials of WR-2721 with radiation therapy

The radioprotector with clinical potential, S-2-(3 aminopropylamino)-ethylphosphorothioic acid (WR-2721) is undergoing two Phase I trials. The objectives of these trials are 1) to determine the maximum tolerated dose (MTD) of WR-2721 in a single dose and 2) to determine the highest dose of WR-2721 that can be tolerated daily in the greatest number of fractions per week. A total of 65 patients have been treated. The single maximum tolerated dose has not yet been reached, though 740 mg/m 2 is well tolerated. A single dose of 910 mg/m 2 has been successfully administered to one patient. The multiple dose MTD is at an early stage with patients currently receiving 170 mg/m 2 four times a week. Among the toxicities noted in both trials are hypotension, hypertension, emesis and somnolence. In addition, in the multiple dose trial there have been three patients who have had allergic reactions including one which was life-threatening. Phase II studies are planned and will begin when the maximum tolerated dose is established from each Phase I trial.

Chemical protection against gastrointestinal radiation injury in mice by WR 2822, WR 2823, OR WR 109342 after 4 MeV X ray or fission neutron irradiation

Three compounds were tested for their radioprotective properties against the effects of 4 MeV X rays or fission neutron irradiation. The endpoints tested were lethality, intestinal crypt survival, and DNA synthesizing cellularity. Two of the compounds tested; S-2(4-aminobutylamino) ethylphosphorothioic acid (WR 2822) and the aminopentylamino derivative (WR 2823) are closely related to WR 2721. The third agent was the iminothiol derivative of 1-methyl-aminoadamantine (WR 109342). All drugs were administered via intraperitoneal injections at their approximately maximum tolerated dose. WR 2822 was shown to have a slight protective effect against X rays and neutrons. The dose modification factor (DMF) for gastrointestinal death (LD 50(6)) was 1.23 for X rays and 1.51 for neutrons. The assay for intestinal crypt survival produced DMF's of 1.44 (X rays) and 1.4 (neutrons). WR 2823 also showed a protective action in these assays. The DMF for (LD 50(6)) was 1.32 (X rays) and 1.42 (neutrons). WR 109342 was found to be extremely toxic and had no significant protective effects. All three drugs were more toxic and demonstrated less protection in most of these assays than the benchmark radioprotective agent WR 2721, although WR 2822 protected against lethal effects of fission neutrons almost as well as WR 2721. Both WR 2822 and WR 2823 produced greater protection in the crypt survival assays for fission neutron irradiation than WR 2721.

Combined radiosensitization and radioprotection for oral cavity tumors: Study with an oral cavity tumor model

Adjuvant therapy with combined radioprotectors and radiosensitizers has not been extensively examined. We investigated the use of combined S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR-2721), misonidazole (Ro-07–0582) and single dose radiotherapy in treating the EMT 6/Ky tumor implanted in the buccal mucosa of Balb/c mice. Our results demonstrate that single dose irradiation plus WR-2721 or misonidazole prolonged the mean survival time (MST) of EMT 6/Ky bearing Balb/c mice significantly compared to either controls or single dose irradiation plus WR-2721 and misonidazole.

The effect of s-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR2721) on the toxicity of melphalan on normal and tumor tissue in mice

The effect of s-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR2721) on the toxicity of melphalan on normal and tumor tissue in mice

Protective effects of WR-2721 against radiation-induced injury of murine gut, testis, lung, and lung tumor nodules

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 radiationinduced indancement of tumor metastases formation generated by intravenously injected FSa cells.

Phase I trials of WR2721 in combination with radiation therapy and with the alkylating agents cyclophosphamide and cis-platinum

Three phase I trials of the radioprotector S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR2721) have accessed 60 patients. Study 1 is devised to determine the maximum tolerated dose (MTD) of a single dose of the protector 15 to 30 minutes before a single radiation treatment of a size used routinely in palliative management. Study 2 plans to determine the MTD for up to 15 daily doses of the drug over 3 weeks during palliative radiotherapy. Also, the multipe-dose study will establish the MTD before palliative irradiation for fewer than five fractions a week. Study 3 uses the existing single-dose MTD determined in Study 1 as pretreatment 15 to 30 minutes before cyclophosphamide or cis-platinum. Toxic symptoms include emesis, hypotension, hypertension, somnolence, and sneezing. Only one serious episode of hypotension, considered idiosyncratic, and one instance of moderate to severe vomiting have occurred. Forty-one patients have been entered in Study 1 and a dose of 600 mg/m2 has been reached. The next step is to proceed to the planned highest level of 740 mg/m2. Of five patients in the multiple-dose study, one has been given, without toxicity, WR2721 at the level of 100 mg/m2 for 15 fractions over 3 weeks. Fourteen patients are accessed to the alkylating agent study. Using protector doses of 450 mg/m2, a cyclophosphamide level of 1500 mg/m2 has been accomplished. However, two of three patients who received 450 mg/m2 of WR2721 before 120 mg/m2 of cis-platinum have shown moderate elevation of the serum creatinine, both of which returned to normal.

A Comparative Study of the Radioprotective Effects of Cysteamine, WR-2721, and WR-1065 in Cultured Human Cells

The compounds, 2-mercaptoethylamine (cysteamine), S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721), and N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were tested for toxicity and radioprotective effect in cultured human cells using viability (reproductive death) as an indicator. WR-2721 and WR-1065 had no harmful effect at concentrations of 4 and 10 mM for periods up to 3 hr whereas cysteamine was toxic if left in contact with the cells for more than 30 min. The response of the cells to radiation was measured after a 30-min treatment with 4 mM of each protective agent. Survival curves were constructed and the following dose reduction factors (DRFs) were calculated: WR-2721, 1.3; cysteamine, 2.3; WR-1065, 2.9. Increasing the concentration of protective agent did not increase the DRF under these conditions except in the case of cysteamine. Extending the period of treatment with drug before irradiation increased the DRF for WR-2721 and WR-1065; values up to 1.8 and 3.4 were obtained with 10 mM WR-2721 and WR-1065, respectively. With these two compounds, the rate of development of protection with time is almost independent of concentration until the maximum is attained; it is probably controlled by the rate of transport across the cell membrane, in the case of WR-1065, and by

Antagonism of cadmium-induced pulmonary toxicity by WR2721

Rats exposed to aerosols of cadmium oxide (⩾ 60 μg l for 30 min) develop severe and frequently fatal pulmonary edema. Electron muscopic examination of lung tissue revealed a progressive destruction of type I alveolar epithelial cells preceding death due to edema. Pre-exposure treatment of rats with a radioprotective agent, WR2721 ( S-2-aminopropylamino)ethyl phosphorothioic acid hydrate) reduced edema formation, as measured by leakage of serum protein into the air spaces, and acute mortality.

Radioprotection of immunologically reactive T lymphocytes by WR-2721

We examined the ability of S-2-(3-aminopropylamino) ethyl phosphorothioic acid (WR-2721) to protect those splenic T lymphocytes that respond to alloantigen in mixed leukocyte culture by differentiating into immunologically specific cytolytic T lymphocytes (CTL). Injection of WR-2721 (400 mg/kg) 20 min before whole-body X-irradiation protected viable-cell recovery and CTL activity, both measured on day 4 of culture. The dose-modifying factor (DMF) for viable-cell recovery was 1.6 (at 10% survival) and that for CTL activity was 1.54 or 1.69, depending on whether the ratio of the slopes or the actual activities at 10% survival were compared. Full protection was obtained 10 min after injection of the drug, and a high degree of protection persisted for at least 90 min. Our results indicate that WR-2721 provides a significant degree of protection to cells of the immune system that are significant in tumor suppression and suggest that the compound may prevent or moderate the immunosuppression that follows radiation therapy.

FAILURE OF PRETREATMENT WITH WR-2721 TO PROTECT RABBIT LUNG FROM IRRADIATIATION DAMAGE

Radioprotective drugs such as S-2 [3-Aminopropylamino] Ethylphosphorothioic acid (WR-2721) have been suggested for use to prevent radiation pneumonitis complicating radiotherapy of primary and metastatic tumors of lung, mediastinum, and chest wall. Juvenile female New Zealand White rabbits (R) between 9 and 12 months of age were either given saline or WR-2721 (36.5 mg/Kg) intravenously 15 minutes before irradiation. Low dose R (n=7) received 250 rads × 4 days to the thorax while high dose R (n=7) were given 375 rads × 4 days. Two, four and six weeks after initiating irradiation, animals were killed and pulmonary pressure volume studies performed. No significant differences between treated or control R (n=7) were seen in body weight change, food intake, water consumption, respirations, PaCO2, WBC, Hct, rectal temp, lung weight, lung distensibility, lung compliance, minimal surface tension or histologic changes. Pretreatment with WR-2721 does not seem to protect juvenile rabbit lung from irradiation damage.

Chemoprotection against Fractionated Radiation Exposures with WR-2721: Skin Injury

These studies extend the analysis of the potential application of WR-2721 (S-2-(3-aminopropylamino)ethylphosphorothioic acid) to radiation therapy by determining the ability of the drug to protect mice against hair loss induced by multiple exposures to radiation. In the two mouse strains tested, RFM and BALB/c, the relatively low drug dose used (200 mg/kg) protected the mice against hair loss from single exposures by factors of 1.67 and 1.73. The dose necessary to produce a given level of hair loss (ED/sub 50/) increases with increasing number of fractions. The ED/sub 50/ increases as N/sup 0.52/ and N/sup 0.59/ in the two strains of mice when they are not drug-treated, but as N/sup 0.39/ and N/sup 0.50/ in mice that are drug-treated. Thus, the protective effectiveness of WR-2721 decreases with increasing numbers of fractions, but the drug is still able to give a dose-modifying factor of about 1.25 when the total exposure is given as a series of 9 or 10 fractions. WR-2721 appears to offer the possibility of improving the efficiency of radiotherapy under clinical conditions.

Prediction of the Effective Radioprotective Dose of WR-2721 in Humans through an Interspecies Tissue Distribution Study

WASHBURN, L. C., RAFTER, J. J., HAYES, R. L., AND YUHAS, J. M. Prediction of the Effective Radioprotective Dose of WR-2721 in Humans through an Interspecies Tissue Distribution Study. Radiat. Res. 66, 100-105 (1976). On the basis of earlier tissue distribution data, obtained in mice and rats, we proposed that the tissue distribution of intravenously injected S-2-[3-aminopropylamino]-ethylphosphorothioic acid (WR-2721) was more closely correlated with the injected dose per unit surface area than it was with the injected dose per unit body weight. To test this proposal, we have compared the tissue distribution of 35S-labeled WR-2721 at 15 and 30 min after intravenous injection in mice, rats, rabbits, and dogs. The results of this study demonstrate that the tissue distribution is not a strict function of either the body weight or the surface area, but rather falls intermediate to the predictions of these parameters. By extrapolation, this observation would predict that a dose of 20 mg of WR-2721 per kilogram of body weight would provide humans with the same amount of protection that

The Effect of S-2-(3-Aminopropylamino)ethylphosphorothioic Acid (WR-2721) on Intestinal Crypt Survival: II. Fission Neutrons

The effect of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) was tested in the intestine for its ability to protect against fission neutron irradiation. The parameters tested were lethality, intestinal crypt survival, and DNA synthesizing cellularity. The results showed a dose modification factor (DMF) of 1.6 for lethality in mice treated with WR-2721 prior to fission neutron irradiation. This resulted in the shifting of the dose--mortality probit curve to the right by 149 rad. The crypt survival curve in protected mice was found to have a slope which was significantly different from unprotected controls. The DMF calculated at 50 percent crypt survival was found to be 1.3. The dose-dependent DNA synthesizing cellularity in the intestine 3 days after irradiation was tested in protected and unprotected animals. WR-2721 was shown to protect the intestine from about 120 to 140 rad of fission neutrons. This resulted in a DMF between 1.4 and 1.5. (auth)

The Effect of S-2-(3-Aminopropylamino)Ethylphosphorothioic Acid (WR-2721) on Intestinal Crypt Survival I. 4 MeV X-Rays

SIGDESTAD, C. P., CONNOR, A. M., AND SCOTT, R. M. The Effect of S-2- (3-aminopropylamino)ethylphosphorothioic Acid (WR-2721) on Intestinal Crypt survival I. 4 MeV X-Rays. Radiat. Res. 62, 267-275 (1975). The effectiveness of S-2-(3-aminopropylamino)ethylphosphorothiotic acid (WR2721) to protect against 4 MeV X-irradiation was tested on the intestinal epithelium of the mouse. The agent was found to be most effective when injected 15 min prior to irradiation. The protective agent increased the LDso(6) by 816 rads. This resulted in a dose-modification factor of 1.64. WR-2721 did not modify the inherent radiosensitivity of the intestinal crypts, but displaced the curve to the right by 758 rads. The total and per crypt cellularity in protected and unportected mice exposed to X-irradiation is described.

Rapid Screening of Radioprotective Drugs in Vivo

The traditional test for radioprotection, a measurement of the ${\rm LD}_{50/30}$ , requires a 1-mo wait between treatment and result. An alternative method is proposed which requires only a 3-day wait. It requires fewer mice but more man-hours than ${\rm LD}_{50/30}$ measurement. The frequency of micronuclei which result from chromosomal aberrations is the criterion of damage. It is shown that a thiophosphate compound S-2-(3-aminopropylamino) ethylphosphorothioic acid (WR 2721) produced a similar protection measured in this way as had been found by the traditional method.

Distribution of WR-2721 in Normal and Malignant Tissues of Mice and Rats Bearing Solid Tumors: Dependence on Tumor Type, Drug Dose and Species

WASHBURN, L. C., CARLTON, J. E., HAYES, R. L., AND YUHAS, J. M. Distribution of WR-2721 in Normal and Malignant Tissues of Mice and Rats Bearing Solid Tumors: Dependence on Tumor Type, Drug Dose and Species. Radiat. Res. 59, 475-483 (1974). S-2-[3-Aminopropylamino]ethylphosphorothioic acid (WR-2721) preferentially protects the normal, as opposed to the malignant, tissues of tumor-bearing mice from radiation injury. The major factor responsible for this difference in response appears to be the relatively poor absorption of the drug by the tumor. In order to determine whether deficient drug absorption was characteristic of solid tumors and to extend the study of the absorption patterns to the problems which might be encountered in clinical radiotherapy, normal and tumor tissue concentrations of 35S-labeled WR-2721 were determined 30 min after injection of graded doses of the drug (1, 50, 100, or 300 mg/kg) in two species (mice and rats) bearing 4 different solid tumors (P-1798 lymphosarcoma and CA-755 adenocarcinoma in mice and the RFT tumor and Morris 7777 hepatoma in rats). It has been demonstrated that: (1) 3 of the 4 tumors studied evidenced deficient drug absorption, but the Morris 7777 hepatoma absorbed WR-2721 as readily as the host's normal tissues; (2) the relative concentration of the drug in normal and tumor tissues was independent of the injected dose between 50 and 300 mg/kg; (3) the injection of tracer levels of WR-2721 (1 mg/kg) can be used to identify those tumors which show a deficient absorption, but not to quantitate the degree of this deficiency; and (4) the relationship between the injected dose and tissue concentration, after correction for species differences in surface area, was species independent for the lungs, small intestine and spleen, but species dependent for kidney and liver. We conclude therefore that WR-2721 is a promising agent for increasing the efficiency of solid tumor radiotherapy, but that each tumor must be considered individually.

Some Pharmacologic Effects of WR-2721: Their Role in Toxicity and Radioprotection

YUHAS, J. M., PROCTOR, J. O. AND SMITH, L. H. Some Pharmacological Effects of WR2721: Their Role in Toxicity and Radioprotection. Radiat. Res. 54, pp. 222-233 (1973). Injection of S-2-[3-aminopropylamino]ethylphosphorothioic acid (WR-2721) into BALB/c or BC3Fi mice induces a dramatic vasodilation, which is most pronounced in the spleen. This response is a component of the drug's toxic mechanism, as evidenced by the fact that splenectomy increases the tolerance of the mice to the toxic effects of the drug. Further, removal of the spleen is associated with a reduced effectiveness of the drug in terms of radioprotection, which is similar to the effects of reduced oxygen tension. Thus, at least part of the superiority of WR-2721 as a radioprotectant is due to its ability to reduce peripheral oxygen tension. Analysis of a number of phosphorothioates has demonstrated that they fall into two general categories, those that can induce the pharmacologic response and those that cannot. Each class is described by a single curve of protection versus amount of sulfur injected. The pharmacologically active drugs produce a two-component curve-an initial rapid rise followed by a less rapid increase. The second component parallels the linear curve that characterizes the entire dose range for the drugs that are not pharmacologically active. We conclude that the radioprotective effectiveness of phosphorothioates in general is determined by two factors, the amount of sulfur that can be injected and the presence or absence of the pharmacologic reaction.

Exposure-Response Curve for Radiation-Induced Lung Tumors in the Mouse

Male RFM mice were given localized chest exposures of 750-3000 R of x-rays, with and without injection of 400 mg of WR-2721 (S-2-[3-aminopropylamino]ethylphosphorothioic acid) before exposure, and were killed 11 months later for analysis of lung tumor patterns. By a technique of clearing and staining, lung tumors as small as 0.1 mm in diameter could be detected. Essentially no mortality occurred over this 11-month interval, so the data could be interpreted without the complication of premature removal; i.e., all mice were available for analysis. The incidence of lung tumors (and the mean number of tumors per mouse) was directly related to exposure over the range of 750-1500 R and then declined to control levels over the range of 1750-3000 R. Tumors occurred randomly among the members of an exposed group and were typical adenomas with various degress of invasiveness. Injection of WR-2721 before exposure did not affect the incidence between 750 and 1250 R but was associated with a peak at 1250 R, as opposed to 1500 R in the nontreated mice; and the incidence in drug-treated mice was lower than that in nontreated mice at all higher exposures. The tracheobronchial lymph nodes were protected by WR-2721, and this protection may account for at least part of the effect of the drug on tumor incidence.

Radioprotective and toxic effects of S-2-(3-aminopropylaminojethylphosphorothioic acid (WR-2721) on the development of immunocompetent cells

The toxic and radioprotective effects of S-2-(3-aminopropylammo)ethylphosphorothioic acid, or WR-2721, on the subsequent development of direct plaque-forming cells (DPFC) in the spleen of BALB/c mice following immunization with sheep red blood cells (SRBC) has been studied. A single injection of the maximally tolerated dose of this drug, 500 mg/kg, accelerates the appearance of DPFC but reduces the eventually attained peak levels to as low as 20% of those observed in mice not receiving the drug. Analysis of closely related analogs of WR-2721 failed to uncover another radioprotective thiophosphate with similar effects on the immune response. Suppression of the peak DPFC levels persists for 4 days after drug injection, followed by a gradual return toward normal responsiveness, which is attained by 10 days postinjection. Injection of the drug after the SRBC suppresses the development of DPFC, but the level of suppression decreases as the time between SRBC injection and drug injection increases. This suppression persists for too long a period to be accounted for by the drug itself or one of its metabolites. We conclude therefore that the drug affects a specific component of the immune response, that it requires 10 days for the integrity of the component to be reconstituted, and that the importance of this component declines as the immune response matures. A quantitative estimate of the protection which the drug affords the immune system against radiation injury was determined by exposing the mice to graded gamma ray exposures, with and without prior drug injection, and assaying the DPFC levels 90 days later, i.e., after sufficient time had been allowed for recovery from drug toxicity and radiation injury. From the regressions of DPFC on radiation dose, it has been shown that WR-2721, if given 15 min before radiation exposure, increases the resistance of the immune response to radiation injury by a factor of 3.4.