Wayne R. Hanson

Topical or Systemic 16,16 Dm Prostaglandin E2 or WR-2721 (WR-1065) Protects Mice from Alopecia after Fractionated Irradiation

Our previous studies in mice demonstrated that systemic or topical 16,16 dm PGE2 protected against single dose radiation-induced hair loss. We have now investigated prostaglandin, or WR-2721, protection against murine alopecia produced by varying doses and schedules of fractionated radiation. On days one to eight after hair was plucked from the thighs of B6D2F1 mice, groups of 6 animals each were given daily exposures of 4.0 or 4.5 Gy for 5 days; 2.5, 3.5, 4.5 or 5.5 Gy for 10 days; or 2 Gy for 15 days. One hour before irradiation each mouse received 10 microgram 16,16 dm PGE2, either by subcutaneous injection into the neck or topical application, 8 mg WR-2721 by injection, or 0.3 mg WR-1065 by topical application. Three weeks later counts of regrowing hairs were recorded from excised skin samples. For the radioprotectors used, hair regrowth was increased 25-100% in the various radiation groups in comparison to irradiated-only control sites. In some studies with the radioprotector given systemically, WR-2721 afforded slightly greater radioprotection than 16,16 dm PGE2. The two compounds were essentially equally radioprotective in the topical application studies. Since both systemic and topical applications of the agents tested enhanced hair regrowth following radiation, we conclude that clinical use of these compounds may provide some protection of hair follicles, and perhaps other tissues, lying within a radiation therapy field.

Subcutaneous or topical administration of 16,16 dimethyl prostaglandin E2 protects from radiation-induced alopecia in mice

Alopecia, a common sequel of radiation treatment of brain tumors, increases patient stress to the extent that refusal of treatment may occur. The expectation that loss of hair will be prevented, or that regrowth will occur, is extremely important to patients. To investigate prostaglandin-induced radiation protection against alopecia, the hair of B6D2F1 male mice was plucked from the right thigh and surrounding area to induce anagen. Fourteen days later, mice were injected subcutaneously in the neck with 10 micrograms 16,16 dm PGE2 in 0.2 ml of vehicle, or with the vehicle alone. In another group of previously plucked mice, 16,16 dm PGE2 in the same concentration, or the vehicle was applied topically. One hour later, graded single doses from 6.5 to 12.5 Gy 137Cs gamma irradiation were given to groups of six animals. On day 21 post-plucking, all animals were killed and a portion of the irradiated site was excised. The average hair counts per field in irradiated animals were 85 +/- 4 (6.5 Gy), 25 +/- 5 (8.5 Gy), and 5.5 +/- 0.7 (10 Gy). Animals receiving the prostaglandin systemically had values of 60 +/- 10 (6.5 Gy), 54 +/- 3 (8.5 Gy), 66 +/- 6 (10 Gy), and 30.1 +/- 8 (12.5 Gy). Topical application of the prostaglandin resulted in protection that yielded 52 +/- 3 (8.5 Gy), 34 +/- 4 (10 Gy), and 3.2 +/- 0.9 (12.5 Gy) hairs per field. Both systemic and topical application of 16,16 dm PGE2 protected from some degree of radiation-induced alopecia, which supports the conclusion that prostaglandins may be useful in the protection of hair follicles in patients treated with radiation for brain tumors.

The prostaglandin E1 analog, misoprostol, a normal tissue protector, does not protect four murine tumors in vivo from radiation injury

The clinical development of radioprotectors, such as misoprostol, to protect normal tissue during cancer treatment must proceed with the assurance that tumors are not protected similarly or significantly. To provide data on this critical question, radiation-induced growth delay with or without the presence of misoprostol was measured in four murine tumors grown in the flanks of mice: the Lewis lung carcinoma, M-5076 ovarian sarcoma, FSA and NFSA. The effect of misoprostol on the tumor control dose (TCD50) of radiation was measured in FSA-bearing mice with or without prior treatment with the nonsteroidal anti-inflammatory agent, indomethacin. Misoprostol did not influence the in vivo growth of any of the four tumors, nor did it protect any of the tumors from radiation-induced growth delay. Likewise, there was no increase in the radiation TCD50 to treat the FSA in vivo in control or indomethacin-treated tumor-bearing mice. To measure any possible influence of tumor burden on the protective effect of misoprostol on normal tissue in mice, the protective effect of misoprostol on the survival of intestinal clonogenic cells was measured in M-5076-bearing mice and found to be the same as in non-tumor-bearing mice. These data suggest that misoprostol protects normal tissue in mice without protecting at least four experimental murine tumors. The data support the contention that misoprostol can achieve therapeutic gain by protecting normal tissues without protecting tumors.

Potential radioprotective agents—VI. Chalcones, benzophenones, acid hydrazides, nitro amines and chloro compounds. radioprotection of murine intestinal stem cells

There is an interest and need for new compounds that protect tissues from radiation injury. In cancer therapy, the protection of normal tissue without protecting tumors is one way to increase the therapeutic gain. Thiol compounds are currently in clinical trials, but are limited to some extent by their human toxicities including hypotension, nausea, and emesis. Several new aminochalcones and aminobenzophenones were synthesized and tested for radioprotective activity in mice. All were less active than p-aminobenzophenone itself. Several acid hydrazides were synthesized and tested similarly, but none exhibited significant activity. The high radioprotective activity of 4-nitroaniline was confirmed, but other nitro amines were substantially less active. 4-Chloro-N-methylaniline is as active as 4-chloroaniline, but other chloro aromatics are devoid of significant activity. When compared with the phosphorothioate amyfostine (WR-2721) using the intestinal clonogenic cell survival assay, 1-(p-aminophenyl)-1-propanol (15), p-aminopropiophenone (16), its ethylene ketal (14), and a mixture of the two (17) protected to a great extent, though slightly less than WR-2721. These results suggest that there is direct cellular radioprotection by these non-thiol compounds. The studies further suggest that preclinical toxicity testing of the most protective agents is warranted.

Misoprostol-induced radioprotection of Syrian hamster embryo cells in utero from cell death and oncogenic transformation.

Misoprostol, a PGE1 analog, is an effective radioprotector of murine intestine and hematopoietic and hair cell renewal systems. The radioprotective nature of misoprostol was extended to examine its ability to influence clonogenic cell survival and induction of oncogenic transformation in Syrian hamster embryo cells exposed to X rays in utero and assayed in vitro. Hamsters in their 12th day of pregnancy were injected subcutaneously with misoprostol, and 2 h later the pregnant hamsters were exposed to graded doses of X rays. Immediately after irradiation, hamsters were euthanized and embryonic tissue was explanted into culture dishes containing complete growth medium. After a 2-week incubation period, clonogenic cell survival and morphologically transformed foci were determined. Survival of misoprostol-treated SHE cells was increased and yielded a dose reduction factor of 1.5 compared to SHE cells treated with X rays alone. In contrast, radiation-induced oncogenic transformation of misoprostol-treated cells was reduced by a factor of 20 compared to cells treated with X rays alone. These studies suggest that misoprostol not only protects normal tissues in vivo from acute radiation injury, but also protects cells, to a large extent, from injury leading to transforming events.

Effect of track structure and radioprotectors on the induction of oncogenic transformation in murine fibroblasts by heavy ions

The oncogenic potential of high-energy 56Fe particles (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at the Brookhaven National Laboratory was examined utilizing the mouse C3H 10T1/2 cell model. The dose-averaged LET for high-energy 56Fe is estimated to be 143 keV/micrometer with the exposure conditions used in this study. For 56Fe ions, the maximum relative biological effectiveness (RBEmax) values for cell survival and oncogenic transformation were 7.71 and 16.5 respectively. Compared to 150 keV/micrometer 4He nuclei, high-energy 56Fe nuclei were significantly less effective in cell killing and oncogenic induction. The prostaglandin E1 analog misoprostol, an effective oncoprotector of C3H 10T1/2 cells exposed to X rays, was evaluated for its potential as a radioprotector of oncogenic transformation with high-energy 56Fe. Exposure of cells to misoprostol did not alter 56Fe cytotoxicity or the rate of 56Fe-induced oncogenic transformation.

Misoprostol-induced radioprotection of oncogenic transformation

PURPOSE Prostaglandins are associated with a variety of both pathologic and normal physiological effects in mammals. Among this broad array of effects, prostaglandins have been shown to provide protection to tissues from a variety of injurious agents including ionizing radiation. Of the prostaglandins tested to date, an analogue of prostaglandin E1, misoprostol (cytotec) was found to be a very effective radioprotector. The purpose of this study was to assess the ability of misoprostol to protect cells from the cytotoxic and oncogenic effects of ionizing radiation. METHODS AND MATERIALS Pregnant Syrian hamsters were injected subcutaneously with 125 micrograms misoprostol/100 g body weight 2 h before being exposed to graded doses of X rays. Embryos were excised immediately after irradiation and cells were explanted into culture dishes. Following 14 days of incubation, cells were fixed in formalin and stained with giemsa for examination of cell clonogenicity and morphological transformation. RESULTS First, misoprostol protected cells from some degree of radiation toxicity. A reduction in cell killing by a factor of 1.5 was seen at 10% cell survival. Second, based on transformation studies, a higher frequency of oncogenic transformation is seen for cells exposed in utero to graded doses of X rays alone than for cells exposed to the combination of misoprostol followed by radiation. In the presence of misoprostol, transformation is reduced by a factor of 20 at the level of 10(-3) transformants per surviving cell. CONCLUSION Misoprostol may have clinical utility, not only in protecting selected normal tissues during cancer therapy, but it may also be useful in protecting cells from secondary tumors caused by ionizing radiation.

Prostaglandin-induced radioprotection of murine intestinal crypts and villi by a PGE diene analog (SC-44932) and a PGI analog (Iloprost)

The aminothiols exemplified by WR-2721 are effective radioprotectors; however, their toxicity associated with hypotension, nausea, and emesis has limited their development for applications to medicine or in harzardous radiation environments. There is a need for new radioprotectors that have fewer toxic side effects when given alone or combined with reduced amounts of thiols. A variety of prostaglandins (PGs) have been shown to be radioprotective agents and some appear to have fewer toxic side effects than the aminothiols. Iloprost, a stable PGI, analog protects the clonogenic epithelial cells of intestinal crypts but does not protect epithelial cells of the villi. In contrast, an E-series omega chain diene analog designated SC-44932 protects epithelial cells of both crypts and villi. When the two are combined, protection of the crypts is additive and the villi are protected to the same degree as when SC-44932 is given alone. Since radioprotection for some PGs has been shown to be dependent upon receptors, we suggest that the pattern of radioprotection seen with these two analogs depend on the location of the respective receptors or on the ability of differentiated villus cells to respond to PGs. By studying different analogs, we hope to identify mechanisms associated with PG-induced radioprotection and to identify the most protective PG analogs for applications of radioprotection.