**4. Aminothiols as radioprotectors**

Of the thousands of compounds synthesised and tested at the Walter Reed Army Institute of Research in the 1960's search for radioprotectors, aminothiols emerged as the most promising compounds. The persistent motif associated with radioprotective activity of aminothiols is a thiol separated from an aliphatic amino group by a two carbon chain (Brown et al., 1982). The simplest example is cysteamine (chemical formula H2N-CH2-CH2- SH). One of the most studied aminothiols is the radioprotector WR1065 (2- [aminopropyl)amino]ethanethiol, Figure 1), which is the active thiol metabolite of amifostine (WR2721).

Fig. 1. Structure of WR1065 (R = H) and its prodrug amifostine (R = H2PO3).

WR1065 protects cultured cells against radiation induced clonogenic death. A dose modification factor (DMF) of 1.9 is achieved for V79 cells pre-incubated 30 min with 4mM of WR1065 before irradiation (Grdina et al., 1985). DMF is defined as the ratio of radiation doses producing the same degree of radiation effect, in the presence and absence of the radiomodifier. In the context of radioprotection, and particularly for *in vivo* endpoints, DRF, dose reduction factor is often used. It has been shown using neutral elution technique that

DNA-Binding Radioprotectors 503

testing as a potential adjuvant to radiotherapy and chemotherapy. The drug has been shown conclusively to have protective activity against both radiation and cisplatin induced toxicity without demonstrable protection of tumours (Wasserman, 1994). One randomised trial of amifostine in patients with inoperable, unresectable, or recurrent rectal cancers (Liu et al., 1992), showed a significant reduction in morbidity in the treated group. Despite these results, and those of subsequent clinical studies, including differing routes of administration, the drug has not found wide clinical acceptance in radiation oncology, because of its toxicity especially hypotension and severe malaise, and the requirement that it be administered systemically (with monitoring of blood pressure) before each radiation treatment. The topical application of WR2721 to rat colon (France et al., 1986) conferred substantial protection, namely a DMF of 1.8. Subsequent clinical trials, the most recent in 2008, employing amifostine doses up to 2 g in a 30 ml enema, did report some clinical benefit, especially for the higher of two doses (Simone et

Methylproamine is a radioprotector, which belongs to a family of DNA minor groove binders featuring a common bi-benzimidazole structure (Figure 2). Two commercially available bi-benzimidazoles Hoechst 33258 and Hoechst 33342 are widely used as

Fig. 2. Chemical structure of bibenzimidazoles. R1 = H and R2 = OCH2CH3 (Hoechst 33342); R1 = CH3 and R2 = N(CH3)2 (methylproamine); R1 = H and R2 = N(CH3)2 (proamine).

Some protective activity against IR was initially discovered for Hoechst 33342 in cultured cells (Smith & Anderson, 1984; Young & Hill, 1989) and followed by reports of radioprotection of isolated DNA (Denison et al., 1992; Martin & Denison, 1992) and *in vivo* radioprotection of mouse lung (Martin et al., 1996) and brain (Lyubimova et al., 2001). New analogues of Hoechst 33342 were designed to improve the radioprotective activity, resulting in synthesis of more efficient compounds proamine (Figure 2) (Martin et al., 1996) and methylproamine (Figure 2) (Martin et al., 2004). Incubation of V79 Chinese Hamster cells in 30 M of methylproamine before and during -irradiation increases clonogenic survival with a DMF of 2.1 (Martin et al., 2004). *In vivo* radioprotection by methylproamine has been

Methylproamine, like other DNA binding bi-benzimidazoles, has a binding preference for AT-rich sequences, the consensus binding site being 3-4 consecutive AT base pairs as

demonstrated in mouse jejunum using the Withers assay with a DMF of 1.2 – 1.3.

al., 2008). These results underline the low potency of this agent.

**5. Radioprotection by methylproamine** 

fluorescent DNA binding dyes.

**5.1 Methylproamine as a DNA binding antioxidant** 

the number of radiation induced DNA DSB in V79 cells is reduced by 4 mM WR1065 with a DMF of 1.8 (Sigdestad et al., 1987). WR1065 also protects against the mutagenic effect of radiation as demonstrated for the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells (Grdina et al., 1985). Radioprotection by WR1065 and WR2721 *in vivo* has been demonstrated using the Withers assay that is based on histological staining and counting of the repopulating crypt clonogens in mouse jejunum (Withers & Elkind, 1969; Withers & Elkind, 1970). A DMF of 1.8 – 2.0 has been reported for this assay (Murray et al., 1988a), however much smaller DMF values of 1.1 – 1.3 have been obtained for the DNA SSB induction end point in the same system. Reduction of the radiation induced phosphorylated histone H2AX (H2AX) level by WR1065 has been observed in human endothelial cells in accordance with increasing clonogenic survival (Kataoka et al., 2007). The phosphorylation of the histone H2AX occurs in response to IR exposure in the regions of chromatin adjacent to the sites of radiation induced DNA DSB (Rogakou et al., 1998; Rogakou et al., 1999; Sedelnikova et al., 2003) and is considered as a marker for DNA DSB (Sedelnikova et al., 2002; Sedelnikova et al., 2003).

Amongst the different mechanisms that have been suggested for radioprotection by WR1065 and other aminothiols, the most likely are the scavenging of hydroxyl radicals, the chemical repair of DNA radicals and the depletion of oxygen (Purdie et al., 1983; Smoluk et al., 1988a). It has been demonstrated that the radioprotective ability of aminothiols is dependent on their positive charge (Aguilera et al., 1992; Zheng et al., 1992). This observation is attributed to the phenomenon of the counterion condensation that results in high local concentration of cationic aminothiols near DNA (Smoluk et al., 1988b). At neutral pH, the WR1065 molecule has a positive charge of +2 and therefore protects better than cysteamine with a charge of +1. Experiments with plasmid DNA demonstrated however, that radioprotection by aminothiols cannot be accounted solely by scavenging of hydroxyl radicals (Zheng et al., 1992). This follows from the fact that WR1065 protects DNA much better than cystamine (a disulfide form of cysteamine, chemical formula H2N-(CH2)2-S-S- (CH2)2-NH2) which has the same positive charge and higher hydroxyl radical scavenging capacity (Zheng et al., 1992). Investigators comparing radioprotective effects of aminothiols on DNA damage endpoints, with clonogenic survival (Murray et al., 1988b; Aguilera et al., 1992) or repopulating crypt clonogens in the *in vivo* mouse jejunum model (Murray et al., 1988a), also conclude that the radioprotective mechanism is more complex than just scavenging of hydroxyl radicals. Studies aimed at investigating the role of chemical repair of DNA in radioprotection of V79 cells suggest that this becomes the dominant mechanism for aminothiols with increasing positive charge (Aguilera et al., 1992). The oxygen depletion hypothesis emerged from the studies of radioprotection in mouse skin by WR2721 under different oxygen tension that demonstrated decrease in radioprotection from a DMF of 1.95 in air, down to 1.1 and less, at 5% oxygen and less (Denekamp et al., 1982). This hypothesis has been further supported by the finding of the rapid oxygen consumption in cell culture medium following addition of WR1065 and WR2721 (Purdie et al., 1983). Cell culture studies with V79 cells have also indicated the decrease in radioprotection by WR1065 under hypoxia (DMF of 1.4) as compared to oxic conditions (DMF of 1.9) (Grdina et al., 1989).

With regard to clinical application, attention has focussed on WR2721 (amifostine, Ethyol), which is a phosphorylated form of the WR1065 (Figure 1). Amifostine has FDA approval for use as a radioprotector for a subgroup of patients undergoing radiation therapy. Following administration, amifostine is dephosphorylated by alkaline phosphatase to convert it to WR1065, which actually affords protection against IR. Amifostine has undergone extensive

the number of radiation induced DNA DSB in V79 cells is reduced by 4 mM WR1065 with a DMF of 1.8 (Sigdestad et al., 1987). WR1065 also protects against the mutagenic effect of radiation as demonstrated for the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells (Grdina et al., 1985). Radioprotection by WR1065 and WR2721 *in vivo* has been demonstrated using the Withers assay that is based on histological staining and counting of the repopulating crypt clonogens in mouse jejunum (Withers & Elkind, 1969; Withers & Elkind, 1970). A DMF of 1.8 – 2.0 has been reported for this assay (Murray et al., 1988a), however much smaller DMF values of 1.1 – 1.3 have been obtained for the DNA SSB induction end point in the same system. Reduction of the radiation induced phosphorylated histone H2AX (H2AX) level by WR1065 has been observed in human endothelial cells in accordance with increasing clonogenic survival (Kataoka et al., 2007). The phosphorylation of the histone H2AX occurs in response to IR exposure in the regions of chromatin adjacent to the sites of radiation induced DNA DSB (Rogakou et al., 1998; Rogakou et al., 1999; Sedelnikova et al., 2003) and is considered as a marker for DNA DSB (Sedelnikova et al.,

Amongst the different mechanisms that have been suggested for radioprotection by WR1065 and other aminothiols, the most likely are the scavenging of hydroxyl radicals, the chemical repair of DNA radicals and the depletion of oxygen (Purdie et al., 1983; Smoluk et al., 1988a). It has been demonstrated that the radioprotective ability of aminothiols is dependent on their positive charge (Aguilera et al., 1992; Zheng et al., 1992). This observation is attributed to the phenomenon of the counterion condensation that results in high local concentration of cationic aminothiols near DNA (Smoluk et al., 1988b). At neutral pH, the WR1065 molecule has a positive charge of +2 and therefore protects better than cysteamine with a charge of +1. Experiments with plasmid DNA demonstrated however, that radioprotection by aminothiols cannot be accounted solely by scavenging of hydroxyl radicals (Zheng et al., 1992). This follows from the fact that WR1065 protects DNA much better than cystamine (a disulfide form of cysteamine, chemical formula H2N-(CH2)2-S-S- (CH2)2-NH2) which has the same positive charge and higher hydroxyl radical scavenging capacity (Zheng et al., 1992). Investigators comparing radioprotective effects of aminothiols on DNA damage endpoints, with clonogenic survival (Murray et al., 1988b; Aguilera et al., 1992) or repopulating crypt clonogens in the *in vivo* mouse jejunum model (Murray et al., 1988a), also conclude that the radioprotective mechanism is more complex than just scavenging of hydroxyl radicals. Studies aimed at investigating the role of chemical repair of DNA in radioprotection of V79 cells suggest that this becomes the dominant mechanism for aminothiols with increasing positive charge (Aguilera et al., 1992). The oxygen depletion hypothesis emerged from the studies of radioprotection in mouse skin by WR2721 under different oxygen tension that demonstrated decrease in radioprotection from a DMF of 1.95 in air, down to 1.1 and less, at 5% oxygen and less (Denekamp et al., 1982). This hypothesis has been further supported by the finding of the rapid oxygen consumption in cell culture medium following addition of WR1065 and WR2721 (Purdie et al., 1983). Cell culture studies with V79 cells have also indicated the decrease in radioprotection by WR1065 under hypoxia (DMF of 1.4) as compared to oxic conditions (DMF of 1.9) (Grdina et al., 1989). With regard to clinical application, attention has focussed on WR2721 (amifostine, Ethyol), which is a phosphorylated form of the WR1065 (Figure 1). Amifostine has FDA approval for use as a radioprotector for a subgroup of patients undergoing radiation therapy. Following administration, amifostine is dephosphorylated by alkaline phosphatase to convert it to WR1065, which actually affords protection against IR. Amifostine has undergone extensive

2002; Sedelnikova et al., 2003).

testing as a potential adjuvant to radiotherapy and chemotherapy. The drug has been shown conclusively to have protective activity against both radiation and cisplatin induced toxicity without demonstrable protection of tumours (Wasserman, 1994). One randomised trial of amifostine in patients with inoperable, unresectable, or recurrent rectal cancers (Liu et al., 1992), showed a significant reduction in morbidity in the treated group. Despite these results, and those of subsequent clinical studies, including differing routes of administration, the drug has not found wide clinical acceptance in radiation oncology, because of its toxicity especially hypotension and severe malaise, and the requirement that it be administered systemically (with monitoring of blood pressure) before each radiation treatment. The topical application of WR2721 to rat colon (France et al., 1986) conferred substantial protection, namely a DMF of 1.8. Subsequent clinical trials, the most recent in 2008, employing amifostine doses up to 2 g in a 30 ml enema, did report some clinical benefit, especially for the higher of two doses (Simone et al., 2008). These results underline the low potency of this agent.
