I.J. Stratford

RSU 1069, a 2-nitroimidazole containing an alkylating group: High efficiency as a radio- and chemosensitizer in vitro and in vivo

Electron affinity as measured by the one-electron reduction potentian, E7(1), is the major factor influencing radiosensitizing efficiency in vitro. RSU 1069 has an electron affinity (E7(1) = 398 mV) similar to misonidazole; however, the ability of this compound to sensitize hypoxic cells is considerably greater in vitro than that of misonidazole, e.g., 0.2 mM RSU 1069 gives an enhancement ratio of greater than 2.0 compared to 1.4 for the same concentration of misonidazole. Radiosensitization studies with the MT tumor in vivo also showed RSU 1069 to be a more efficient sensitizer than misonidazole. An administered dose of 0.08 mg/g RSU 1069 yielded an enhancement of 1.8 to 1.9 using tumor cell survival and tumor cure as end-points. At least a 10-fold higher dose of misonidazole is required for a similar degree of sensitization. Low doses of RSU 1069 also radiosensitize the Lewis lung and B16 experimental tumors. The ability of RSU 1069 to potentiate the cytotoxic action of melphalan and other cytotoxic drugs towards the MT tumor was also examined. RSU 1069 (0.08 mg/g) given to mice 1 hour before melphalan gave an enhancement of 2.8.

Analogues of RSU-1069: radiosensitization and toxicity in vitro and in vivo

A series of nitroimidazoles containing aziridine and alkyl-substituted aziridine functions has been synthesized. The 2-nitroimidazole compounds examined all show greater radiosensitizing efficiency in vitro than misonidazole. The 4- and 5-nitroimidazole analogues are also more efficient than equivalent compounds which do not contain the alkylating aziridine moiety. All the compounds show increased toxicity towards hypoxic cells relative to aerobic cells, but this toxicity is reduced by alkyl-substitution of the aziridine ring. In vivo toxicity can also be reduced by modification of the aziridine function, but such alterations appear to have less effect upon the high radiosensitizing efficiency of these compounds in vivo. As a consequence of this study, compounds of potentially improved therapeutic utility compared to RSU-1069, the first reported member of this class, have been identified.

Bioreductive drugs as post-irradiation sensitizers: Comparison of dual function agents with SR 4233 and the mitomycin C analogue EO9

Various bioreductive drugs that are potent hypoxic cell cytotoxins can also function as effective potentiators of radiation action when administered in vivo post irradiation. There is evidence that a contributory mechanism to this potentiation is enhanced sensitivity to the bioreductive drugs exhibited by cells that are damaged sublethally by radiation.

The radiosensitizing and toxic effects of RSU-1069 on hypoxic cells in a murine tumor

RSU-1069 is one of a group of compounds of particular interest in radiobiology, since it combines the nitroimidazole ring with a side chain bearing a monofunctional alkylating agent. This compound has been shown to be a potent radiosensitizer both in vitro and in vivo. Furthermore, it has recently been shown to be an effective hypoxic cell cytotoxin in vitro. Our studies have been carried out using the SCCVII squamous carcinoma implanted subcutaneously in C3H mice, using a technique we recently developed which facilitates isolation of tumor cell subpopulations from known locations relative to the tumor blood supply. The response of the separated tumor subpopulations was assessed using a soft agar clonogenic assay. For radiosensitization studies, RSU-1069 was administered i.p. at 0.5 mumol/g 20 min before irradiation and the tumors excised 20 min after irradiation. For toxicity studies, tumors were excised 16-18 hr after RSU-1069 administration. The results obtained to date clearly demonstrate that RSU-1069 is an efficient hypoxic cell radiosensitizer and cytotoxin in this murine tumor and has little effect on well perfused (i.e., oxic) cells.

Induction of severe tumor hypoxia by modifiers of the oxygen affinity of hemoglobin.

Methods have been compared for inducing severe hypoxia in experimental tumors. Hypoxic fractions in the tumors were obtained from measurements of the displacement of cellular survival plots in vitro following tumor irradiation in vivo. Two compounds that displace to the left, oxygen-hemoglobin association curves greatly increase the hypoxic fractions in the tumors. The compound BW12C increases the hypoxic fractions in the KHT and Lewis-Lung tumors from about 10% to between 50-100%. The longer acting analogue BW589C increases hypoxic fraction in the KHT tumor to the same level achievable by treatment with the vaso-active drug hydralazine. The effect is also observed in the RIF-1 tumor even though the hypoxic fraction in this tumor is normally only about 1-3%. The kinetics for hypoxia induction by BW589C and its subsequent return to normal levels are comparable to those for the left-shifting of the oxy-hemoglobin association curve observable up to about 2 days post treatment.

Induction of hypoxia in the KHT sarcoma by tumour necrosis factor and flavone acetic acid.

The interaction of FAA or TNF with radiation was studied in the murine KHT sarcoma. When used alone both agents showed a dose- and time-dependent toxicity towards the tumour cells and significantly reduced tumour blood flow within 1 h of treatment. When used in combination with radiation, both TNF and FAA caused an increase in the fraction of hypoxic cells in the KHT tumour. This was assessed by an in vivo/in vitro clonogenic assay and by a comparison with the radioprotection provided by clamping tumours prior to and during irradiation. When TNF was given at a dose of 2.5 X 10(5) U/kg an increase in tumour hypoxia was seen after 30 min. Close to 100% radiobiological hypoxia was reached by 1 h after treatment, lasting for up to 16 h. Doses of TNF below 0.25 X 10(5) U/kg did not induce levels of hypoxia comparable to clamping when administered 3 h prior to irradiation. Similarly, FAA produced a rapid increase in tumour hypoxia: a dose of 200 mg/kg induced close to 100% radiobiological hypoxia when give 1 h prior to irradiation. Complete tumour hypoxia was still apparent 18 h after treatment with FAA. Administered doses of FAA below 100 mg/kg did not produce close to 100% radiobiological hypoxia when administered 3 h prior to irradiation.

Oral (po) dosing with RSU 1069 or RB 6145 maintains their potency as hypoxic cell radiosensitizers and cytotoxins but reduces systemic toxicity compared with parenteral (ip) administration in mice

RB 6145 is a pro-drug of the hypoxic cell radiosensitizer RSU 1069 with reduced systemic toxicity. The maximum tolerated dose (MTD) of RSU 1069 for C3H/He mice was 80 mg/kg (0.38 mmol/kg) ip but 320 mg/kg (1.5 mmol/kg) following po administration. The MTD values of RB 6145 were 350 mg/kg (0.94 mmol/kg) ip and 1 g/kg (2.67 mmol/kg) po. Toxicity of RSU 1069 toward bone marrow stem cells was also less after po administration than after ip administration; 0.1 mmol/kg ip RSU 1069 and 0.38 mmol/kg po RSU 1069 both reduced the surviving fraction of clonogenic CFU-A cells by 50%. Oral administration of RSU 1069 resulted in lower spermatogenic toxicity. No loss of intestinal crypts was detected after ip or po administration of RSU 1069. Some nephrotoxicity was observed in half of the mice given the highest po dose of 1.5 mmol/kg of RSU 1069; this was not observed following the highest ip dose of drug. For RSU 1069 and RB 6145, administered by either route, the maximum hypoxic cell radiosensitization in murine KHT sarcomas, occurred when the drugs were given 45-60 min before 10 Gy of X rays. The degree of radiosensitization produced by a particular dose of either compound was largely independent of the route of administration. Preliminary pharmacokinetic studies, using 3H-RSU 1069, suggested that anti-tumor efficacy correlated with peak blood level of label and concentration in the tumor at the time of irradiation, which were not reduced by po compared with ip administration. Normal tissue toxicity tended to correlate with total exposure over time, which was reduced approximately two-fold by po administration. Oral administration of RSU 1069 or RB 6145, as well as being convenient, may give therapeutic benefit since dose-limiting toxicity in mice was reduced compared with parenteral administration, whereas radiosensitizing activity was less affected.

Dual-function Radiation Sensitizers and Bioreductive Drugs: Factors Affecting Cellular Uptake and Sensitizing Efficiency in Analogues of RSU 1069

Alkyl aziridine analogues of the hypoxic cell radiosensitizer RSU 1069 have been synthesized and one of these, RB 7040, containing the tetramethyl substituted aziridine, is a more efficient sensitizer in vitro than RSU 1069 (Ahmed et al., 1986). The extent to which variation in drug uptake can influence the sensitizing efficiency of RSU 1069 and its analogues has been investigated by determining the cellular uptake of these weakly basic sensitizers as a function of the pH of the extracellular medium (pHe) over the range 5.4-8.4. Following exposure of V79 cells to these agents for 1 h at room temperature, the ratio of intra- to extracellular concentration (Ci/Ce) was near unity at pH 5.4. Increasing pHe to 8.4 resulted in no change in the ratio Ci/Ce for RSU 1069 (pKa = 6.04). In contrast, the values of Ci/Ce increased three-fold for RSU 1165 (pKa = 7.38) and eleven-fold for RB 7040 (pKa = 8.45). Radiosensitization by RSU 1069 showed little dependence on pHe over the range studied, whereas increasing pH caused an apparent increase in sensitizing efficiency of both RSU 1165 and RB 7040. However, when the enhancement ratios for sensitization were normalized to take account of the effect of extracellular pH on drug uptake, efficiency of sensitization was independent of pHe. This study suggests that changes in basicity (pKa) may have wider potential for therapeutic exploitation on the basis of selective tumour uptake for this type of agent.

Chemopotentiation by CB 1954: The importance of postincubations and the possible involvement of poly(ADP-ribosylation)☆

CB 1954 potentiates the cytotoxic action of the bifunctional alkylating agent melphalan (L-PAM). In vitro, this potentiation does not require the preincubation in hypoxia normally needed for other nitroaromatic compounds such as misonidazole. Chemopotentiation is observed when cells are held in CB 1954 in air after treatment with L-PAM. This may reflect an inhibition of DNA repair process(es). Structural considerations suggested that CB 1954 might be acting as an inhibitor of poly(ADP-ribosylation). However, an inhibition of the drop in NAD levels consequent on exposure to melphalan was not obtained. Furthermore, unlike the known poly(ADP-ribose) inhibitor, 3-aminobenzamide, CB 1954 does not potentiate the cytotoxicity of the monofunctional alkylator N-methyl-N nitro N-nitrosoguanidine, or inhibit NAD depletion caused by this agent. Therefore the evidence suggests that CB 1954 is not an inhibitor of poly(ADP ribosylation).

Radiosensitization by the 2,4-dinitro-5-aziridinyl Benzamide CB 1954: A Structure/Activity Study

CB 1954 (2,4-dinitro-5-aziridinyl benzamide) is a radiosensitizer which is up to 10 times more efficient in vitro than would be predicted on the basis of its electron affinity. In order to determine the contribution of the various functional groups comprising the molecule to overall sensitizing efficiency, nine structural analogues have been studied. The redox potential, E7(1), and sensitizing efficiency, C1.6, were obtained for each compound. The value of C1.6 depends on both redox potential and the magnitude of an additional component defined by C1.6/C1.6, where C1.6 is derived from a structure/activity relationship (Adams et al. 1979 b, Wardman 1982) described by the equation: log (C1.6/mol dm-3) = (6.96 +/- 0.22) + (9.54 +/- 0.56)E7(1)V. The magnitude of C1.6/C1.6 for CB 1954 and its analogues depends on alkyl substitution of the amide, the presence/absence and position of the nitro groups and is independent of the presence of the aziridine group. Holding cells in the presence of the drug post-irradiation marginally enhanced sensitization by CB 1954, CB 10-107 and by CB 10-092 but the largest effect was seen with the mononitro compound CB 7060 which also has a value of 26 for C1.6/C1.6. This compound was also interesting in that when combined with 2-phenyl-4(5)amino-5(4)-imidazole carboxamide (phenyl AIC) an enhancement of sensitization was obtained. In contrast, phenyl AIC protected against radiosensitization by CB 1954. Taken together, the data suggest that multiple mechanisms of radiosensitization may contribute to the abnormal radiosensitizing efficiency of CB 1954 and its analogues. This has implications for the further design and development of novel radiosensitizing drugs.