The combination of multiple doses of etanidazole and pimonidazole in 48 patients: a toxicity and pharmacokinetic study

Bleehen, Norman M.; Maughan, T; Workman, Paul; Newman, H; Stenning, Sally; Ward, R.

doi:10.1016/0167-8140(91)90201-q

“…Most importantly, failure to locally control primary tumours has been attributed to the presence of these oxygen-deprived cells, as it has been previously shown that hypoxic cells are resistant to the lethal effects of ionizing radiation and certain chemotherapeutic agents (Koch and Kruuv, 1971;Sutherland et al, 1982;Wilson et al, 1989). The advent of combining chemical compounds with ionizing radiation, such that these compounds could mimic the effect(s) of oxygen in hypoxic tumour regions, allowed for the possibility of increased therapeutic efficacy after radiotherapy (Bleehen et al, 1991;Overgaard, 1994). A class of these compounds are the 2-nitroimidazoles, which have demonstrated enhanced radiosensitization of hypoxic cells in vitro and in vivo (Coleman et al, 1988).…”

mentioning

confidence: 99%

Apoptosis and 1-methyl-2-nitroimidazole toxicity in CHO cells

Brezden

¹

,

McClelland²,

Rauth³

1997

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Summary The time course and characteristics of the selective hypoxic cytotoxicity of the 2-nitroimidazole model compound 1 -methyl-2-nitroimidazole (IN02) were analysed during prolonged time periods (up to 5 days post treatment). When control populations were seeded at the same cell density as drug-treated cells, they entered confluency at day 3 and underwent apoptosis at day 5, which appeared to be mediated by an autocrine mechanism. In subsequent studies of drug-treated cells, the seeding density of treated cells was adjusted to avoid this cell confluency effect. Treatment with a low INO2 concentration (2.5 mM) resulted in apoptotic DNA fragmentation (ladders), which was observed 4-5 days after an acute 6-h hypoxic drug exposure. In contrast, at a high INO2 concentration (40 mM) for 2 h, which was equitoxic to the low concentration, no characteristic DNA ladders were observed. Fluorescence microscopy revealed apoptotic bodies and pyknotic nuclei 5 days following hypoxic 2.5 mm INO2 exposure, whereas 40 mm INO2 hypoxic treatment produced cellular ghosts devoid of DNA 5 days after exposure, consistent with the DNA ladder results. However, characteristic apoptotic morphology was previously observed immediately after the acute hypoxic exposure of 40 mm INO2. Cell cycle analysis and DNA fragmentation as measured by the TdT assay suggested that dose-dependent differences in the apoptotic response occur post exposure after an equitoxic acute hypoxic exposure to either the low or the high INO2 concentration. This dose-dependent differential in response may be attributed to the degree of initial DNA damage as measured by the comet assay.

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“…Initial interest focused on use in radiation therapy, investigating their ability to act as oxygen mimetics to sensitize hypoxic tumours to ionizing radiation. Drugs trialled under this application included the prototypical misonidazole (Kogelnik et al, 1978), and its analogues etanidazole and pimonidazole (Bleehen et al, 1991). Unfortunately in large scale human trials the compounds demonstrated dose limiting neurotoxicity and failed to show clear clinical benefit (Wardman, 2007).…”

Section: -Nitroimidazole Hypoxia Imaging Agentsmentioning

confidence: 99%

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

Williams¹

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<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A. This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents. In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity. Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

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“…Initial interest focused on use in radiation therapy, investigating their ability to act as oxygen mimetics to sensitize hypoxic tumours to ionizing radiation. Drugs trialled under this application included the prototypical misonidazole (Kogelnik et al, 1978), and its analogues etanidazole and pimonidazole (Bleehen et al, 1991). Unfortunately in large scale human trials the compounds demonstrated dose limiting neurotoxicity and failed to show clear clinical benefit (Wardman, 2007).…”

Section: -Nitroimidazole Hypoxia Imaging Agentsmentioning

confidence: 99%

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

Williams¹

0

View full text Add to dashboard Cite

<p>There is strong interest in developing novel targeted cancer therapies. It has been known for over a century that certain viruses and bacteria can preferentially infect and lyse cancerous cells. Clinical utility has lagged behind the initial promise of the idea; however three therapeutic agents from the oncolytic virus field are currently in Phase IIB/Phase III clinical trials. The development path of such therapies would be substantially smoothed by an ability to nonin vasively monitor the ir location in the patient’s body post-administration. This would allay fears that viral/bacterial distribution may not be confined to the tumour and provide real time information on vector localisation and replication. This could be achieved by positron emission tomography (PET) scanning if the vector expressed a reporter protein which could activate a PET suitable imaging agent. Furthermore the potency of such therapies could be increased by if this reporter protein could also act therapeutically by converting a systemically delivered benign prodrug into a potent chemotherapeutic – thus targeting the toxicity of the prodrug specifically to cancerous cells. A promising enzyme/prodrug combination is the use of bacterial nitroreductase (NTR) enzymes to activate DNA damaging prodrugs, such as the dinitrobenzamides CB1954 and PR-104A. This thesis presents work aimed at developing the ability to noninvasively image bacterial NTR expression so that these enzymes can act as both therapeutic and reporter proteins. The primary focus of this study was to achieve this by repurposing pre-existing 2-nitroimidazole (NI) PET imaging agents, originally developed for imaging tumour hypoxia. Microplate based screening strategies were developed to enable detection of 2-NI bioreductive activation by different bacterial NTRs over-expressed heterologously in Escherichia coli, and these technologies were used to screen a 58-membered library of nitroreductase candidates. Although the most widely studied NTR for enzyme/prodrug therapy - NfsB from E. coli - was found to lack activity with 2-NI substrates, numerous NTRs from the NfsA family were able to metabolise these molecules to the cell entrapped form required for PET imaging. Following this discovery, a directed evolution study was conducted to improve the native activity of the enzyme NfsA from E. coli. In this study targeted mutagenesis of active site residues was carried out, resulting in identification of several NfsA multi-site mutants that were substantially improved in their ability to activate a range of 2-NI imaging agents. In addition to repurposing existing PET probes, this work sought to identify and engineer NTRs for efficient activation of a next - generation PET probe that is designed to be substantially less responsive to hypoxia and hence give a cleaner signal for NTR imaging (i.e. low to no background resulting from tumour hypoxia). SN 33623, a novel 5-NI analogue of the existing 2-NI PET probe EF5, was designed and synthesised by our University of Auckland collaborators. It was found that this novel probe was not only activated by NfsA enzymes, but also by a subset of NfsB enzymes. Although this subset did not include E. coli NfsB, sequence alignment and site-directed mutagenesis were used to identify two key mutations that can be introduced into E. coli NfsB (as well as engineered variants thereof) to confer high levels of SN 33623 activity. Finally work was carried out, as part of a wider collaborative project, to generate NfsA mutants that retained the ability to metabolise 2-NI imaging agents while also showing increased activation of the nitroaromatic prodrug PR-104A. Ongoing evaluation of these enzymes will include assessment of their therapeutic effect in preclinical models and their ability to be noninvasively imaged (by microPET) when expressed from the tumour targeting bacterial strain Clostridium sporogenes.</p>

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The combination of multiple doses of etanidazole and pimonidazole in 48 patients: a toxicity and pharmacokinetic study

Cited by 8 publications

References 7 publications

Apoptosis and 1-methyl-2-nitroimidazole toxicity in CHO cells

Apoptosis and 1-methyl-2-nitroimidazole toxicity in CHO cells

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

Development of bacterial nitroreductase enzymes for noninvasive imaging in cancer gene therapy

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