TY - JOUR
T1 - Time dependent HPLC analysis of the product ratio of enzymatically reduced prodrug CB1954 by a modified and immobilised nitroreductase
AU - Ball, Patrick
AU - Thompson, Emma
AU - Anderson, Simon
AU - Gwenin, Vanessa
AU - Gwenin, Chris
N1 - Funding Information:
The authors would like to thank the School of Chemistry at Bangor University for their support throughout this project as well as funding from the Life Sciences Research Network Wales , Welsh Government A4B programme the EU-backed Knowledge Economy Skills Scholarships 2 (KESS 2) and Cancer Research Wales.
Publisher Copyright:
© 2018
PY - 2019/1/15
Y1 - 2019/1/15
N2 - Directed enzyme prodrug therapy is a chemotherapy strategy that utilises prodrug-activating enzymes to activate prodrugs at the tumour location, thus reducing off-target effects. The most commonly investigated enzyme for use with the CB1954 prodrug is the NfnB nitroreductase from E. coli. Literature states that CB1954 is reduced by NfnB at the 2- or 4-position at a 1:1 ratio; deviation from this ratio has been observed in the literature, but not further investigated. The kinetic parameters for the genetically-modified enzymes; NfnB-his, NfnB-cys and AuNP-NfnB-cys were assessed and HPLC analysis was used to determine the hydroxylamine product ratios formed when reacted with CB1954. Time-dependent HPLC studies were carried out to assess how this ratio changes over time. It was shown that the hydroxylamine ratio formed by the reduction of CB1954 by a nitroreductase changes over time and that this change in ratio relates directly to the kinetics of the reaction. Thus, the hydroxylamine ratio measured using HPLC at a given time point was not a true indication of the preference of the nitroreductase enzymes during catalysis. These results question how nitroreductases are evaluated in terms of the hydroxylamine ratio and it is suspected that this phenomenon may also apply to other enzyme/prodrug combinations.
AB - Directed enzyme prodrug therapy is a chemotherapy strategy that utilises prodrug-activating enzymes to activate prodrugs at the tumour location, thus reducing off-target effects. The most commonly investigated enzyme for use with the CB1954 prodrug is the NfnB nitroreductase from E. coli. Literature states that CB1954 is reduced by NfnB at the 2- or 4-position at a 1:1 ratio; deviation from this ratio has been observed in the literature, but not further investigated. The kinetic parameters for the genetically-modified enzymes; NfnB-his, NfnB-cys and AuNP-NfnB-cys were assessed and HPLC analysis was used to determine the hydroxylamine product ratios formed when reacted with CB1954. Time-dependent HPLC studies were carried out to assess how this ratio changes over time. It was shown that the hydroxylamine ratio formed by the reduction of CB1954 by a nitroreductase changes over time and that this change in ratio relates directly to the kinetics of the reaction. Thus, the hydroxylamine ratio measured using HPLC at a given time point was not a true indication of the preference of the nitroreductase enzymes during catalysis. These results question how nitroreductases are evaluated in terms of the hydroxylamine ratio and it is suspected that this phenomenon may also apply to other enzyme/prodrug combinations.
U2 - 10.1016/j.ejps.2018.11.001
DO - 10.1016/j.ejps.2018.11.001
M3 - Article
C2 - 30414836
AN - SCOPUS:85056275879
VL - 127
SP - 217
EP - 224
JO - European Journal of Pharmaceutical Sciences
JF - European Journal of Pharmaceutical Sciences
SN - 0928-0987
ER -