Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

L H Patterson, S R McKeown, T Robson, R Gallagher, S M Raleigh, S Orr

Research output: Contribution to journalReview article

75 Citations (Scopus)

Abstract

An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

Original languageEnglish
Pages (from-to)473-86
Number of pages14
JournalAnti-Cancer Drug Design
Volume14
Issue number6
Publication statusPublished - Dec 1999

Fingerprint

Prodrugs
Cytochrome P-450 Enzyme System
Tumors
Chemical activation
Cytochrome P-450 CYP3A
Neoplasms
Proteins
Cells
Messenger RNA
Protein Isoforms
Air
NADP
Oxides
Enzymes
AQ4N
Oxygen
NADPH-Ferrihemoprotein Reductase
Enzyme Therapy
Kidney
Cell Hypoxia

Keywords

  • Animals
  • Antineoplastic Agents
  • Biotransformation
  • Cytochrome P-450 Enzyme System
  • Drug Design
  • Genetic Therapy
  • Humans
  • Neoplasms
  • Prodrugs
  • Journal Article
  • Review

Cite this

Antitumour prodrug development using cytochrome P450 (CYP) mediated activation. / Patterson, L H; McKeown, S R; Robson, T; Gallagher, R; Raleigh, S M; Orr, S.

In: Anti-Cancer Drug Design, Vol. 14, No. 6, 12.1999, p. 473-86.

Research output: Contribution to journalReview article

Patterson, LH, McKeown, SR, Robson, T, Gallagher, R, Raleigh, SM & Orr, S 1999, 'Antitumour prodrug development using cytochrome P450 (CYP) mediated activation' Anti-Cancer Drug Design, vol. 14, no. 6, pp. 473-86.
Patterson, L H ; McKeown, S R ; Robson, T ; Gallagher, R ; Raleigh, S M ; Orr, S. / Antitumour prodrug development using cytochrome P450 (CYP) mediated activation. In: Anti-Cancer Drug Design. 1999 ; Vol. 14, No. 6. pp. 473-86.
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TY - JOUR

T1 - Antitumour prodrug development using cytochrome P450 (CYP) mediated activation

AU - Patterson, L H

AU - McKeown, S R

AU - Robson, T

AU - Gallagher, R

AU - Raleigh, S M

AU - Orr, S

PY - 1999/12

Y1 - 1999/12

N2 - An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

AB - An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.

KW - Animals

KW - Antineoplastic Agents

KW - Biotransformation

KW - Cytochrome P-450 Enzyme System

KW - Drug Design

KW - Genetic Therapy

KW - Humans

KW - Neoplasms

KW - Prodrugs

KW - Journal Article

KW - Review

M3 - Review article

VL - 14

SP - 473

EP - 486

JO - Anti-Cancer Drug Design

JF - Anti-Cancer Drug Design

SN - 0266-9536

IS - 6

ER -