Activity of the DNA minor groove cross-linking agent SG2000 (SJG-136) against canine tumours.
BACKGROUND
Cancer is the leading cause of death in older dogs and its prevalence is increasing. There is clearly a need to develop more effective anti-cancer drugs in dogs. SG2000 (SJG-136) is a sequence selective DNA minor groove cross-linking agent. Based on its in vitro potency, the spectrum of in vivo and clinical activity against human tumours, and its tolerability in human patients, SG2000 has potential as a novel therapeutic against spontaneously occurring canine malignancies.


RESULTS
In vitro cytotoxicity was assessed using SRB and MTT assays, and in vivo activity was assessed using canine tumour xenografts. DNA interstrand cross-linking (ICL) was determined using a modification of the single cell gel electrophoresis (comet) assay. Effects on cell cycle distribution were assessed by flow cytometry and measurement of γ-H2AX by immunofluorescence and immunohistochemistry. SG2000 had a multi-log differential cytotoxic profile against a panel of 12 canine tumour cell lines representing a range of common tumour types in dogs. In the CMeC-1 melanoma cell line, DNA ICLs increased linearly with dose following a 1 h treatment. Peak ICL was achieved within 1 h and no removal was observed over 48 h. A relationship between DNA ICL formation and cytotoxicity was observed across cell lines. The formation of γ-H2AX foci was slow, becoming evident after 4 h and reaching a peak at 24 h. SG2000 exhibited significant anti-tumour activity against two canine melanoma tumour models in vivo. Anti-tumour activity was observed at 0.15 and 0.3 mg/kg given i.v. either once, or weekly x 3. Dose-dependent DNA ICL was observed in tumours (and to a lower level in peripheral blood mononuclear cells) at 2 h and persisted at 24 h. ICL increased following the second and third doses in a repeated dose schedule. At 24 h, dose dependent γ-H2AX foci were more numerous than at 2 h, and greater in tumours than in peripheral blood mononuclear cells. SG2000-induced H2AX phosphorylation measured by immunohistochemistry showed good correspondence, but less sensitivity, than measurement of foci.


CONCLUSIONS
SG2000 displayed potent activity in vitro against canine cancer cell lines as a result of the formation and persistence of DNA ICLs. SG2000 also had significant in vivo antitumour activity against canine melanoma xenografts, and the comet and γ-H2AX foci methods were relevant pharmacodynamic assays. The clinical testing of SG2000 against spontaneous canine cancer is warranted.
CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London, WC1E 6BT, UK. Maria.Mellinas-Gomez@spirogen.com.|Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK. Maria.Mellinas-Gomez@spirogen.com.
Maria
M
Mellinas-Gomez
CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London, WC1E 6BT, UK. v.spanswick@ucl.ac.uk.
Victoria J
VJ
Spanswick
CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London, WC1E 6BT, UK. solange.moscosso.10@ucl.ac.uk.
Solange R
SR
Paredes-Moscosso
UCL Cancer Institute, Paul O'Gorman Building, 72 Huntley Street, London, WC1E 6BT, UK. mathew.robson@ucl.ac.uk.
Matthew
M
Robson
UCL Cancer Institute, Paul O'Gorman Building, 72 Huntley Street, London, WC1E 6BT, UK. r.b.pedley@ucl.ac.uk.
R Barbara
RB
Pedley
The School of Pharmacy, University College London, Brunswick Square, London, WC1E 6BT, UK. david.thurston@kcl.ac.uk.|Present address: Institute of Pharmaceutical Science, King's College London, Britannia House, 7 Trinity Street, London, SE1 1DB, UK. david.thurston@kcl.ac.uk.
David E
DE
Thurston
Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK. stephenbaines@gmail.com.|Present address: Willows Referral Service, Highlands Road, Shirley, Solihull, West Midlands, B90 4NH, UK. stephenbaines@gmail.com.
Stephen J
SJ
Baines
Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Herts, AL9 7TA, UK. astell@RVC.AC.UK.
Anneliese
A
Stell
CR-UK Drug-DNA Interactions Research Group, UCL Cancer Institute, Paul O'Gorman Building, University College London, 72 Huntley Street, London, WC1E 6BT, UK. john.hartley@ucl.ac.uk.
John A
JA
Hartley
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001570:Benzodiazepinones / Q000494:pharmacology*; D045744:Cell Line, Tumor; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004247:DNA; D004283:Dog Diseases / Q000188:drug therapy*; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004334:Drug Administration Schedule; D004354:Drug Screening Assays, Antitumor; D005260:Female; D005434:Flow Cytometry; D051379:Mice; D008819:Mice, Nude; D011758:Pyrroles / Q000494:pharmacology*; D023041:Xenograft Model Antitumor Assays
C423343:1,1'-((propane-1,3-diyl)dioxy)bis(7-methoxy-2-methylidene-1,2,3,10,11,11a-hexahydro-5H-pyrrolo(2,1-c)(1,4)benzodiazepin-5,11-dione); D000970:Antineoplastic Agents; D001570:Benzodiazepinones; D003432:Cross-Linking Reagents; D011758:Pyrroles; D004247:DNA