Cytometry of Anticancer Prodrug OCT1002 Activation and Targeting Using In Vitro and In Vivo Models of Tumour Hypoxia

Background: Tumour microenvironment hypoxia (TMH)facilitates disease progression and therapeutic resistance,presenting challenges for the treatment of prostate and pancreaticcancer. The non-toxic anthraquinone prodrug OCT1002(OncoTherics, UK) is designed to distribute widely in tissues,become irreversibly activated to OCT1001, a potent DNAtopoisomerase II inhibitor, thereby acting as unidirectionalhypoxia activated prodrug (uHAP). We have investigated therelationship between intracellular generation and persistence ofTMH-generated OCT1001 and biological responses both in vitroand in vivo.Methods: Confocal imaging and FCM exploited the intrinsic farredfluorescence of the uHAP. Cancer cell lines were exposed toclinically relevant prodrug levels at 1-100 nM under hypoxia (4d;1-3% oxygen); studied for cell proliferation, OCT1002bioreduction and cell cycle arrest. Human prostate cancerxenografts expressing a luciferase reporter (LNCaP-luc) in Balb/cSCID mice were used to monitor growth delay, lung metastasisand vascular changes in relation to OCT1002 targeting in dorsalskin-flap/window chambers. pO2 of tumours was recorded usingan Oxylite 2000 system and assessed in tissues by Glut1 staining.Xenograft induced hypoxia was achieved by exposure tobicalutamide (Casodex™; a non-steroidal anti-androgen).Pancreatic cancer human BX-PC3/SCID mouse xenografts,presenting intrinsic hypoxia, were used to assess uHAPmonotherapy.Results: Hypoxia-dependent growth arrest induced by OCT1002was found in a wide range cancer cell lines irrespective of p53status. Cytostasis and sustained G2 cell cycle arrest was achievedwithin 1 population doubling at 100 nM [% cells arrested at1%>3%>>air]. OCT1002 and hypoxia co-exposure generatedpersistent intracellular fluorescence attributable to the metaboliteOCT1001 while the prodrug OCT1002 was not retained.OCT1001 located within LNCaP-luc tumours at regions distantfrom blood vessels, persisted for >7 days, slowed tumour growthand reduced lung metastasis. A pilot study showed that a singledose OCT1002 induced a growth delay in pancreatic cancer BXPC3/SCIDmice xenografts, which are hypoxic, comparable withthat achieved by fractionated Gemcitabine treatment.Conclusion: Cytometric analyses have shown effective OCT1002activation in vitro. A quantitative relationship was found betweenthe degree of hypoxia, extent of uHAP bioreduction, persistenceof OCT1001, growth delay and first cycle G2 arrest. In vivoimaging, in a prostate cancer model that recapitulates TMHinduced by anti-androgen treatment, showed that uHAPactivation at TMH locations. The slowing of tumour growth andreduction in metastatic potential in prostate cancer together withmonotherapy efficacy in a pancreatic cancer model suggest thatOCT1002 has potential for tumour indications and in combinedmodalities where TMH is considered to be a bar