title: Prodrug bioactivation and oncolysis of diffuse liver metastases by a herpes simplex virus 1 mutant that expresses the CYP2B1 transgene.
abstract: BACKGROUND
  Herpes simplex virus 1 (HSV-1) infection of cancer cells results in viral replication with cell destruction and liberation of progeny virion that infect adjacent tumor cells. rRp450 is a novel replication-conditional HSV-1 mutant that expresses both the endogenous herpes viral thymidine kinase gene and the rat p450 CYP2B1 transgene; p450 bioactivates such cancer prodrugs as cyclophosphamide.
  
  
  METHODS
  Viral cytotoxicity and replication assays were performed in colon carcinoma cells as well as primary human hepatocytes. For in vivo studies, diffuse liver metastases were generated by inoculating MC26 cells into the portal system of BALB/c mice. Mice were treated with control media, rRp450, or rRp450 plus cyclophosphamide.
  
  
  RESULTS
  Cytopathic effects induced by rRp450 replication in colon carcinoma cells were equivalent to those induced by wild type HSV-1 in vitro. Assays developed to separate cytotoxicity mediated by viral replication from cytotoxicity mediated by chemotherapy confirmed that HSV-1 thymidine kinase bioactivates ganciclovir and CYP2B1 bioactivates cyclophosphamide in rRp450-infected cells. rRp450 mediated cytotoxicity in the presence of cyclophosphamide was increased by 21% to 30% above that achieved by viral replication alone. Cyclophosphamide bioactivation produced bystander killing of colon carcinoma cells but not hepatocytes. In contrast to these effects of cyclophosphamide, rRp450 mediated cytotoxicity was reduced in the presence of ganciclovir. These findings are explained by further experiments showing that bioactivation of cyclophosphamide only minimally affected HSV-1 replication in colon carcinoma cells, whereas bioactivation of ganciclovir markedly attenuated HSV-1 replication. In vivo studies revealed a substantial decrease in hepatic tumor burden in all rRp450-treated animals compared to controls. The addition of cyclophosphamide augmented rRp450's in vivo anti-neoplastic effect.
  
  
  CONCLUSIONS
  The rRp450 mutant HSV-1 is highly oncolytic against colon carcinoma cells both in vitro and in vivo. rRp450 displays preferential replication in colon carcinoma cells compared to normal hepatocytes. Activation of cyclophosphamide by the p450 transgene augmented the anti-neoplastic effects of rRp450 without simultaneously decreasing viral replication. Oncolysis induced by HSV-1 replication combined with cyclophosphamide prodrug activation warrants further investigation as a potential therapy for colon carcinoma liver metastases.
authors.affiliation: Division of Surgical Oncology, Department of Surgery, Massachusetts General Hospital, Harvard Medical School, Boston 02114-2696, USA.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Pawlik
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakamura
authors.forename: John T
authors.initials: JT
authors.lastname: Mullen
authors.forename: Hideki
authors.initials: H
authors.lastname: Kasuya
authors.forename: Sam S
authors.initials: SS
authors.lastname: Yoon
authors.forename: Soundararajalu
authors.initials: S
authors.lastname: Chandrasekhar
authors.forename: E Antonio
authors.initials: EA
authors.lastname: Chiocca
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Tanabe
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000378:metabolism* / Q000494:pharmacology*; D002277:Carcinoma / Q000473:pathology*; D016923:Cell Death; D003110:Colonic Neoplasms / Q000473:pathology*; D003520:Cyclophosphamide / Q000378:metabolism* / Q000494:pharmacology*; D019362:Cytochrome P-450 CYP2B1 / Q000096:biosynthesis* / Q000494:pharmacology; D015316:Genetic Therapy; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000472:pathogenicity*; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology / Q000556:secondary*; D011355:Prodrugs; D019076:Transgenes; D014407:Tumor Cells, Cultured; D014779:Virus Replication
chemical_list: D018906:Antineoplastic Agents, Alkylating; D011355:Prodrugs; D003520:Cyclophosphamide; D019362:Cytochrome P-450 CYP2B1