title: Extravascular diffusion of tirapazamine: effect of metabolic consumption assessed using the multicellular layer model.
abstract: OBJECTIVE
  Hypoxia-selective cytotoxic agents, like tirapazamine (TPZ), must diffuse considerable distances in tumors to reach their target cell population. This study uses a new three-dimensional tissue culture model, in which cells are grown as multicellular layers (MCL), to investigate whether metabolic consumption of TPZ is sufficiently rapid to compromise its extravascular diffusion in tumors.
  
  
  METHODS
  V79-171b and MGH-U1 cells were grown as MCL to thicknesses of approximately 120 and 360 microm respectively. The extent of hypoxia in MCL, as assessed by EF5 binding, was modulated by altering gas-phase O2 content, and flux of TPZ through MCL was investigated by high-performance liquid chromatography (HPLC). Data were fitted to a diffusion-reaction mathematical model to determine the diffusion coefficient of TPZ in the MCL (DM) and the rate of its metabolic consumption under anoxia. These parameters were used to simulate TPZ transport in tumors.
  
  
  RESULTS
  The flux of TPZ through well-oxygenated MCL (equilibrated with 95% O2) was well fitted as Fickian diffusion without reaction, with a D(M) of 7.4 x 10(-7) cm2s(-1) (12-fold lower than in culture medium) for V79 and 1.3 x 10(-6) cm2s(-1) for MGH-U1 MCL. Flux of TPZ was suppressed under anoxia, and fitting the data required inclusion of a reaction term with a rate constant for metabolic consumption of TPZ of 0.52 min(-1) for V79 and 0.31 min(-1) for MGH-U1 MCL. These transport parameters would translate into a 43% or 30% decrease respectively in TPZ exposure, as a result of drug metabolism, in the center of a slab of anoxic tissue 100 microm in thickness.
  
  
  CONCLUSIONS
  MCL cultures provide an in vitro model for investigating the interaction between metabolic consumption and diffusion of bioreductive drugs. If rates of diffusion and metabolism similar to those measured in V79 and MGH-U1 MCL apply in tumors, then cells in large confluent regions of hypoxia would be partially protected by failure of TPZ penetration. Simulation of extravascular transport of TPZ-like bioreductive drugs demonstrates that the optimum metabolic rate constant is determined by two competing requirements: it should be high enough to ensure potent cytotoxicity under hypoxia, yet low enough that penetration is not severely compromised.
authors.affiliation: Department of Pathology, The University of Auckland, New Zealand.
authors.forename: K O
authors.initials: KO
authors.lastname: Hicks
authors.forename: Y
authors.initials: Y
authors.lastname: Fleming
authors.forename: B G
authors.initials: BG
authors.lastname: Siim
authors.forename: C J
authors.initials: CJ
authors.lastname: Koch
authors.forename: W R
authors.initials: WR
authors.lastname: Wilson
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D015687:Cell Hypoxia; D002478:Cells, Cultured / Q000378:metabolism; D006224:Cricetinae; D004058:Diffusion; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D011838:Radiation-Sensitizing Agents / Q000378:metabolism / Q000493:pharmacokinetics*; D018874:Spheroids, Cellular; D000077704:Tirapazamine; D014227:Triazines / Q000378:metabolism / Q000493:pharmacokinetics*; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D011838:Radiation-Sensitizing Agents; D014227:Triazines; D000077704:Tirapazamine