title: Factors influencing the cellular accumulation of SN-38 and camptothecin.
abstract: OBJECTIVE
  The influence of biophysical factors (drug metabolism, transport proteins, and chemical stability) on the cellular accumulation of camptothecin (CPT) and SN-38 was examined.
  
  
  METHODS
  Drug transporter RNA transcript levels were measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular drug accumulation, metabolism, and drug stability studies were all performed by HPLC.
  
  
  RESULTS
  A panel of three human cell lines exhibiting different drug resistant phenotypes was investigated. HT29 colon cells glucuronidated SN-38 but did not express P-gp or MRP1 or 2. HCT116 colon cells expressed P-gp and MRP2 but did not catalyse conjugation. A2780 ovarian cells neither catalysed drug metabolism nor contained these drug transporters. In all lines, SN-38 lactone was rapidly taken up achieving peak concentrations at the earliest time point studied (5 min, 3.3-4.1 ng/10(6) cells). Subsequently, a fall in intracellular lactone concentration occurred, stabilising after 4 h at 0.48-1.18 ng/10(6) cells. No significant differences in intracellular levels of lactone were observed between the three cell lines with one exception: a twofold increase in HCT116 cells at 24 h. Stability studies in culture medium revealed that SN-38 lactone concentrations disappeared at the same rate regardless of whether cells were present, initially falling to reach equilibrium with the hydroxy acid by 4 h. Indeed, changes in intracellular lactone concentrations followed closely chemical stability profiles in media. Similar patterns of cellular retention and chemical degradation were observed with CPT.
  
  
  CONCLUSIONS
  The major determinant of drug accumulation in three diverse cell line phenotypes was lactone chemical stability in culture medium.
authors.affiliation: Imperial Cancer Research Fund, Medical Oncology Unit, Western General Hospital, Edinburgh, EH4 2XU, UK. j.cummings@icrf.icnet.uk
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Cummings
authors.forename: Gary
authors.initials: G
authors.lastname: Boyd
authors.forename: Janet S
authors.initials: JS
authors.lastname: Macpherson
authors.forename: Helga
authors.initials: H
authors.lastname: Wolf
authors.forename: Gillian
authors.initials: G
authors.lastname: Smith
authors.forename: John F
authors.initials: JF
authors.lastname: Smyth
authors.forename: Duncan I
authors.initials: DI
authors.lastname: Jodrell
mesh_terms: D000230:Adenocarcinoma; D001692:Biological Transport; D002166:Camptothecin / Q000031:analogs & derivatives / Q000493:pharmacokinetics*; D003110:Colonic Neoplasms; D018432:Drug Resistance, Multiple; D004791:Enzyme Inhibitors / Q000493:pharmacokinetics; D005260:Female; D019450:Genes, MDR; D006801:Humans; D000077146:Irinotecan; D007700:Kinetics; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics; D010051:Ovarian Neoplasms; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D059004:Topoisomerase I Inhibitors; D014407:Tumor Cells, Cultured
chemical_list: D002166:Camptothecin; D004791:Enzyme Inhibitors; D000077146:Irinotecan; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D059004:Topoisomerase I Inhibitors; C000715087:ABCC2 protein, human; C421109:multidrug resistance-associated protein 1