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Phototoxicity, Redox Behavior, and Pharmacokinetics of Benzophenoxazine Analogues in EMT-6 Murine Sarcoma Cells¹

Rowland Institute for Science, Cambridge 02142 [L. C., J. W. F.], and Wellman Laboratories of Photomedicine, Massachusetts General Hospital, Harvard Medical School, Department of Dermatology, Boston 02114 [A. H. C.], Massachusetts

Structural modifications to the photoinactive benzophenoxazine Nile blue A have led to three novel derivatives which include 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium (EtNBA), and 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium (EtNBSe) chlorides. The incorporation of sulfur and selenium into the benzophenoxazine moiety results in lipophilic, red-absorbing (650–660 nm) chromophores which possess significantly increased singlet oxygen yields (0.025 and 0.65, respectively, compared to 0.005 for EtNBA). This study examines the photosensitizing efficacies and pharmacokinetics in vitro in the EMT-6 murine mammary sarcoma cell line as well as the physicochemical, photochemical, and redox properties of these new analogues. Comparisons with Photofrin II, the only photosensitizer available clinically, were made in an attempt to highlight their different pharmacological characteristics. The photodynamic activity of the benzophenoxazine dyes correlates with their ability to generate the phototoxin singlet oxygen and increases in the following order: EtNBA < EtNBS << EtNBSe. At an extracellular dye concentration of 0.5 µM, the light dose required to kill approximately 50% of the cells was 2.0 and <0.5 J/cm² for the sulfur and selenium dyes, respectively. The light dose required to kill approximately 50% of the cells for both EtNBA and Photofrin II could not be determined because of their weak phototoxic effect under these conditions. At a light dose of 3.3 J/cm², EtNBSe is approximately 1000 times more phototoxic than Photofrin II. All three benzophenoxazine derivatives are characterized by a similar uptake/efflux pattern in vitro consisting of a rapid and extensive cellular accumulation followed by a slow efflux rate. Contrary to their rapid uptake, 50% of the accumulated EtNBS and EtNBSe is retained intracellularly after a 6-h period in dye-free medium. Video-enhanced fluorescence microscopy corroborates the rapid uptake measurements as well as indicating the intracellular localization of the dyes in both living and thermally inactivated cells. Low extracellular dye concentrations (0.05 µM) result in a punctate fluorescence pattern in the perinuclear region, while higher dye concentrations (>0.1 µM) lead to additional fluorescence in the cytoplasm, cytomembranes, and other organelles but apparently not the nucleus. Absorption spectrometry revealed that living cells rapidly reduce the dyes to their colorless leuko form (photoinactive) if oxygen is not readily available in the environment. It is shown that the cellular reduction is an enzymatic process and that an oxygen-free and cell-free medium containing both the coenzyme NADH and the hydride transfer enzyme diaphorase is capable of reducing the dyes to the colorless leuko form. EtNBSe exhibited the fastest rate of reduction under these conditions. In addition, the benzophenoxazine derivatives also undergo a light-induced reduction under anaerobic conditions in the presence of NADH; the rates increase in the following order: EtNBA < EtNBS << EtNBSe. Reoxidation of the leuko form occurs upon introduction of oxygen. This investigation demonstrates that the benzophenoxazine chalcogen analogues are a unique class of photochemotherapeutic agents.

¹ Funded by the Rowland Institute for Science and the Wellman Laboratories for Photomedicine, Massachusetts General Hospital.

² To whom requests for reprints should be addressed, at the Rowland Institute for Science, 100 Edwin Land Boulevard, Cambridge, MA 02142.

Received 12/15/92. Accepted 3/26/93.

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