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Novel Photodynamic Effects of a Benzophenothiazine on Two Different Murine Sarcomas¹

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

The photochemotherapeutic properties of a novel benzophenothiazine, 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride, were assessed in vitro and in vivo against two murine mammary sarcoma models (EMT-6 and RIF). Photodynamic therapy (PDT) of EMT-6 and RIF cells following a 30-min incubation with dye (0.4 µg/ml) and a light dose of 3.3 J/cm² killed 87.0 and 99.6% of the cells, respectively. Over this same time period, RIF cells accumulate more than twice the amount of dye than the EMT-6 cell line [7.54 ± 0.17 (SD) versus 3.11 ± 0.15 nmol/10⁶ cells which probably accounts for their increased sensitivity to PDT. Conversely, in vivo, the EMT-6 tumor accumulates 3 times more dye (34.66 ± 2.16 µg/g dry weight) than the RIF tumor (12.28 ± 1.27 µg of dye/g) 3 h post-s.c. injection of dye (15 mg/kg). A study of the concentration dependent uptake of dye (following s.c. injection) in the tumor and plasma of mice bearing the EMT-6 tumor indicated a nonlinear relationship for both compartments. Maximum tissue uptake of dye and discrimination between tumor and skin or muscle occur 3–8 h following s.c. injection of dye. The ratios of dye in the tumor to the dye in surrounding skin and gastrocnemius muscle 8 h following dye injection were 4:1 and 8:1, respectively. At 24 h after dye injection, the dye was not detectable by absorption spectroscopy in the tumor, skin, or muscle. Decreasing the fluence rate from 200 to 50 mW/cm² at a total light dose of 100 J/cm² optimized the PDT effect. At 3 h following s.c. administration of dye, PDT of EMT-6 (7.5 mg of dye/kg; 50 mW/cm²; 100 J/cm²) and RIF tumors (15 mg dye/kg; 50 mW/cm²; 150 J/cm²) resulted in 100 and 70% cures, respectively. Histology at 24 and 72 h post-PDT showed minimal or no damage to the surrounding tissue (skin) while 70–90% of the tumor cells were destroyed or damaged. Moreover, 50–60% of the tumor cells isolated and cultured immediately following PDT were found to be nonviable. Similarly, the administration of 60 mg 5-ethylamino-9-diethylaminobenzo[a]phenothiazinium chloride/kg also resulted in no damage to the skin 24 h following PDT. It is suggested that the redox properties of the dye coupled with the differing metabolic states of the tumor and skin, which increase the amount of photoactive, oxidized dye present in the tumor and decrease it in the skin, are responsible for this unique differential PDT effect. Histological and fluorescein dye exclusion data 24 and 72 h post-PDT indicated that there is minimal damage to the irradiated vasculature within and surrounding the tumor under conditions which lead to high cure rates. This correlates with the predominantly intracellular tumor localization of the dye, demonstrated by fluorescence microscopy, and the low levels of dye (54 ng/ml plasma) found in the plasma at the time of light exposure. Taken together, these results indicate a novel PDT effect in that direct tumor cell killing (without skin photosensitization) rather than destruction of the supporting vasculature is the primary mode of tumor irradication.

¹ This work was funded by the Rowland Institute for Science, the Wellman Laboratories for Photomedicine, Massachusetts General Hospital, and Grant N00014-91-C-0084 from the Office of Naval Research.

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

Received 10/18/93. Accepted 12/28/93.