In-vivo singlet oxygen threshold doses for PDT.

Objective: Dosimetry of singlet oxygen (¹O₂) is of particular interest because it is the major cytotoxic agent causing biological effects for type-II photosensitizers during photodynamic therapy (PDT). An in-vivo model to determine the singlet oxygen threshold dose, [¹O₂]_rx,sh, for PDT was developed.

Material and methods: An in-vivo radiation-induced fibrosarcoma (RIF) tumor mouse model was used to correlate the radius of necrosis to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include five photosensitizer-specific photochemical parameters along with [¹O₂]_rx,sh. Photosensitizer-specific model parameters were determined for benzoporphyrin derivative monoacid ring A (BPD) and compared with two other type-II photosensitizers, Photofrin^® and m-tetrahydroxyphenylchlorin (mTHPC) from the literature.

Results: The mean values (standard deviation) of the in-vivo [¹O₂]_rx,sh are approximately 0.56 (0.26) and 0.72 (0.21) mM (or 3.6×10⁷ and 4.6×10⁷ singlet oxygen per cell to reduce the cell survival to 1/e) for Photofrin^® and BPD, respectively, assuming that the fraction of generated singlet oxygen that interacts with the cell is 1. While the values for the photochemical parameters (ξ, σ, g, β) used for BPD were preliminary and may need further refinement, there is reasonable confidence for the values of the singlet oxygen threshold doses.

Discussion: In comparison, the [¹O₂]_rx,sh value derived from in-vivo mouse study was reported to be 0.4 mM for mTHPC-PDT. However, the singlet oxygen required per cell is reported to be 9×10⁸ per cell per 1/e fractional kill in an in-vitro mTHPC-PDT study on a rat prostate cancer cell line (MLL cells) and is reported to be 7.9 mM for a multicell in-vitro EMT6/Ro spheroid model for mTHPC-PDT. A theoretical analysis is provided to relate the number of in-vitro singlet oxygen required per cell to reach cell killing of 1/e to in-vivo singlet oxygen threshold dose (in mM). The sensitivity of threshold singlet oxygen dose for our experiment is examined. The possible influence of vascular vs. apoptotic cell killing mechanisms on the singlet oxygen threshold dose is discussed by comparing [¹O₂]_rx,sh for BPD with 3 hr and 15 min drug-light-intervals, with the later being known to have a dominantly vascular effect.

Conclusions: The experimental results of threshold singlet oxygen concentration in an in-vivo RIF tumor model for Photofrin^®, BPD, and mTHPC are about 20 times smaller than those observed in vitro. These results are consistent with knowledge that factors other than singlet oxygen-mediated tumor cell killing can contribute to PDT damage in-vivo.