Photodynamic therapy, although used effectively for the treatment of many types of solid tumors, still relies on empirically derived treatment parameters that include photosensitizer dose, fluence, and fluence rate. These are usually adjusted so that the duration of patient treatment can be kept short for convenience and patient comfort. However, it has been recognized for some time that the current treatment parameters utilized in clinical settings may not be optimal. For their optimization, a full understanding of the different components that determine PDT dosimetry and effectiveness is necessary. This is a difficult task because of the enormous complexity created by the interplay of the factors that determine tumor destruction and the heterogeneity in the tissue of these factors, especially the availability of oxygen required for the formation of the cytotoxic product. In the present study, we identify the duration of photodynamic exposure, which is a function of fluence rate and fluence, as an important independent factor influencing treatment outcome. Our study further shows that treatment duration affects tumor destruction largely independently of a wide range of photosensitizer doses and fluences, i.e., the drug-light products.
The established benefit of PDT light exposure at low fluence rates in preclinical models has been explained by the conservation of tumor oxygenation during illumination (11
), the current paradigm being that high fluence rate leads to photochemical oxygen consumption that overwhelms the oxygen re-supply from the microvasculature. Such a mechanism has been proven in preclinical and clinical settings (11
). Of particular relevance to our study is a report by Foster et al.
), which investigated tumor response to various irradiation regimes comprised of constant 2-hour duration. Fractionated irradiation schedules with dark intervals of 30–60 seconds provided the most durable tumor control, while constant irradiance and more rapid fractionation were less efficacious given identical fluences and treatment durations. Because of the existing evidence for photochemical oxygen depletion as a PDT limiting factor, it was the first focus of this study. Simulation of the tumor oxygenation status based on theoretical assumptions for oxygen consumption (14
) during light exposure of the tissue predicted extensive oxygen depletion at high fluence rate that was highly photosensitizer concentration dependent. Simulation of the instantaneous singlet oxygen distribution in the tissue emphasized this drug concentration dependence even more. Increasing drug doses should have shifted the fluence rate dependence towards increasingly lower fluence rates for achieving maximum benefit. It was therefore surprising to observe that in spite of a 30-fold difference in HPPH concentration and 15-fold difference in drug-light product, dependence on the treatment laser power over a range from 3 to 150 mW was strikingly similar.
While photochemical oxygen depletion is likely to have occurred, neither of the methods we employed to determine tumor oxygenation was able to clearly identify its existence. Non-invasive fMRI based on BOLD-contrast has previously been utilized to monitor changes in tumor oxygenation during PDT (39
). In the previous study by Gross et al.
, it was shown that the observed change in BOLD contrast during light treatment was a result of contributions from both photochemical and hemodynamic components (39
). Similarly, in our study, while the use of BOLD fMRI allowed for non-invasive monitoring of changes in oxygenation status during light exposure, it did not distinguish whether these were the consequences of photochemical oxygen depletion or vascular disruption. The direct, invasive oxygen measurements (Oxylite®) could only be taken after the light was turned off due to probe limitations and therefore could not determine photochemical oxygen depletion that occurs only during illumination. However, both approaches clearly indicated that acute decreases in tumor oxygenation occurred that were not immediately reversible after cessation of light exposure, indicating that these were not due to purely photochemical processes. Focusing therefore on possible vascular changes, it is noteworthy that following just one minute of 150 mW light under conditions of 3 mg/kg HPPH, oxygen measurements closely approached anoxic levels (0.85 ± 0.14 mm Hg). Given the predicted very high singlet oxygen levels near the vessel wall under high fluence rate conditions (, right panel
), the data suggest acute but transient vascular spasm. The transient nature of this effect is indicated by the eventual recovery of tumor oxygenation observed by BOLD fMRI and the patency of the tumor vasculature to the Evans Blue dye delivered 72 h after completion of treatment (). Also, by 2 hrs after the end of a 6 min (50 J) treatment, tumor oxygenation was measured at ~3 mm Hg (), indicating recovery. It is of interest that we have previously observed in a mouse model that normal, but not tumor vasculature, was somewhat protected by brief high fluence rate PDT using Photofrin (40
). This protection could be counteracted by administration of the nitric oxide synthase (NOS) inhibitor L-NNA. These and the current observations may reflect differences in the tumor physiology of the animal models used.
The key finding of this study is the dramatic progression of tumor hypoxia during and after light delivery at low incident laser power and long duration ( & ) that is largely independent of drug-light product. We have identified, however, a threshold photosensitizer dose (in this model < 0.1 mg/kg) below which even extended illumination fails to elicit any tumor necrosis. Above this dose, the effects of long duration PDT are striking. As computed in , extremely low levels of singlet oxygen generated throughout the tissue volume over extended periods of time appear sufficient for tissue destruction. It has to be noted that these computations are based on a theoretical model that assumes optimal conditions for 1
production, i.e., optimal vascular supply, drug distribution etc., and neglect any photobleaching of the photosensitizer during illumination, which might shift oxygenation and therefore 1
production to even lower levels. A comprehensive theoretical model describing microscopic dose deposition during PDT, which takes into account temporal and spatial variations in blood flow, oxygen concentration, and photosensitizer distribution, has recently been developed (15
) and could be utilized in future studies to create more rigorous estimates of photodynamic dose under a wide range of treatment conditions.
The most puzzling result may be found in . Here it is predicted that the conditions of 0.1 mg/kg HPPH under 150 mW cm−2
fluence rate and 3 mg/kg HPPH under 7 mW cm−2
fluence rate should produce roughly equal 1
amounts and distribution, ranging from ~4 pM at the vessel to ~1 pM at distance. Yet, the actual 150 mW regimen is ineffective, while the 7 mW regimen is effective. We propose that the reason for this phenomenon may lie in the intermittent nature of acute hypoxia in tumors. Evidence for fluctuating tumor blood flow has been provided by numerous groups suggesting that tumor blood vessels are often temporarily occluded, resulting in acute hypoxia (41
). In a previous report by Woodhans et al.
) it was shown that PDT of normal rat livers using low power (25 mW) treatment regimen results in a greater reduction in hemoglobin oxygen saturation and increased necrosis compared to high power illumination (100 mW). Fluorescence angiography measurements showed that the increased necrosis following low power light treatment was likely due to ischemia/reperfusion injury (41
). It is estimated that at least 20% of the surface area of the tumor vasculature is subject to hypoxic changes, which last for at least several minutes (42
). Such acute hypoxia would not only protect cells adjacent to the vessels from PDT, but also the vasculature itself. This might occur during brief light exposures (such as 5–6 min), and this protection would be largely independent of PDT dose. In contrast, extended light exposures would allow for opening and closing of vessels, thus exposing a much larger fraction of the vasculature to PDT damage, “averaging” the PDT dose over time and resulting in more effective tumor destruction. . The immediate translation of this paradigm faces the significant limitation of prolonged treatment times that are frequently not clinically feasible. It therefore must await the development of novel, possibly portable and/or implantable light sources. Such light sources are currently under development (4