Henriëtte S. de Bruijn

Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5‐aminolaevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT‐induced dam‐age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo‐bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo‐bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT‐induced dam‐age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo‐bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Protoporphyrin IX Fluorescence Photobleaching during ALA-Mediated Photodynamic Therapy of UVB-Induced Tumors in Hairless Mouse Skin

Abstract— Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB‐induced tumor tissue in the SKH‐HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT‐induced damage were examined. Results show that the rate of PpIX photobleaching (i.e. fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB‐induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB‐induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.

Oncologic Photodynamic Therapy: Basic Principles, Current Clinical Status and Future Directions

Photodynamic therapy (PDT) is a clinically approved cancer therapy, based on a photochemical reaction between a light activatable molecule or photosensitizer, light, and molecular oxygen. When these three harmless components are present together, reactive oxygen species are formed. These can directly damage cells and/or vasculature, and induce inflammatory and immune responses. PDT is a two-stage procedure, which starts with photosensitizer administration followed by a locally directed light exposure, with the aim of confined tumor destruction. Since its regulatory approval, over 30 years ago, PDT has been the subject of numerous studies and has proven to be an effective form of cancer therapy. This review provides an overview of the clinical trials conducted over the last 10 years, illustrating how PDT is applied in the clinic today. Furthermore, examples from ongoing clinical trials and the most recent preclinical studies are presented, to show the directions, in which PDT is headed, in the near and distant future. Despite the clinical success reported, PDT is still currently underutilized in the clinic. We also discuss the factors that hamper the exploration of this effective therapy and what should be changed to render it a more effective and more widely available option for patients.

Topical 5-Aminolevulinic Acid-photodynamic Therapy of Hairless Mouse Skin Using Two-fold Illumination Schemes: PpIX Fluorescence Kinetics, Photobleaching and Biological Effect†¶

Abstract Light fractionation with dark periods of the order of hours has been shown to considerably increase the efficacy of 5-aminolevulinic acid-photodynamic therapy (ALA-PDT). Recent investigations have suggested that this increase may be due to the resynthesis of protoporphyrin IX (PpIX) during the dark period following the first illumination that is then utilized in the second light fraction. We have investigated the kinetics of PpIX fluorescence and PDT-induced damage during PDT in the normal skin of the SKH1 HR hairless mouse. A single illumination (514 nm), with light fluences of 5, 10 and 50 J cm−2 was performed 4 h after the application of 20% ALA, to determine the effect of PDT on the synthesis of PpIX. Results show that the kinetics of PpIX fluorescence after illumination are dependent on the fluence delivered; the resynthesis of PpIX is progressively inhibited following fluences above 10 J cm−2. In order to determine the influence of the PpIX fluorescence intensity at the time of the second illumination on the visual skin damage, 5 + 95 and 50 + 50 J cm−2 (when significantly less PpIX fluorescence is present before the second illumination), were delivered with a dark interval of 2 h between light fractions. Each scheme was compared to illumination with 100 J cm−2 in a single fraction delivered 4 or 6 h after the application of ALA. As we have shown previously greater skin damage results when an equal light fluence is delivered in two fractions. However, significantly more damage results when 5 J cm−2 is delivered in the first light fraction. Also, delivering 5 J cm−2 at 5 mW cm−2 + 95 J cm−2 at 50 mW cm−2 results in a reduction in visual skin damage from that obtained with 5 + 95 J cm−2 at 50 mW cm−2. A similar reduction in damage is observed if 5 + 45 J cm−2 are delivered at 50 mW cm−2. PpIX photoproducts are formed during illumination and subsequently photobleached. PpIX photoproducts do not dissipate in the 2 h dark interval between illuminations.

Photobleaching during and re-appearance after photodynamic therapy of topical ALA-induced fluorescence in UVB-treated mouse skin

Photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5‐aminolevulinic acid (ALA) seems a promising alternative for the treatment of superficial non‐melanoma skin cancer and actinic keratosis. In this study, the kinetics of new PpIX fluorescence arising after a PDT treatment that had photobleached the original fluorescence were determined. Our purpose was to examine the feasibility of multiple irradiations, following a single topical ALA application, to increase PDT efficacy. In addition, photobleaching during PDT and the fluorescence spectra during and after PDT were studied. As a model we used hairless mice with and without UVB‐induced skin lesions. ALA was applied to the skin for 4 hr. An illumination was delivered either immediately after application or 6 hr after the end of the application (at interval of maximum fluorescence). During PDT, the fluorescence of normal skin decreased at a faster rate than the fluorescence of the skin lesions. In the fluorescence study after PDT, the areas treated immediately post‐application showed a fluorescence increase over time similar to that in non‐treated areas on the same mice. A remarkable result was that the fluorescence of areas treated at maximum fluorescence increased, whereas the fluorescence of non‐treated areas did not increase over time. With both treatment intervals the new fluorescence showed a characteristic PpIX spectrum. Our results demonstrate that a second illumination, when new PpIX fluorescence has been formed, may increase PDT efficacy after topical ALA application. This finding has been demonstrated previously for systemic ALA administration. Int. J. Cancer 72:110–118, 1997.

Dose and Timing of the First Light Fraction in Two-fold Illumination Schemes for Topical ALA-mediated Photodynamic Therapy of Hairless Mouse Skin¶

Abstract A fractionated illumination scheme in which a cumulative fluence of 100 J cm−2 is delivered in two equal light fractions separated by a dark interval of 2 h has been shown to considerably increase the efficacy of 5-aminolevulinic acid (ALA)–photodynamic therapy (PDT). The efficacy of such a scheme is further increased if the fluence of the first light fraction is reduced to 5 J cm−2. We have investigated the relationship between the PDT response and the kinetics of protoporphyrin IX (PpIX) fluorescence in the SKH1 HR hairless mouse for first fraction fluences below 5 J cm−2 delivered 4 h after the application of ALA and 10 J cm−2 delivered 2 h after the application of ALA. Illumination is performed using 514 nm at a fluence rate of 50 mW cm−2. Reducing the fluence of the first fraction to 2.5 J cm−2 does not result in significantly different visual skin damage. The PDT response, however, is significantly reduced if the fluence is lowered to 1 J cm−2, but this illumination scheme (1 + 99 J cm−2) remains significantly more effective than a single illumination of 100 J cm−2. A first light fraction of 10 J cm−2 can be delivered 2 h earlier, 2 h after the application of ALA, without significant reduction in the PDT response compared with 5 + 95 J cm−2 delivered 4 and 6 h after the application of ALA. The kinetics of PpIX fluorescence are consistent with those reported previously by us and do not explain the significant increase in PDT response with a two-fold illumination scheme. Histological sections of the illuminated volume showed a trend toward increasing extent and depth of necrosis for the two-fold illumination scheme in which the first light fraction is 5 J cm−2, compared with a single illumination scheme.

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48 h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mast cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2h found after ALA but not after MAL-PDT.

In vivo monitoring of protein-bound and free NADH during ischemia by nonlinear spectral imaging microscopy

Nonlinear spectral imaging microscopy (NSIM) allows simultaneous morphological and spectroscopic investigation of intercellular events within living animals. In this study we used NSIM for in vivo time-lapse in-depth spectral imaging and monitoring of protein-bound and free reduced nicotinamide adenine dinucleotide (NADH) in mouse keratinocytes following total acute ischemia for 3.3 h at ~3 min time intervals. The high spectral resolution of NSIM images allows discrimination between the two-photon excited fluorescence emission of protein-bound and free NAD(P)H by applying linear spectral unmixing to the spectral image data. Results reveal the difference in the dynamic response between protein-bound and free NAD(P)H to ischemia-induced hypoxia/anoxia. Our results demonstrate the capability of nonlinear spectral imaging microscopy in unraveling dynamic cellular metabolic events within living animals for long periods of time.

Monitoring ALA-induced PpIX Photodynamic Therapy in the Rat Esophagus Using Fluorescence and Reflectance Spectroscopy

The presence of phased protoporphyrin IX (PpIX) bleach kinetics has been shown to correlate with esophageal response to 5‐aminolevulinic acid‐based photodynamic therapy (ALA‐PDT) in animal models. Here we confirm the existence of phased PpIX photobleaching by increasing the temporal resolution of the fluorescence measurements using the therapeutic illumination and long wavelength fluorescence detection. Furthermore fluorescence differential pathlength spectroscopy (FDPS) was incorporated to provide information on the effects of PpIX and tissue oxygenation distribution on the PpIX bleach kinetics during illumination. ALA at a dose of 200 mg kg−1 was orally administered to 15 rats, five rats served as control animals. PDT was performed at an in situ measured fluence rate of 75 mW cm−2 using a total fluence of 54 J cm−2. Forty‐eight hours after PDT the esophagus was excised and histologically examined for PDT‐induced damage. Fluence rate and PpIX photobleaching at 705 nm were monitored during therapeutic illumination with the same isotropic probe. A new method, FDPS, was used for superficial measurement on saturation, blood volume, scattering characteristics and PpIX fluorescence. Results showed two‐phased PpIX photobleaching that was not related to a (systematic) change in esophageal oxygenation but was associated with an increase in average blood volume. PpIX fluorescence photobleaching measured using FDPS, in which fluorescence signals are only acquired from the superficial layers of the esophagus, showed lower rates of photobleaching and no distinct phases. No clear correlation between two‐phased photobleaching and histologic tissue response was found. This study demonstrates the feasibility of measuring fluence rate, PpIX fluorescence and FDPS during PDT in the esophagus. We conclude that the spatial distribution of PpIX significantly influences the kinetics of photobleaching and that there is a complex interrelationship between the distribution of PpIX and the supply of oxygen to the illuminated tissue volume.

Protoporphyrin IX Fluorescence Kinetics and Localization after Topical Application of ALA Pentyl Ester and ALA on Hairless Mouse Skin with UVB‐Induced Early Skin Cancer

Abstract In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.