Results

After 1-h application of the cream with ALA-Me, the fluorescence of PpIX in the skin increased for about 1 h, remained constant for another hour, and then decreased (Figure 1). The PpIX fluorescence was similar at 0.5 and at 3 h. Therefore, these two time points were chosen for further experiments.

Figure 1.

Production of protoporphyrin IX in normal mouse skin at different times after 1 h topical application of 20% 5-aminolevulinic acid methyl ester. The cream with ALA-ME was removed from the skin after the 1-h application. Error bars represent the standard errors for groups of three mice.

Full figure and legend (14K)

The fluence rates of the two light sources were adjusted so that the exposure times needed to reduce the PpIX fluorescence in the skin by 50% were in the same range (Figure 2). For the blue light lamp (420 nm), 1.5 and 2.6 min exposures were needed for 50% reduction 0.5 and 3 h, after cream removal, respectively (Figure 2a). For the light emitting diodes (LED) light (632 nm), the corresponding exposure times were 2.1 and 4.2 min (Figure 2b). In both cases, the photodegradation rate of PpIX was larger 0.5 h after cream removal than 3 h after removal (Figure 2). The rate of PpIX appearance after light exposure was significantly faster 0.5 h after cream removal than 3 h after cream removal. This was true both for exposure to blue and to red light (Figure 3 and 4). Furthermore, the appearance of PpIX 0.5 h after cream removal was faster after blue light exposure than after red light exposure Figure 3 and Figure 4. PpIX reappearance was significantly faster at a skin temperature of 35°C than at a temperature of 23°C (Figure 5). In all cases, the shape of the fluorescence excitation spectra was similar before and after light-induced degradation of PpIX in the skin (data not shown).

Figure 2.

Decay of protoporphyrin IX fluorescence in mouse skin induced by exposure to blue (A) or red (B) light at 0.5 or 3 h after removal of cream. Twenty percent of 5-aminolevulinic acid methyl ester cream was topically applied for 1 h and then removed. Error bars represent standard errors for groups of three mice.

Full figure and legend (29K)

Figure 3.

Rebuilding of protoporphyrin IX fluorescence in mouse skin after exposure to blue light at 0.5 h (A) or at 3 h (B) after removing the cream (20% 5-aminolevulinic acid methyl ester was topically applied for 1 h). The mice were irradiated for 1, 3, or 4 min. Error bars represent standard errors for groups of three mice.

Full figure and legend (31K)

Figure 4.

Rebuilding of protoporphyrin IX fluorescence in mouse skin after exposure to red light at 0.5 h (A) or at 3 h (B) after removing the cream (20% 5-aminolevulinic acid methyl ester was topically applied for 1 h). Mice were irradiated for 2, 5, or 7 min. Error bars represent standard errors for groups of three mice.

Full figure and legend (30K)

Figure 5.

Rebuilding of protoporphyrin IX fluorescence in mouse skin after exposure to red light for 5 min at 0.5 h (A) or for 7 min at 3 h (B) after cream removal. The mice were irradiated at 35°C () or at 23°C (). Topical application of 20% 5-aminolevulinic acid methyl ester was carried out on the mice for 1 h. Then the cream was removed. Error bars represent standard errors for groups of three mice.

Full figure and legend (29K)

Moreover, the reactions of skin to PDT with blue and red light were documented by photos (pictures not shown). In general, for blue light-PDT after 0.5 h, the skin reaction was negligible for light exposures of 1 and 3 min, whereas the skin became pale and significant edema developed for an exposure of 4 min. This reaction was less expressed when the skin was exposed at 3 h than at 0.5 h. For red light-PDT, slight edema could be seen when the skin was exposed for 7 min 0.5 h after removing the cream. No visible skin reaction was found when the red light was imparted at 3 h after removal of the cream.

The rate of PpIX elimination from the volume of detection was faster after blue light irradiation than that after red light irradiation (Figure 6). It was of interest to verify whether ALA-Me itself was stable during PDT with PpIX present. Because of their lipophilicity when added to the cream, both PpIX and ALA-Me should be present mainly in its oil compartments. ALA-Me was hardly destroyed by ¹O₂, even after a 15-min exposure to blue or red light (Table I).

Figure 6.

Rate of elimination of protoporphyrin IX from mouse skin after exposure for 3 min to blue light or 5 min to red light. Cream with 20% 5-aminolevulinic acid methyl ester was topically applied on the skin for 1 h. Then the cream was removed, and light exposure was imparted 3 h after cream removal. These two exposure times gave approximately the same bleaching effect. Error bars represent standard errors for groups of four mice.

Full figure and legend (16K)

Table I - Effect of photodynamic therapy with blue light or red light on ALA-Me.

Full table

Discussion

The kinetics of PpIX formation induced by ALA-Me in mouse skin observed in this work (Figure 1) are in general agreement with earlier findings (^{Moan et al, 2003}). The main purpose of this work was to study PpIX reappearance after light exposures that degrade a substantial fraction of the PpIX present in the skin. Knowledge of the kinetics of reappearance would be valuable in the planning of fractionated PDT.

Since it is known that second-order processes may play a role in porphyrin photodegradation (^{Moan et al, 1997}), we decided to carry out the light exposures at time points when the PpIX fluorescence intensity was similar, either in the accumulation phase (0–1 h, Figure 1) or in the decay phase (after 2 h, Figure 1). At 0.5 and 3 h the intensities are similar: about 70% of the maximal value (Figure 1).

The fact that PpIX synthesis occurs after cream removal shows that a significant amount of ALA-Me is present as a pool in the skin. This pool is not completely depleted even 3 h after cream removal, since some PpIX formation occurs at 35°C (Figure 5b). The ALA-Me in the pool is probably not sensitive to PDT itself. This is indicated by the data shown in Table I: ALA-Me exposed to PpIX and light in the cream induced similar amounts of PpIX in mouse skin as ALA-Me unexposed to light. Because of their lipophilicity, ALA-Me and PpIX can be supposed to be localized close to each other in the oil-containing cream. Therefore, the short diffusion length of ¹O₂ should be of no hindrance for photodegradation of ALA-Me if it were significantly sensitive to light exposures of the same magnitudes as those applied in this work.

The decay kinetics of the PpIX fluorescence in normal mouse skin during light exposure to the blue and red light are shown in Figure 2. As a first approximation, the decay curves can be decomposed into two exponential curves (Figure 2). After 5–6 min (2.5–3 J per cm²) and 5.5–8 min (30–45 J per cm²) light exposure with the blue and red light lamps, respectively, 75% of PpIX fluorescence was bleached. The remaining fluorescence was from PpIX and its photoproducts (^{Juzenas et al, 2001}), and was more photostable. This may be because of depletion of oxygen. From in vitro studies, it is known that the rate of bleaching with a constant fluence rate may depend on the concentration of oxygen.^{Robinson et al (1996)} observed an increase in photobleaching rate when increasing the oxygen or the PpIX concentration in the solution.^{Van Der Veen et al (1997)} did not find any correlation between the initial fluorescence intensity and the photobleaching rate in the mouse skin. This indicates that second-order processes are not dominating.

Faster initial photodegradation of PpIX was observed with blue light for both application times (0.5 and 3 h) after cream removal (Figure 7). The ratio of the light exposure times of the red and the blue light lamps needed to bleach an equal fraction of the PpIX was highest in the beginning of the light exposure and decreased with increasing light exposure time (Figure 7). When about 75% of the PpIX (fluorescence was bleached according to Figure 2), there was no significant difference between the cream removal times 0.5 and 3 h or between the lamps (Figure 7). The differences were largest after 3 h of cream application (Figure 2), which may be related to the depth distribution of PpIX. Exposure to the high fluence rate from the red light lamp (90 mW per cm²) may lead to a faster oxygen depletion compared with the blue light lamp (9 mW per cm²), resulting in slower bleaching. Faster bleaching is observed at low fluence rates (^{Robinson et al, 1999;Finlay et al, 2001}). The photobleaching rate of PpIX depends on the oxygen concentration (^{Robinson et al, 1996}). During light exposure, stasis or occlusion of the microvasculature may arise, and the supply of oxygen is gradually impaired.^{Xu et al (2004)} found that some of these problems could be reduced by using low fluence rates. Non-uniform oxygen bleaching could be the reason why the ratio of the light exposure times of the red and the blue light lamps needed to bleach an equal fraction of the PpIX was highest in the beginning of the light exposure (Figure 7).

Figure 7.

Ratio of the exposure times of the red and blue light to bleach an equal fraction of protoporphyrin IX (PpIX) in normal skin of mouse. The calculations were carried out using data from Figure 2.

Full figure and legend (14K)

The photodegradation rate of PpIX was significantly larger 0.5 h after cream removal than 3 h after removal (Figure 2). Since the exposure time is short, negligible synthesis of PpIX should occur during exposure. We propose that the difference in degradation rates is related to the distribution of PpIX in the skin. It is likely that it is localized deeper at 3 h than at 0.5 h (Figure 2) and, therefore, PpIX may appear more photostable.

The yield of photodegradation per incident photon is about a factor 15–20 larger at 420 nm than at 632 nm, which can be calculated from the data in Figure 2a and b using the measured fluence rates (90 mW per cm² at 632 nm and 9 mW per cm² at 420 nm). The value of the fluorescence excitation spectrum of PpIX is about 20–27 times larger at 420 nm than at 632 nm (^{Juzenas et al, 2002}). Thus, the photodegradation rate in the detection volume is about 1.4–2.3 times greater for exposure to light at 420 nm than for exposure at 632 nm (Figure 7), which is theoretically expected from light diffusion calculations (not shown) if it is assumed that the difference in light scattering in the skin at the two wavelengths is of minor importance.

Following light exposure up to 4 min at 420 nm 0.5 h after cream removal, the appearance of PpIX occurred at almost the same rate as that of the synthesis in unirradiated skin (Figure 3a). But, it was slower after exposure at 632 nm, notably for the largest exposures (Figure 4a). This is in agreement with the observation that ALA-Me seems to be stable in the cream during PDT. We can also conclude that the enzymatic machinery involved in PpIX synthesis is not significantly impaired by PDT at 420 nm (Figure 3a). The fact that some reappearance of PpIX occurs after the largest exposure at 420 nm but not at 632 nm is remarkable and not easy to explain. The only difference between the two exposures is that light penetrates significantly deeper in tissue at 632 nm than at 420 nm. The penetration depth into rat skin is 0.3 mm at 420 nm and 2 mm at 632 nm (^{Juzenas et al, 2002}). According to the data for 420 nm (Figure 3a), it is unlikely that the difference in reappearance rate (Figure 3a and Figure 4a) is related to enzymatic damage or photodegradation of ALA-Me. The amount of PpIX in the detection volume decreases after 2 h in unirradiated skin (Figure 1). Thus, PpIX is either transported away in the blood stream or converted to non-fluorescing products, most likely to heme. PDT at 632 nm acts deeply, and may damage the blood flow and reduce the oxygen concentration in the skin. Vessel damage would therefore lead to reduced removal of PpIX through the circulation and/or through heme synthesis, which is oxygen dependent. Thus, it seems that vessel damage plays no significant role here. The most likely explanation for the difference in reappearance after PDT at 420 and 632 nm (Figure 3a and Figure 4a) is diffusion of PpIX into the volume of measurement. This diffusion of intact PpIX would play a larger role after shallow PDT (420 nm) than after deep PDT (632 nm). More deeply localized PpIX would remain after PDT at 420 nm than after PDT at 632 nm. Removal of PpIX through the circulation would be faster for deeply localized PpIX than for shallowly localized PpIX. This is in agreement with the data shown in Figure 6.

In agreement with earlier work, the rate of PpIX reappearance is larger at 35°C than at 23°C (Figure 5). We have earlier found that the temperature dependence of PpIX synthesis is related to the efficiency of the rate-limiting enzyme porphobilinogen deaminase (^{Moan et al, 1999}).

The photographical documentation of the skin reactions after PDT with red and blue light at different time points are of interest in view of the kinetic data. Generally, the effects were larger after exposure to blue light than after exposure to red light, notably for short ALA-Me application times. Thus, the edema and paleness of skin seem to originate from primary reactions in the upper skin layers where blue light is predominantly absorbed and acts.

In summary, we have shown that the photodegradation rate of PpIX in mouse skin, as measured by its fluorescence, is larger if the light exposure is carried out 0.5 h after removal of the ALA-Me cream than when it is carried out 3 h after cream removal. A different pattern of localization of PpIX in the skin is probably the reason for this. After light exposures carried out early (0.5 h after cream removal), the machinery of PpIX synthesis, as well as the concentration of ALA-Me, seems to be damaged. At this time point, reappearance of PpIX in the volume of detection is faster after PDT at 420 nm than after PDT at 632 nm. This may be related to diffusion of PpIX in the skin. Finally, the reappearance of PpIX was found to be faster at a skin temperature of 35°C than at 23°C. These findings should be taken into account when fractioned PDT is being planned.

Materials and Methods

Animals

Animal studies were approved by the National Animal Research Authority (Norway), and were performed according to the European Convention for the Protection of Vertebrates Used for Scientific Purposes. Female Balb/c athymic nude mice were obtained from The Norwegian Radium Hospital (Oslo, Norway). At the start of each experiment, they were 7–8 wk old and weighed 18–25 g. Three mice were housed per cage with an autoclaved filter covers in a room with subdued light at constant temperature (24°C–26°C) and humidity (30%–50%). Food and bedding were sterilized, and the mice were given water ad libitum in sterilized bottles. For proper application of the cream, anesthesia, Hypnorm (Janssen Pharmaceutica B.V., Tilburg, the Netherlands) and Dormicum (Hoffmann-La Roche AG, Basel, Switzerland) (1:1 vol/vol, approximately 4 mL per kg body weight), was intraperitoneally injected into the mice. The mice awakened within 1 h and appeared normally active during the ALA-Me application.

Chemicals

ALA-Me and PpIX were purchased from Sigma (St Louis, Missouri). Twenty percent (wt/wt) ALA-Me was dissolved in a cream (Unguentum, Merck, Darmstadt, Germany). Approximately 0.2 g of the freshly prepared cream was applied to a single spot of 1 cm diameter on normal skin of the mice. Then the spot was covered with an adhesive dressing (OpSite Flexigrid, Smith and Nephew Medical, Hull, UK). The cream was left on the skin for 1 h and then removed. At different times after cream removal, the PpIX was measured with a fiberoptic probe.

Lamps

Exposure of the skin to light was performed using two lamps. An in-house-produced blue light lamp with four fluorescent tubes (Philips Lighting, TLK 40 W/03, R.V. Roosendaal, the Netherlands) emited light in the wavelength region 400–460 nm with a peak at 420 nm. The fluence rate at the surface of the mouse skin was 90.5 mW per cm² as measured with the photodiode (NewPort, Model 1815-C, Irvine, California). An LED lamp (PhotoCure ASA, Oslo, Norway) possess the spectral range 580–670 nm and a peak wavelength at 632 nm. The fluence rate at the surface of the mouse skin was 904 mW per cm².

Fluorescence measurements

The fluorescence intensity of PpIX in the skin was determined by means of a fiberoptic probe connected to a Perkin Elmer LS50B spectrofluorimeter (Norwalk, Connecticut). The excitation wavelength was set at 407 nm; the slit width corresponded to a resolution of 10 nm. The emission wavelength was scanned from 550 to 750 nm.

Fluorescence kinetics

The fluorescence of PpIX in the mouse skin before and after light exposure was measured. The animals were divided into three experimental groups. Group one: production of PpIX in the skin was measured as a function of the time after removal of ALA-Me; group two: photobleaching and appearance of PpIX in skin irradiated 0.5 h after cream removal; and group three: photobleaching and appearance of PpIX in skin irradiated 3 h after cream removal. ALA-Me was topically applied for 1 h and then removed.

Temperature control

The effect of temperature on the ability of the skin to resynthesize PpIX after light exposure was studied. The temperature of the mouse skin was measured with a calibrated thin thermocouple (Kane-May Ltd. KM457XP, Welwyn Garden City, UK). Before light exposure, the mice were administered different amounts of anesthetics i.p.(0.02–0.07 mL per mouse), which resulted in different skin temperatures.

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Acknowledgments

The work was supported by The Norwegian Cancer Society, Oslo, Norway.