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Changes in human skin after topical PDT with hexyl aminolevulinate
Photodiagnosis and Photodynamic Therapy (2008) 5, 176—181
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pdpdt
Changes in human skin after topical PDT with hexyl aminolevulinate Asta Juzeniene a,∗, Kristian Pagh Nielsen b, Lu Zhao b, Gennady A. Ryzhikov b, Marina S. Biryulina b, Jakob J. Stamnes b,c, Knut Stamnes d, Johan Moan a,e a
Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, 0310 Oslo, Norway b Balter Medical AS, Thormøhlensgt. 55, N-5008 Bergen, Norway c Department of Physics and Technology, University of Bergen, Allégt. 55, 5007 Bergen, Norway d Department of Physics and Engineering Physics, Light and Life Laboratory, Stevens Institute of Technology, Castle Point on Hudson, NJ, USA e Institute of Physics, University of Oslo, Blindern, 0316 Oslo, Norway Available online 28 August 2008
KEYWORDS Photodynamic therapy; 5-Aminolevulinic acid; Hexyl aminolevulinate; Oxygen; Pigmentation; Erythema
Summary Background: Photodynamic therapy (PDT) induces physiological changes in human skin, but details and kinetics are not known. Methods: Changes in human skin induced by PDT with red light in the presence of topically applied cream with the hexyl aminolevulinate (HAL) were investigated in the skin of five healthy volunteers. In addition to testing the effects of HAL-PDT three control studies were performed on the volunteers: (A) the HAL containing cream was applied to the skin without light exposure; (B) the cream without HAL was applied to the skin; (C) the skin was exposed to light in the absence of the cream. Reflectance spectra of the skin were measured in the wavelength region 300—600 nm before and after treatment. An advanced and new inverse radiative transfer model was used to determine changes induced in a number of skin parameters. Results: The main discoveries were that the dermal blood concentration increased immediately after PDT, reached a maximum after 1—2 days, and then decreased. The blood oxygenation increased significantly immediately after PDT and then decreased. After PDT the melanosome concentration in the upper epidermis increased steadily. No such changes were observed in the control sites. Conclusions: Our results imply that HAL-PDT leads to increased vascularisation, oxygenation and melanin formation. © 2008 Elsevier B.V. All rights reserved.
Introduction
∗
Corresponding author. Tel.: +47 22935113; fax: +47 22934270. E-mail address: [email protected] (A. Juzeniene).
Photodynamic therapy (PDT) of skin disorders with 5aminolevulinic acid (ALA) or ALA derivatives is becoming a standard treatment in a number of countries [1,2]. A similar procedure is being used for skin photorejuvenation [2,3].
1572-1000/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2008.07.001
Changes in human skin Furthermore, ALA derivatives have been proposed as ingredients in sun creams since they provide melanin generating and tumour protecting properties [4—6]. It is well known that topical PDT acts on the vascular system in the skin [7—10]. Topical PDT can lead to vasoconstriction, vessel clogging, and vasodilatation, depending on the conditions such as fluence rate, fluence, drug concentration, drug application time and tissue type [8,9]. These effects can in turn lead to changes in the oxygenation. Also, oxygen is consumed in the photodynamic action itself [11,12]. Knowledge of these processes is of great importance for the development of optimal PDT procedures. It is desirable to be able to monitor them non-invasively during and after light exposure. Topical PDT leads to pigmentation [5,13,14], but neither the kinetics nor the mechanisms of this are well known. We here utilize a non-invasive technique, called Optical Transfer Retrieval (OTR), for monitoring of a number of physiological/morphological parameters in the skin, including those mentioned above. Furthermore, we report an application of the OTR technique on human skin after topical HAL-PDT. The aim is to elucidate details of the kinetics of vascular effects and melanogenesis due to HAL-PDT. The OTR technique is based on reflectance spectroscopy and inverse radiative transfer modelling based on the discrete-ordinate radiative-transfer (DISORT) method [15,16].
Materials and methods Chemicals Hexyl aminolevulinate (5-aminolevulinic acid hexyl ester) hydrochloride (HAL HCl, MW = 251.8 g/mol) was synthesized and donated to us by Norbert Lange (Lausanne, Switzerland) or by PhotoCure ASA (Oslo, Norway).
Volunteers Experiments were carried out on five healthy volunteers (age from 27 to 59 years) with skin types II or III. The study was approved by the local ethical committee (Regional komite for medisinsk forskningsetikk Sør-Norge, Ref. S-05112). The volunteers did not use any medications or drugs.
Topical application of HAL Details of the experimental procedures were described earlier [17]. Cream was prepared using 10% (w/w) of HAL in a standard oil-in-water base (Unguentum Merck, Darmstad, Germany). As the test area, the inner part of the right forearm of each volunteer was covered with a transparent adhesive dressing (OpSite Flexifix, Smith & Nephew Medical Ltd., Hull, UK). As the control area, the inner part of the left forearm of each volunteer was also covered with a transparent adhesive dressing of the same type. On each of these two dressings, three openings were cut, each being 1 cm × 1 cm in area. The distance between adjacent openings was approximately 1.5 cm. Freshly prepared cream with approximately 75 mg/cm2 ± 10 mg/cm2 of HAL was topically applied on the three areas of skin under the three openings
177 of the dressing on the right forearm. The three areas of skin under the dressing on the left forearm were used for control while real changes were observed at the test area. On the left forearm, one of the three skin control areas was applied cream with HAL, one was applied cream without HAL, and the other one was applied no cream at all. Then the same type dressing was applied on all areas to prevent cream diffusion to adjacent areas. The creams and the dressings were kept for 24 h.
Light exposure The dressings and the creams were removed after 24 h from all areas. Afterwards, the three test areas and one control (light) area were exposed separately with red light for 2 min. A Curelight lamp (Photocure ASA, Norway) with an emission peak at 632 ± 11 nm was used for light exposure of the three test areas and the three control areas. The fluence rate was 90 mW/cm2 as measured 5 cm below the lamp, i.e., at the position of the test and control areas.
Reflectance spectroscopy Reflectance measurements were conducted with a luminescence spectrometer (PerkinElmer LS50B, Norwalk, CT, USA). The spectrometer was equipped with two scanning grating monochromators, one in front of the light source (a pulsed xenon lamp) and the other in front of the detector. Reflectance spectra were measured in synchronous scans in which both gratings were set at the same wavelength and band pass (5 nm). Thus, fluorescence artefacts were avoided. The area exposed to the excitation light of the spectrometer and the area from which the reflected flux was detected by the spectrometer were the same. The geometry of the fibre probe was such that both the directly (Fresnel) reflected and the diffusely reflected light from the skin were collected and recorded. These reflectance spectra were dominated by the emission spectrum of the xenon lamp of the spectrometer, the transmission spectrum of the optical system, and the optical properties of the skin. Reflectance spectra from the surface of a TiO2 plate with a single-scattering albedo of approximately equal to 1 and a reflectance close to 1 were recorded and used for calibration. The auto-reflectance spectrum was recorded in a dark room by pointing the probe at darkness. This auto-reflectance spectrum was subtracted from each recorded reflectance spectrum. After subtraction of the auto-reflectance spectrum, all recorded reflectance spectra from the test and control areas were normalized to the reflectance spectrum of TiO2 .
Radiative transfer model The radiative transfer model and the discrete ordinate method have been described in detail elsewhere [15,16,18,19]. With this method given physiological/morphological parameters can be calculated from the reflectance spectra of skin [16]. We refer to this technique as OTR. In Ref. [16] the focus was on testing the feasibility of the mathematical approach, whereas we,
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in this manuscript, focus on the biological implications of retrieved results.
Statistics and curve fitting The same person has performed all measurements. The study was not blinded. In the original dataset there were three repetitions of each measurement. In the inversion analysis the average of these three repetitions was used. Data are presented as mean ± S.E. (standard error). The obtained data were also analysed using standard linear, exponential or sigmoid fitting routines in Sigmaplot 10.0 from Systat Software, Inc. (Richmond, CA, USA).
Results and discussion Figure 1 shows the typical reflectance spectra of the skin from one volunteer (skin type III) before HAL application and light exposure (baseline reflectance spectrum) and immediately after HAL-PDT. The baseline reflectance spectra, measured in the mentioned areas, were relatively consistent for the same volunteer, but they vary between volunteers due to skin thickness, melanin concentration, hemoglobin concentration, etc. There were differences between the test areas (HAL-PDT) of one person and bigger differences between the test areas of different persons, but recorded reflectance spectra from all five volunteers had the same tendency after HAL-PDT. Knowledge of the absorption spectra of melanin, hemoglobin and oxyhemoglobin [20] is essential for OTR-determination of the following seven physiological/morphological parameters of interest: dermal blood concentration and oxygenation, upper and lower epidermal thickness, upper and lower epidermal melanosome concentration, and epidermal keratin concentration. We separate the epidermis into an upper and a lower part because the melanosomes, at least in the lighter skin types, are found mostly in the lower layers of the epidermis. This makes the optical properties of the upper part of the epidermis significantly different from those of the lower part of the epidermis [21,22]. Therefore, it is very reasonable to separate the epidermis into two distinct layers in an opti-
Figure 1 Mean reflectance spectra recorded from three test areas on human forearm skin before HAL application and light exposure (baseline) and immediately after HAL-PDT.
Figure 2 Retrieved percentage of dermal blood concentration before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HALPDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
cal model of the skin. The OTR calculations required for retrieval of the seven physiological/morphological parameters are time consuming. Therefore, since the recorded reflectance spectra for the five persons taking part in the study were found to have the similar tendency after treatment, we chose the recorded reflectance spectra from one volunteer to be representative in the present detailed investigation. Figs. 2—6 show the kinetics of the changes of the mentioned seven parameters in the same one volunteer. The application of cream without HAL alone or exposure to light alone had no influence on the above-mentioned parameters (Figs. 2—6A and C). A slight redness was observed after topical application of 10% HAL alone for 24 h. The dermal blood concentration increased from 2.7 ± 0.2% to 4.2 ± 0.4% after treatment with HAL alone, indicating a slight erythema, and then declined to the baseline concentration within 2 days (Fig. 2B). This erythema induction is probably due to effects of HAL itself on the vasculature system. Similarly, the dermal blood concentration increased immediately after HAL-PDT, but, instead of decreasing as for HAL alone, it continued to increase for 2 days to a maximum level of (7.1 ± 0.4%). Then decayed over the next 10 days, but even at that time it remained above control values (Fig. 2D). The given PDT dose (10.8 J/cm2 ) was smaller than those used in tumour treatment (50—100 J/cm2 ). Nevertheless, similar erythema, lasting for 2—3 days, has been reported by several authors [23—27].
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Figure 3 Retrieved percentage of oxygenated dermal blood before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HALPDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
Figure 4 Retrieved thickness (m) of the upper (䊉) and the lower () epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
The dermal blood oxygenation followed similar kinetics as the dermal blood concentration after HAL treatment (Fig. 3B). An immediate increase of 70% of the dermal blood oxygenation (as for HAL) was observed also for HAL-PDT (Fig. 3D). It remained stable for 2 days, and then decayed, but baseline values were not reached during the 2 weeks of observation (Fig. 3D). The similarity in the kinetics of the dermal blood concentration and in that of the oxygenation for HAL-PDT can probably be explained by vasodilatation, leading to faster blood flow, and, therefore, apparently to less oxygen depletion in the dermal tissues. Since the PDT efficiency increases with oxygenation [28,29], this indicates that a fractionated exposure scheme (light exposure is split into two or more fractions with dark intervals between after a single application of ALA) might be advantageous under the present condition. The OTR method for determining the percent blood oxygenation is non-invasive, and will therefore be useful for optimal timing of fractionated therapy. The thickness of the upper part of the epidermis decreased after HAL treatment, but then started to increase and reached the baseline thickness after 1 week (Fig. 4B). While the thickness of the lower part of the epidermis did not change immediately after HAL treatment, a 1.4-fold increase was observed 2—3 days afterwards. For the rest of the observation time this thickness remained constant (Fig. 4B). The keratin concentration in the upper epidermis decreased immediately after HAL treatment, but later
Figure 5 Retrieved keratin concentration (%) in the upper epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
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Acknowledgements The present work was supported by the Research Foundation of the Norwegian Radium Hospital and the Norwegian Cancer Society (Kreftforeningen).
References
Figure 6 Retrieved melanosome concentration (%) in the upper (䊉) and the lower () epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
increased again (Fig. 5B). The thickness of the lower part of the epidermis decreased during HAL-PDT and continued to decrease the next 2—3 days. After that it started to increase and reached a maximum level around 8 days after PDT. The thickness and keratin concentration of the upper part of epidermis remained constant for 2—4 days after HAL-PDT, but then started to decline (Fig. 4D and Fig. 5D). Figure 6 shows how the melanosome concentration in the upper and lower part of the epidermis changed after HAL application and after HAL-PDT. The melanosome concentration after topical application of HAL remained unchanged in the upper part of the epidermis, while in the lower part of the epidermis it first increased slightly and then decreased (Fig. 6B). There was a steady increase in the melanosome concentration after HAL-PDT in the upper layer of the epidermis (Fig. 6D). In the lower part of the epidermis the concentration of melanosomes was the same for 4 days. Then it started to decrease and reached a constant lower level after 4—5 days (Fig. 6D). These changes may be due to the diffusion of melanin dust to the upper part of the epidermis or to changes in the melanosome size distribution. We, and others, have earlier noted pigment induction after PDT [4,5,13,14,26,30]. The observed pigmentation after PDT may be due to tyrosinase activation through singlet oxygen mediated production of diacylglycerol [5]. This is the first time that the evolution of the pigmentation has been quantitatively followed.
[1] Braathen LR, Szeimies RM, Basset-Seguin N, et al. Guidelines on the use of photodynamic therapy for nonmelanoma skin cancer: an international consensus. International Society for Photodynamic Therapy in Dermatology, 2005. J Am Acad Dermatol 2007;56(1):125—43. [2] Gold MH. Photodynamic therapy with lasers and intense pulsed light. Fac Plast Surg Clin North Am 2007;15(2):145—60. [3] Nootheti PK, Goldman MP. Aminolevulinic acid-photodynamic therapy for photorejuvenation. Dermatol Clin 2007; 25(1):35—45. [4] Moan J, Bissonnette R. Skin preparation. Patent 2001; 10/275,557 US Patent 6,911,194 B2. [5] Monfrecola G, Procaccini EM, D’Onofrio D, et al. Hyperpigmentation induced by topical 5-aminolaevulinic acid plus visible light. J Photochem Photobiol B 2002;68(2—3):147—55. [6] Sharfaei S, Juzenas P, Moan J, Bissonnette R. Weekly topical application of methyl aminolevulinate followed by light exposure delays the appearance of UV-induced skin tumours in mice. Arch Dermatol Res 2002;294(5):237—42. [7] Henderson BW, Vaughan L, Bellnier DA, van Leengoed H, Johnson PG, Oseroff AR. Photosensitization of murine tumor, vasculature and skin by 5-aminolevulinic acid-induced porphyrin. Photochem Photobiol 1995;62(4):780—9. [8] Schacht V, Szeimies RM, Abels C. Photodynamic therapy with 5-aminolevulinic acid induces distinct microcirculatory effects following systemic or topical application. Photochem Photobiol Sci 2006;5(5):452—8. [9] Van der Veen N, Hebeda KM, de Bruijn HS, Star WM. Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination. Photochem Photobiol 1999;70(6):921—9. [10] Wang I, ndersson-Engels S, Nilsson GE, Wardell K, Svanberg K. Superficial blood flow following photodynamic therapy of malignant non-melanoma skin tumours measured by laser Doppler perfusion imaging. Br J Dermatol 1997;136(2):184—9. [11] Foster TH, Murant RS, Bryant RG, Knox RS, Gibson SL, Hilf R. Oxygen consumption and diffusion effects in photodynamic therapy. Radiat Res 1991;126(3):296—303. [12] Tromberg BJ, Orenstein A, Kimel S, et al. In vivo tumor oxygen tension measurements for the evaluation of the efficiency of photodynamic therapy. Photochem Photobiol 1990;52(2):375—85. [13] Bissonnette R, Shapiro J, Zeng H, McLean DI, Lui H. Topical photodynamic therapy with 5-aminolaevulinic acid does not induce hair regrowth in patients with extensive alopecia areata. Br J Dermatol 2000;143(5):1032—5. [14] Itoh Y, Ninomiya Y, Tajima S, Ishibashi A. Photodynamic therapy of acne vulgaris with topical delta-aminolaevulinic acid and incoherent light in Japanese patients. Br J Dermatol 2001;144(3):575—9. [15] Hestenes K, Nielsen KP, Zhao L, Stamnes JJ, Stamnes K. Monte Carlo and discrete-ordinate simulations of spectral radiances in a coupled air-tissue system. Appl Opt 2007;46(12):2333— 50. [16] Nielsen KP, Zhao L, Ryzhikov GA, et al. Retrieval of the physiological state of human skin from UV—VIS reflectance spectra—–a feasibility study. J Photochem Photobiol B 2008;93:23—31. [17] Zhao L, Nielsen KP, Juzeniene A, et al. Spectroscopic measurements of photoinduced processes in human skin after topical
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application of the hexyl ester of 5-aminolevulinic acid. J Environ Pathol Toxicol Oncol 2006;25(1—2):307—20. Nielsen KP, Lu Z, Juzenas P, Stamnes JJ, Stamnes K, Moan J. Reflectance spectra of pigmented and nonpigmented skin in the UV spectral region. Photochem Photobiol 2004;80(3):450—5. Nielsen KP, Juzeniene A, Juzenas P, Stamnes K, Stamnes JJ, Moan J. Choice of optimal wavelength for PDT: the significance of oxygen depletion. Photochem Photobiol 2005;81(5):1190—4. Plaetzer K, Krammer B, Berlanda J, Berr F, Kiesslich T. Photophysics and photochemistry of photodynamic therapy: fundamental aspects. Lasers Med Sci 2008, doi:10.1007/s10103-008-0539-1. Tadokoro T, Yamaguchi Y, Batzer J, et al. Mechanisms of skin tanning in different racial/ethnic groups in response to ultraviolet radiation. J Invest Dermatol 2005;124(6):1326—32. Yamaguchi Y, Hearing VJ. Melanocyte distribution and function in human skin. Effects of ultraviolet radiation. In: Hearing VJ, Leong SPL, editors. Melanocytes to Melanoma: The Progression to Malignancy. 1st ed. Totowa: Humana Press, Inc.; 2005. p. 101—15. Alexiades-Armenakas M. Long-pulsed dye laser-mediated photodynamic therapy combined with topical therapy for mild to severe comedonal, inflammatory, or cystic acne. J Drugs Dermatol 2006;5(1):45—55.
181 [24] Alster TS, Tanzi EL, Welsh EC. Photorejuvenation of facial skin with topical 20% 5-aminolevulinic acid and intense pulsed light treatment: a split-face comparison study. J Drugs Dermatol 2005;4(1):35—8. [25] Goldman MP, Merial-Kieny C, Nocera T, Mery S. Comparative benefit of two thermal spring waters after photodynamic therapy procedure. J Cosmet Dermatol 2007;6(1):31—5. [26] Wiegell SR, Stender IM, Na R, Wulf HC. Pain associated with photodynamic therapy using 5-aminolevulinic acid or 5aminolevulinic acid methylester on tape-stripped normal skin. Arch Dermatol 2003;139(9):1173—7. [27] Wiegell SR, Wulf HC. Photodynamic therapy of acne vulgaris using 5-aminolevulinic acid versus methyl aminolevulinate. J Am Acad Dermatol 2006;54(4):647—51. [28] Henderson BW, Fingar VH. Relationship of tumor hypoxia and response to photodynamic treatment in an experimental mouse tumor. Cancer Res 1987;47(12):3110—4. [29] Moan J, Sommer S. Oxygen dependence of the photosensitizing effect of hematoporphyrin derivative in NHIK 3025 cells. Cancer Res 1985;45(4):1608—10. [30] Pogue BW, O’Hara JA, Goodwin IA, et al. Tumor PO(2) changes during photodynamic therapy depend upon photosensitizer type and time after injection. Comp Biochem Physiol A Mol Integr Physiol 2002;132(1):177—84.
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/pdpdt
Changes in human skin after topical PDT with hexyl aminolevulinate Asta Juzeniene a,∗, Kristian Pagh Nielsen b, Lu Zhao b, Gennady A. Ryzhikov b, Marina S. Biryulina b, Jakob J. Stamnes b,c, Knut Stamnes d, Johan Moan a,e a
Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Rikshospitalet University Hospital, Montebello, 0310 Oslo, Norway b Balter Medical AS, Thormøhlensgt. 55, N-5008 Bergen, Norway c Department of Physics and Technology, University of Bergen, Allégt. 55, 5007 Bergen, Norway d Department of Physics and Engineering Physics, Light and Life Laboratory, Stevens Institute of Technology, Castle Point on Hudson, NJ, USA e Institute of Physics, University of Oslo, Blindern, 0316 Oslo, Norway Available online 28 August 2008
KEYWORDS Photodynamic therapy; 5-Aminolevulinic acid; Hexyl aminolevulinate; Oxygen; Pigmentation; Erythema
Summary Background: Photodynamic therapy (PDT) induces physiological changes in human skin, but details and kinetics are not known. Methods: Changes in human skin induced by PDT with red light in the presence of topically applied cream with the hexyl aminolevulinate (HAL) were investigated in the skin of five healthy volunteers. In addition to testing the effects of HAL-PDT three control studies were performed on the volunteers: (A) the HAL containing cream was applied to the skin without light exposure; (B) the cream without HAL was applied to the skin; (C) the skin was exposed to light in the absence of the cream. Reflectance spectra of the skin were measured in the wavelength region 300—600 nm before and after treatment. An advanced and new inverse radiative transfer model was used to determine changes induced in a number of skin parameters. Results: The main discoveries were that the dermal blood concentration increased immediately after PDT, reached a maximum after 1—2 days, and then decreased. The blood oxygenation increased significantly immediately after PDT and then decreased. After PDT the melanosome concentration in the upper epidermis increased steadily. No such changes were observed in the control sites. Conclusions: Our results imply that HAL-PDT leads to increased vascularisation, oxygenation and melanin formation. © 2008 Elsevier B.V. All rights reserved.
Introduction
∗
Corresponding author. Tel.: +47 22935113; fax: +47 22934270. E-mail address: [email protected] (A. Juzeniene).
Photodynamic therapy (PDT) of skin disorders with 5aminolevulinic acid (ALA) or ALA derivatives is becoming a standard treatment in a number of countries [1,2]. A similar procedure is being used for skin photorejuvenation [2,3].
1572-1000/$ — see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2008.07.001
Changes in human skin Furthermore, ALA derivatives have been proposed as ingredients in sun creams since they provide melanin generating and tumour protecting properties [4—6]. It is well known that topical PDT acts on the vascular system in the skin [7—10]. Topical PDT can lead to vasoconstriction, vessel clogging, and vasodilatation, depending on the conditions such as fluence rate, fluence, drug concentration, drug application time and tissue type [8,9]. These effects can in turn lead to changes in the oxygenation. Also, oxygen is consumed in the photodynamic action itself [11,12]. Knowledge of these processes is of great importance for the development of optimal PDT procedures. It is desirable to be able to monitor them non-invasively during and after light exposure. Topical PDT leads to pigmentation [5,13,14], but neither the kinetics nor the mechanisms of this are well known. We here utilize a non-invasive technique, called Optical Transfer Retrieval (OTR), for monitoring of a number of physiological/morphological parameters in the skin, including those mentioned above. Furthermore, we report an application of the OTR technique on human skin after topical HAL-PDT. The aim is to elucidate details of the kinetics of vascular effects and melanogenesis due to HAL-PDT. The OTR technique is based on reflectance spectroscopy and inverse radiative transfer modelling based on the discrete-ordinate radiative-transfer (DISORT) method [15,16].
Materials and methods Chemicals Hexyl aminolevulinate (5-aminolevulinic acid hexyl ester) hydrochloride (HAL HCl, MW = 251.8 g/mol) was synthesized and donated to us by Norbert Lange (Lausanne, Switzerland) or by PhotoCure ASA (Oslo, Norway).
Volunteers Experiments were carried out on five healthy volunteers (age from 27 to 59 years) with skin types II or III. The study was approved by the local ethical committee (Regional komite for medisinsk forskningsetikk Sør-Norge, Ref. S-05112). The volunteers did not use any medications or drugs.
Topical application of HAL Details of the experimental procedures were described earlier [17]. Cream was prepared using 10% (w/w) of HAL in a standard oil-in-water base (Unguentum Merck, Darmstad, Germany). As the test area, the inner part of the right forearm of each volunteer was covered with a transparent adhesive dressing (OpSite Flexifix, Smith & Nephew Medical Ltd., Hull, UK). As the control area, the inner part of the left forearm of each volunteer was also covered with a transparent adhesive dressing of the same type. On each of these two dressings, three openings were cut, each being 1 cm × 1 cm in area. The distance between adjacent openings was approximately 1.5 cm. Freshly prepared cream with approximately 75 mg/cm2 ± 10 mg/cm2 of HAL was topically applied on the three areas of skin under the three openings
177 of the dressing on the right forearm. The three areas of skin under the dressing on the left forearm were used for control while real changes were observed at the test area. On the left forearm, one of the three skin control areas was applied cream with HAL, one was applied cream without HAL, and the other one was applied no cream at all. Then the same type dressing was applied on all areas to prevent cream diffusion to adjacent areas. The creams and the dressings were kept for 24 h.
Light exposure The dressings and the creams were removed after 24 h from all areas. Afterwards, the three test areas and one control (light) area were exposed separately with red light for 2 min. A Curelight lamp (Photocure ASA, Norway) with an emission peak at 632 ± 11 nm was used for light exposure of the three test areas and the three control areas. The fluence rate was 90 mW/cm2 as measured 5 cm below the lamp, i.e., at the position of the test and control areas.
Reflectance spectroscopy Reflectance measurements were conducted with a luminescence spectrometer (PerkinElmer LS50B, Norwalk, CT, USA). The spectrometer was equipped with two scanning grating monochromators, one in front of the light source (a pulsed xenon lamp) and the other in front of the detector. Reflectance spectra were measured in synchronous scans in which both gratings were set at the same wavelength and band pass (5 nm). Thus, fluorescence artefacts were avoided. The area exposed to the excitation light of the spectrometer and the area from which the reflected flux was detected by the spectrometer were the same. The geometry of the fibre probe was such that both the directly (Fresnel) reflected and the diffusely reflected light from the skin were collected and recorded. These reflectance spectra were dominated by the emission spectrum of the xenon lamp of the spectrometer, the transmission spectrum of the optical system, and the optical properties of the skin. Reflectance spectra from the surface of a TiO2 plate with a single-scattering albedo of approximately equal to 1 and a reflectance close to 1 were recorded and used for calibration. The auto-reflectance spectrum was recorded in a dark room by pointing the probe at darkness. This auto-reflectance spectrum was subtracted from each recorded reflectance spectrum. After subtraction of the auto-reflectance spectrum, all recorded reflectance spectra from the test and control areas were normalized to the reflectance spectrum of TiO2 .
Radiative transfer model The radiative transfer model and the discrete ordinate method have been described in detail elsewhere [15,16,18,19]. With this method given physiological/morphological parameters can be calculated from the reflectance spectra of skin [16]. We refer to this technique as OTR. In Ref. [16] the focus was on testing the feasibility of the mathematical approach, whereas we,
178
A. Juzeniene et al.
in this manuscript, focus on the biological implications of retrieved results.
Statistics and curve fitting The same person has performed all measurements. The study was not blinded. In the original dataset there were three repetitions of each measurement. In the inversion analysis the average of these three repetitions was used. Data are presented as mean ± S.E. (standard error). The obtained data were also analysed using standard linear, exponential or sigmoid fitting routines in Sigmaplot 10.0 from Systat Software, Inc. (Richmond, CA, USA).
Results and discussion Figure 1 shows the typical reflectance spectra of the skin from one volunteer (skin type III) before HAL application and light exposure (baseline reflectance spectrum) and immediately after HAL-PDT. The baseline reflectance spectra, measured in the mentioned areas, were relatively consistent for the same volunteer, but they vary between volunteers due to skin thickness, melanin concentration, hemoglobin concentration, etc. There were differences between the test areas (HAL-PDT) of one person and bigger differences between the test areas of different persons, but recorded reflectance spectra from all five volunteers had the same tendency after HAL-PDT. Knowledge of the absorption spectra of melanin, hemoglobin and oxyhemoglobin [20] is essential for OTR-determination of the following seven physiological/morphological parameters of interest: dermal blood concentration and oxygenation, upper and lower epidermal thickness, upper and lower epidermal melanosome concentration, and epidermal keratin concentration. We separate the epidermis into an upper and a lower part because the melanosomes, at least in the lighter skin types, are found mostly in the lower layers of the epidermis. This makes the optical properties of the upper part of the epidermis significantly different from those of the lower part of the epidermis [21,22]. Therefore, it is very reasonable to separate the epidermis into two distinct layers in an opti-
Figure 1 Mean reflectance spectra recorded from three test areas on human forearm skin before HAL application and light exposure (baseline) and immediately after HAL-PDT.
Figure 2 Retrieved percentage of dermal blood concentration before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HALPDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
cal model of the skin. The OTR calculations required for retrieval of the seven physiological/morphological parameters are time consuming. Therefore, since the recorded reflectance spectra for the five persons taking part in the study were found to have the similar tendency after treatment, we chose the recorded reflectance spectra from one volunteer to be representative in the present detailed investigation. Figs. 2—6 show the kinetics of the changes of the mentioned seven parameters in the same one volunteer. The application of cream without HAL alone or exposure to light alone had no influence on the above-mentioned parameters (Figs. 2—6A and C). A slight redness was observed after topical application of 10% HAL alone for 24 h. The dermal blood concentration increased from 2.7 ± 0.2% to 4.2 ± 0.4% after treatment with HAL alone, indicating a slight erythema, and then declined to the baseline concentration within 2 days (Fig. 2B). This erythema induction is probably due to effects of HAL itself on the vasculature system. Similarly, the dermal blood concentration increased immediately after HAL-PDT, but, instead of decreasing as for HAL alone, it continued to increase for 2 days to a maximum level of (7.1 ± 0.4%). Then decayed over the next 10 days, but even at that time it remained above control values (Fig. 2D). The given PDT dose (10.8 J/cm2 ) was smaller than those used in tumour treatment (50—100 J/cm2 ). Nevertheless, similar erythema, lasting for 2—3 days, has been reported by several authors [23—27].
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Figure 3 Retrieved percentage of oxygenated dermal blood before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HALPDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
Figure 4 Retrieved thickness (m) of the upper (䊉) and the lower () epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
The dermal blood oxygenation followed similar kinetics as the dermal blood concentration after HAL treatment (Fig. 3B). An immediate increase of 70% of the dermal blood oxygenation (as for HAL) was observed also for HAL-PDT (Fig. 3D). It remained stable for 2 days, and then decayed, but baseline values were not reached during the 2 weeks of observation (Fig. 3D). The similarity in the kinetics of the dermal blood concentration and in that of the oxygenation for HAL-PDT can probably be explained by vasodilatation, leading to faster blood flow, and, therefore, apparently to less oxygen depletion in the dermal tissues. Since the PDT efficiency increases with oxygenation [28,29], this indicates that a fractionated exposure scheme (light exposure is split into two or more fractions with dark intervals between after a single application of ALA) might be advantageous under the present condition. The OTR method for determining the percent blood oxygenation is non-invasive, and will therefore be useful for optimal timing of fractionated therapy. The thickness of the upper part of the epidermis decreased after HAL treatment, but then started to increase and reached the baseline thickness after 1 week (Fig. 4B). While the thickness of the lower part of the epidermis did not change immediately after HAL treatment, a 1.4-fold increase was observed 2—3 days afterwards. For the rest of the observation time this thickness remained constant (Fig. 4B). The keratin concentration in the upper epidermis decreased immediately after HAL treatment, but later
Figure 5 Retrieved keratin concentration (%) in the upper epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
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Acknowledgements The present work was supported by the Research Foundation of the Norwegian Radium Hospital and the Norwegian Cancer Society (Kreftforeningen).
References
Figure 6 Retrieved melanosome concentration (%) in the upper (䊉) and the lower () epidermis before and for 15 days after application of cream alone (A), cream with HAL alone (B), exposure to light alone (C), and HAL-PDT (D). Data are presented as mean ± S.E. (n = 3 for figures A—C and n = 9 for figure D).
increased again (Fig. 5B). The thickness of the lower part of the epidermis decreased during HAL-PDT and continued to decrease the next 2—3 days. After that it started to increase and reached a maximum level around 8 days after PDT. The thickness and keratin concentration of the upper part of epidermis remained constant for 2—4 days after HAL-PDT, but then started to decline (Fig. 4D and Fig. 5D). Figure 6 shows how the melanosome concentration in the upper and lower part of the epidermis changed after HAL application and after HAL-PDT. The melanosome concentration after topical application of HAL remained unchanged in the upper part of the epidermis, while in the lower part of the epidermis it first increased slightly and then decreased (Fig. 6B). There was a steady increase in the melanosome concentration after HAL-PDT in the upper layer of the epidermis (Fig. 6D). In the lower part of the epidermis the concentration of melanosomes was the same for 4 days. Then it started to decrease and reached a constant lower level after 4—5 days (Fig. 6D). These changes may be due to the diffusion of melanin dust to the upper part of the epidermis or to changes in the melanosome size distribution. We, and others, have earlier noted pigment induction after PDT [4,5,13,14,26,30]. The observed pigmentation after PDT may be due to tyrosinase activation through singlet oxygen mediated production of diacylglycerol [5]. This is the first time that the evolution of the pigmentation has been quantitatively followed.
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