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Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate Anne Lise Ording Helgesen a,b,c,∗, Petter Gjersvik b,c, Qian Peng d, Vlada Vasovic d, Are Hugo Pripp e, Peter Jebsen d, Tom Tanbo c,f, Trond Warloe g a
National Resource Centre for Women’s Health, Division for Women’s and Children’s Health, Oslo University Hospital, 0424 Oslo, Norway b Department of Dermatology, Oslo University Hospital, 0424 Oslo, Norway c Institute for Clinical Medicine, University of Oslo, Box 1171 Blindern, 0318 Oslo, Norway d Department of Pathology, Oslo University Hospital, 0424 Oslo, Norway e Department of Biostatistics and Epidemiology, Oslo University Hospital, 0424 Oslo, Norway f Department of Gynaecology, Oslo University Hospital, 0424 Oslo, Norway g Department of Internal Medicine, Oslo University Hospital, 0424 Oslo, Norway
KEYWORDS Hexylaminolevulinate; Erosive lichen planus; Genital
Summary Genital erosive lichen planus (GELP) is a chronic inflammatory disease, in women characterized by painful vulvar and vaginal erosions. To prepare for a clinical trial on photodynamic treatment (PDT), we applied hexyl 5-aminolevulinate hydrochloride (HAL) in clinically normal and affected mucosa in 12 women with GELP using two different doses (6.25 or 50 mg/ml). Biopsies were taken after 30 min and 3 h. The biodistribution of HAL, measured as photoactive protoporphyrin IX (PpIX), was studied using non-invasive superficial fluorescence measurements and microscopic fluorescence photometry. More PpIX was detected after application of 12.5 mg HAL than after 100 mg, with large inter-individual variations. PpIX levels after 3 h were overall higher than after 30 min. PpIX fluorescence was not detected in skin distant to the genital area. In conclusion, 6.25 mg/ml HAL applied for 3 h seems adequate for HAL absorption and conversion to PpIX in submucosal inflammatory and epithelial cells and can be used in a PDT trial of GELP. © 2014 Elsevier B.V. All rights reserved.
Introduction ∗
Corresponding author at: National Resource Centre for Women’s Health, Division for Women’s and Children’s Health, Oslo University Hospital, 0424 Oslo, Norway. Tel.: +47 23072683; fax: +47 23072684. E-mail address: [email protected] (A.L.O. Helgesen).
Lichen planus is a mucocutaneous, inflammatory and autoimmune disease with many subtypes, often affecting both oral and genital mucosa. Genital erosive lichen planus (GELP) is characterized by painful vulvar and vaginal lesions
http://dx.doi.org/10.1016/j.pdpdt.2014.01.005 1572-1000/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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[1], which may lead to major anatomical scarring. Treatment of GELP is difficult and the effect poorly documented [2,3]. Photodynamic therapy (PDT) is based upon the use of a photosensitizer and tissue accumulation of a photoactive substance that upon illumination generate selective damage [4,5]. Systemic and/or topical application of several photosensitizers, including aminolevulinic acid (ALA), has been used in treating cancer and precancerous lesions [6], as well as various inflammatory conditions including oral lichen planus [7—9]. Hexyl 5-aminolevulinate (HAL) is a more lipophilic ester of ALA and approved for the diagnosis of bladder cancer. PDT has been suggested as a possible treatment in cervical precancerous lesions [10] and HAL-PDT has been found to be more effective in cervical intraepithelial neoplasia (CIN) 1—3 than the less lipophilic methyl aminolevulinate (MAL)PDT [11]. In this study, we have explored the biodistribution and safety of topically applied HAL in clinically normal and affected mucosal areas in 12 women with GELP, using two different doses without subsequent illumination.
Materials and methods Twelve adult women (median age 51 years; range 26—78) with clinical and histological verified GELP were recruited from the Vulva Clinic, Rikshospitalet, Oslo. The patients had been diagnosed according to established criteria [12]. Median disease duration was 8 years (range 3—21). All patients had a treatment wash-out period for at least 4 weeks before trial start. Women with known or suspected porphyries or allergy to HAL, as well as pregnant and lactating women, were excluded. At first visit, a complete medical history and clinical examination with location and size of GELP-lesions were recorded. In most of the patients, the lesion areas were multiple and scattered and often located both in the introital vulva and the vagina. For the purpose of this study we selected affected areas in vulva and vagina in seven and five patients, respectively. The patients were randomized to application of a 2 ml vaginal suppository with either 12.5 mg or 100 mg HAL totally (6.25 or 50 mg/ml HAL (Photocure ASA, Oslo, Norway). The suppositories were prepared by the hospital pharmacy with identical packaging and patient number. Patients and investigators were blinded for the dose given until analyses were completed. In the vagina, the suppositories became liquid after some minutes, and an occlusive and self-adhesive dressing (Allevyn Thin, Smith & Nephew, Hull, UK) was used to cover the vulva. For patients with vulvar involvement, the suppositories were melted at 37 ◦ C and applied to the area with an application thickness of approximately 1 mm. A similar dressing was then applied. The patients lied down horizontally during the first 30 min. Patients were contacted one week after HAL application in order to register possible side effects.
Surface fluorescence Surface fluorescence from HAL-induced protoporphyrin IX (PpIX) was assessed using a fiber optic probe oriented
perpendicular to the tissue surface and measuring fluorescence within an area of 3 mm in diameter. The probe emits blue excitation light of 405 nm to the tissue, and measures the fluorescence in the area of red light of 550—750 nm [13—15]. Each value at any time point represents the mean value of a series of at least five repeated measurements with a small shift of area to avoid the photobleaching effect. Such measurements were performed repeatedly in the vulva and the vagina at baseline, after 30 min, 1 h and 3 h in clinically normal and affected tissue. Fluorescence was also measured in normal skin in the head-and-neck area after 3 h.
Microscopic fluorescence photometry In every patient, three 4 mm punch or forceps biopsies were taken, two from affected mucosa after 30 min and 3 h, respectively, and one from clinically normal mucosa after 3 h exposure to HAL. Biopsies were immersed in liquid nitrogen and stored at −80 ◦ C. The tissue blocks were mounted in a medium (Tissue Tek II embedding compound, BDH, Poole, UK) and cut with a cryostat microtome to slices with 8 m thickness and mounted on clean glass slides. Three different microscopy images were available from each biopsy: a white light image, and a hematoxylin and eosin-stained image of an adjacent section, as well as a fluorescence image of HALinduced PpIX, made by means of microscopic fluorescence photometry. PpIX fluorescence measurements were done by microscopic fluorescence photometry using a microscope (Nikon Eclipse E800) with a 100 W mercury lamp and a highly lightsensitive thermo-electrically cooled charge-coupled device camera (ORCAII -ER, Hamamatsu, Japan) [16]. The fluorescence intensity within the selected area was quantified and plotted as a function of depth of the lesion. The area under the curve (AUC) of each plot was measured and presented as AUC per m in depth because of a diverse depth of epithelial and submucosal tissue in different lesion samples. Measurements were carried out in a blinded manner with respect to the concentration and application time of HAL.
Ethics The study was performed in accordance with the Helsinki Declaration of 1964 and was reviewed and approved by the Norwegian Medicines Control Authority, the Regional Ethics Committee and the Institutional Review Board. Written informed consent was obtained from all patients. The study is part of a treatment study registered at www.clinicaltrials.org as study ALH ELP 2/10.
Statistical analysis Descriptive statistics are mean (standard deviation) or number of patients (percentage) as appropriate. Results were analyzed with linear mixed models for repeated measured data using a random intercept for each patients and post hoc-tests with Bonferroni corrections. This statistical method uses all available data for estimations, including those patients with missing data for one of the repeated
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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Figure 1 Hematoxylin and eosin-stained images (a, d) and fluorescence images (b, e) from adjacent tissue sections of the biopsies obtained from clinically normal (a, b) and affected mucosa (d, e) of female genital erosive lichen planus (GELP) at 3 h after topical hexaminolevulinate (HAL) application. HAL-induced protoporphyrin IX (PpIX) fluorescence intensity as a function of the tissue depth in the red rectangular areas (b, e) was quantitatively measured by means of microscopic fluorescence photometry (c, f). In clinically normal mucosa, some weak PpIX fluorescence is seen in the epithelial layer (a, b), but not in the submucosal layer. In affected mucosa, much stronger PpIX fluorescence was found in both epithelial cells and infiltrating lymphocytes in the submucosal layer.
measurements. The significance level was set to 5% (i.e., P < .05). All analyses were performed using PASW Statistics 18 (IBM, Armonk, New York).
Results The superficial PpIX fluorescence increased significantly. For affected areas, the increase in PpIX fluorescence was statistically non-significant after 30 min, but statistically significant after 3 h, with mean difference between baseline and 3 h being 71 (95% CI 37—104; P < .001) and between 30 min and 3 h being 40 (95% CI 5—76; P = .018). No significant differences in PpIX fluorescence between clinically normal and affected mucosa, nor between the different doses, were found (P = .149 and P = .827, respectively). There were no differences in PpIX fluorescence between measurements after 30 and 60 min; results at 60 min were therefore omitted from further analyses. There was a considerable patient-to-patient fluorescence variability, but this was included in the statistical modeling. No PpIX fluorescence was found in the head-and-neck area after 3 h. No general skin phototoxicity was observed. Five patients reported some local burning sensation up to 30 min after HAL application in the vulva, but not after
vaginal application, unrelated to dosage. No serious adverse events were registered. In all, 36 biopsies from GELP-affected mucosa and clinically normal mucosa were sampled. Of these, 26 samples were technically suitable for further analysis. Fluorescence microscopy of the biopsies revealed differences in the biodistribution of PpIX: After 3 h, PpIX was found in the epithelium of both GELP-affected mucosa and clinically normal mucosa, the former also having strong fluorescence in submucosal inflammatory cells just below the basal cell membrane (Fig. 1). This was confirmed by a statistically significant difference in PpIX fluorescence between lichen planus and clinically normal mucosa after 3 h (mean difference 272; 95% CI 44—500; P = .014). The PpIX fluorescence in affected mucosa increased from 30 min to the highest value after 3 h (Fig. 2) with no significant difference between epithelial tissue (marginal mean 500; 95% CI 384—615) and submucosal tissue (marginal mean 441; 95% CI 325—556) with mean difference 59; 95% CI −93 to 211; P = 0.441. PpIX fluorescence in epithelial and submucosal tissue at different time points is shown in Fig. 3. There was a significant difference in PpIX fluorescence between biopsies taken from patients using different concentrations of HAL (P = .033), both in epithelial and submucosal tissue, favoring the lowest dose, and not modified by time. Mean
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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A.L.O. Helgesen et al. did not change, supporting the superficial measurements and the fluorometric results.
Discussion
Figure 2 Hexaminolevulinate (HAL)-derived protoporphyrin IX fluorescence intensity as a function of tissue depth quantified in biopsies from affected mucosa in female genital erosive lichen planus (GELP), taken at 30 min (low curve) and 3 h (upper curve), and in a biopsy of clinically normal mucosa at 3 h (middle curve). The red arrows indicate the borderline between the epithelial layer and submucosal layer.
difference between the two doses, estimated by the linear mixed model, was 211 (95% CI 21—401; P = .033). There was a substantial difference in PpIX fluorescence between 50 mg/ml HAL at 30 min (mean 134.8) and 6.25 mg/ml HAL at 3 h (mean 783.8), favoring the lowest dose at 3 h (Fig. 3). With both HAL concentrations, we found a high peak level of fluorescence between 1500 and 2500 in both lower epithelial tissue and submucosal tissue after 3 h in affected mucosa (Fig. 3). Using peak values instead of AUC, the conclusions
In this study, 6.25 mg/ml HAL applied for 3 h seemed to be sufficiently absorbed and converted to PpIX in epithelial and submucosal inflammatory cells in GELP. No measurable systemic up-take of HAL or side effects was registered. Superficial fluorescence measurement is a recognized tool for measuring photoactive porphyrins, such as PpIX. In such measurements, excitation light at 405 nm penetrating the tissue about 0.1—0.2 mm, similar to the thickness of vulvar mucosal epithelium, is used [12]. In our study, such measurements showed significant differences between normal and affected mucosa, as well as between patients using different concentrations of HAL. We used the superficial fluorescence primarily to confirm the biological PpIX process locally and to detect any systemic uptake of HAL. Other studies have shown greater inter-individual differences in surface fluorescence measurements than in microscopic fluorescence photometry [15]. Both fluorescence measurement methods demonstrated increased PpIX fluorescence in epithelial cells and a peak level in basal cells, possibly due to the high proliferation in this area (Fig. 1). There was no statistical significant difference between clinically normal and affected mucosal epithelium. This is in contrast to the pattern seen in nonmelanoma skin cancer [16], where PpIX accumulation is almost exclusively seen in pathological epidermis. This may
Figure 3 Comparative distribution of hexaminolevulinate (HAL)-induced protoporphyrin IX (PpIX) fluorescence in biopsies from female genital erosive lichen planus (GELP) after topical application of 12.5 mg HAL or 100 mg HAL for 30 min (affected mucosa) and 3 h (affected and clinically normal mucosa). The PpIX fluorescence is described separately for the epithelial and submucosal tissue. The accumulation of PpIX fluorescence is reported as area under the curve (AUC), as explained in the methods section. Each bar point represents mean value with 95% confidence intervals.
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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HAL-induced PpIX biodistribution in genital erosive lichen planus be explained by the very thin mucosal epithelium composed of only a few layers of keratin cells. Microscopic fluorescence photometry demonstrated PpIX fluorescence both in mucosal epithelium and submucosal tissue with high inflammatory activity. Typically, there is a linear dermal lymphocyte infiltrate in lichen planus, although there may be variations in mucosal lesions [12]. Recent immunohistochemical studies in GELP have shown a submucosal band of lymphocytes consisting of different immunoactive cells [17,18]. Photoactive PpIX in these submucosal immunoactive cells may indicate a possible role for red light PDT with HAL in GELP. Both doses of HAL, especially of 6.25 mg, induced increasing PpIX fluorescence in submucosal inflammatory cells with longer application time. All biopsies were small, soft, easily bent and difficult to orientate for tissue sectioning. Therefore, ten biopsies could not be included in the microscopic fluorescence photometry analyses. In GELP patients with a scattered and patchy network of lesions, it may be difficult to distinguish affected and non-affected mucosa. Surprisingly, we found a higher PpIX fluorescence in both epithelial and submucosal tissue when using the lower HAL dose. This could be explained by a possible saturation and inhibitory overdosage of the heme biosynthetic pathway from a higher HAL dose. It could also be due to an individual variability and small sample size. The statistical analyses should be interpreted with caution due to this small number of patients. In conclusion, topical mucosal application of 6.25 mg/ml HAL for 3 h seems to be sufficient for HAL absorption and conversion to PpIX in vaginal and vulvar epithelial cells and in adjacent submucosal inflammatory cells in women with GELP. This indicates that red light should be used in photodynamic treatment of this disease. No detectable general skin photosensitizing was observed by skin fluorescence measurement in distant head and neck area.
Conflicts of interest None.
Acknowledgements We thank Ellen Hellesylt for excellent technical assistance and Photocure AS for support. The study was financed by the National Resource Centre for Women’s Health, Oslo University Hospital.
References [1] Helgesen AL, Gjersvik P, Jebsen P, Kirschner R, Tanbo T. Vaginal involvement in genital erosive lichen planus. Acta Obstet Gynecol Scand 2010;89:966—70.
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[2] Cheng S, Kirtschig G, Cooper S, Thornhill M, LeonardiBee J, Murphy R. Interventions for erosive lichen planus affecting mucosal sites. Cochrane Database Syst Rev 2012;2(February):CD008092. [3] Simpson RC, Littlewood SM, Cooper SM, et al., Cruickshank ME, Green CM, Derrick E. Real-life experience of managing vulval erosive lichen planus: a case-based review and U.K. multicentre case note audit. Br J Dermatol 2012;167:85—91. [4] Peng Q, Warloe T, Berg K, et al. 5-Aminolevulinic acid-based photodynamic therapy. Clinical research and future challenges. Cancer 1997;79:2282—303. [5] Peng Q. Photodynamic therapy and detection. J Environ Pathol Toxicol Oncol 2006;25:1—5. [6] Ibbotson SH. An overview of topical photodynamical therapy in dermatology. Photodiagnosis Photodyn Ther 2010;7:16—23. [7] Aghahosseini F, Arbabi-Kalati F, Fashtami LA, Djavid GE, Fateh M, Beitollahi JM. Methylene blue-mediated photodynamic therapy: a possible alternative treatment for oral lichen planus. Lasers Surg Med 2006;38:33—8. [8] Olejek A, Steplewska K, Gabriel A, et al. Efficacy of photodynamic therapy in vulvar lichen sclerosus treatment based on immunohistochemical analysis of CD34, CD44, myelin basic protein, and Ki67 antibodies. Int J Gynecol Cancer 2010;20:879—87. [9] Kvaal SI, Angell-Petersen E, Warloe T. Photodynamic treatment of oral lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:62—70. [10] Soergel P, Wang X, Stepp H, Hertel H, Hillemanns P. Photodynamic therapy of cervical intraepithelial neoplasia with hexaminolevulinate. Lasers Surg Med 2008;40:611—5. [11] Soergel P, Dahl GF, Onsrud M, Hillemanns P. Photodynamic therapy of cervical intraepithelial neoplasia 1-3 and human papilloma virus (HMV) infection with methylaminolevulinate and hexaminolevulinate — a double-blind, dose-finding study. Lasers Surg Med 2012;44:468—74. [12] Wilkinson EJ, Massol NA. Benign diseases of the vulva. In: Kurman RJ, editor. Blaustein’s pathology of the female genital tract. 5th ed. New York: Springer; 2011. p. 1—53. [13] Pottier RH, Chow YF, LaPlante JP, Truscott TG, Kennedy JC, Beiner LA. Non-invasive technique for obtaining fluorescence excitation and emission spectra in vivo. Photochem Photobiol 1986;44:679—87. [14] Angell-Petersen E, Sørensen R, Warloe T, et al. Porphyrin formation in actinic keratosis and basal cell carcinoma after topical application of methyl 5-aminolevulinate. J Invest Dermatol 2006;126:265—71. [15] Angell-Petersen E, Christensen C, Müller CR, Warloe T. Phototoxic reaction and porphyrin fluorescence in skin after topical application of methyl aminolaevulinate. Br J Dermatol 2007;156:301—7. [16] Peng Q, Soler A, Warloe T, Nesland JM, Giercksky KE. Selective distribution of porphyrins in thick basal cell carcinoma after topical application of methyl 5-aminolevulinate. J Photochem Photobiol 2001;62:140—5. [17] Pereira JS, Monteiro BV, Nonaka CF, Silveira ÉJ, Miguel MC. FoxP3(+) T regulatory cells in oral lichen planus and its correlation with the distinct clinical appearance of the lesions. Int J Exp Pathol 2012;93:287—94. [18] Vered M, Fürth E, Shalev Y, Dayan D. Inflammatory cells of immunosuppressive phenotypes in oral lichen planus have a proinflammatory pattern of expression and are associated with clinical parameters. Clin Oral Investig 2013;17:1365—73.
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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Photodiagnosis and Photodynamic Therapy (2014) xxx, xxx—xxx
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/pdpdt
Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate Anne Lise Ording Helgesen a,b,c,∗, Petter Gjersvik b,c, Qian Peng d, Vlada Vasovic d, Are Hugo Pripp e, Peter Jebsen d, Tom Tanbo c,f, Trond Warloe g a
National Resource Centre for Women’s Health, Division for Women’s and Children’s Health, Oslo University Hospital, 0424 Oslo, Norway b Department of Dermatology, Oslo University Hospital, 0424 Oslo, Norway c Institute for Clinical Medicine, University of Oslo, Box 1171 Blindern, 0318 Oslo, Norway d Department of Pathology, Oslo University Hospital, 0424 Oslo, Norway e Department of Biostatistics and Epidemiology, Oslo University Hospital, 0424 Oslo, Norway f Department of Gynaecology, Oslo University Hospital, 0424 Oslo, Norway g Department of Internal Medicine, Oslo University Hospital, 0424 Oslo, Norway
KEYWORDS Hexylaminolevulinate; Erosive lichen planus; Genital
Summary Genital erosive lichen planus (GELP) is a chronic inflammatory disease, in women characterized by painful vulvar and vaginal erosions. To prepare for a clinical trial on photodynamic treatment (PDT), we applied hexyl 5-aminolevulinate hydrochloride (HAL) in clinically normal and affected mucosa in 12 women with GELP using two different doses (6.25 or 50 mg/ml). Biopsies were taken after 30 min and 3 h. The biodistribution of HAL, measured as photoactive protoporphyrin IX (PpIX), was studied using non-invasive superficial fluorescence measurements and microscopic fluorescence photometry. More PpIX was detected after application of 12.5 mg HAL than after 100 mg, with large inter-individual variations. PpIX levels after 3 h were overall higher than after 30 min. PpIX fluorescence was not detected in skin distant to the genital area. In conclusion, 6.25 mg/ml HAL applied for 3 h seems adequate for HAL absorption and conversion to PpIX in submucosal inflammatory and epithelial cells and can be used in a PDT trial of GELP. © 2014 Elsevier B.V. All rights reserved.
Introduction ∗
Corresponding author at: National Resource Centre for Women’s Health, Division for Women’s and Children’s Health, Oslo University Hospital, 0424 Oslo, Norway. Tel.: +47 23072683; fax: +47 23072684. E-mail address: [email protected] (A.L.O. Helgesen).
Lichen planus is a mucocutaneous, inflammatory and autoimmune disease with many subtypes, often affecting both oral and genital mucosa. Genital erosive lichen planus (GELP) is characterized by painful vulvar and vaginal lesions
http://dx.doi.org/10.1016/j.pdpdt.2014.01.005 1572-1000/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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[1], which may lead to major anatomical scarring. Treatment of GELP is difficult and the effect poorly documented [2,3]. Photodynamic therapy (PDT) is based upon the use of a photosensitizer and tissue accumulation of a photoactive substance that upon illumination generate selective damage [4,5]. Systemic and/or topical application of several photosensitizers, including aminolevulinic acid (ALA), has been used in treating cancer and precancerous lesions [6], as well as various inflammatory conditions including oral lichen planus [7—9]. Hexyl 5-aminolevulinate (HAL) is a more lipophilic ester of ALA and approved for the diagnosis of bladder cancer. PDT has been suggested as a possible treatment in cervical precancerous lesions [10] and HAL-PDT has been found to be more effective in cervical intraepithelial neoplasia (CIN) 1—3 than the less lipophilic methyl aminolevulinate (MAL)PDT [11]. In this study, we have explored the biodistribution and safety of topically applied HAL in clinically normal and affected mucosal areas in 12 women with GELP, using two different doses without subsequent illumination.
Materials and methods Twelve adult women (median age 51 years; range 26—78) with clinical and histological verified GELP were recruited from the Vulva Clinic, Rikshospitalet, Oslo. The patients had been diagnosed according to established criteria [12]. Median disease duration was 8 years (range 3—21). All patients had a treatment wash-out period for at least 4 weeks before trial start. Women with known or suspected porphyries or allergy to HAL, as well as pregnant and lactating women, were excluded. At first visit, a complete medical history and clinical examination with location and size of GELP-lesions were recorded. In most of the patients, the lesion areas were multiple and scattered and often located both in the introital vulva and the vagina. For the purpose of this study we selected affected areas in vulva and vagina in seven and five patients, respectively. The patients were randomized to application of a 2 ml vaginal suppository with either 12.5 mg or 100 mg HAL totally (6.25 or 50 mg/ml HAL (Photocure ASA, Oslo, Norway). The suppositories were prepared by the hospital pharmacy with identical packaging and patient number. Patients and investigators were blinded for the dose given until analyses were completed. In the vagina, the suppositories became liquid after some minutes, and an occlusive and self-adhesive dressing (Allevyn Thin, Smith & Nephew, Hull, UK) was used to cover the vulva. For patients with vulvar involvement, the suppositories were melted at 37 ◦ C and applied to the area with an application thickness of approximately 1 mm. A similar dressing was then applied. The patients lied down horizontally during the first 30 min. Patients were contacted one week after HAL application in order to register possible side effects.
Surface fluorescence Surface fluorescence from HAL-induced protoporphyrin IX (PpIX) was assessed using a fiber optic probe oriented
perpendicular to the tissue surface and measuring fluorescence within an area of 3 mm in diameter. The probe emits blue excitation light of 405 nm to the tissue, and measures the fluorescence in the area of red light of 550—750 nm [13—15]. Each value at any time point represents the mean value of a series of at least five repeated measurements with a small shift of area to avoid the photobleaching effect. Such measurements were performed repeatedly in the vulva and the vagina at baseline, after 30 min, 1 h and 3 h in clinically normal and affected tissue. Fluorescence was also measured in normal skin in the head-and-neck area after 3 h.
Microscopic fluorescence photometry In every patient, three 4 mm punch or forceps biopsies were taken, two from affected mucosa after 30 min and 3 h, respectively, and one from clinically normal mucosa after 3 h exposure to HAL. Biopsies were immersed in liquid nitrogen and stored at −80 ◦ C. The tissue blocks were mounted in a medium (Tissue Tek II embedding compound, BDH, Poole, UK) and cut with a cryostat microtome to slices with 8 m thickness and mounted on clean glass slides. Three different microscopy images were available from each biopsy: a white light image, and a hematoxylin and eosin-stained image of an adjacent section, as well as a fluorescence image of HALinduced PpIX, made by means of microscopic fluorescence photometry. PpIX fluorescence measurements were done by microscopic fluorescence photometry using a microscope (Nikon Eclipse E800) with a 100 W mercury lamp and a highly lightsensitive thermo-electrically cooled charge-coupled device camera (ORCAII -ER, Hamamatsu, Japan) [16]. The fluorescence intensity within the selected area was quantified and plotted as a function of depth of the lesion. The area under the curve (AUC) of each plot was measured and presented as AUC per m in depth because of a diverse depth of epithelial and submucosal tissue in different lesion samples. Measurements were carried out in a blinded manner with respect to the concentration and application time of HAL.
Ethics The study was performed in accordance with the Helsinki Declaration of 1964 and was reviewed and approved by the Norwegian Medicines Control Authority, the Regional Ethics Committee and the Institutional Review Board. Written informed consent was obtained from all patients. The study is part of a treatment study registered at www.clinicaltrials.org as study ALH ELP 2/10.
Statistical analysis Descriptive statistics are mean (standard deviation) or number of patients (percentage) as appropriate. Results were analyzed with linear mixed models for repeated measured data using a random intercept for each patients and post hoc-tests with Bonferroni corrections. This statistical method uses all available data for estimations, including those patients with missing data for one of the repeated
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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Figure 1 Hematoxylin and eosin-stained images (a, d) and fluorescence images (b, e) from adjacent tissue sections of the biopsies obtained from clinically normal (a, b) and affected mucosa (d, e) of female genital erosive lichen planus (GELP) at 3 h after topical hexaminolevulinate (HAL) application. HAL-induced protoporphyrin IX (PpIX) fluorescence intensity as a function of the tissue depth in the red rectangular areas (b, e) was quantitatively measured by means of microscopic fluorescence photometry (c, f). In clinically normal mucosa, some weak PpIX fluorescence is seen in the epithelial layer (a, b), but not in the submucosal layer. In affected mucosa, much stronger PpIX fluorescence was found in both epithelial cells and infiltrating lymphocytes in the submucosal layer.
measurements. The significance level was set to 5% (i.e., P < .05). All analyses were performed using PASW Statistics 18 (IBM, Armonk, New York).
Results The superficial PpIX fluorescence increased significantly. For affected areas, the increase in PpIX fluorescence was statistically non-significant after 30 min, but statistically significant after 3 h, with mean difference between baseline and 3 h being 71 (95% CI 37—104; P < .001) and between 30 min and 3 h being 40 (95% CI 5—76; P = .018). No significant differences in PpIX fluorescence between clinically normal and affected mucosa, nor between the different doses, were found (P = .149 and P = .827, respectively). There were no differences in PpIX fluorescence between measurements after 30 and 60 min; results at 60 min were therefore omitted from further analyses. There was a considerable patient-to-patient fluorescence variability, but this was included in the statistical modeling. No PpIX fluorescence was found in the head-and-neck area after 3 h. No general skin phototoxicity was observed. Five patients reported some local burning sensation up to 30 min after HAL application in the vulva, but not after
vaginal application, unrelated to dosage. No serious adverse events were registered. In all, 36 biopsies from GELP-affected mucosa and clinically normal mucosa were sampled. Of these, 26 samples were technically suitable for further analysis. Fluorescence microscopy of the biopsies revealed differences in the biodistribution of PpIX: After 3 h, PpIX was found in the epithelium of both GELP-affected mucosa and clinically normal mucosa, the former also having strong fluorescence in submucosal inflammatory cells just below the basal cell membrane (Fig. 1). This was confirmed by a statistically significant difference in PpIX fluorescence between lichen planus and clinically normal mucosa after 3 h (mean difference 272; 95% CI 44—500; P = .014). The PpIX fluorescence in affected mucosa increased from 30 min to the highest value after 3 h (Fig. 2) with no significant difference between epithelial tissue (marginal mean 500; 95% CI 384—615) and submucosal tissue (marginal mean 441; 95% CI 325—556) with mean difference 59; 95% CI −93 to 211; P = 0.441. PpIX fluorescence in epithelial and submucosal tissue at different time points is shown in Fig. 3. There was a significant difference in PpIX fluorescence between biopsies taken from patients using different concentrations of HAL (P = .033), both in epithelial and submucosal tissue, favoring the lowest dose, and not modified by time. Mean
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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A.L.O. Helgesen et al. did not change, supporting the superficial measurements and the fluorometric results.
Discussion
Figure 2 Hexaminolevulinate (HAL)-derived protoporphyrin IX fluorescence intensity as a function of tissue depth quantified in biopsies from affected mucosa in female genital erosive lichen planus (GELP), taken at 30 min (low curve) and 3 h (upper curve), and in a biopsy of clinically normal mucosa at 3 h (middle curve). The red arrows indicate the borderline between the epithelial layer and submucosal layer.
difference between the two doses, estimated by the linear mixed model, was 211 (95% CI 21—401; P = .033). There was a substantial difference in PpIX fluorescence between 50 mg/ml HAL at 30 min (mean 134.8) and 6.25 mg/ml HAL at 3 h (mean 783.8), favoring the lowest dose at 3 h (Fig. 3). With both HAL concentrations, we found a high peak level of fluorescence between 1500 and 2500 in both lower epithelial tissue and submucosal tissue after 3 h in affected mucosa (Fig. 3). Using peak values instead of AUC, the conclusions
In this study, 6.25 mg/ml HAL applied for 3 h seemed to be sufficiently absorbed and converted to PpIX in epithelial and submucosal inflammatory cells in GELP. No measurable systemic up-take of HAL or side effects was registered. Superficial fluorescence measurement is a recognized tool for measuring photoactive porphyrins, such as PpIX. In such measurements, excitation light at 405 nm penetrating the tissue about 0.1—0.2 mm, similar to the thickness of vulvar mucosal epithelium, is used [12]. In our study, such measurements showed significant differences between normal and affected mucosa, as well as between patients using different concentrations of HAL. We used the superficial fluorescence primarily to confirm the biological PpIX process locally and to detect any systemic uptake of HAL. Other studies have shown greater inter-individual differences in surface fluorescence measurements than in microscopic fluorescence photometry [15]. Both fluorescence measurement methods demonstrated increased PpIX fluorescence in epithelial cells and a peak level in basal cells, possibly due to the high proliferation in this area (Fig. 1). There was no statistical significant difference between clinically normal and affected mucosal epithelium. This is in contrast to the pattern seen in nonmelanoma skin cancer [16], where PpIX accumulation is almost exclusively seen in pathological epidermis. This may
Figure 3 Comparative distribution of hexaminolevulinate (HAL)-induced protoporphyrin IX (PpIX) fluorescence in biopsies from female genital erosive lichen planus (GELP) after topical application of 12.5 mg HAL or 100 mg HAL for 30 min (affected mucosa) and 3 h (affected and clinically normal mucosa). The PpIX fluorescence is described separately for the epithelial and submucosal tissue. The accumulation of PpIX fluorescence is reported as area under the curve (AUC), as explained in the methods section. Each bar point represents mean value with 95% confidence intervals.
Please cite this article in press as: Helgesen ALO, et al. Biodistribution of protoporphyrin IX in female genital erosive lichen planus after topical application of hexaminolevulinate. Photodiagnosis and Photodynamic Therapy (2014), http://dx.doi.org/10.1016/j.pdpdt.2014.01.005
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HAL-induced PpIX biodistribution in genital erosive lichen planus be explained by the very thin mucosal epithelium composed of only a few layers of keratin cells. Microscopic fluorescence photometry demonstrated PpIX fluorescence both in mucosal epithelium and submucosal tissue with high inflammatory activity. Typically, there is a linear dermal lymphocyte infiltrate in lichen planus, although there may be variations in mucosal lesions [12]. Recent immunohistochemical studies in GELP have shown a submucosal band of lymphocytes consisting of different immunoactive cells [17,18]. Photoactive PpIX in these submucosal immunoactive cells may indicate a possible role for red light PDT with HAL in GELP. Both doses of HAL, especially of 6.25 mg, induced increasing PpIX fluorescence in submucosal inflammatory cells with longer application time. All biopsies were small, soft, easily bent and difficult to orientate for tissue sectioning. Therefore, ten biopsies could not be included in the microscopic fluorescence photometry analyses. In GELP patients with a scattered and patchy network of lesions, it may be difficult to distinguish affected and non-affected mucosa. Surprisingly, we found a higher PpIX fluorescence in both epithelial and submucosal tissue when using the lower HAL dose. This could be explained by a possible saturation and inhibitory overdosage of the heme biosynthetic pathway from a higher HAL dose. It could also be due to an individual variability and small sample size. The statistical analyses should be interpreted with caution due to this small number of patients. In conclusion, topical mucosal application of 6.25 mg/ml HAL for 3 h seems to be sufficient for HAL absorption and conversion to PpIX in vaginal and vulvar epithelial cells and in adjacent submucosal inflammatory cells in women with GELP. This indicates that red light should be used in photodynamic treatment of this disease. No detectable general skin photosensitizing was observed by skin fluorescence measurement in distant head and neck area.
Conflicts of interest None.
Acknowledgements We thank Ellen Hellesylt for excellent technical assistance and Photocure AS for support. The study was financed by the National Resource Centre for Women’s Health, Oslo University Hospital.
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