Arch Dermatol Res (2008) 300:53–60
`DOI 10.1007/s00403-007-0787-0
`
`HOT CLINI CAL STUDY
`
`Fluorescence characteristics and pharmacokinetic properties
`of a novel self-adhesive 5-ALA patch for photodynamic therapy
`of actinic keratoses
`
`Jan-Dirk Fauteck · Günther Ackermann ·
`Manfred Birkel · Marion Breuer · Anne C. E. Moor ·
`Andrea Ebeling · Christoph Ortland
`
`Received: 29 May 2007 / Revised: 29 June 2007 / Accepted: 21 August 2007 / Published online: 25 October 2007
` Springer-Verlag 2007
`
`Abstract Actinic keratosis (AK) can be treated by photo-
`dynamic therapy (PDT), which is becoming a well-estab-
`lished tool in dermatology. Normally a precursor of the
`photosensitiser is applied topically and converted into pro-
`toporphyrin IX (PPIX) in the cells. By activating PPIX with
`light, the dysplastic cells will be destroyed. We report the
`results of two clinical studies investigating the properties of
`a novel self-adhesive 5-ALA-patch (PD P 506 A) intended
`for PDT of mild to moderate AK on the face and head. The
`studies investigated the inXuence of patch application dura-
`tion on PPIX-speciWc Xuorescence and the pharmacokinetic
`properties of the 5-ALA patch. The PPIX Xuorescence in
`AK lesions and normal skin after patch application (intrain-
`dividual comparison; application for 2, 3, 4, 5 h) was inves-
`tigated in 13 patients using DYADERM Professional
`(Biocam). In the subsequent pharmacokinetic study 12
`patients were treated with 8 patches each (4 h application).
`5-ALA and PPIX were analysed in plasma (over 24 h) and
`urine (over 12 h). PPIX-speciWc Xuorescence measured
`immediately after patch removal increased with increasing
`
`J.-D. Fauteck · M. Breuer
`PROVERUM GmbH, Münsterstraße 111,
`48155 Münster, Germany
`
`G. Ackermann
`Biocam GmbH, Friedenstraße 30, 93053 Regensburg, Germany
`
`M. Birkel
`CRS Clinical Research Services Mannheim GmbH,
`Richard-Wagner-Str. 20, 67269 Grünstadt, Germany
`
`A. C. E. Moor · A. Ebeling · C. Ortland (&)
`photonamic GmbH & Co. KG,
`Theaterstraße 6, 22880 Wedel, Germany
`e-mail: c.ortland@photonamic.de
`
`application duration to a maximum at 4-h application. The
`Xuorescence in AK lesions was more intense than in normal
`skin. A small increase of 5-ALA plasma concentrations was
`observed in 10 of 12 patients after applying 8 patches for
`4 h, which rapidly declined to normal values after patch
`removal. The maximum increase was 3.7-fold of the pre-
`dose 5-ALA plasma concentration. No PPIX-concentra-
`tions above the lower limit of quantiWcation were observed.
`PPIX-speciWc Xuorescence in AK lesions can be steered by
`application duration of this novel 5-ALA patch. Applica-
`tion is safe and well tolerable. The observed small rise in
`5-ALA plasma concentrations is regarded clinically irrelevant.
`Clinical eYcacy of the patch in PDT will be investigated in
`further clinical trials.
`
`Keywords Aminolevulinic acid · Fluorescence ·
`Pharmacokinetics · PPIX
`
`Introduction
`
`Actinic keratosis (AK) mainly occurs in people with fair
`skin. Today, AK is considered as in situ carcinoma, which
`usually is caused by sun exposure. The head, hands and
`lower legs, are primarily aVected. It is standard of care to
`treat AK when diagnosed which can be achieved by either
`physical ablation, chemotherapeutic agents or photody-
`namic therapy (PDT). For PDT in dermatology, the most
`frequently used substances are 5-ALA (5-aminolevulinic
`acid) and its derivative methyl aminolevulinic acid. Studies
`have proven the eVectiveness of topical 5-ALA-PDT in the
`treatment of actinic keratoses and other superWcial skin
`tumours [3, 8, 14, 15, 18, 19, 25].
`The procedure for PDT of AK normally involves the appli-
`cation of 5-ALA or its ester in a dermatological preparation
`
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`
`©
`

`

`54
`
`Arch Dermatol Res (2008) 300:53–60
`
`(e.g. alcoholic solution or cream) for several hours. After
`application the lesion has to be occluded and protected
`from light for the whole application period. 5-ALA pene-
`trates the skin and is metabolised to protoporphyrin IX
`(PPIX). PPIX concentrations resulting from 5-ALA appli-
`cation are higher in tumour cells than in normal cells [10].
`PPIX is the actual photosensitiser and will, when excited by
`light of an appropriate wavelength, lead to the generation of
`singlet oxygen, which will damage tumour cells and Wnally
`lead to cell death [4].
`In the clinical studies presented here we tested a newly
`developed self-adhesive thin 5-ALA patch (PD P 506 A),
`which has the capability to bypass current handling disad-
`vantages in PDT procedures due to its easy use. The patch
`has the advantage to deliver 5-ALA to the lesion and pro-
`tect the newly formed PPIX from bleaching at the same
`time [16]. The primary aim of the Xuorescence analysis
`study (study code AK 01) was the assessment of the inXu-
`ence of application duration of PD P 506 A on the PPIX
`speciWc Xuorescence in AK lesions. The second study
`(study code AK 05) aimed at evaluating the pharmacoki-
`netic parameters of 5-ALA and PPIX when applying eight
`5-ALA patches simultaneously to AK lesions for 4 h.
`
`Methods
`
`Both studies were performed as prospective open monocen-
`tre study. The Xuorescence analysis study (FA study) was
`designed as intra-individual comparison of diVerent appli-
`cation times of the study patch. The FA study was clinically
`conducted from February 2004 to April 2004 and the phar-
`macokinetic study (PK study), from April 2005 to June
`2005. The study centre, which was the same for both stud-
`ies (PROVERUM), is located in Münster, Germany.
`PROVERUM holds a department, which specialises on the
`conduct of clinical phase I studies in dermatology. Derma-
`tologists from the surrounding areas send suitable patients
`to the study centre.
`
`Ethics
`
`Both study protocols and associated information had been
`submitted to and approved by the Independent Ethics Com-
`mittee responsible for the principal investigator prior to the
`starting of the study. The investigations were carried out in
`accordance with the German Drug Law, national and inter-
`national GCP-Guidelines and the Declaration of Helsinki.
`
`Study medication
`
`PD P 506 A is a rectangular, skin coloured, self-adhesive
`patch with rounded corners. One patch contains 2 mg
`
`123
`
`5-aminolevulinic acid (present as 5-aminolevulinic acid
`hydrochloride) per cm2 and has a size of 4 cm2. Four
`patches, all on a single removable protective Wlm, are
`packed in a heat-sealed pouch. The pouch provides
`protection against moisture, light and environmental con-
`tamination.
`
`Study population
`
`The aim of each study was to involve 12 patients with
`mild to moderate AK on the head or face. The following is
`a list of the most important inclusion and exclusion crite-
`ria. Females of childbearing potential were not eligible for
`the studies. Known or suspect, acute or chronic-hepatic
`diseases or renal dysfunction led to exclusion of the
`patients. Patients with dermatologic conditions jeopardis-
`ing the aim of the study (e.g. scalp psoriasis) were
`excluded. Any treatment being able to inXuence the dis-
`ease status of AK was not permitted 4 weeks prior to or
`during the study, urea and salicylic-acid containing for-
`mulations, 2 weeks prior to or during the study. Patients
`suitable for the PK study had furthermore to be in good
`condition with pulse and blood pressure reading within
`the normal ranges. The patient’s alcohol and caVeine con-
`sumption characteristics were deWned by the study proto-
`col. Post organ transplantation condition, any major
`concomitant disease or any indication of drug abuse dis-
`qualiWed the patients. In both studies, the studied AK
`lesions had to be either of mild (deWned as Xat, pink mac-
`ulae or patches on sun-damaged skin, background mot-
`tling, no roughness or hyperkeratosis) or moderate grade
`(deWned as pink to red papules or plaques with rough,
`hyperkeratotic surface, variable induration) [5]. The iden-
`tiWcation of suitable AK lesions and the classiWcation of
`their severity were done on clinical grounds.
`
`Fluorescence analysis (FA) study
`
`The primary aim of the study was the assessment of the
`inXuence of patch application duration (2, 3, 4 or 5 h) on
`the PPIX-speciWc Xuorescence in AK. The primary parame-
`ter was the Xuorescence of the patched areas determined
`directly after patch removal.
`In the FA study, four distinct AK study lesions were
`chosen per patient. The application times of the 5-ALA
`patch (2, 3, 4, 5 h) were randomly allocated to the lesions.
`Two lesions of non-aVected skin were patched (2, 5 h). No
`preparation of the study lesions prior to patch application as
`e.g. curettage was performed. The Xuorescence data of all
`studied areas were referenced to baseline data (background
`Xuorescence prior to patch application). PPIX-speciWc Xuo-
`rescence of the study areas was determined at given inter-
`vals for all selected skin areas (directly after removal (0 h)
`
`

`

`Arch Dermatol Res (2008) 300:53–60
`
`55
`
`and 2, 4, 6, 24 and 48 h after removal of the 5-ALA patch).
`In the time between the measurements, all study lesions
`were protected from ambient light by cotton wool and alu-
`minium foil to prevent Xuorescence bleaching. A Xuores-
`cent foil was used as Xuorescence standard.
`Fluorescence measurements were performed with
`DYADERM, a highly sensitive digital Xuorescence imag-
`ing system (Biocam GmbH, Regensburg, Germany).
`DYADERM is a diagnostic medical product for Xuores-
`cence diagnostics and serves the early diagnosis of dermal
`changes [1]. The light source emits light in the range of
`370–440 nm. A picture of the Xuorescence is taken by an
`integrated CCD camera and stored electronically. The
`Xuorescence data were analysed by a person blinded to
`the application duration. After correction of shading and
`determination of the reference value the Xuorescence was
`determined for the AK area, where the study medication
`was applied.
`
`Pharmacokinetic (PK) study
`
`The primary aim of the study was the evaluation of the
`pharmacokinetics of 5-ALA after application of 8 patches
`for 4 h to mild to moderate AK lesions. The primary param-
`eters were AUC0–1, AUC0–t, Cmax, tmax, Ae,ur and urinary
`recovery for 5-ALA. Secondary aims of the study were the
`evaluation of the pharmacokinetics of PPIX and safety of
`the patch application.
`In this study, eight AK lesions of each patient were
`patched with PD P 506 A for 4 h. The application duration
`of 4 h had been determined on the basis of the results of the
`FA study. Again, no curettage of the study lesions was per-
`formed prior to patch application. For the determination of
`the PK parameters, blood and urine samples were collected
`prior to and during 12 h after start of the patch application.
`Body weight, body temperature, pulse and blood pressure
`were measured prior to administration, pulse and blood
`pressure also at 2, 4 and 8 h after administration. A Wnal
`blood sample was taken 24 h after the application of the
`study medication.
`For the analysis of 5-ALA in human plasma and human
`urine and for the analysis of PPIX in human plasma, a chro-
`matographic method (HPLC/Xuorescence method) devel-
`oped and validated by CRS-Mannheim GmbH (Grünstadt,
`Germany) was used [23]. The bioanalytical procedures of
`the laboratories were GLP-certiWed. 5-ALA was deter-
`mined in plasma by derivatisation with acetylacetone/form-
`aldehyde and separation by high-performance
`liquid
`chromatography. The limit of quantiWcation was 11.69 ♯g/l
`for 5-ALA. PPIX was isolated from plasma by acidic pro-
`tein precipitation followed by high performance liquid
`chromatography with Xuorescence detection. The limit of
`quantiWcation was 4.47 ♯g/l for PPIX.
`
`Statistical analysis
`
`FA study
`
`The inXuence of the application duration of the 5-ALA patch
`on the PPIX Xuorescence was statistically evaluated by
`regarding the observed value of Xuorescence directly after
`patch removal (F0h). The log-transformed Xuorescence char-
`acteristic F0h was submitted to analysis of variance including
`the eVects of patient and application (four levels with regard
`to the AK areas) in the model. In order to assess the inXu-
`ence of application duration in a conWrmative manner, the
`multiple test procedure REGWQ was applied. Point esti-
`mates with 95% conWdence intervals were calculated for the
`following pair-wise diVerences of application duration: “3 h
`minus 2 h”, “4 h minus 2 h”, “5 h minus 2 h”, “4 h minus
`3 h”, “5 h minus 3 h” and “5 h minus 4 h”. An additional
`analysis was run in an exploratory manner by extending the
`above-mentioned ANOVA to six treatments levels, i.e. the
`results obtained for normal skin were incorporated.
`
`PK study
`
`All patients having an evaluable treatment period were
`included in the PK analysis. Calculation of the pharmacoki-
`netic characteristics was based on actual blood-sampling
`time [h] (relative to the corresponding administration time)
`rounded to two decimal digits and negative pre-dose times
`set to zero. Descriptive statistics of concentrations were cal-
`culated if at least half of the individual data were quantiW-
`able, concentrations below the LLOQ were calculated as
`half of the LLOQ. For calculation of the pharmacokinetic
`characteristics the following rules were applied: At time
`points in the lag-time between time zero and the Wrst con-
`centration equal or above LLOQ, concentrations below
`LLOQ were calculated as zero. Concentrations below
`LLOQ between two quantiWable concentrations were calcu-
`lated with half the LLOQ. Trailing concentrations below
`LLOQ were not used in calculations. The 5-ALA plasma
`concentrations were corrected for pre-dose concentrations
`(Ct - C0, in the case of C0 < LLOQ C0 was replaced by
`LLOQ). The primary model-independent pharmacokinetic
`characteristics for 5-ALA in plasma were calculated using
`non-compartmental procedures.
`Furthermore, a subgroup with an absorption of 5-ALA
`with Cmax concentrations > 1.5*C0 was evaluated statisti-
`cally in addition to the evaluation of the 5-ALA plasma
`pharmacokinetics of all 12 patients.
`
`Safety and tolerability
`
`In both studies adverse events were documented. The
`investigator judged their relation to the study medication.
`
`123
`
`

`

`56
`
`Arch Dermatol Res (2008) 300:53–60
`
`Local phototoxic reactions were prevented by applying a
`cover to the study lesions for the 48–72 h after removal of
`the 5-ALA-patch.
`
`Results
`
`The patient population as well as the selected study lesions
`shows comparable characteristics
`for both
`studies
`(Tables 1, 2). The majority of study lesions were located on
`the scalp and forehead. Most of the study lesions (70%)
`were of mild intensity.
`
`Fluorescence analysis study (FA study)
`
`Thirteen patients were recruited into the study. PPIX Xuo-
`rescence data are based on the analysis of 48 AK study
`lesions and 24 normal skin areas (12 patients) which had
`been patched with the 5-ALA patch as speciWed in the
`study protocol and which provided valid Xuorescence data.
`The patient who did not show valid Xuorescence data was
`identiWed being a “PDT non-responder” retrospectively. All
`13 patients were valid for safety evaluation.
`
`Table 1 Summary of patient characteristics of the safety samples of
`both studies
`
`FA study N = 13
`(safety sample)
`
`PK study N = 12
`(safety sample)
`
`Age (years)
`Mean § SD
`Median (range)
`Sex
`Female
`Male
`
`69.6 § 9.87
`73 (49–80)
`
`2 (15%)
`11 (85%)
`
`69.8 § 7.78
`71 (50–81)
`
`2 (17%)
`10 (83%)
`
`FA Fluorescence Analysis; PK pharmacokinetic; SD Standard
`deviation
`
`Table 3 FA Study: Xuorescence at the moment of patch removal (F0h;
`N = 12) and summary statistics for primary variable
`
`Study area
`
`Application
`duration (h)
`
`Fluorescence
`mean (SD)
`
`Fluorescence
`median (range)
`
`AK lesions
`
`Normal skin
`
`2
`3
`4
`5
`2
`5
`
`1.558 (0.403)
`2.496 (0.856)
`3.183 (1.041)
`3.407 (0.980)
`0.823 (0.157)
`1.598 (0.649)
`
`1.635 (0.81–2.17)
`2.580 (0.69–3.76)
`3.220 (1.55–4.95)
`3.260 (1.42–4.79)
`0.805 (0.56–1.15)
`1.430 (0.75–2.77)
`
`Baseline Xuorescence was well comparable between
`study lesions. Mean values ranged from 0.66 to 0.70 for all
`except one AK lesions as well as normal skin areas.
`The analysis of variance and multiple test procedure for
`treatment comparisons yielded the following results for the
`PPIX Xuorescence in AK lesions (Tables 3, 4):
`• The 2 h-duration is signiWcantly diVerent from each of
`the 3, 4 and 5-h durations
`• The 3-h duration is signiWcantly diVerent from each of
`the 4 and 5-h durations
`• The 4 and 5-h durations do not show a statistically sig-
`niWcant diVerence.
`
`By applying a repeated measurement model of analysis of
`variance, a statistically signiWcant interaction between
`application duration and time was detected. DiVerences in
`mean Xuorescence levels between the application durations
`changed over time. The mean proWles for Xuorescence data
`are presented in Fig. 1a. Figure 1b presents the data in ref-
`erence to the start of patch application. Since there is no
`diVerence in the Xuorescence proWles depicted from start of
`the application for the application durations 3, 4 and 5 h, it
`can be postulated that a saturation of the biological system
`responsible for the conversion of 5-ALA to PPIX takes
`place.
`
`Table 2 Summary of lesion
`characteristics of the safety
`samples of both studies
`
`Application
`duration (h)
`
`FA study N = 13 (52 lesions)
`
`PK study N = 12
`(96 lesions)
`
`2 h
`
`3 h
`
`4 h
`
`5 h
`
`4 h
`
`Site
`Scalp
`Forehead
`Cheek
`Ear
`Temple
`Severity
`Mild
`Moderate
`
`5 (38%)
`6 (46%)
`2 (15%)
`0
`0
`
`10 (77%)
`3 (23%)
`
`6 (46%)
`4 (31%)
`3 (23%)
`0
`0
`
`8 (62%)
`5 (38%)
`
`4 (31%)
`9 (69%)
`0
`0
`0
`
`11 (85%)
`2 (15%)
`
`6 (46%)
`5 (38%)
`1 (7%)
`1 (7%)
`0
`
`9 (69%)
`4 (31%)
`
`7 (7%)
`68 (71%)
`19 (20%)
`1 (1%)
`1 (1%)
`
`66 (69%)
`30 (31%)
`
`FA Fluorescence Analysis;
`PK pharmacokinetic;
`SD Standard deviation
`
`123
`
`

`

`Arch Dermatol Res (2008) 300:53–60
`
`57
`
`Table 4 FA Study: Xuores-
`cence at the moment of
`patch removal (F0h; N = 12):
`comparison of application
`durations
`
`Comparison
`of AK study
`areas (application
`duration)
`
`p value
`
`Pharmacokinetic study (PK study)
`
`In the PK study, none of the 144 blood samples (pre-dose
`samples excluded) deviated from the planned schedule by
`more than 10 min. The actual blood-sampling times were
`used for calculations. There were no missing concentration-
`time data for 5-ALA and PPIX.
`5-ALA plasma concentrations increased from endoge-
`nous 11.23 ♯g/l (measured pre-dose) to a maximum of
`27.57 ♯g/l (geometric means of 12 patients; values not
`baseline corrected). A tmax of 3.99 h after start of the appli-
`cation and an AUC0–t of 393.8 ♯g h/l (median value of 12
`patients) were calculated. Descriptive statistics of plasma
`concentrations of 5-ALA as well as summaries of the
`results for the pharmacokinetic parameters for 5-ALA (with
`and without baseline correction) are given in Tables 5, 6, 7.
`The primary parameter AUC(0–1) could not be calculated
`for non-baseline corrected values due to the endogenous 5-
`ALA concentrations mimicking a longer elimination half-
`life. Figure 2 presents the synoptic plots of the geometric
`mean concentration-time proWles of 5-ALA plasma concen-
`trations and individual proWles.
`
`Table 5 PK Study: descriptive statistics of 5-ALA plasma concentra-
`tions (♯g/l) (N = 12)
`
`Nominal time
`after application (h)
`
`Geometric
`mean (SD)
`
`Median (range)
`
`0.00
`0.25
`0.50
`0.75
`1.00
`1.50
`2.00
`3.00
`4.00
`6.00
`8.00
`12.00
`24.00
`
`11.23 (1.49)
`14.22 (1.10)
`14.06 (1.36)
`15.99 (1.14)
`16.37 (1.44)
`18.59 (1.31)
`21.65 (1.38)
`25.16 (1.46)
`27.57 (1.47)
`19.75 (1.31)
`16.83 (1.24)
`14.81 (1.39)
`11.93 (1.60)
`
`13.75 (<LLOQ¡15.42)
`14.26 (12.04–16.49)
`15.06 (<LLOQ¡20.11)
`15.86 (12.86–20.92)
`16.81 (<LLOQ–27.95)
`17.20 (12.94–32.67)
`21.47 (12.77–37.63)
`22.22 (15.29–50.69)
`24.87 (16.98– 53.08)
`20.31 (13.56–31.96)
`17.44 (12.34–23.48)
`15.65 (<LLOQ¡20.98)
`13.72 (<LLOQ¡25.10)
`
`Table 6 PK study: descriptive characteristics of pharmacokinetic
`parameters for 5-ALA (N = 12)
`
`Parameter
`
`Cmax (♯g/l)
`AUC(0–t) (♯g*h/l)
`Tmax (h)
`
`Geometric
`mean (SD)
`
`28.36 (1.44)
`350.00 (1.60)
`4.58 (1.73)
`
`Median (range)
`
`25.64 (18.05–53.08)
`393.81 (109.37–565.64)
`3.99 (3.00–23.97)
`
`123
`
`3–2 h
`4–2 h
`5–2 h
`4–3 h
`5–3 h
`5–4 h
`
`0.0005
`<0.0001
`<0.0001
`0.0200
`0.0036
`0.4932
`
`baseline
`
`0
`
`4
`6
`2
` TIME after patch removal [h]
`
`24
`
`48
`
`0
`
`2
`
`4
`
`6
`TIME [h]
`
`8
`
`10
`
`12
`
`0123456
`
`0123456
`
`(a)
`
`ecnecseroulF .leR
`
`(b)
`
`ecnecseroulF .leR
`
`Fig. 1 FA study. a Mean proWles for PPIX Xuorescence values. b
`Fluorescence values referenced to start of patch application. Dotted
`lines: 3, 4, 5 h application on AK lesions; solid line: 2 h application on
`AK lesions; dashed line: 2, 5 h application on normal skin (Normal
`skin 2 h: open triangle; normal skin 5 h: open square; AK lesion 2 h:
`closed triangle; AK lesion 3 h: closed rhomb; AK lesion 4 h: closed
`circle; AK lesion 5 h: closed square)
`
`The Xuorescence ratio between AK lesions and normal
`skin at the time of patch removal is about 2:1 for the 2 and
`5-h applications. Exploratory statistical analyses for the
`comparison of the two application durations 2 and 5 h, on
`normal skin, provided signiWcant diVerences at each of the
`time-points after patch removal besides the 48-h value
`which was not statistically signiWcantly diVerent (p = 0.36).
`Fluorescence values on AK lesions, 48 h after patch
`removal, still signiWcantly exceeded the baseline value for
`all application durations (p < 0.001) showing at least 3-fold
`mean diVerences between 48 h and baseline (Fig. 1a).
`
`

`

`58
`
`Arch Dermatol Res (2008) 300:53–60
`
`Table 7 PK study: descriptive characteristics of baseline corrected
`pharmacokinetic parameters for 5-ALA (N = 12)
`
`Parameter
`
`Cmax (♯g/l)
`AUC(0–t) (♯g*h/l)
`Tmax (h)
`
`Geometric
`mean (SD)
`
`16.45 (2.02)
`101.37 (2.56)
`4.58 (1.73)
`
`Median
`
`19.92 (3.57–39.42)
`120.13 (11.53–377.19)
`3.99 (3.00–23.97)
`
`5-ALA Cmax concentrations of 10 of the 12 patients
`exceeded 1.5*baseline (C0). The maximum increase in 5-
`ALA plasma concentrations was 3.7-fold of the pre-dose
`concentration (Cmax 53.08 ♯g/l in one patient). The excre-
`tion of 5-ALA in urine in the Wrst 12 h after application was
`low with a maximum of 2.06% of the dose administered
`and a median of 1.39%.
`For PPIX all analysis results were below the lower limit
`of quantiWcation (4.470 ♯g/l).
`
`Safety and tolerability
`
`In both studies, the 5-ALA patch PD P 506 A proved to be
`a safe medication. None of the patients of the FA study
`experienced any adverse event, which was rated as being
`causally related to the study medication. Local reactions at
`
`the application site (erythema and pruritus), which were
`observed in three patients, most probably have their origin
`in the application of the protection against PPIX bleaching.
`In the PK study, 7 of 12 patients experienced adverse
`events. These were reactions at the application site of the
`study medication (6 patients) and glucose in urine (2
`patients). All adverse events were described as mild to
`moderate. Four patients experienced adverse events at the
`application site, which were rated by the investigator as
`being related to the study medication (erythema, irritation
`and/or pruritus). They all started during (3 patients) or after
`application (2 patients) of the 5-ALA patch. None of the
`events in both studies required any intervention.
`No serious adverse events were observed. Transami-
`nases and ♤GT levels were monitored in all patients. In
`none of the patients a change in liver enzymes was
`observed when comparing pre- and post-dose values. No
`clinically signiWcant changes in vital signs or physical Wnd-
`ings were reported.
`
`Discussion
`
`PD P 506 A, a self-adhesive 5-ALA patch, was invented to
`facilitate the application of the photosensitiser’s precursor
`
`(a)
`
`30
`
`25
`
`20
`
`15
`
`-ALA [µg/L]
`
`105
`
`Fig. 2 a Geometric mean
`(N = 12) concentrations of
`5-ALA (♯g/l) versus time (h)
`following topical administration
`of 5-ALA patch. b All individual
`(N = 12) concentrations of
`5-ALA (♯g/l) versus time (h)
`
`0 1
`
`2
`
`3 4
`
`5
`
`6 7
`
`8
`
`9 10
`
`11 12 13 14 15 16 17 18 19 20 21 22 23 24
`TIME [h]
`
`(b)
`
`5 0
`
`60
`
`50
`
`40
`
`30
`
`20
`
`10
`
`0
`
`5-ALA [µg/L]
`
`0 1
`
`2
`
`3 4
`
`5
`
`6 7
`
`8
`
`9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
`TIME [h]
`
`123
`
`

`

`Arch Dermatol Res (2008) 300:53–60
`
`59
`
`in PDT. The application and subsequent occlusion of e.g. a
`cream is not only time-consuming for dermatologists and
`staV but also cosmetically disturbing for the patient. Prior
`to the start of clinical eYcacy studies where the application
`of the 5-ALA patch would be followed by illumination with
`light of an appropriate wavelength the basic clinical proper-
`ties of the 5-ALA patch should be investigated. Two stud-
`ies were performed: one study had the aim to investigate
`the inXuence of the application duration on the PPIX spe-
`ciWc Xuorescence in AK lesions, the underlying question
`being whether dosing can be achieved via application dura-
`tion. After having identiWed the maximum reasonable
`application duration for clinical eYcacy trials this applica-
`tion duration was investigated for pharmacokinetic charac-
`teristics of the patch in the PK study.
`Fritsch et al. [11] had shown that Xuorescence after
`application of 5-ALA could be quantitatively linked to
`PPIX contents in tissue biopsies. A basic assumption of the
`FA study therefore, was that the Xuorescence would reXect
`the PPIX contents in the studied lesions. In fact, levels of
`PPIX Xuorescence and therefore dosing of 5-ALA can be
`achieved via variation of duration of patch application. The
`results furthermore support the previously reported notion
`that PPIX production is speciWcally increased in tumour
`cells as opposed to normal cells [2, 11, 26]. Furthermore, it
`was shown that the DYADERM system is capable in diVer-
`entiating between the tested treatments and thus proves to
`be a sensible method for Xuorescence diagnosis.
`PPIX generation does not stop when the 5-ALA patch is
`removed but a 5-ALA deposit in skin might have formed
`which is subsequently converted to PPIX. However, after
`3 h of patch application the biological system, which con-
`verts 5-ALA
`into PPIX,
`is saturated. Fluorescence
`increases after this time-point primarily because of the con-
`version of 5-ALA into PPIX, and probably not because of
`additional 5-ALA supply (Fig. 1).
`The PPIX Xuorescence levels after 48 h do not necessar-
`ily reXect the clinical setting of PDT. Illumination would
`take place immediately after patch removal leading to the
`desired phototoxic eVect and cell destruction as well as
`photo-induced destruction of the photosensitiser (photoble-
`aching) [17]. Some residual 5-ALA in the skin, however,
`will most probably be available for conversion into PPIX
`after the end of illumination and can theoretically increase
`the local phototoxic reaction. It therefore seems reasonable
`to instruct the patients to protect the treated lesion from
`light after PDT.
`So far, no direct quantitative link between Xuorescence
`intensity and clinical eYcacy has been published. Fink-
`Puches and colleagues observed a strong and statistically
`signiWcant correlation between “moderate” to “very strong”
`Xuorescence and lesion clearance [8]. Since there was no
`signiWcant increase of Xuorescence between the 4- and 5-h
`
`applications at the time of patch removal, 4 h was chosen as
`application duration for the PK study and will be the maxi-
`mum application in clinical eYcacy studies.
`Studies on the absorption of 5-ALA after topical applica-
`tion using diVerent formulations in diVerent indications
`have been described in literature. Results vary between
`studies and strongly depend on formulation and indication.
`In a study by Rick et al. [22], 5-ALA plasma concentrations
`did not exceed the detection limit of 0.1 mg/l after topical
`administration (10% 5-ALA in cream, 6 h incubation). Frit-
`sch et al. did not Wnd a signiWcant increase in 5-ALA and
`total porphyrin levels in erythrocytes and plasma of patients
`with AK and basal cell carcinoma (BCC) after topical
`administration of 5-ALA in total doses varying between
`0.02 and 7.0 g [9]. In patients with AK and BCC, no alter-
`ation in 5-ALA and porphobilinogen levels in 24 h urine
`samples were found after topical administration of 5-ALA
`[30]. However, spontaneous urine samples collected imme-
`diately after topical 5-ALA administration for the treatment
`of AK showed higher 5-ALA concentrations than control
`samples in 7 out of 10 patients in another study [25]. Traces
`of porphyrins were found in urine samples taken directly
`after 5-ALA-PDT in 3 out of 48 patients with epithelial
`skin tumours [29]. Since a very sensitive detection method
`for 5-ALA in plasma was used in the present study the
`results are interpreted as being consistent with published
`data.
`The patient population of PK studies often consists of
`healthy volunteers. We decided to investigate the PK prop-
`erties of the 5-ALA patch on damaged skin. Damaged skin
`facilitates the penetration of externally applied molecules
`due to its lower barrier [20, 27] and as was shown in the FA
`study the conversion of 5-ALA to PPIX is faster in AK
`lesions than in normal skin. A patient study therefore was
`considered superior to a study in healthy volunteers.
`The observed increase in 5-ALA plasma concentrations
`is considered low in comparison to 5-ALA plasma concen-
`trations recorded after exogenous administration 5-ALA
`[6]. They are much lower than the plasma concentrations, at
`which side eVects have been reported in literature (oral
`administration of 10–60 mg 5-ALA/kg BW). These side
`eVects include nausea with or without vomiting, haemody-
`namic alterations (hypotension), generalised skin phototox-
`icity and abnormalities in liver function [7, 12, 13, 21, 24,
`28]. Since the reported side eVects are dose-dependent, it is
`highly unlikely that any of these reactions will occur after
`dermal application of PD P 506 A.
`In the PK study, PPIX levels were below the lower limit
`of quantiWcation in all cases. This indicates that there is no
`danger of phototoxic reactions on any part of the body other
`than on areas treated with the 5-ALA patch.
`The good local tolerability of the 5-ALA patch together
`with the fact that PPIX was not detectable in plasma
`
`123
`
`

`

`60
`
`Arch Dermatol Res (2008) 300:53–60
`
`samples prove PD P 506 A being a safe medication. Its
`application results in the generation of PPIX-speciWc Xuo-
`rescence in AK lesions with 4 h application resulting in
`maximum PPIX Xuorescence at patch removal. It has been
`planned to conduct further clinical trials focussing on the
`eYcacy of the 5-ALA patch after diVerent duration of
`application in combination with illumination.
`
`Acknowledgments We thank Corinna Gronover and Silke Haller-
`berg from Proverum GmbH for their support in the accurate realisation
`of the study protocols. We furthermore thank Manfred Wargenau,
`Ph.D., M.A.R.C.O. GmbH & Co. KG, and Karl-Ernst Siegler, Ph.D.,
`CRS-Mannheim GmbH, for statistical support. The studies were fund-
`ed by photonamic GmbH & Co. KG.
`
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