Topical application of 5-aminolaevulinic acid, methyl 5-aminolaevulinate and hexyl 5-aminolaevulinate on normal human skin

PHOTOBIOLOGY DOI 10.1111/j .1365-2133.2006.07484.x

Topical application of 5-aminolaevulinic acid, methyl5-aminolaevulinate and hexyl 5-aminolaevulinate onnormal human skinA. Juzeniene,* P. Juzenas,* L-W. Ma,* V. Iani* and J. Moan*�

*Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, 0310 Montebello, Oslo, Norway

�Department of Physics, Oslo University, 0316 Blindern, Oslo, Norway

CorrespondenceAsta Juzeniene.

E-mail: [email protected]

Accepted for publication22 May 2006

Key words5-aminolaevulinic acid, hexyl 5-aminolaevulinate,

methyl 5-aminolaevulinate, photodynamic therapy,

protoporphyrin IX

Conflicts of interestNone declared.

Summary

Background 5-Aminolaevulinic acid (ALA) and its ester derivatives are used in photo-dynamic therapy. Despite extensive investigations, the differences in biodistribu-tion and pharmacokinetics of protoporphyrin IX (PpIX) induced by ALA and itsderivatives are still not well understood, notably for humans.Objectives To study porphyrin accumulation after topical application of ALA andtwo of its ester derivatives in normal human skin.Methods Creams containing 0Æ2%, 2% and 20% (w/w) of ALA, methyl 5-amino-laevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) were applied on normalhuman skin of six volunteers. The amount and distribution of porphyrins formedin the skin was investigated noninvasively by means of fluorescencespectroscopy.Results Fluorescence emission and excitation spectra exhibited similar spectralshapes for the all drugs, indicating that mainly PpIX was formed. Low concentra-tions (0Æ2% and 2%) of MAL induced considerably less PpIX in normal humanskin than similar concentrations of ALA and HAL. A high concentration (20%) ofALA gave higher PpIX fluorescence in normal human skin than was found forMAL and HAL.Conclusions The concentrations inducing half of the maximal PpIX fluorescence arearound 2% for ALA, 8% for MAL and 1% for HAL.

Photodynamic therapy (PDT) combines the administration of

a photosensitizer and light exposure. In many cases photosen-

sitizers are preferentially localized in neoplastic tissue.1 Light

exposure initiates photosensitizing reactions, resulting in pho-

todamage and elimination of the tumour. Porphyrin-type dyes

are commonly used as photosensitizers in PDT.2 Under excita-

tion with visible light porphyrins fluoresce, and their charac-

teristic red emission has been suggested for use in

fluorescence diagnosis of neoplastic tissues.3 Most importantly,

fluorescence spectroscopy enables noninvasive investigation of

neoplastic and normal tissues labelled with porphyrins.2 Dur-

ing the last two decades tissue imaging using fluorescence has

been extensively exploited in vivo.4,5

A new approach in PDT, namely to use the endogenous

photosensitizer protoporphyrin IX (PpIX), has been introduced

during the past decade. Accumulation of PpIX in tissues is

achieved by exogenous administration of its natural precursor

5-aminolaevulinic acid (ALA). Normally, every living cell syn-

thesizes PpIX from endogenous ALA. The haem biosynthesis

cycle tightly regulates the endogenous levels of ALA, and con-

centrations of PpIX high enough to induce tissue photosensiti-

zation normally do not accumulate. ALA administered

exogenously bypasses this feedback control and consequently

free PpIX accumulates in the cells. Destruction of cells by

endogenous porphyrins was suggested by Malik and Lugaci.6

PpIX formation in animal tumours was studied by Qian et al.7

Kennedy et al. applied ALA in the clinics, and so-called ALA-

PDT was proposed for clinical practice by several investiga-

tors.8–11 ALA-induced PpIX is also intended for use in fluores-

cence diagnosis.12 ALA can be administered either systemically

(intravenously or orally) or topically in an ointment applied

directly on lesions.10,11,13 Basically, topical application is

advantageous over systemic administration in ALA-PDT as sys-

temic photosensitization is avoided.

Ester derivatives of ALA have been proposed for use in

PDT.14–16 These derivatives are more lipophilic17 and were

expected to penetrate deeper than ALA.18 However, it appears

that ALA goes more readily into circulation than ALA esters,19

which is an opposing argument. Despite extensive investiga-

tions, the differences in biodistribution and pharmacokinetics

� 2006 British Association of Dermatologists • British Journal of Dermatology 2006 155, pp791–799 791

of ALA and its derivatives are still not well understood, especi-

ally in humans. Therefore, the aim of the present work was to

study the accumulation of porphyrin after topical application

of ALA and two of its ester derivatives in normal human skin.

The amount and distribution of PpIX formed in the skin was

investigated noninvasively by means of fluorescence spectros-

copy. Considerations for dosimetry (drug concentration, appli-

cation time) are discussed on the basis of the experimental

results.

Materials and methods

Chemicals

ALA and methyl 5-aminolaevulinate (MAL) were purchased

from Sigma Chemical Co. (St Louis, MO, U.S.A.). Hexyl 5-

aminolaevulinate (HAL) was kindly supplied by PhotoCure

ASA (Oslo, Norway).

Human volunteers

Fluorescence measurements were carried out in six healthy

white-skinned volunteers (age range 29–57 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). Three volunteers had visually lighter

skin and three volunteers had visually darker skin.

Topical application of 5-aminolaevulinic acid, methyl

5-aminolaevulinate and hexyl 5-aminolaevulinate

For the experiments intended to study fluorescence kinetics,

creams were prepared using 0Æ2%, 2% and 20% (w/w) of

ALA, MAL and HAL in a standard ointment (Unguentum;

Merck, Darmstadt, Germany). Transparent adhesive dressings

(OpSite Flexifix; Smith & Nephew Medical Ltd, Hull, U.K.)

with defined cut areas of 1 cm2 were applied on the upper

right arm of six volunteers with a distance of approximately

1 cm between adjacent application spots. Approximately

75 ± 10 mg cm)2 of the freshly prepared cream formulations

were applied topically on a single spot of defined area 1 cm2

(Fig. 1a). After cream application, the whole area was covered

with another dressing of the same type. The creams were

maintained continuously on the test spots for the duration of

the experiment (24 h).

Fluorescence excitation spectroscopy was performed on

three volunteers. Twenty per cent ALA (1Æ2 mmol g)1), 20%

MAL (1Æ1 mmol g)1), 20% HAL (0Æ8 mmol g)1) creams and

(a) (b)

Fig 1. Set of application areas on the upper arm of the volunteers used for fluorescence kinetics (a) and fluorescence excitation spectroscopy

(b). Numbers denote percentage of 5-aminolaevulinic acid (A), methyl 5-aminolaevulinate (M) and hexyl 5-aminolaevulinate (H) in the cream

and C is the control (ointment only).

� 2006 British Association of Dermatologists • British Journal of Dermatology 2006 155, pp791–799

792 Topical ALA and its esters on human skin, A. Juzeniene et al.

ointment only were applied topically on defined application

areas (Fig. 1b) on the upper left arm. The dressings were

removed and the creams were gently wiped off after 6 h of

application. The fluorescence excitation spectra were measured

on the application areas immediately after cream removal

(corresponding to 6 h after application) and at 9, 12 and

24 h after application.

Fluorescence measurements

The fluorescence of porphyrins was measured noninvasively

with a fibre-optic probe coupled to a luminescence spectro-

meter (LS50B; Perkin Elmer, Norwalk, CT, U.S.A.). The probe

is a commercially available fibre accessory (Perkin-Elmer, two

1-m fused silica fibre bundles joined in parallel at the measur-

ing tip) fitted with a cylinder-shaped aluminium spacer

(6Æ5 mm diameter), which provides a constant fixed distance

of 10 mm between the fibre ends and the sample surface. This

assures a relatively uniform distribution of the excitation light

over the area to be measured and provides the maximum

fluorescence signal for a given set-up.

For kinetic measurements, fluorescence emission was meas-

ured at 635 nm, following application of 407-nm excitation

light from the luminescence spectrometer. This light had a

low intensity (< 1 mW cm)2) and did not induce any signifi-

cant photobleaching of PpIX. A 515-nm cut-off filter built in

the luminescence spectrometer was used on the emission side

of the spectrometer. In addition, complete emission spectra in

the range of 550–800 nm were measured. The fluorescence

measurements were carried out through the transparent occlu-

sive dressing, which did not alter the fluorescence signal.

For fluorescence excitation spectroscopy, the emission

wavelength was set at 705 nm, corresponding to the second

emission maximum of PpIX. The complete excitation spectra

were recorded in the range of 300–685 nm. An interference

filter (Ealing Electro-Optics, Inc., Holliston, MA, U.S.A.) with

a narrow bandpass (700–720 nm) was used on the emission

side of the luminescence spectrometer.

The autofluorescence background, i.e. the fluorescence of

the skin measured before the application of the drugs, was

subtracted from the fluorescence data. Individual fluorescence

data were calculated as means of three recordings and the pre-

sented data are means from all volunteers within the groups.

Error bars represent SEM.

Light transmission through human skin

A specimen of healthy human skin, excised from the abdom-

inal region of a patient, was kindly supplied by Dr J. Evensen

(Oncology Department of the Norwegian Radium Hospital,

Oslo). Light emitted from a 10-W halogen lamp was guided

on to the skin surface and the light transmitted through the

skin was collected with a quartz fibre (diameter 1 mm)

attached to the other side of the skin. The distance between

the skin surface and the fibre, i.e. the thickness of the skin,

was set by a micrometric screw.20 The fibre was connected to

the luminescence spectrometer and emission spectra of the

transmitted light were recorded in the range of 300–900 nm

for different skin thicknesses. The light fluence followed an

exponential decay with increasing skin thickness and the light

penetration depth d was determined as described earlier.20

Kinetics analysis and statistics

The kinetics of PpIX fluorescence were plotted as a function of

application time. The significance (P < 0Æ05) of differences in

kinetics was tested with Student’s t-test. Concentration

dependence curves were fitted (P < 0Æ01) with the equation

describing a saturating process:

F ¼ FmaxC

C1=2 þ Cð1Þ

where F is measured fluorescence intensity of PpIX, C is con-

centration (%) of ALA or its derivatives in the applied cream,

Fmax is the maximal intensity of PpIX fluorescence that can be

achieved after application of the cream, and C1/2 is the con-

stant for the process showing the concentration of the drug in

the applied cream that will induce 50% of the maximal

amount of a fluorophore (in our case, that will induce half of

the maximal fluorescence, F ¼ ½Fmax).

Spectroscopic determination of the penetration depth

Comparison of the spectral shape of the excitation spectra, by

normalizing their intensity to 1 at 633 nm, can be used for

estimation of PpIX fluorescence depth:21

FðkexÞ ¼ F0ðkexÞs�1ð1� e�dsÞ ð2Þ

in which s ¼ 1dðkexÞ þ

1dðkemÞ and F0 is the excitation spectrum

recorded in cells in vitro under dilute conditions, taken from

our previous work,21 F is the excitation spectrum measured

with the fibre probe on the surface of the skin, kex ¼ 300–

685 nm, kem ¼ 705 nm and d(k) is the light penetration

depth measured in the human skin for a certain wavelength k.The depth d of PpIX fluorescence is then varied to obtain the

best fit. The depth d is expressed in comparative units (comp.

u.) as the excitation spectra compare relative penetration of

the drugs, and not the actual penetration depth.

Results

Protoporphyrin IX accumulation in normal human skin

Typical fluorescence emission and excitation spectra of PpIX

were observed in normal human skin after topical application

of the creams containing ALA or its derivatives (Fig. 2). Top-

ical application of ALA, MAL and HAL resulted in gradual

increase of PpIX fluorescence, which depended on the concen-

tration (Fig. 2). Low concentration (0Æ2%) induced negligible

amounts of PpIX in the case of MAL, whereas ALA and HAL

gave significantly higher fluorescence (Fig. 2a). An intermedi-

ate concentration (2%) of MAL induced PpIX but significantly


Topical ALA and its esters on human skin, A. Juzeniene et al. 793

less compared with ALA and HAL (Fig. 2b). The difference

was not statistically significant between 2% ALA and 2% HAL.

The highest PpIX fluorescence was achieved using 20% of

the drugs in the cream. ALA at 20% gave more PpIX in nor-

mal skin than 20% MAL and 20% HAL (Fig. 2c), but differ-

ences were not statistically significant. The variations of PpIX

fluorescence between volunteers are reflected in large error

bars (Fig. 2).

Fluorescence images taken in one of the volunteers showed

a strong fluorescence in normal skin after a 12-h topical appli-

cation of 20% ALA, 20% MAL and 20% HAL cream (Fig. 3b).

In this case the strongest fluorescence was observed with ALA

and MAL cream. HAL (20%) cream had lower viscosity and

was distributed over a larger area than ALA or MAL (Fig. 3b).

Fluorescence measurements of PpIX from the same patch

showed that after a 12-h cream application ALA gave the

strongest fluorescence, while for MAL and HAL the fluores-

cence was 10% and 25% weaker, respectively, compared with

ALA (Fig. 3b). Fluorescence images of the same area at 24 h

after cream removal showed the strongest fluorescence for

ALA cream and weaker for MAL and HAL (Fig. 3c). At 24 h

after the cream removal the fluorescence had decreased by

30% for ALA, by 85% for MAL and by 30% for HAL

(Fig. 3c). No visible skin reactions were found after vehicle

cream application.

Influence of drug concentration on protoporphyrin IX

accumulation

We suppose that measured fluorescence is proportional to

PpIX concentration in the upper layers of skin. This fluores-

cence increased with time (1–14 h) for all drugs studied

(Fig. 4), and at prolonged application times (14–24 h) the

PpIX fluorescence had similar profiles (Fig. 4).

The accumulation of PpIX after topical application of ALA

and HAL showed saturation kinetics (Fig. 4a,c). The accumu-

lation of PpIX by MAL was delayed at low concentrations

(Fig. 4b) and seemed to reach saturation at higher concentra-

tions than those of ALA and HAL. Higher concentrations than

20% were not studied, because they increased the fluidity of

the cream.

The fit parameter (C1/2) was calculated using Equation 1.

Half of the maximal PpIX fluorescence was achieved at much

lower concentrations using ALA and HAL than MAL (Fig. 5a).

Concentrations inducing half of the maximal PpIX fluorescence

were around 1Æ5 ± 0Æ2% for ALA (0Æ09 ± 0Æ02 mmol g)1)

(a)

(b)

(c)

Fig 2. Kinetics of protoporphyrin IX (PpIX) fluorescence at 636 nm

during topical application of 5-aminolaevulinic acid (ALA), methyl 5-

aminolaevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) at

different concentrations: (a) 0Æ2%, (b) 2% and (c) 20%. Results are

shown as mean ± SEM; rel. u., relative units. Inset: the fluorescence

emission spectrum (corrected for the spectral sensitivity of the

luminescence spectrometer) of PpIX recorded in human skin. Only

one fluorescence spectrum is shown because the spectral shape was

identical in all cases.

(a)

(b)

(c)

Fig 3. Set of application areas on the upper arm of a volunteer used

for 20% hexyl 5-aminolaevulinate (HAL), 20% methyl 5-

aminolaevulinate (MAL), 20% 5-aminolaevulinic acid (ALA) and

vehicle cream only (a). Fluorescence images of the same patch area

after 12 h topical application (b) and after 24 h following cream

removal (c).



and around 0Æ8 ± 0Æ3% for HAL (0Æ03 ± 0Æ01 mmol g)1).

For MAL they were around 8Æ0 ± 3Æ5% (0Æ44 ±

0Æ19 mmol g)1). Calculations of the cream concentrations on

a molar basis show a slightly different pattern, due to differ-

ences in the molecular weights of the different compounds

used (Figs 4 and 5b).

Penetration of 5-aminolaevulinic acid and ester

derivatives

The calculated fluorescence excitation spectra (Equation 2)

were fitted with the spectra measured at 6, 9, 12 and 24 h

after topical application of 20% ALA, MAL and HAL. This pro-

cedure is illustrated in Figure 6 for the case of ALA (12 h). In

all other cases the same procedure was applied. The results of

the calculations show large variations from person to person

in the penetration depths, being in the range of 0Æ7–1Æ7comp. u. for ALA, 0Æ5–1Æ9 comp. u. for MAL and 0Æ3–1Æ5comp. u. for HAL. However, on average, there was no signifi-

cant difference in penetration depths estimated in this way for

ALA and its derivatives (Fig. 7). For application times < 5 h,

(a)

(b)

(c)

Fig 4. Accumulation of protoporphyrin IX for different application

times (1–24 h) of 5-aminolaevulinic acid (a), methyl 5-

aminolaevulinate (b) and hexyl 5-aminolaevulinate (c). Results are

shown as mean ± SEM; rel. u., relative units. Arrows show the

abscissa values corresponding to 1 mmol g)1 concentrations.

(a)

(b)

Fig 5. Concentrations of 5-aminolaevulinic acid (ALA), methyl 5-

aminolaevulinate (MAL) and hexyl 5-aminolaevulinate (HAL) in the

applied cream sufficient to induce half of the maximal protoporphyrin

IX fluorescence in normal human skin obtained by fitting with

Equation 1 curves presented in Figure 4. Results are shown as

mean ± SEM. Concentrations are given in percentages (a) and in

mmol g)1 (b).

Fig 6. Excitation spectrum of protoporphyrin IX fluorescence at

705 nm measured in human skin (solid line; error bar shows SEM)

and excitation spectra calculated with Equation 2 for various depths d

(broken lines). comp. u., comparative units. Inset: light penetration in

human skin.



the fluorescence intensity at 705 nm was low and the excita-

tion spectra were noisy, with only the Soret band distinct.

According to Equation 2, this leads to high values of the ratio

F408/F633, and, in turn, low penetration values (d < 0Æ5 comp.

u.) are estimated for t < 5 h.

The spectral shape of the calculated spectrum differs slightly

from the measured spectrum (Fig. 6); the difference may be

due to different environments in cells in vitro and in human

skin in vivo, to different conditions (cuvette vs. fibre probe)

used to record these spectra and to differences in PpIX aggre-

gation. The fit is mainly weighted by the ratio of the Soret

peak (408 nm) to the long wavelength peak (633 nm), which

is practically independent of the spectral shape in this case.

Discussion

Topical ALA-PDT has been used to treat different skin

neoplasms.22–36 However, the limited penetration depth of

topically applied ALA is a major problem16,37,38 resulting in

low cure rates, particularly for nodular and thick tumours.10,11

The distribution of ALA in skin is dependent on many param-

eters such as drug penetration through the stratum corneum,

diffusion through the epidermis and dermis, drug clearance

rate and conversion of ALA into PpIX.39

ALA derivatives, which are more lipophilic than ALA,17

have been proposed as new PDT drugs under the assumption

that they should penetrate deeper than ALA.18,40 ALA deriva-

tives were found to induce PpIX up to 100-fold more effi-

ciently than ALA in cell cultures in vitro.14,16 So the assumption

is certainly fulfilled for layers as thin as a cell membrane.

However, in animal models ALA derivatives do not induce

widely different amounts of PpIX.41,42 In most cases ALA

esters induced similar or slightly higher PpIX levels compared

with ALA18 in normal mouse skin,42–44 while in humans the

opposite result was found.45,46 The structures of the hair folli-

cles and of the stratum corneum in mouse skin differ from

those in human skin.47,48 These differences may influence

drug penetration route and ability to penetrate the stratum

corneum and produce endogenous porphyrins. Therefore, the

permeation of ALA and its ester across the stratum corneum

and following porphyrin production in the mouse model will

not always be the same as in human skin.

ALA was the most efficient at inducing PpIX in normal

human skin (Fig. 2). This is in agreement with the data of

Fritsch et al.45 and Wiegell et al.46 Considerably higher doses of

MAL must be applied to achieve the same effect (Figs 2, 4

and 5). HAL at 20% gave lower PpIX fluorescence than ALA

and MAL because it had a lower viscosity and was distributed

over a larger area compared with ALA and MAL (Fig. 3b).

Accumulation of PpIX after topical application of ALA and

HAL followed saturation kinetics (Fig. 2). However, higher

concentrations of MAL (> 20%) were not tested. It seems that

MAL has to overcome a concentration threshold before it can

induce significant amounts of PpIX in normal human skin

(Fig. 4). Some ALA derivatives were found to induce higher

amounts of PpIX than ALA in human cancers in vivo.49,50 Expo-

sure of rat skin explants to ALA, MAL and HAL41 led to almost

similar PpIX levels, as we found in human skin (Fig. 4). The

use of MAL, rather than that of ALA, preferentially enriched

solar keratoses with porphyrins, although lower porphyrin

levels were found after application of MAL as compared with

ALA.45 Topical application of ALA or its esters on human skin

probably does not induce any systemic action in contrast to

what was observed for ALA in mice,19,42 as the fluorescence

was located in the areas of cream application. This is most

likely to be because a large area relative to the body mass was

used in the case of mice, whereas a very small area relative to

the body mass was employed in the human situation.

The stratum corneum is three to five orders of magnitude

less permeable than the dermis.51 The thickness of the stratum

corneum (10–50 lm), epidermis (50–100 lm) and the entire

dermis (2000 lm) varies greatly from person to person and

from one location to another in the same person.51 There are

large differences in the rates of penetration of different

Fig 7. Apparent penetration depths, as determined by fluorescence

excitation spectroscopy. Error bars show SEM; comp. u., comparative

units. ALA, 5-aminolaevulinic acid; MAL, methyl 5-aminolaevulinate;

HAL, hexyl 5-aminolaevulinate.



substances.52 From such facts and from our experimental data

it is clear that the stratum corneum of normal human skin is

the main barrier for topically applied ALA and its derivatives,

especially for low concentrations (0Æ2%) of MAL (Figs 2 and

4). Fluorescence excitation spectroscopy indicates that ALA

and its derivatives penetrate down to similar depths after 6 h

of topical application (Fig. 7). The depth remains nearly con-

stant up to 12 h. The constant presence of PpIX after cream

removal (Fig. 7) indicates bioavailability of ALA and its deriv-

atives in the skin. This ‘reservoir effect’ has been observed

after topical application, and the drug reservoirs seem to be

located mainly in the stratum corneum.52 Spectroscopic deter-

minations of the penetration are in reasonable agreement with

data obtained by other methods. ALA, being a small hydrophi-

lic molecule, might be expected to penetrate about 3 mm after

3–15 h of topical application.53 Application times of more

than 4 h seem to be necessary for penetration down into thick

tumours.37 Martin et al.38 found that after topical application

of ALA on nodular basal cell carcinomas the fluorescence of

PpIX was distributed through the whole thickness in thinner

tumours (0Æ42 mm), while in thicker tumours (1Æ25 mm)

fluorescence was seen only in upper layers. Fluorescence micro-

scopy shows that the penetration depth of ALA in basal cell

carcinoma is around 1–2 mm.54 The application of 16% MAL

for 3 h can induce porphyrin formation throughout the depth

of thick basal cell carcinoma lesions up to 2 mm with high

selectivity.55 The number of publications related to ALA and

its esters has steadily been increasing during the last decade.

However, hardly any attempts have been made to compare

these compounds systematically in vivo.

ALA and MAL induced more homogeneous PpIX distribu-

tion after 12 h application compared with HAL (Fig. 3b). Fur-

thermore, at the same time we found higher fluorescence of

PpIX by fluorescence measurements after ALA and MAL appli-

cation than after HAL. Small spots with enhanced PpIX fluores-

cence (probably from hair follicles56 or sebaceous glands57)

were seen after HAL application. After topical application of

ALA on human skin, high levels of PpIX fluorescence were

found in the epidermis, with little PpIX in the dermis, cutane-

ous musculature and vasculature.38 The hair follicles and seba-

ceous glands are localized relatively deep in human skin.

Therefore, the fraction of the PpIX fluorescence around the

hair follicles and sebaceous glands measured at the skin sur-

face is considerably less than the fraction of the fluorescence

measured from the epidermis.

The esters have generally been reported to give a more

homogeneous PpIX distribution than ALA.58 Gerscher et al.59

found more homogeneous tissue distribution of PpIX with

ALA-n-pentyl ester than with ALA in healthy human skin. At

the same time they found greater phototoxicity with ALA-n-

pentyl ester than with ALA but no difference in surface-detec-

ted PpIX levels and no difference in depth of PpIX production

induced by these compounds. This implies that the distribu-

tion of PpIX in skin is important.

ALA-, MAL- and HAL-induced PpIX fluorescence shows

large variability among different volunteers (Fig. 2). This may

be related to different skin thickness of different volunteers.

Human skin exposed frequently to solar radiation produces

less PpIX than skin exposed more rarely to the sun.60 Ultravi-

olet radiation introduces persistent changes in the skin, cer-

tainly relevant in view of its capability to produce PpIX from

ALA. Thickening of the stratum corneum and the viable epi-

dermis after sun exposure61 may be one of the reasons for

this. Experiments show that the stratum corneum acts as a bar-

rier for ALA and its esters: use of penetration enhancer or tape

stripping enhanced PpIX production after ALA and HAL appli-

cation in normal mouse skin.62 Higher fluorescence of PpIX

from ALA, MAL and HAL was recorded for volunteers with

light skin than for those with visually darker skin.

In conclusion, topical application to normal human skin of

creams containing ALA, MAL and HAL induced production of

PpIX with some pharmacokinetic differences. Detectable PpIX

fluorescence appears 1–2 h after application. From the present

data the following practical conclusions can be drawn, relevant

for dosimetry in normal human skin: half of the maximal

amount of PpIX is achieved already at concentrations of the

order of 2% (0Æ12 mmol g)1) for ALA, 8% (0Æ44 mmol g)1)

for MAL and 1% (0Æ04 mmol g)1) for HAL. On average, as

estimated by fluorescence excitation spectroscopy, there is no

significant difference in the penetration depths of ALA and its

derivatives in normal skin.

Acknowledgments

The present work was supported by the Research Foundation

of the Norwegian Radium Hospital and by the Norwegian

Cancer Society (Kreftforeningen).

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Topical application of 5-aminolaevulinic acid, methyl 5-aminolaevulinate and hexyl 5-aminolaevulinate on normal human skin