Systemic component of protoporphyrin IX production in nude mouse skin upon topical application of aminolevulinic acid depends on the application conditions

Topical application of 5-aminolevulinic acid (ALA) for protoporphyrin IX (PpIX)-based photodynamic therapy of skin cancer is generally considered not to induce systemic side effects because PpIX is supposed to be formed locally. However, earlier studies with topically applied ALA have revealed that in mice PpIX is not only produced in the application area but also in other organs including skin outside the application area, whereas esterified ALA does not. From these results, it was concluded that it is not redistribution of circulating PpIX that causes the fluorescence distant from the ALA application site, but rather, local PpIX production induced by circulating ALA. In the present study we investigate the effects of the ALA concentration in the cream, the application time, the presence of a penetration enhancer, the presence of the stratum corneum and esterification of ALA on the PpIX production in nude mouse skin outside the area where ALA is applied. For this purpose, ALA and ALA hexyl ester (ALAHE) were applied to one flank, and the PpIX fluorescence was measured in the contralateral flank. During a 24 h application of ALA, PpIX was produced in the contralateral flank. No PpIX could be detected in the contralateral flank after ALA application times ranging from 1 to 60 min. Tape-stripping the skin prior to short-term ALA application, but not the addition of a penetration enhancer, resulted in PpIX production in the contralateral flank. When ALAHE was applied, no PpIX fluorescence was measured in the contralateral flank under any application condition. The results suggest that the systemic component of PpIX production outside the ALA application area plays a minor or no role in relevant clinical situations, when the duration of ALA (ester) application is relatively short and a penetration enhancer is possibly added.     

Temperature effect on accumulation of protoporphyrin IX after topical application of 5-aminolevulinic acid and its methylester and hexylester derivatives in normal mouse skin

Significant amounts of protoporphyrin IX (PpIX) are formed after 6 min of topical application of 5-aminolevulinic acid (ALA) and its hexylester derivative, whereas PpIX is formed after 10 min of topical application of ALA-methylester derivative in normal mouse skin at 37 degrees C. Lowering the skin temperature to 28-32 degrees C by the administration of the anesthetic Hypnorm-Dormicum reduces the PpIX fluorescence by a factor of 2-3. Practically no PpIX was formed as long as the skin temperature was kept at 12-18 degrees C. At around 30 degrees C PpIX fluorescence appears later after application of ALA-ester derivatives (14-20 min) than after application of ALA (8 min), indicating differences in their bioavailability (delayed penetration through the stratum corneum, cellular uptake, conversion to ALA, PpIX production) in mouse skin in vivo. The difference in lag time in the PpIX formation after application of ALA and ALA-esters may be partly related to deesterification of the ALA-ester molecules. The temperature dependence of PpIX production may be used for improvement of photodynamic therapy with ALA and ALA-ester derivatives, where accumulation of PpIX can be selectively enhanced by increasing the temperature of the target tissue.     

The influence of UV exposure on 5-aminolevulinic acid-induced protoporphyrin IX production in skin.

The skin of nude mice was exposed to erythemogenic doses of UV radiation, which resulted in erythema with edema. An ointment containing 5-aminolevulinic acid (ALA) was topically applied on mouse and human skin. Differences in the kinetics of protoporphyrin accumulation were investigated in normal and UV-exposed skin. At 24 and 48 h after UV exposure, skin produced significantly less protoporphyrin IX (PpIX) than skin unexposed to UV. Human skin on body sites frequently exposed to solar radiation (the lower arm) also produced less PpIX than skin exposed more rarely to the sun (the upper arm). It is concluded that UV radiation introduces persisting changes in the skin, relevant to its capability of producing PpIX from ALA. The observed differences in ALA-induced PpIX fluorescence may be the result of altered penetration of ALA through the stratum corneum or altered metabolizing ability of normal and UV-exposed skin (or both).     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

Topical application of 5-aminolevulinic acid and its methylester,hexylester and octylester derivatives: considerations for dosimetry in mouse skin model

Ester derivatives of 5-aminolevulinic acid (ALA-esters) have been proposed as alternative drugs for ALA in photodynamic therapy. After topical application of creams containing ALA, ALA methylester (ALA-Me), ALA hexylester (ALA-Hex) and ALA octylester (ALA-Oct) on mouse skin, typical fluorescence excitation and emission spectra of protoporphyrin IX (PpIX) were recorded, exhibiting a similar spectral shape for all the drugs in the range of concentrations (0.5-20%) studied. The accumulation kinetics of PpIX followed nearly a similar profile for all the drug formulations. The fluorescence of PpIX peaked at around 6-12 h of continuous cream application. Nevertheless, some differences in pharmacokinetics were noticed. For ALA cream, the highest PpIX fluorescence was achieved using 20% of ALA in an ointment. Conversely, 10% of ALA-Me and ALA-Hex, but not of ALA-Oct, in the cream was more efficient (P < 0.05) than was 20%. The cream becomes rather fluid when 20% of any of these ALA-esters is used in ointment, whereas 10% and lower concentrations of ALA-esters do not significantly increase fluidity of the cream. The dependence of PpIX accumulation on the concentration of ALA and ALA-ester in the applied cream followed (P < 0.002) kinetics as described by a mathematical model based on the Michaelis-Menten equation for enzymatic processes. Under the present conditions, the PpIX amount in the skin increased by around 50% by the application of ALA-Me, ALA-Hex or ALA-Oct for 4-12 h as compared with ALA for the same period. Observations of the mice under exposure to blue light showed that after 8-24 h of continuous application of ALA, the whole mouse was fluorescent, whereas in the case of ALA-Me, ALA-Hex and ALA-Oct the fluorescence of PpIX was located only at the area of initial cream application. The amount of the active compound in the applied cream necessary to induce 90% of the maximal amount of PpIX was determined for normal mouse skin. Optimal PpIX fluorescence can be attained using around 5% ALA, 10% ALA-Me and 5% ALA-Hex creams during short application times (2-4 h). Topical application of ALA-Oct may not gain optimal PpIX accumulation for short applications (<5 h). For long application times (8-12 h), it seems that around 1% ALA, 4% ALA-Me, 6% ALA-Hex and 16% ALA-Oct can give optimal PpIX fluorescence. But for long application times and high concentrations, systemic effect of ALA applied topically on relatively large areas should be considered.     

Protoporphyrin IX fluorescence kinetics and localization after topical application of ALA pentyl ester and ALA on hairless mouse skin with UVB-induced early skin cancer

In order to improve the efficacy of 5-aminolevulinic acid-based (ALA) photodynamic therapy (PDT), different ALA derivatives are presently being investigated. ALA esters are more lipophilic and therefore may have better skin penetration properties than ALA, possibly resulting in enhanced protoporphyrin IX (PpIX) production. In previous studies it was shown that ALA pentyl ester (ALAPE) does considerably enhance the PpIX production in cells in vitro compared with ALA. We investigated the in vivo PpIX fluorescence kinetics after application of ALA and ALAPE to hairless mice with and without UVB-induced early skin cancer. ALA and ALAPE (20% wt/wt) were applied topically to the mouse skin and after 30 min, the solvent was wiped off and PpIX fluorescence was followed in time with in vivo fluorescence spectroscopy and imaging. At 6 and 12 h after the 30 min application, skin samples of visible lesions and adjacent altered skin (UVB-exposed mouse skin) and normal mouse skin were collected for fluorescence microscopy. From each sample, frozen sections were made and phase contrast images and fluorescence images were recorded. The in vivo fluorescence kinetics showed that ALAPE induced more PpIX in visible lesions and altered skin of the UVB-exposed mouse skin, but not in the normal mouse skin. In the microscopic fluorescence images, higher ALAPE-induced PpIX levels were measured in the stratum corneum, but not in the dysplastic layer of the epidermis. In deeper layers of the skin, PpIX levels were the same after ALA and ALAPE application. In conclusion, ALAPE does induce higher PpIX fluorescence levels in vivo in our early skin cancer model, but these higher PpIX levels are not located in the dysplastic layer of the epidermis.     

Kinetics of protoporphyrin IX formation in rat oral mucosa and skin after application of 5-aminolevulinic acid and its methylester

The kinetics of accumulation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its methylester (5-aminolevulinic acid methylester [ALA-Me]) was studied on rat oral mucosa. The accumulation of PpIX in mucosa and skin after intravenous injection of ALA and ALA-Me was also studied. The elimination rate of PpIX was dependent on drug and dose as well as on administration route. Application of ALA on rat oral mucosa and skin caused a systemic effect with PpIX building up in remote skin sites not exposed to the drugs. No such systemic effect was seen after application of ALA-Me either in mucosa or on skin. Intravenous injection of the drugs (0.2 g/kg) leads to more fluorescence in the skin than topical application of the drug (20%). For mucosa, the opposite is true. Maximal PpIX fluorescence appeared later after application of high concentrations of the drugs (around 8 h for 5% and 20% wt/wt) than after application of low concentrations (around 3-5 h for 1% and 2% wt/wt).     

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insuffici?nt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.     

Uptake of topically applied 5-aminolevulinic acid and production of protoporphyrin IX in normal mouse skin: dependence on skin temperature

The temperature dependence of the uptake phase of 5-aminolevulinic acid (ALA) and the following production phase of protoporphyrin IX (PpIX) in normal mouse skin was investigated. A cream containing 20% ALA was topically applied on the skin for 10 min. The amount of ALA-induced PpIX was evaluated by measuring the fluorescence of PpIX from the treated skin. No measurable amount of PpIX was found in the skin immediately after 10 min application of ALA. The penetration of ALA into the skin was almost temperature independent while the following production of PpIX was found to be a strongly temperature-dependent process. Practically no PpIX was formed in the skin as long as skin temperature was kept low (12 degrees C).     

Electrically enhanced percutaneous delivery of delta-aminolevulinic acid using electric pulses and a DC potential

Selectivity of photodynamic therapy can be improved with localized photosensitizer delivery, but topical administration is restricted by poor diffusion across the stratum corneum. We used electric pulses to increase transdermal transport of delta-aminolevulinic acid (ALA), a precursor to the photosensitizer protoporphyrin IX (PpIX). ALA-filled electrodes were attached to the surface of excised porcine skin or the dorsal surface of mice. Pulses were administered and, in some in vivo cases, a continuous DC potential (6 V) was concomitantly applied. For in vitro 14C ALA penetration, 10 microm layers parallel to the stratum corneum were assayed by liquid scintillation analysis, and 10 microm cross sections were examined autoradiographically. As the electrical dose (voltage x frequency x pulse width x treatment duration) increased, there was an increase in penetration depth. In vivo delivery was assayed by measuring the fluorescence of PpIX in skin samples. A greater than two-fold enhancement of PpIX production with electroporative delivery was seen versus that obtained with passive delivery. Superimposition of a DC potential resulted in a nearly three-fold enhancement of PpIX production versus passive delivery. Levels were higher than the sum of PpIX detected after pulse-alone and DC-alone delivery. Electroporation and electrophoresis are likely factors in electrically enhanced delivery.     

Comparison of the pharmacokinetics and phototoxicity of protoporphyrin IX metabolized from 5-aminolevulinic acid and two derivatives in human skin in vivo

Our novel approach was to compare the pharmacokinetics of 5-aminolevulinic acid (ALA), ALA-n-butyl and ALA-n-hexylester induced protoporphyrin IX (PpIX), together with the phototoxicity after photodynamic therapy (PDT) in human skin in vivo, using iontophoresis as a dose-control system. A series of four increasing doses of each compound was iontophoresed into healthy skin of 10 volunteers. The kinetics of PpIX metabolism (n = 4) and the response to PDT (n = 6) performed 5 h after iontophoresis, were assessed by surface PpIX fluorescence and post-irradiation erythema. Whilst ALA-induced PpIX peaked at 7.5 h, highest PpIX fluorescence induced by ALA-n-hexylester was observed at 3-6 h and no clear peak was seen with ALA-n-butylester. With ALA-n-hexylester, more PpIX was formed after 3 (P < 0.05) and 4.5 h, than with ALA or ALA-n-butylester. All compounds showed a linear correlation between logarithm of dose and PpIX fluorescence/phototoxicity at 5 h, with R-values ranging from 0.87 to 1. In addition, the ALA-n-hexylester showed the tendency to cause greater erythema than ALA and ALA-n-butylester. Fluorescence microscopy (n = 2) showed similar PpIX distributions and penetration depths for the three drugs, although both ALA esters led to a more homogeneous PpIX localization. Hence, ALA-n-hexylester appears to have slightly more favorable characteristics for PDT than ALA or ALA-n-butylester.     

Microscopic localisation of protoporphyrin IX in normal mouse skin after topical application of 5-aminolevulinic acid or methyl 5-aminolevulinate

Light fractionation does not enhance the response to photodynamic therapy (PDT) after topical methyl-aminolevulinate (MAL) application, whereas it is after topical 5-aminolevulinic acid (ALA). The differences in biophysical and biochemical characteristics between MAL and ALA may result in differences in localisation that cause the differences in response to PDT. We therefore investigated the spatial distribution of protoporphyrin IX (PpIX) fluorescence in normal mouse skin using fluorescence microscopy and correlated that with the PDT response histologically observed at 2.5, 24 and 48h after PDT. As expected high fluorescence intensities were observed in the epidermis and pilosebaceous units and no fluorescence in the cutaneous musculature after both MAL and ALA application. The dermis showed localised fluorescence that corresponds to the cytoplasma of dermal cells like fibroblast and mast cells. Spectral analysis showed a typical PpIX fluorescence spectrum confirming that it is PpIX fluorescence. There was no clear difference in the depth and spatial distribution of PpIX fluorescence between the two precursors in these normal mouse skin samples. This result combined with the conclusion of Moan et al. that ALA but not MAL is systemically distributed after topical application on mouse skin [Moan et al., Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters, Int. J. Cancer 103 (2003) 132-135] suggests that endothelial cells are involved in increased response of tissues to ALA-PDT using light fractionation. Histological analysis 2.5h after PDT showed more edema formation after ALA-PDT compared to MAL-PDT that was not accompanied by a difference in the inflammatory response. This suggests that endothelial cells respond differently to ALA and MAL-PDT. Further investigation is needed to determine the role of endothelial cells in ALA-PDT and the underlying mechanism behind the increased effectiveness of light fractionation using a dark interval of 2h found after ALA but not after MAL-PDT.     

Topical bioadhesive patch systems enhance selectivity of protoporphyrin IX accumulation

In clinical 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) of skin tumors it is desirable to develop vehicles that minimize the penetration of ALA through normal stratum corneum and maximize it through the compromised stratum corneum of the tumors to improve tumor selectivity. We have designed a bioadhesive patch, which may be able to achieve this aim. It induces levels of protoporphyrin IX (PpIX) in skin overlying tumors similar to those induced by the proprietary cream (Porphin) but at the same time induces less PpIX to form in normal skin and at distant sites. The mechanisms of action of the patch, as compared with that of the cream, were studied by means of Cuprophan barriers that mimic compromised tumor stratum corneum and in a mouse model with transplanted tumors.     

Comparison of ALA- and ALA hexyl-ester-induced PpIX depth distribution in human skin carcinoma

Photodynamic therapy (PDT) based on the use of photoactivable porphyrins, such as protoporphyrin IX (PpIX), induced by the topical application of amino-levulinic acid (ALA) or its derivatives, ALA methyl-ester (m-ALA), is a treatment for superficial basal cell carcinoma (BCC), with complete response rates of over 80%. However, in the case of deep, nodular-ulcerative lesions, the complete response rates are lower, possibly related to a lower bioavailability of PpIX. Previous in vitro skin permeation studies demonstrated an increased penetration of amino-levulinic acid hexyl-ester (h-ALA) over ALA. In this study, we tested the validity of this approach in vivo on human BCCs. An emulsion containing 20% ALA (w/w) and preparations of h-ALA at different concentrations were applied topically to the normal skin of Caucasian volunteers to compare the PpIX fluorescence intensities with an optical fiber-based spectrofluorometer. In addition, the PpIX depth distribution and fluorescence intensity in 26 BCCs were investigated by fluorescence microscopy following topical application of 20% ALA and 1% h-ALA. We found that, for application times up to 24h, h-ALA is identical to ALA as a PpIX precursor with respect to PpIX fluorescence intensity, depth of penetration, and distribution in basal cell carcinoma, but has the added advantage that much smaller h-ALA concentrations can be used (up to a factor 13). We observed a non-homogenous distribution in BCCs with both precursors, independent of the histological type and depth of invasion in the dermis.     

Quantitative model calculation of the time-dependent protoporphyrin IX concentration in normal human epidermis after delivery of ALA by passive topical application or lontophoresis

We present a mathematical layer model to quantitatively calculate the diffusion of 5-aminolevulinic acid (ALA) in the skin in vivo, its uptake into the cells and its conversion to protoporphyrin IX (PpIX) and subsequently to heme. The model is a modification and extension of a recently presented three-compartment model. The diffusion of ALA in the skin (epidermis, dermis) is described by the time-dependent diffusion equation, and the sink in this equation accounts for ALA uptake in the cells. As boundary conditions, we use the ALA flux across the human stratum corneum (SC) in vitro during passive or iontophoretic ALA delivery as measured in vitro. Besides the diffusion equation, the model includes three additional equations, similar in form to those of the three-compartment model but with a different interpretation. Our additional equations are supposed to describe, respectively, the conversion of ALA in the cytoplasm to some intermediate compound in the mitochondria and the conversion of the latter to PpIX and of PpIX to heme. The first conversion is a process of the Michaelis-Menten type, the other two are first-order rate processes. When fitted to the published data of PpIX fluorescence from normal human skin following iontophoresis of ALA, the model yields the tissue concentration of PpIX as a function of time after ALA application. The computed concentrations are in good agreement with the published phototoxic concentrations of PpIX in the tissues obtained from extraction. The model parameters obtained from the fit are subsequently used to compute the PpIX concentration in normal human skin after 4 h topical application of 10, 20 and 40% ALA. This again yields the PpIX concentrations in tissue, in good agreement with the published values. The saturation of the PpIX concentration as a function of applied ALA concentration is calculated and agrees with clinical observations on the effectiveness of photodynamic therapy. Photobleaching is simulated, with subsequent resynthesis of PpIX in qualitative agreement with experiment. Finally, the model predicts that only 2.5-3.5% of the ALA entering the skin after passing the SC is converted to PpIX. The layered model is a considerable simplification of real skin, but its successful qualitative and quantitative reproduction of experimental data may encourage further studies to test and refine the model to improve our understanding of the kinetics of ALA and the synthesis of PpIX in the skin.     

Protoporphyrin IX fluorescence photobleaching and the response of rat Barrett's esophagus following 5-aminolevulinic acid photodynamic therapy

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre)malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg(-1) ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm(-2) and a fluence of 54 J cm(-2). Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.     

Photodynamic effectiveness and vasoconstriction in hairless mouse skin after topical 5-aminolevulinic acid and single- or two-fold illumination

Several options were investigated to increase the efficacy of photodynamic therapy (PDT) using protoporphyrin IX (PpIX) induced by topically applied 5-aminolevulinic acid (ALA). Hairless mice with normal skin or UVB-light-induced skin changes were used as a model. In the first part of the study animals were illuminated immediately (t = 4) or 6 h (t = 10, PpIX fluorescence maximum) after the end of a 4 h ALA application. A total incident light fluence of 100 J/cm2 (514.5 nm) was delivered at a fluence rate of 100 or 50 mW/cm2. The PDT-induced damage to normal skin was more severe after treatment at t = 10 than at t = 4. Illumination at 50 mW/cm2 caused significantly more visible damage than the same light fluence given at 100 mW/cm2. For UVB-illuminated skin, different intervals or fluence rates made no significant difference in the severity of damage, although some qualitative differences occurred. In situ fluence rate measurements during PDT indicated vasoconstriction almost immediately after the start of the illumination. A fluorescein exclusion assay after PDT demonstrated vasoconstriction that was more pronounced in UVB-treated skin than in normal skin. The second part of the study examined the effect of two illuminations. The first illumination bleaches the PpIX fluorescence. At the start of the second illumination, new PpIX had been formed. Light of 514.5 nm was delivered at 100 mW/cm2 to a total incident light fluence of 200 J/cm2 at t = 4 (single illumination) or 100 J/cm2 at t = 4 plus 100 J/cm2 at t = 10. There was no visual difference in skin damage between 100 and 200 J/cm2 single illumination. Two-fold illumination (100 + 100 J/cm2) caused significantly more skin damage, indicating a potentially successful option for increasing the efficacy of topical ALA-PDT.     

Effect of mammalian cell differentiation on response to exogenous 5-aminolevulinic acid

Many different types of mammalian cells accumulate fluorescing and photosensitizing concentrations of protoporphyrin IX (PpIX) when exposed to exogenous 5-aminolevulinic acid (ALA) in vivo or in vitro. Most types of malignant cells accumulate substantially more ALA-induced PpIX than do the normal cells from which they arose. Most types of malignant cells also are less differentiated than their normal counterparts. We therefore considered the possibility that malignant cells demonstrate a malignant ALA phenotype (accumulate abnormally large amounts of PpIX when exposed to exogenous ALA) as a direct consequence of their less differentiated state. Human promyelocyte cell line HL-60 and mouse preadipocyte cell line 3T3 L1 were induced to differentiate by exposing them to inducing agents in vitro. The HL-60 cells accumulated less ALA-induced PpIX when differentiated, but the 3T3 L1 cells accumulated more. It appears then that changes in the ALA phenotype with changes in the state of differentiation are cell-type specific. The decreased accumulation of ALA-induced PpIX that accompanied differentiation of the promyelocytic leukemia cells may have clinical application for rapid quantitation of the response of myelocytic leukemia patients to differentiation therapy.     

Quantification of topically delivered 5-aminolevulinic acid by lontophoresis across ex vivo human stratum corneum

Iontophoretic transport of the prodrug 5-aminolevulinic acid (ALA), which is used for photodynamic therapy (PDT), across human stratum corneum (SC) was studied quantitatively in vitro. The experiments were carried out in a three-compartment iontophoresis cell consisting of two electrode chambers equipped with Ag-AgCl electrodes, each separated from a central acceptor chamber by a sheet of SC, supported by a dialysis membrane, to mimic the side-by-side configuration normally used in vivo. Acceptor fluid samples were collected every hour for a period of 30 h in a fraction collector and analyzed by high-performance liquid chromatography-fluorometry after derivatization of the ALA. The iontophoretic ALA flux was studied as a function of the applied current density and the ALA concentration in the donor solution (1, 2.5 or 10% ALA). Depending on the ALA concentration in the donor cell, iontophoresis enhances the flux from close to the detection limit of 0.23 nmol cm(-2) h(-1) at zero current density (passive diffusion) to several hundred or thousand nanomoles per square centimeter per hour at current densities ranging from 100 to 1000 microA cm(-2). For example, interpolating our data we find that with an ALA concentration of 2% in the donor chamber, a current density of 0.255 mA cm(-2) transports 0.065 micromol cm(-2) ALA across the SC in 10 min (conditions of Rhodes et al. (1997), J. Invest. Dermatol. 108, 87-91). For passive diffusion we find that a 5 h topical application of 20% ALA results in the transport of 0.05 micromol cm(-2). Thus, the amount of ALA that passively diffuses through the SC in several hours, leading to therapeutic levels of protoporphyrin IX (PpIX) in the epidermis, can be delivered by iontophoresis in 10 min or less. However, because the formation of sufficient PpIX also requires several hours and also because the SC overlying skin lesions such as basal cell carcinoma (BCC) is not intact, the clinical benefit of topical ALA delivery by iontophoresis for PDT of BCC is yet to be established.     

Study of protoporphyrin IX (PpIX) pharmacokinetics after topical application of 5-aminolevulinic acid in urethral condylomata acuminata

The pharmacokinetics of 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) in lesions of urethral condylomata acuminata were investigated. Sixty patients (20 to 60 years old, 48 male and 12 female) were divided randomly into five groups and received topic application of different concentrations of ALA solution (0.5%, 1%, 3%, 5% or 10%). Biopsy was performed between 1 and 7 h and specimens were subjected to histological, PpIX fluorescence and human papillomavirus (HPV) DNA typing analyses. Fluorescence examination confirmed that ALA-induced PpIX fluorescence was dominantly distributed in the HPV-infected epidermis. In contrast, only a minimal amount of PpIX fluorescence was detected in the dermis. The maximal fluorescence intensity was detected at 5 h incubation. Higher ALA concentration (e.g. 5% and 10%) produced a stronger intensity. These results suggest that the topical application of 5-10% ALA solution for 3-5 h is the optimal condition for the photodynamic therapy of urethral condylomata acuminata. The selective damage of the condylomata acuminata lesions in the epidermis without damaging the dermis ensures a better control of recurrence and side effects such as ulceration or scarring. DNA typing showed that all patients were positive for low risk-HPV DNA and among them 18.3% of patients harbored high risk-HPV DNA.