5-Aminolevulinic acid and its derivatives: physical chemical properties and protoporphyrin IX formation in cultured cells

Protoporphyrin IX (PpIX) is used as a fluorescence marker and photosensitizing agent in photodynamic therapy (PDT). A temporary increase of PpIX in tissues can be obtained by administration of 5-aminolevulinic acid (ALA). Lipophilicity is one of the key parameters defining the bioavailability of a topically applied drug. In the present work, octanol-water partition coefficients of ALA and several of its esters have been determined to obtain a parameter related to their lipophilicity. The influence of parameters such as lipophilicity, concentration, time, and pH value on PpIX formation induced by ALA and its esters is then investigated in human cell lines originating from the lung and bladder. ALA esters are found to be more lipophilic than the free acid. The optimal concentration (c(opt), precursor concentration at which maximal PpIX accumulation is observed) is then measured for each precursor. Long-chained ALA esters are found to decrease the c(opt) value by up to two orders of magnitude as compared to ALA. The reduction of PpIX formation observed at higher concentrations than c(opt) is correlated to reduced cell viability as determined by measuring the mitochondrial activity. Under optimal conditions, the PpIX formation rate induced by the longer-chained esters is higher than that of ALA or the shorter-chained esters. A biphasic pH dependence on PpIX generation is observed for ALA and its derivatives. Maximal PpIX formation is measured under physiological conditions (pH 7.0-7.6), indicating that further enhancement of intracellular PpIX content may be achieved by adjusting the pharmaceutical formulation of ALA or its derivatives to these pH levels.     

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.     

Detection of dysplastic lesions by fluorescence in a model of chronic colitis in rats after local application of 5-aminolevulinic acid and its esterified derivatives

5-Aminolevulinic acid (ALA) and ALA ester-induced protoporphyrin IX (PPIX) fluorescence are used for photodynamic diagnosis and therapy with promising results. The aim of the present study was to investigate the detection of dysplastic lesions by fluorescence after topical application of ALA and different esterified derivatives in a model of chronic colitis in rats. In female CD rats chronic colitis was induced by oral application of 5% dextrane sulfate sodium. ALA was used at different concentrations (0.072 and 0.036 mol/L). ALA-methylester (m-ALA), ALA-hexylester (h-ALA) and ALA-benzylester (b-ALA) were used at a concentration of 0.003, 0.002 and 0.002 mol/L, respectively. Fluorescence was examined under blue light, and histological findings of fluorescent and nonfluorescent biopsy specimens were recorded. Using ALA at a concentration of 0.072 mol/L, all dysplastic lesions (8/8) showed fluorescence (sensitivity 100%). Specificity was low at 57%. Reducing the concentration to 0.036 mol/L resulted in a sensitivity of only 56% (5/9) with an increase in specificity to 76%. On using h-ALA, sensitivity was 60% (3/5) with a specificity of 51%. Using m-ALA and b-ALA, sensitivity values were 25% and 33%, and values for specificity were 62% and 63%, respectively. Despite a low number of dysplastic lesions, the results of this study indicate that ALA ester-induced PPIX fluorescence has the potential for the detection of premaligant lesions but was not superior to ALA. ALA esters were used in 18- to 36-fold lower concentrations compared with ALA.     

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.     

Derivatives of 5-Aminolevulinic Acid for Photodynamic Therapy: Enzymatic Conversion into Protoporphyrin

In recent years, 5-aminolevulinic acid (ALA) has become a widespread agent for photodynamic therapy (PDT). In nucleated cells, ALA is converted into the endogenous photosensitizer protoporphyrin IX (PpIX). A major drawback of ALA is its low bioavailability. As a result, high doses of ALA must be administered in order to reach clinically relevant levels of PpIX. Moreover, only superficially located lesions can be treated as a result of the poor penetration of ALA into tissues. A possible solution for this problem may be provided by the prod rug concept. In the present study, prodrugs of ALA have been synthesized. These ALA prodrugs are shown to result in higher PpIX levels in cells than does ALA itself. Of a range of ester prodrugs of ALA, the ALA-pentyl ester elicits the highest fluorescence. Further-more, the enzymatic conversion of the derivatives into ALA and PpIX has been studied in lysed cells. Under these circumstances, the esters with the shorter alkyl chains induce the highest fluorescence. The alcohols that arise as side products from enzymatic conversion of the prodrugs are shown to have no influence on the experiments.     

Endogenous protoporphyrin IX, a clinically useful photosensitizer for photodynamic therapy.

The tissue photosensitizer protoporphyrin IX (PpIX) is an immediate precursor of heme in the biosynthetic pathway for heme. In certain types of cells and tissues, the rate of synthesis of PpIX is determined by the rate of synthesis of 5-aminolevulinic acid (ALA), which in turn is regulated via a feedback control mechanism governed by the concentration of free heme. The presence of exogenous ALA bypasses the feedback control, and thus may induce the intracellular accumulation of photosensitizing concentrations of PpIX. However, this occurs only in certain types of cells and tissues. The resulting tissue-specific photosensitization provides a basis for using ALA-induced PpIX for photodynamic therapy. The topical application of ALA to certain malignant and non-malignant lesions of the skin can induce a clinically useful degree of lesion-specific photosensitization. Superficial basal cell carcinomas showed a complete response rate of approximately 79% following a single exposure to light. Recent preclinical studies in experimental animals and human volunteers indicate that ALA can induce a localized tissue-specific photosensitization if administered by intradermal injection. A generalized but still quite tissue-specific photosensitization may be induced if ALA is administered by either subcutaneous or intraperitoneal injection or by mouth. This opens the possibility of using ALA-induced PpIX to treat tumors that are too thick or that lie too deep to be accessible to either topical or locally injected ALA.     

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.     

Protoporphyrin IX–β‐Cyclodextrin Bimodal Conjugate: Nanosized Drug Transporter and Potent Phototoxin

Topical or systemic administration of 5‐aminolevulinic acid (ALA) and its esters results in increased production and accumulation of protoporphyrin IX (PpIX) in cancerous lesions allowing effective application of photodynamic therapy (PDT). The large concentrations of exogenous ALA practically required to bypass the negative feedback control exerted by heme on enzymatic ALA synthesis and the strong dimerization propensity of ALA are shortcomings of the otherwise attractive PpIX biosynthesis. To circumvent these limitations and possibly enhance the phototoxicity of PpIX by adjuvant chemotherapy, covalent bonding of PpIX with a drug carrier, β‐cyclodextrin (βCD) was implemented. The resulting PpIX + βCD product had both carboxylic termini of PpIX connected to the CD. PpIX + βCD was water soluble, was found to preferentially localize in mitochondria rather than in lysosomes both in MCF7 and DU145 cell lines while its phototoxiciy was comparable to that of PpIX. Moreover, PpIX + βCD effectively solubilized the breast cancer drug tamoxifen metabolite N‐desmethyltamoxifen (NDMTAM) in water. The PpIX + βCD/NDMTAM complex was readily internalized by both cell lines employed. Furthermore, the multimodal action of PpIX + βCD was demonstrated in MCF7 cells: while it retains the phototoxic profile of PpIX and its fluorescence for imaging purposes, PpIX + βCD can efficiently transport tamoxifen citrate intracellularly and confer cell death through a synergy of photo‐ and chemotoxicity.     

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.     

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.     

5-Aminolaevulinic acid-mediated photodynamic therapy in multidrug resistant leukemia cells

To verify if photodynamic therapy (PDT) could overcome multidrug resistance (MDR) when it it applied to eradicate minimal residual disease in patients with leukemia, we investigated the fluorescence kinetics of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) and the effect of subsequent photodynamic therapy on MDR leukemia cells, which express P-glycoprotein (P-gp), as well as on their parent cells. Evaluation of PpIX accumulation by flow cytometry showed that PpIX accumulated at higher levels in mdr-1 gene-transduced MDR cells (NB4/MDR) and at lower levels in doxorubicin-induced MDR cells (NOMO-1/ADR) than in their parent cells. A P-gp inhibitor could not increase PpIX accumulation. Measurement of extracellular PpIX concentration by fluorescence spectrometry showed that P-gp did not mediate the fluorescence kinetics of ALA-induced PpIX production. Assessment of ferrochelatase activity using high-performance liquid chromatography indicated that PpIX accumulation in drug-induced MDR cells was probably regulated by this enzyme. Assessment of phototoxicity of PDT using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that PDT was effective in NB4, NB4/MDR, NOMO-1 and NOMO-1/ADR cells, which accumulated high levels of PpIX, but not effective in K562 and K562/ADR cell lines, which accumulated relatively low levels of PpIX. These findings demonstrate that P-gp does not mediate the ALA-fluorescence kinetics, and multidrug resistant leukemia cells do not have cross-resistance to ALA-PDT.     

Ferrochelatase Deficiency Abrogated the Enhancement of Aminolevulinic Acid‐mediated Protoporphyrin IX by Iron Chelator Deferoxamine

Aminolevulinic acid (ALA) is a prodrug that is metabolized in the heme biosynthesis pathway to produce protoporphyrin IX (PpIX) for tumor fluorescence detection and photodynamic therapy (PDT). The iron chelator deferoxamine (DFO) has been widely used to enhance PpIX accumulation by inhibiting the iron‐dependent bioconversion of PpIX to heme, a reaction catalyzed by ferrochelatase (FECH). Tumor response to DFO treatment is known to be highly variable, and some tumors even show no response. Given the fact that tumors often exhibit reduced FECH expression/enzymatic activity, we examined how reducing FECH level affected the DFO enhancement effect. Our results showed that reducing FECH level by silencing FECH in SkBr3 breast cancer cells completely abrogated the enhancement effect of DFO. Although DFO enhanced ALA‐PpIX fluorescence and PDT response in SkBr3 vector control cells, it caused a similar increase in MCF10A breast epithelial cells, resulting in no net gain in the selectivity toward tumor cells. We also found that DFO treatment induced less increase in ALA‐PpIX fluorescence in tumor cells with lower FECH activity (MDA‐MB‐231, Hs 578T) than in tumor cells with higher FECH activity (MDA‐MB‐453). Our study demonstrates that FECH activity is an important determinant of tumor response to DFO treatment.     

Subcellular Localization of Photofrin and Aminolevulinic Acid and Photodynamic Cross-Resistance in Vitro in Radiation-Induced Fibrosarcoma Cells Sensitive or Resistant to Photofrin-Mediated Photodynamic Therapy

Abstract— The subcellular and, specifically, mitochondrial localization of the photodynamic sensitizers Photofrin and aminolevulinic acid (ALA)-induced protoporphyrin-IX (PpIX) has been investigated in vitro in radiation-induced fibrosarcoma (RIF) tumor cells. Comparisons were made of parental RIF-1 cells and cells (RIF-8A) in which resistance to Photofrin-mediated photodynamic therapy (PDT) had been induced. The effect on the uptake kinetics of Photofrin of coincubation with one of the mitochondria-specific probes 10N-Nonyl acridine orange (NAO) or rhodamine-123 (Rh-123) and vice versa was examined. The subcellular colocalization of Photofrin and PpIX with Rh-123 was determined by double-label confocal fluorescence microscopy. Clonogenic cell survival after ALA-mediated PDT was determined in RIF-1 and RIF-8A cells to investigate cross-resistance with Photofrin-mediated PDT. At long (18 h) Photofrin incubation times, stronger colocalization of Photofrin and Rh-123 was seen in RIF-1 than in RIF-8A cells. Differences between RIF-1 and RIF-8A in the competitive mitochondrial binding of NAO or Rh-123 with Photofrin suggest that the inner mitochondrial membrane is a significant Photofrin binding site. The differences in this binding may account for the PDT resistance in RIF-8A cells. With ALA, the peak accumulations of PpIX occurred at 5 h for both cells, and followed a diffuse cytoplasmic distribution compared to mitochondrial localization at 1 h ALA incubation. There was rapid efflux of PpIX from both RIF-1 and RIF-8A. As with Photofrin, ALA-induced PpIX exhibited weaker mitochondrial localization in RIF-8A than in RIF-1 cells. Clonogenic survival demonstrated cross-resistance to incubation in PpIX but not to ALA-induced PpIX, implying differences in mitochondrial localization and/or binding, depending on the source of the PpIX within the cells.     

Protoporphyrin IX level correlates with number of mitochondria, but increase in production correlates with tumor cell size

Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway, from the substrate aminolevulinic acid (ALA), and can be used for tumor detection, monitoring or photodynamic therapy. PpIX production varies considerably between tumor cell types, and determining the cell types and methods to optimize production is a central issue in properly utilizing this drug. A panel of eight cancer cell types was examined for PpIX production capacity, including breast, prostate, and brain cancer tumors, and the production varied up to 10-fold among cell types. A positive correlation was seen between mitochondrial content and naturally occurring PpIX prior to ALA administration, but mitochondrial content did not correlate to the yield of PpIX resulting from the addition of ALA. Interestingly, total cell size was positively correlated to the yield of PpIX from ALA administration. Addition of an iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed, thereby further increasing the yield of PpIX. Those cell types that had the lowest ALA to PpIX production without L1 showed the largest percentage increase in production with L1. The study indicates that use of L1 in tumors with a lower innate production of PpIX with ALA alone may be the most productive approach to this combined delivery.     

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.     

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.     

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.     

Characterization of Endogenous Protoporphyrin IX Induced by δ-Aminolevulinic Acid in Resting and Activated Peripheral Blood Lymphocytes by Four-Color Flow Cytometry

Lymphocytes treated with δ-aminolevulinic acid (ALA) can accumulate the photoactive, fluorescent heme precursor, protoporphyrin IX (PpIX). With visible light illumination, PpIX can be used in photodynamic therapy (ALA-PDT) to kill or functionally alter cells. The aim of this study was to characterize the effects of ALA and ALA-PDT on resting and activated human peripheral blood T lymphocytes. Accumulation of PpIX depends inversely on the rate of its iron-dependent conversion into heme. Activated, replicating lymphocytes have low intracellular iron levels, with corresponding increases in the transferrin receptor (CD71). Thus, we expected activated lymphocytes would preferentially accumulate PpIX. Using four-color flow cytometry, we examined ALA-induced PpIX levels in T-cell subsets of resting and activated human peripheral blood mononuclear cells and the relationship between CD71 and PpIX. Peripheral blood mononuclear cells stimulated by phytohemagglutinin (PHA) were simultaneously phenotyped for PpIX, CD71 and the T-cell markers CD3 and CD4 or CDS. In activated cells treated with 0-6mM ALA for 4 h, PpIX fluorescence was maximal at 1 mM ALA. On a single cell basis, there was a strong correlation between PpIX ac-cumulation and CD71 expression. The ALA-treated, PHA-stimulated, CD71⁺ lymphocytes had an eight-fold greater mean PpIX fluorescence than nonactivated, CD71- cells. Approximately 87% of the CD4* and 85% of the CD8⁺ T cells accumulated PpIX. The PpIX levels of CDS⁺ cells were about 5% greater than CD4⁺ cells. In addition, mixed lymphocyte reaction-stimulated cells treated with ALA accumulated more PpIX than controls. Thus, activated cells preferentially accumulate endogenous PpIX when exogenous ALA is administered. Cytotoxicity studies showed that the majority of the activated cells following ALA-PDT were killed but resting cells were spared. Also, in examining activation markers by flow cytometry the number of cells that were positive for activation markers CD38 or CD71 dramatically decreased after ALA and light treatment in activated populations. The data suggest a role for ALA-PDT as an immunomodulator or photocytotoxic agent targeting activated lymphocytes.     

Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Aminolevulinic acid (ALA)‐mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA‐mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA‐mediated PDT. Silencing PBGS or PBGD significantly reduced ALA‐stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA‐stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA‐stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA‐PDT, while increased sensitivity to ALA‐PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA‐mediated PpIX fluorescence and PDT efficacy.     

Direct comparison of delta-aminolevulinic acid and methyl-aminolevulinate-derived protoporphyrin IX accumulations potentiated by desferrioxamine or the novel hydroxypyridinone iron chelator CP94 in cultured human cells

Aminolevulinic acid photodynamic therapy (ALA-PDT) is a cancer therapy that combines the selective accumulation of a photosensitizer in tumor tissue with visible light (and tissue oxygen) to produce reactive oxygen species. This results in cellular damage and ablation of tumor tissue. The use of iron chelators in combination with ALA has the potential to increase the accumulation of the photosensitizer protoporphyrin IX (PpIX) by reducing its bioconversion to heme. This study compares directly for the first time the effects of the novel hydroxypyridinone iron chelating agent CP94 and the more clinically established iron chelator desferrioxamine (DFO) on the enhancement of ALA and methyl-aminolevulinate (MAL)-induced PpIX accumulations in cultured human cells. Cultured human cells were incubated with a combination of ALA, MAL, CP94 and DFO concentrations; the resulting PpIX accumulations being quantified fluorometrically. The use of iron chelators in combination with ALA or MAL was shown to significantly increase the amount of PpIX accumulating in the fetal lung fibroblasts and epidermal carcinoma cells; while minimal enhancement was observed in the normal skin cells investigated (fibroblasts and keratinocytes). Where enhancement was observed CP94 was shown to be significantly superior to DFO in the enhancement of PpIX accumulation.