Comparative in vivo study of precursors of PpIX (ALA and MAL) used topically in Photodynamic Therapy

Raquel Ferreira Rego1, Natalia Mayumi Inada2, Juliana Ferreira2, Fernando Manuel Araújo-Moreira1, Vanderlei Salvador Bagnato2.

1Universidade Federal de São Carlos – Programa de Pós-graduação em Biotecnologia – Rodovia Washington Luís, Km 235 – SP 310 – São Carlos – SP –

Brazil – CEP 13565- 905 2 Universidade de São Paulo – Instituto de Física de São Carlos – Av. Trabalhador

São-carlense, 400 – São Carlos – SP – Brazil – CEP: 13666-590

ABSTRACT

The efficacy of Photodynamic Therapy (PDT) combined with aminolevulinic acid (ALA) or methyl

aminolevulinate (MAL) in treatment of cancer has been studied for over ten years. However, there is no

established dose for the topical use of these drugs in PDT. The purpose of this study was the comparison

of induced PDT response of ALAsense (5-aminolevulinic acid - ALA) and Metvix (methyl

aminolevulinate - MAL). Depth of necrosis induced by PDT was analyzed in normal liver of male Wistar

rats, using different light doses and topical application of both PpIX precursors – ALA and MAL. PDT

was performed with a diode laser at 630 nm with different doses of light (20, 50, 100 and 200 J/cm2), and

intensity of 250 mW/cm2. Depth of necrosis analysis was used to calculate the threshold dose for each

drug. The results showed that MAL-PDT presented a better response than ALA-PDT, mainly due to

formulation differences. Moreover, the ability of the ALA PpIX production was more efficient.

Key - words: PDT, ALA, MAL, topical photosensitization, necrosis, dosimetry.

1. INTRODUCTION

Photodynamic therapy (PDT) is a technique used for photodetection and treatment of cancer and other

clinical conditions. It is based on the application of a photosensitizer (PS) that accumulates in tumour

cells. When this PS is illuminated by a light source of specific wavelength, it causes a chemical reaction

with molecular oxygen, and generates reactive oxygen species (ROS) which are highly toxic for cellular

constituents and, therefore, responsible for tissue destruction [1].

However, the effectiveness of PDT is dependent on the type of cancer and used PS [2-4].

Photodynamic Therapy: Back to the Future, edited by David H. Kessel, Proc. of SPIE Vol. 7380,73805I · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.823012

Proc. of SPIE Vol. 7380 73805I-1

Porphyrins, chlorines, phthalocyanine and porphycens are the four main groups of studied

photosinsitizers. Porphyrins are the most frequently used PSs, but its systemic administration is an

adverse factor in dermatology. Due to the high accumulation and slow clearance of the skin, porphyrins

lead to prolonged photosensitization of the organism – from 6 and 10 weeks after application [5]. The

systemic photosensitization has been replaced by topical administration of the porphyrin precursors,

which became popular not only with the therapeutic purpose, but also for dermatological diagnosis [6].

For over ten years, topical application of protoporphyrin IX (PpIX) precursors associated with PDT, such

as 5-aminolevulinic acid (ALA) (Figure 1A) and Methyl aminolevulinate (MAL) (Figure 1B), have been

studied in a variety of tumours and other cancerous conditions [7]. Several studies reported in the

literature the topical use of ALA and MAL associated with PDT [8-15].

The 5-aminolevulinic acid is a hydrophilic compound at physiological pH, so it has a limited capacity to

cross biological barriers like skin. This penetration is a prerequisite for ALA conversion into PpIX [16].

The methyl-aminolevulinate (MAL) is an ester derivative of ALA, which is lipophilic in nature and,

therefore, has better penetration in the keratinized layer and the ability to achieve greater depth compared

to ALA. This difference is even more evident when it comes to malignant cells [7,17,18].

Soon after its penetration into the cell, MAL is rapidly demethylated forming ALA and from there, both

follow the same metabolic pathway, leading to the synthesis of PpIX [19].

The ALA topical photosensitization, as well as their derivatives, can improve: frequency of target site

reach, frequency of intracellular space reach, and enzymatic photoactive conversion rate. These variables

change depending on nature of compound [20].

Although ALA and MAL are very similar drugs, they differ on chemical structure and formulation, which

may influence its response to PDT, since drugs penetration can be increased by varying the composition

of the vehicle [21].

In principle, the use of ALA derivates promises several advantages over ALA, such as the increase of

photoactive compounds generation, improvement in depth of penetration, more homogeneous distribution

of photoactive porphyrins, reduction in applications times, decrease in drug doses, less adverse effects

and improvement in stability [20].

The objective of this study was to compare the efficiency between two photodynamic substances

frequently used in topical PDT: ALAsens (5-aminolevulinic acid - ALA), from Russia, and Metvix®,

(methyl aminolevulinate - MAL) from United Kingdom, varying light doses.

2. METHODOLOGY

2.1. Animals

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Thirty-eight male Wistar rats, weighting 350 g, from the Medical School of Ribeirão Preto of the

University of Sao Paulo (Faculdade de Medicina de Ribeirão Preto, FMRP/USP) were used. The animals

underwent a preparation procedure (twelve hours of fasting, with free access to water). Animals were

weighted and anesthetized by intramuscular injection of ketamine hydrochloride 5% (Vetanarcol ® -

Konig). A dose of 0.08 ml/100g body weight was associated with a muscle relaxant, analgesic and

sedative of xilasina 2% (Coopazine ® - Coopers) at a dose of 0.04 ml/100g body weight. Through a

median incision in the abdominal region, the right lobe of the animal liver was exposed and isolated in

gauze soaked with saline. Then, 0.08 ml was applied topically to ALA or MAL.

The ALA used was 5-aminolevulinic acid powder (ALAsens - Russia) at 16% incorporated in emulsion

water / oil specifically for topical application with the following composition: Polawax (alcohol + fatty

alcohol ethoxylate); Crodalan LA (alcohol of acetylated lanolin), Isopropyl myristate, BHT (butyl

hydroxytoluene), uniphen (phenoxyethanol + parahydroxybenzoate of methyl, ethyl, propyl and butyl),

Propylene glycol, EDTA, distilled water.

Metvix® commercial cream (for methyl aminolevulinate hydrochloride 16%) of the UK, acquired by

Galderma Brasil Ltda, is composed of 160 mg/g of methyl aminolevulinate (as hydrochloride), glyceryl

monostearate self-emulsifier, cetostearyl alcohol, stearate 40 polioxil, parahydroxybenzoate Methyl (E

218), parahydroxybenzoate propionate (E 216), Edetato disodium, glycerol, white Vaseline, Cholesterol,

Isopropyl myristate, Groundnut oil, refined oil, almonds, and Oleyl Alcohol purified water.

2.2. Determination of Drug Light Interval (DLI)

The drug light interval (DLI) is the determination of the optimum time between the application of each

photosensitizers (PS) and maximum concentration of PpIX in the tissue, which was obtained by

fluorescence spectroscopy in four animals (two animals for each drug). It followed a protocol described

by Rego [22]. Five measurements were performed for each time of investigation. Measurements data

were processed, and generated graphs of fluorescence intensity versus time.

In order to assess the drug depth of penetration in liver, the PpIX produced in rat liver after DLI for each

drug was monitored by fluorescence spectroscopy. Two of the four animals received topical application

of ALA, and the other two received MAL application. Detection was performed on the surface and for

three levels of depth.

2.3. Photodynamic Therapy

The PDT application was performed in 24 animals, divided in two equal groups (ALA and MAL).

Animals were divided in four groups with three animals for each dose of light. The animals were

previously prepared and photosensitizers as described in section 2.1. After 4 hours, fluorescence spectra

Proc. of SPIE Vol. 7380 73805I-3

were collected to detect the presence of PpIX locally. An area of 1 cm2 of liver was illuminated with

diode laser 630 nm Ceramoptec ®, Germany, in light doses of 20, 50, 100 and 200 J/cm2, total intensity

of 250 mW/cm2, and respective times of 80, 200, 400 and 800 seconds.

After irradiation, liver was carefully reinserted in the abdominal cavity; the animals were sutured, placed

in their cages and taken to accommodation. After 30 hours, the animals were killed by intracardiac

anaesthesia overdose. The right lobe of the liver was removed and necrosis area was cut into slices with

approximately 1 mm in anterior-posterior direction to allow analysis of depth. The slices of tissue were

placed in plastic vials containing 40% formaldehyde solution (Formol - Merck ®), mixed solution of

sodium monophosphate hydrate (NaH2PO4H2O - Merck ®) and hydrated sodium diphosphate

(Na2HPO42H2O - Merck ®) diluted in distilled water for 24 hours. The fragments were then included in

paraffin and sliced by microtome at thickness of 4 µm. The process of staining was performed by

standard methodology with hematoxylin-eosin (HE).

Histological preparations were used in optical microscopy study. Liver epithelium changed was

morphologically and morphometrically examined. With the aid of a microscope (40x increase)

illuminated from below by LED with two micromanipulators attached (one that performs steps towards

the abscissa and other measures in order to place), was used to determine the depth of necrosis, i.e.,

distance between necrosis surface and adjacent normal tissue. For each animal, four to six slices were

obtained of the necrosis region, and for each cut, there were four measurements of depth of necrosis.

The values of average depth of necrosis were analyzed for each experimental group (fluence and drugs)

and generated graphs of depth of necrosis versus fluence.

Based on a mathematical model proposed in literature [23], the threshold dose was calculated for the

studied substances.

3. RESULTS AND DISCUSSION

3.1 Obtaining DLI

Experiments were carried out to establish the DLI for each product in order to eliminate possible

interference on the pharmacokinetics. From these data, the spectral analysis was performed according to

the waiting time, taking as reference the main peak for each PS.

The DLI was determined by collection of PpIX fluorescence in liver after 16% ALA and MAL

application with fifteen min intervals. Higher accumulation of PpIX was observed between 150 and 200

min for ALA and between 230 and 260 minutes for MAL. Thus, DLI adopted was 2 hours and 45

minutes for ALA and 4 hours for MAL. After this time, there was a reduction in PpIX concentration to

both drugs used. Figure 1A and B show the average change in PpIX production by ALA and MAL,

respectively, in rats in the range from 0 to 360 seconds.

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It should be noticed in Figure 1 that porphyrin formation time is drug-administering dependent; therefore,

variations in processes of PpIX formation can be related to differences between the formulations of ALA

and MAL creams.

Maximum time of porphyrin formation was significantly different for both precursors and the reduction in

fluorescence emission occurred gradually in both cases.

Uehling et al. [14], observed in their experiments that porphyrin generation present a great concentration

in which the intensity of fluorescence is maximal. Beyond this value, the formation of porphyrin

decreases dramatically. This fact can be consequence of cytotoxic effects generated by the compound or

its metabolic products. Moreover, disruption of the cell membrane after exposure to high doses of 5-ALA

lipophilic esters has been reported [24].

About DLI, MAL topic values obtained in this work are consistent with what is described in literature for

maximum PpIX concentration in tissue. Other findings show that topical application of MAL in normal

tissues and lesions causes an increase in fluorescence versus time, reaching a plateau around 3 to 8 hours

[25, 26].

For ALA, PpIX fluorescence emission peak in rats liver occurred around 2 hours and 45 minutes. This

value is lower than expected, since other studies show a DLI between 4 and 14 hours [7]. However, this

time can vary with drug formulation, application time and tissue photosensitizers.

These data contrast to considerations in literature claim that derivatives of ALA like MAL have DLI

smaller than ALA [20].

However, concentration of PpIX in liver reached a higher level for ALA than MAL (Figure 1). This result

is consistent with Lopez et al. [27] report. These authors showed that substances derived from ALA

produce less PpIX than the ALA. These can probably be due to the lipophilic character that provide high

affinity for cell membrane, which may be retained in this local and prevent their conversion to PpIX.

Another factor can influence amount of PpIX produced is transport mechanism that can be inhibited by

presence of non-polar amino acids such as alanine, methionine, tryptophan and glycine [20].

Wiegell & Wulf [28] found similar results about PpIX concentration in skin when compared the

fluorescence emitted after topical application of ALA and MAL in acne vulgaris.

Another study of different cell types culture also showed that MAL produced almost half of PpIX

fluorescence produced by ALA [29].

However, this superiority in the production of porphyrin by ALA occurs regardless of drugs formulation.

Furthermore, study in which ALA and their derivatives were expressed in a standardized way as a

lipophilic cream, similar levels of porphyrin were observed when the ointment was applied to the skin of

nude mice for 24 h or 10 min after removal of corneum stratum [30].

3.3 Study of Necrosis

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In this study, measure of the liver depth of necrosis was used to evaluate the photodynamic action of ALA

and MAL.

These measures obtained showed that values found for MAL were higher than ALA at all light doses used

(Figure 3).

Results are consistent with findings in the literature, pointing the difficulty of ALA to cross biological

barriers. The MAL has lipophilic character, therefore, has greater ability to penetrate tissue and reach

deeper levels [7,16,17,18].

Furthermore, systemic PS are distributed across the entire area of lesion, while topics are able to penetrate

only a few millimeters [7,31].

Morton, et al. [32], in their work with basal cell carcinomas (BCC) surface, showed that there could be an

inverse relationship between tumour thickness and its response to PDT, showing that effectiveness of

PDT in nodular and ulcerative nodular lesions is drastically reduced when they have more than 2 or 3

mm.

Greater penetration of light in tissue is on red and near infrared wavelength. However, in this absorption

spectrum, the maximum depth of tissue necrosis achieved is less than 5 mm [33].

In this study, the wavelength used was 630 nm (red region) and the maximum depth reached was

approximately 1.5 mm to 2.2 mm for ALA and MAL respectively. Thus, necrosis occurred only

superficially, consequently, the use of these drugs should be restricted to the treatment of local,

superficial and non-invasive tumours.

Fluence is another determining factor to outcomes in PDT. Oseroff [34] in their study topic associated

with ALA PDT for basal cell carcinoma treatment emphasized the importance of the light dose. Results

showed that frequency of clinical response was 95% for 200 J/cm2 and less than 70% for 150 J/cm2 (both

with intensity of 150 mW/cm2).

Results of this study showed that both ALA and MAL after PDT caused liver necrosis from light dose of

20 J/cm2. Groènlund-Pakkanen et al. [35], in their study with intravenous application of ALA PDT with

the combined dose of 20 J/cm2, found a similar result, with superficial and irregular necrosis in mucous.

In depth of necrosis versus fluence graphs (Figure 3) depth of necrosis was greater for ALA at 200 J/cm2

dose, when compared to lower light doses. For MAL, dose of 200 J/cm2 also caused a deeper necrosis,

however, very similar to the values obtained for dose of 100 J/cm2. This result to MAL was similar found

by Ferreira [36] for Photogem®, an intravenous PS hematoporphyrin derivative.

However, the threshold dose of light for the ALA (3.45 ± 0.75 J/cm2) was higher compared to MAL (1.2

± 1.35 J/cm2). Thus, minimum dose needed to causes necrosis in liver using MAL is significantly lower

than minimum dose for ALA in equal conditions, to reach the same goal (Figure 4), suggesting that MAL

has a tendency to provide better response to the ALA photodynamic effect. However, this disparity can be

attributed to differences in drugs formulations, especially for type of vehicle used.

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PDT using ALA can be limited by amount of PS that reaches target cells, whereas this molecule is highly

hydrophilic [37].

Fotinos et al. [20] consider that PS choice is essential for effective therapy. This step involves not only

the delivery of compounds to the target tissue, but also understanding the drug action mechanism

considering the disease etiology.

Although two substances used topically follow the same metabolic pathway after its penetration in tissue

[19], MAL is more lipophilic than ALA due of methyl radical presence, thus it has better penetration and

reaches deeper levels of tissue compared to ALA. Therefore it is expected that necrosis caused by PDT

with MAL tends to be deeper [17,18, 38].

Another determining factor with respect to drug penetration in tissue concerns on physical-chemical

properties of ALA. In physiological conditions, over 90% of molecules of ALA are present in zwitterions

form and carry a positive charge in amine terminal and a negative charge on the carboxyl terminal. These

compounds have limited capacity to penetrate into biological environment and achieving target cell. This

deficiency results in low depth of penetration and uneven distribution of PpIX after topical application

[20]. Saturated and unsaturated fatty acids use as promoters for drugs permeation is of interest both in

topical and transdermic release of drugs, because this class promoters are endogenous components of

human skin including corneum stratum.

However, the necrosis caused by the PpIX precursor substances analyzed, only occurred at surface,

reaching only 2.2 mm.

These findings showed that MAL promoted better photodynamic response than ALA. This may be

attributed to differences in drugs formulations, and allows one to suggest the use of absorption promoters

of the skin, such as oleic acid or 9-cis octadecendico, which is long chain fatty acid (18 carbons) and has

been reported to increase permeation of many drugs [39,40].

Moreover, topical use of these compounds should be restricted to superficial, local, and non-invasive

tumours treatment.

ACKNOWLEDGMENTS

We acknowledge Opto and the Instituto de Física de São Carlos of the University of Sao Paulo

for the research support, and José Dirceu Vollet-Filho for the suggestions and text corrections.

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FIGURES

Figure 1: Chemical Structures of ALA (A) and MAL (B).

Figure 2: PpIX fluorescence produced in rats livers by ALA (A) and MAL (B). After topical administration of PpIX precursors, ALA and MAL, both at 16%, PpIX formation in rats livers was monitored by fluorescence spectra for 6 hours.

Figure 3: Depth of necrosis versus fluence for ALA (A) and MAL (B).

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Figure 4: Graph comparing threshold dose of ALA and MAL.

ALA MAL0

1

2

3

4

5

Thre

shol

d do

se (J

/cm

²)

Proc. of SPIE Vol. 7380 73805I-12