Biological activity of 5-aminolevulinic acid and its methyl ester after storage under different conditions

Journal of Photochemistry and Photobiology B: Biology 87 (2007) 67–72 www.elsevier.com/locate/jphotobiol

Biological activity of 5-aminolevulinic acid and its methyl ester after storage under different conditions Miron Kaliszewski a,b,*, Miroslaw Kwasny b, Asta Juzeniene a, Petras Juzenas a, Alfreda Graczyk b, Li-Wei Ma a, Vladimir Iani a, Patrycja Mikolajewska a, Johan Moan b

a,c

a Department of Radiation Biology, Institute for Cancer Research, 0310 Montebello, Oslo, Norway Institute of Optoelectronics, Military University of Technology, ul. Gen. S. Kaliskiego 2, 00-908 Warsaw, Poland c Department of Physics, Oslo University, 0316 Blindern, Oslo, Norway

Received 14 November 2006; received in revised form 30 January 2007; accepted 30 January 2007 Available online 4 February 2007

Abstract 5-Aminolevulinic acid (ALA) is a natural precursor of protoporphyrin IX (PpIX) and heme in cells. Photodynamic therapy (PDT) utilizes a metabolic imbalance in cancer cells, leading to increased PpIX generation from exogenous ALA. Due to chemical instability of ALA in therapeutic concentrations at pH values larger than 5.0 and at high temperatures, it looses its activity by spontaneous dimerization to 2,5-dicarboxyethyl-3,6-dihydropyrazine (DHPY). ALA esters are now supplementing ALA in PDT, but little is known about their stability. We have studied the stability of ALA and its methyl ester (MAL) stored under different conditions (temperatures, pH values) by measuring their ability to generate PpIX. 100 mM solutions of both compounds were found to be stable at pH 4 and at 4 C. However, at pH 5.5 they lost almost 10% of the initial activity during 5 days of storage at 4 C. The fastest decay of ALA and MAL was seen at pH 7.4 and at 37 C, and followed first order kinetics. At pH 7.4 and at 4 C MAL lost its PpIX producing ability more slowly than at 37 C. Our work shows that solutions should be prepared immediately before use and stored at low temperatures. The pH of stock solutions should not exceed 5.  2007 Elsevier B.V. All rights reserved. Keywords: Photodynamic therapy; Stability; 5-Aminolevulinic acid; ALA esters; Protoporphyrin IX

1. Introduction Photodynamic therapy with 5-aminolevulinic acid (ALA-PDT) is accepted world wide for treatment of skin cancers and non-cancerous diseases [1–3]. In normal and tumour cells enzymatic transformation of ALA, via the heme cycle, leads to production of protoporphyrin IX (PpIX) and then of heme. In some tumours this process is perturbed, and application of excess exogenous ALA

* Corresponding author. Address: Institute of Optoelectronics, Military University of Technology, ul. Gen. S. Kaliskiego 2, 00-908 Warsaw, Poland. Tel.: +48 22 683 74 33; fax: +48 22 666 89 50. E-mail address: [email protected] (M. Kaliszewski).

1011-1344/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2007.01.003

induces preferential accumulation of PpIX that is a strong photosensitizer. Due to the hydrophilic properties of ALA, its penetration through the plasma membrane of cells is slow. The penetration is expected to be better for lipophilic ester derivatives of ALA. These esters are supposed to be converted to a biologically active form of ALA, possibly ALA itself, by non-specific esterases [4–6]. ALA and its esters have significant advantages over exogenous photosensitizers. They give transient skin photosensitization, lasting only 24–48 h, compared to 6 weeks for photosensitizers [7]. Moreover, they can be applied topically. ALA is an acid, and like methyl 5-aminolevulinate (MAL), it is applied in the form of its hydrochloric salt

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at a low pH value (about 2.5). For minimal discomfort of the patients the solutions are buffered to a pH value close to the physiological one. Under such conditions ALA undergoes dimerization, resulting in loss of activity. Two molecules of ALA form a cyclic pyrazine product [7–13]. The stability of ALA in different formulations has been investigated [14,15], while that of the commonly used esters is still scarce. In the present study we have investigated the influence of storage conditions on the stability of ALA and MAL. The stabilities were assessed by determining the abilities of the drugs to induce PpIX. Gadmar et al. [16] presented data on the influence of storage conditions of ALA solutions on its ability to induce PpIX in cells. The method applied in the present work is similar to that applied by Gadmar et al. [16]. 2. Materials and methods 2.1. Chemicals 5-Aminolevulinic acid hydrochloride (ALA, ClNH3CH2CO(CH2)2COOH, 167.6 g/mol), methyl 5-aminolevulinate hydrochloride (MAL, ClNH3CH2CO(CH2)2COOCH3, 181.7 g/mol), RPMI-1640 medium, Penicillin/Streptomycin Solution, L-Glutamine, Trypsin-EDTA, phosphate-buffered saline (PBS), sodium phosphate dibasic, sodium phosphate monobasic and other chemicals were obtained from Sigma Chemical Co, St. Louis, MO. Foetal calf serum (FCS) was obtained from PAA Laboratories Gmbh, Linz, Austria. All chemicals were of the highest purity commercially available. 2.2. Stock solutions Solutions (100 mM) were prepared by dissolving ALA or MAL in 0.4 M buffer made from Na2HPO4 and NaH2PO4. The pH of the solutions was checked with an electronic pH meter (Metrohm, Switzerland) and adjusted by addition of 3 M HCl or NaOH to pH 4.0, 5.5 and 7.4. During storage a slight decrease in pH was compensated by addition of 3 M NaOH. The resulting dilution was less than 3%. One set of samples, in closed vials, was stored at 4 C, and another at 37 C in an incubator. Stock solutions were diluted in medium without serum to a final concentration of 0.5 mM. 2.3. Cell cultivation WiDr cells, derived from a primary adenocarcinoma of the human recto-sigmoid colon [17], were maintained in exponential growth in RPMI 1640 medium with 10% FCS, 100 units/ml penicillin and 100 lg/ml streptomycin and 2 mM L-glutamine. The cells were grown and incubated in cell culture flasks (Nunc, Roskilde, Denmark) at 37 C in a humidified atmosphere 5% CO2 and subcultured twice a week using 0.01% trypsin in 0.02% EDTA. Fortyeight hours before the experiments the WiDr cells were

seeded in 6 well plates to a density of 2 · 105 cells per well and 3 ml of medium per well. 2.4. Determination of PpIX production in vitro After 4 h incubation with ALA or MAL the cells were rinsed twice with 1 ml of ice cold PBS and one ml of porphyrin extracting solution of 1% sodium dodecyl sulphate (SDS) in 1 N perchloric acid – methanol (1:1 vol/vol) [18] was added per well. The cells were detached by means of a cell scrapper (Costar, Cambridge, MA) and collected into 1.5 ml tubes. The samples were kept frozen at 80 C. Just before PpIX measurements the samples were thawed, and the fluorescence of PpIX was measured in a 1 cm path length cuvette with a Perkin–Elmer LS-50B spectrofluorometer (Norwalk, CT). The excitation wavelength was set at 407 nm and emission was scanned from 550 to 750 nm, and showed maximum fluorescence at 606 nm. This corresponds to the maximum emission of PpIX in acidic medium [18]. The excitation and emission slits were set at 10 and 15 nm, respectively. A long-pass cut-off filter (515 nm) was used on the emission side to block scattered excitation light. The effective ALA/MAL concentration was determined by comparing the fluorescence of PpIX of the sample with that of standard solutions. 2.5. Standard curve WiDr cells were incubated for 4 h with 0–0.5 mM solutions of ALA and MAL in six well plates. After that the cells were rinsed twice with 1 ml of ice cold PBS and treated with 1 ml of porphyrin extracting solution. The cells were scraped and collected in 1.5 ml tube. Fluorescence was measured at kex = 407 nm and kem = 606 nm. 2.6. Dark toxicity About 104 cells were seeded out in 24 well plates. After 48 h the medium was removed, and 1 ml of medium without serum containing 0.5 mM ALA or MAL (from 100 mM solutions stored at different conditions) was added to each well. After 4 h incubation with the drugs the cells were washed with cold PBS, and fresh medium with serum was added for 24 h. The survival of the cells was measured using the colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)2,5 diphenyl) assay [19]. MTT was dissolved in phosphate-buffered saline (PBS; pH 7.4) at 2 mg/ml, filtered to become sterile and stored at 4 C. 50 ll of the stock solution was added to each well containing 1 ml of medium, and the well plates were incubated at 37 C for 4 h. After that the medium was removed, and the cells were washed with ice-cold PBS. The formazan crystals were dissolved by adding 600 ll of isopropanol per well. Samples (75 ll) were transferred from each well into a 96-well micro plate with 200 ll of isopropanol. The optical density was read on a Multiskan MS (type 352, Labsys-

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tems, Helsinki, Finland) plate reader using a 570 nm bandpass filter. Cell viability (cell survival) was expressed as the percentage of viable treated cells relative to that of untreated control cells. 2.7. Topical application of ALA and MAL on mice skin Female BALB/c athymic nude mice were bred at the animal department of the Norwegian Radium Hospital (Oslo, Norway). They were kept under pathogen free conditions. Water and food was given ad libitum. All procedures involving mice were carried out in agreement with the protocols approved by the animal care committee at the Norwegian Radium Hospital, under control by the National Ethical Committee’s guidelines on animal welfare. At the start of the experiments the mice were 7–8 weeks old, and had an average body weight of 20– 25 g. Eight different creams, based on an ointment (Unguentum, Merck, Darmstad, Germany) were prepared for topical application (see Section 2.8). The creams were applied continuously on the skin of both flanks of two mice per group and covered with a transparent adhesive dressing (OpSite Flexfix, Smith & Nephew Medical, Hull, UK). 2.8. Cream preparation Creams were made from Unguentum Merck by mixing the cream base with the proper amount of 100 mM stock solution of ALA or MAL to obtain final concentrations of 33.3 mM (0.56% (w/w) and 0.61% (w/w), respectively). The stock solutions were stored at different pH for 6 days at 37 C and 4 days at 4 C (1-st not buffered pH about 2, 2-nd pH 4, 3-rd pH 5.5, 4-th pH 7.4) before being mixed into the cream base. 2.9. Determination of PpIX production in vivo The PpIX fluorescence in the skin of the mice was measured by means of a fibre-optic probe (a tip diameter 6 mm) coupled to the luminescence spectrometer. The fluorescence spectrum was found to be identical with that of PpIX. The fluorescence was excited at 407 nm and monitored at 636 nm, corresponding to the maximum of fluorescence emission of PpIX in mouse skin in vivo [20]. The excitation and emission slits were set at 10 and 15 nm, respectively. A long-pass cut-off filter (515 nm) was used on the emission side of the luminescence spectrometer to block scattered excitation light.

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after incubation with solutions stored at 4 C up to 120 h. PpIX formation decreased about 5% for solutions stored for 120 h at 37 C (Fig. 1a and b). At pH 5.5 and 4 C both ALA and MAL lost less than 10% of activity over the time course of the experiment (5 days). Analogous tests at 37 C revealed a 40% decrease in activity for both substances (Fig. 1c and d). ALA and MAL solutions stored at pH 7.4 and 4 C had apparently longer half-lives than samples stored at 37 C (Table 2). Also shelf life, parameter defining pharmaceutical acceptability to use of the drug, was evidently longer at low temperatures. The fastest degradation rate of the drugs was during first 30 h of storage at pH 7.4 and 37 C (Fig. 1e and f). Under these conditions they lost more than 90% of their ability to induce PpIX formation. The solution turned yellow in a few minutes. The samples turned brown at towards the end of the experiments. The same solutions stored at 4 C were more stable and about 60% ALA and 70% MAL did not dimerize as judged from their abilities to induce PpIX formation. During the first 70 h of storage, MAL was able to induce (after normalization) relatively higher PpIX levels than did ALA (Fig. 1e and f). 3.2. Assay for stability of ALA and MAL after topical application on mouse skin Storage for 6 days at 37 C and pH 7.4 resulted in a complete loss of the PpIX forming abilities of ALA and MAL, while at pH 4, the activity of both drugs was not reduced comparing to those of freshly prepared references (Fig. 2). At pH 5.5 ALA induced lower levels of PpIX than a fresh reference, whereas MAL induced the same amounts of PpIX as the reference (Fig. 2). Storage of ALA and MAL for 4 days at 4 C at pH 4 and 5.5 did not significantly reduce their abilities to form PpIX in mouse skin comparing to freshly prepared solutions. At pH 7.4 the degradation of ALA/MAL was about 60% (data not shown). 3.3. Dark toxicity For dark toxicity, the MTT assay was performed after storage ALA and MAL for 50 h under different conditions. Since DHPY has been found to promote oxidative stress [13], it was necessary to test its influence on cell viability. The dimerization product of ALA did not influence cell viability (data not shown), probably due to short incubation time and/or enzymatic cellular red-ox system.

3. Results 4. Discussion 3.1. Cell line assay for stability of ALA and MAL Solutions of ALA and MAL stored at pH 4 did not turn yellow at any temperature during these experiments. Similarly, cell line tests showed no difference in PpIX formation

ALA is not stable in solution and its degradation rate strongly depends on concentration, temperature and pH [11,7]. However, little is known about stability of the commonly used ALA esters. From the chemical point of view

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Fig. 1. Apparent concentrations of ALA and MAL remaining after storage of 100 mM stock solutions at different pH values, temperatures and times. Stock solutions were diluted before use. WiDr cells were incubated with 0.5 mM drug for 4 h and the abilities of the drugs to induce PpIX formation were measured. Error bars show the standard error of the mean, n = 3.

Fig. 2. Kinetics of PpIX formation in normal skin of mice after application of creams containing 33.3 mM of the drugs (corresponding to 0.56% (w/w) ALA and 0.61% (w/w) MAL). The creams were prepared from 100 mM water stock solutions of ALA and MAL stored for 6 days at 37 C at the indicated pHs. Creams were applied on normal mouse skin. Reference pH was 2.3 and 3.8 for ALA and Me-ALA, respectively. Error bars show the standard error of the mean, n = 4.

there is no reason why ALA carboxyl esters should be more stable than the parent compound. Our previous studies showed that the stability of ALA at increased pH values could be obtained by masking amino group involved in the first steps of dimerization [5].

In the present study we compared the ability of ALA and its methyl ester stored at different pH values and temperatures to induce PpIX. Our approach allowed us to determine the effective concentration of the substance with respect to PpIX generation.

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Table 1 The correlation coefficients of linear regression analysis for different models of reaction order Derivative

pH

Temperature (C)

Zero order

First order

Second order

Third order

ALA ALA** ALA ALA ALA* MAL MAL MAL* MAL MAL*

7.4 7.4 5.5 5.5 4 7.4 7.4 5.5 5.5 4

4 37 4 37 37 4 37 4 37 37

0.8813 0.4652 0.9728 0.8889 0.9296 0.9625 0.7184 0.7943 0.8337 0.7182

0.9942 0.9630 0.9737 0.8752 0.9308 0.9594 0.9660 0.7992 0.8296 0.7183

0.8759 0.8612 0.9745 0.8534 0.9319 0.7930 0.6098 0.8041 0.8190 0.7183

0.7462 0.8414 0.9752 0.8265 0.9327 0.6977 0.5615 0.8088 0.8037 0.7182

Stars indicate points removed due to low correlation: ( 50 h)*, ( 72 h)**.

The experimental data (Table 1) show that reaction orders of ALA/MAL dimerization differs depending on experimental conditions. The decay curves of ALA and MAL at pH 7.4 fit those of a first order reaction. However, MAL stored at 4 C showed equally good correlation to zero and first order reaction kinetics. Previous studies by Elfsson et al., Bunke et al., de Blois et al., indicate second order kinetics regardless of concentration (0.006–0.6 M) and pH (5.0–7.4) [12,8,9]. The first order kinetics seen in our experiments is related to lower temperatures used. A similar pattern of kinetics was demonstrated by McCarron et al. [10], where the stability of ALA was tested in non-aqueous gels. Depending on storage temperature, 5 C or 60 C, the degradation kinetics was either zero or second order, respectively [10]. First order kinetics was also postulated by Gadmar et al. [16] in similar experiments, where PpIX production from ALA was tested in WiDr cell lines (pH 7.4, 37 C). An unequivocal determination of reaction kinetics at pH 4 and 5.5 is not possible, since correlation coefficients are similar for 0–3rd reaction order. Nevertheless, under such conditions one might expect zero or first order due to the low temperature. At pH 5.5 the ability of ALA to form PpIX (100 mM– 1.67%) and MAL (1.81%) decreased by 10% during roughly 150 and 45 h at 4 and 37 C, respectively. According to de Blois [9] (pH 5, 21 C, 2% ALA) 10% decay was found after 150 days. Such a difference can be attributed to the lower pH used by them. At pH values higher than 5.0 ALA dimerizes rapidly [7]. Moreover, in our experiments the pH was corrected 2–3 times per day by addition of 3 M NaOH, while de Blois et al. [9] noticed a small decrease in pH during storage. Such a spontaneous decrease in pH protects ALA against dimerization.

The in vivo tests at pH 4 and 7.4 are in agreement with the in vitro experiments (Figs. 1 and 2). ALA and MAL stored at pH 7.4, applied on normal mouse skin induced negligible amounts of PpIX (Fig. 2). At pH 4 both drugs induced the same amounts of PpIX in normal animal skin as a fresh reference did (Fig. 2). In vitro test showed that when ALA was stored at pH 5.5 the amount of PpIX was reduced about 30%. Comparable results were obtained with animal skin tests. A somewhat different pattern was found for MAL where storage at pH 5.5 gave the same fluorescence levels as a fresh reference. The reason for the discrepancy between cell and animal tests may be different methods of data acquisition. While in the in vitro experiments we measured average fluorescence, in the in vivo experiments we measured the maximal values. ALA and MAL solutions showed comparable stability, except at pH 7.4 (4 C). In these conditions we found a faster decay in PpIX forming ability for ALA than for MAL (the reaction rate constant for ALA and MAL are 0.192 l/h and 0.157 l/h, respectively) (Table 2). MAL is less acidic than ALA, thus lower amounts of basic sodium phosphate are necessary to increase its pH. This could result in slower dimerization rate of MAL. It is also possible that mobility of MAL, due to larger dimensions, results in lower rate of creation of molecular bounds. Identical decay of both drugs at 37 C (pH 7.4) may result from rapid depletion of the ALA/MAL comparing to number of data acquisition points. In our experiments at pH 7.4 and 37 C we found that PpIX formation by ALA and MAL decreased faster than found by Gadmar et al. [16]. While in our case a 90% decrease in PpIX formation was observed after 24 h (Fig. 1e and f), Gadmar found two times higher PpIX levels

Table 2 Half (t0.5), shelf life (t0.9), reaction constants (k) of 100 mM ALA and MAL solutions, calculated from first order equation, pH 7.4 Temperature (C)

Decay time

ALA time (h)

MAL time (h)

ALA k(l/h)

MAL k(l/h)

4 4 37 37

t0.5 t0.9 t0.5 t0.9

36.10 5.49 9.34 1.42

44.15 6.71 8.58 1.30

0.0192 ± 0.0016 – 0.0742 ± 0.0212 –

0.0157 ± 0.0032 – 0.0808 ± 0.0127 –

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after 14 days. Such a difference may be attributed to 100 times higher concentration of MAL and ALA in our experiments. Di Venosa et al. provided evidence that deestrification of ALA hexyl ester is not required for condensation to pyrrole ring [1]. The same group found that no spontaneous hydrolysis occurred during storage of Und-ALA and THP-ALA esters for 6 h at pH 7.4. They concluded hydrolysis of ALA esters is esterase dependent process [21]. Similar results were obtained with preliminary HPLCMS tests. MAL (30 mM) stored at room temperature for 24 h at pH ranged from 4.0–7.4 confirmed no occurrence of pH dependant hydrolysis (Kaliszewski M., Kwas´ny M., Szyposzyn´ska M., Laszczak J. Graczyk A. – unpublished data). Although after 24 h at pH 7.2 small amount of DHPY appeared. This suggests that at this pH dimerization and hydrolysis may occur concurrently. From our experimental data it is difficult to make precise determination of reaction order. Nevertheless, it shows the actual impact of ALA/MAL degradation on PpIX formation. Our approach can be useful, especially for clinicians who are not familiar with advanced methods of chemical analysis. Presented work shows that certain conditions (increased temperatures and pH values) should be avoided if higher stability is desired. Solutions should be prepared immediately before use and stored at low temperatures. The pH should not exceed 5. 5. Abbreviations ALA MAL PpIX PDT DHPY

5-aminolevulinic acid methyl 5-aminolevulinate protoporphyrin IX photodynamic therapy 2,5-dicarboxyethyl-3,6-dihydropyrazine

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