- Email: [email protected]
Accumulation of porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters
Accepted Manuscript Title: Accumulation of Porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters. Authors: Arisa Ogata, Yuya Hasunuma, Emii Kikuchi, Takuya Ishii, Masahiro Ishizuka, Yoshikazu Tokuoka PII: DOI: Reference:
S1572-1000(17)30306-X http://dx.doi.org/doi:10.1016/j.pdpdt.2017.06.004 PDPDT 975
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Accepted date:
14-5-2017 7-6-2017
Please cite this article as: Ogata Arisa, Hasunuma Yuya, Kikuchi Emii, Ishii Takuya, Ishizuka Masahiro, Tokuoka Yoshikazu.Accumulation of Porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.06.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Accumulation of Porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters. Arisa Ogata1, Yuya Hasunuma1, 2, Emii Kikuchi2, Takuya Ishii3, Masahiro Ishizuka3, and Yoshikazu Tokuoka1,2
1:
Graduate School of Engineering, Toin University of Yokohama,
1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan.
2:
Faculty of Biomedical Engineering, Toin University of Yokohama,
1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan.
3:
SBI Pharmaceuticals Co., Ltd.
Izumi Garden Tower 20F, 1-6-1, Roppongi, Minato-ku, Tokyo 106-6020, Japan.
Corresponding author: Yoshikazu Tokuoka
E-mail: [email protected]
Highlights
ALA is superior to ALA esters in accumulating porphyrins in P.acnes.
PDT efficacy for P.acnes is greater than by ALA than by ALA esters.
Peptidoglycans in cell wall of P.acnes could make uptake of ALA esters difficult.
1
Abstract We have investigated the accumulation of porphyrins in Propionibacterium acnes (P.acnes) by 5-aminolevulinic acid hydrochloride (ALA) and its esters, ALA methyl ester hydrochloride (mALA), ALA octyl ester hydrochloride (oALA), and ALA benzyl ester hydrochloride (bALA). From the fluorescence spectra of porphyrins accumulated in P.acnes, the order of porphyrin accumulation is as follows: ALA >> mALA ≈ bALA > oALA (≈ 0). Moreover, the PDT efficacy is reduced in the order of ALA > mALA ≈ bALA > oALA (≈ without additives). These results confirm that ALA is superior to ALA esters in accumulating porphyrins in P.acnes.
Keywords: 5-aminolevulinic acid; 5-aminolevulinic acid esters; P.acnes; porphyrin accumulation; and PDT efficacy
2
Introduction Propionibacterium acnes (P.acnes) is one of the major causes of the pathogenesis of acne vulgaris. Photodynamic therapy (PDT) with 5-aminolevulinic acid hydrochloride (ALA) has attracted attention to treat acne vulgaris. When ALA is incorporated into P.acnes, porphyrins are produced, and the photoactivated porphyrin forms cytotoxic active oxygen species, leading to photochemical and photobiological processes that cause irreversible damage to P.acnes. An ALA ester is a well-known method to enhance penetrability of ALA through plasma membranes and accumulation of porphyrins, protoporphyrin IX (PpIX), in cancer cells [1]. Thus, there are many biochemical and clinical studies on ALA-based PDT for cancer treatments using ALA esters [2-6]. In the present study, we investigate the accumulation of porphyrins in P.acnes by ALA and its esters, ALA methyl ester hydrochloride (mALA), ALA octyl ester hydrochloride (oALA), and ALA benzyl ester hydrochloride (bALA).
3
Materials and Methods Materials
ALA was provided from SBI Pharmaceuticals Co., Ltd., and mALA was purchased
from Cosmo Bio Co., Ltd. We synthesized oALA and bALA according to previous paper [7].
Accumulation of porphyrins A control strain, P.acnes ATCC 6919, was allowed to grow anaerobically in modified GAM agar with 6x10-4 mol/L of ALA and its esters for 48 hours at 37 °C. After being grown, P.acnes was dispersed in a physiological saline solution, and the fluorescence spectrum of the dispersion was measured using a fluorescence spectrometer (FP-6500/6000, JASCO). Here, the excitation wavelength was 410 nm, and the emission wavelength ranged from 550 to 700 nm. Also, the P.acnes dispersion was diluted and was cultured in modified GAM agar for 72 hours to measure cell count in the dispersion. We then evaluated accumulation of porphyrins in P.acnes by the fluorescence peak area per cell.
PDT efficacy
P.acnes grown anaerobically in modified GAM agar with ALA and its esters, as
mentioned above, was dispersed and was diluted with a physiological saline solution at McFarland No. between 0.5 and 0.8. The P.acnes dispersion was inoculated into modified GAM agar and was irradiated aerobically for an hour by LED light of which the wavelength ranged from 400 to 800 nm. Its total fluence rate was 6.54 mW/cm2. After the irradiation, the medium with P.acnes was incubated anaerobically at 37 °C for 72 hours, and the viable cell count was measured. P.acnes dispersion without light irradiation was also set as a control group. The PDT efficacy is defined as the survival rate of viable cell count with and without LED light irradiation; the smaller the survival rate is, the greater the PDT efficacy is.
4
Results Figures 1 are fluorescence spectra of P.acnes cultured with ALA and its esters. No fluorescence peak is observed in P.acnes cultured without additives (Fig. 1 A). In P.acnes cultured with ALA, two fluorescence peaks are recognized at 580 and 620 nm (Fig. 1 B). We suggest that the peak at 580 nm is derived from metalloporphyrins and that the peak at 620 nm is ascribed to coproporphyrin III and/or uroporphyrin III [8]. Moreover, the fluorescence peak of porphyrins in P.acnes is observed in the case with mALA and bALA (Fig. 1 C and D), but not in the case with oALA (Fig. 1 E). Figure 2 shows the fluorescence peak area per cell. Since there is no peak in the case with oALA, we neglect its fluorescence peak area per cell. Figure 2 provides that the fluorescence peak area per cell is as follows: ALA >> mALA ≈ bALA.
The survival rate of P.acnes by PDT with ALA and its esters is depicted in Fig. 3. This figure indicates that the PDT efficacy without additives is almost equal to zero. Also, the PDT efficacy is the greatest in the case with ALA and is reduced as follows: ALA > mALA ≈ bALA > oALA (≈ without additives). The tendency of PDT efficacy is consistent with that of porphyrin accumulation in P.acnes.
5
Discussion Lipophilicity by esterification is one of the most effective methods for pharmacologically active agents to enhance their uptake into animal cells, because of increment in passive permeability of the agent through plasma membranes. In fact, ALA esters are more lipophilic than ALA and penetrate plasma membranes more readily, thus leading to the biosynthesis of porphyrins, PpIX, in a large quantity in animal cells. However, surprisingly, Figs. 2 and 3 indicate that ALA is superior to ALA esters in accumulating porphyrins in P.acnes. In contrast to animal cells, P.accnes is a gram-positive bacterium, having a thick cell wall of which the skeleton is composed of a peptidoglycan [9]. Since peptidoglycan is hydrophilic, the cell wall makes uptake of lipophilic agents difficult. This may be the
reason why porphyrin accumulation is reduced by ALA esters.
According to our result of porphyrin accumulation in P.acnes, we expect that clinical PDT treatment for acne vulgaris is more effective by ALA than by ALA esters. Unfortunately, Wiegell et al demonstrated that there were no significant differences in the PDT efficacy for inflammatory acne vulgaris between ALA and mALA that were applied topically [10]. We can infer that mALA permeates through stratum corneum more readily than ALA because of its lipophilicity, resulting in similar efficacy to ALA on the PDT treatment [11]. We suggest therefore that enhancement techniques for improving ALA skin permeation promote PDT efficacy on acne vulgaris treatment. This interest warrants further investigation.
6
References [1]
V. Zenzen, H. Zankl, In vitro evaluation of the cytotoxic and mutagenic potential of the 5aminolevulinic acid hexylester-mediated photodynamic therapy, Mutat. Res., 561 (2004) 91-100.
[2]
O.A. Gederaas, A. Holroyd, S.B. Brown, D. Vernon, J. Moan, K. Berg, 5-Aminolaevulinic acid methyl ester transport on amino acid carriers in a human colon adenocarcinoma cell line, Photochem. Photobiol., 73 (2001) 164-9.
[3]
S. El Khatib, J. Didelon, A. Leroux, L. Bezdetnaya, D. Notter, M. D'Hallewin, Kinetics, biodistribution and therapeutic efficacy of hexylester 5-aminolevulinate induced photodynamic therapy in an orthotopic rat bladder tumor model, J. Urol., 172 (2004) 2013-2017.
[4]
T. Smits, A.C. Moor, New aspects in photodynamic therapy of actinic keratoses, J. Photochem. Photobiol. B., 96 (2009) 159-69.
[5]
M. Wakui, Y. Yokoyama, H. Wang, T. Shigeto, M. Futagami, H. Mizunuma, Efficacy of a methyl ester of 5-aminolevulinic acid in photodynamic therapy for ovarian cancers, J Cancer Res. Clin. Oncol., 136 (2010) 1143-50.
[6]
M.J. Bader, H. Stepp, W. Beyer, T. Pongratz, R. Sroka, M. Kriegmair, D. Zaak, M. Welschof, D. Tilki, C.G. Stief, R. Waidelich, Photodynamic therapy of bladder cancer - a phase I study using hexaminolevulinate (HAL), Urol Oncol., 31 (2013) 1178-83.
[7]
N. Miyachi, T. Tanaka, S. Nishikawa, H. Takeya, Y. Hotta, Preparation and chemical properties of 5-aminolevulinic acid and its derivatives, Porphyrins, 7 (1998) 342-347.
[8]
S. Ramstad, N.L. Anh-Vu, A. Johnsson, The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA), Photochem. Photobiol. Sci., 5 (2006) 66-72.
[9]
W.W. Navarre, O. Schneewind, Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope, Microbiol. Mol. Biol. Rev., 63 (1999) 174-229.
7
[10] S.R. Wiegell, H.C. Wulf, Photodynamic therapy of acne vulgaris using 5-aminolevulinic acid versus methyl aminolevulinate, J. Am. Acad. Dermatol., 54 (2006) 647-51. [11] L.W Zhang, Y.P. Fang, J.Y. Fang, Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin, Dermatol. Sin., 29 (2011) 1-7.
8
Figure captions Figure 1
Fluorescence spectra of porphyrins induced by ALA and its esters. A: non-additives, B: ALA, C: mALA, D: oALA, and E: bALA.
Figure 2
Fluorescence peak area per cell. P1: at 580 nm and P2: at 620 nm. Values are mean ± SD (n=3).
Figure 3
Survival rate of P.acnes by PDT with ALA and its esters. Values are mean ± SD (n=3).
9
600
650
700
550
600
Wavelength (nm)
650
700
5
550
600
Wavelength (nm)
Fluorescence intensity (a.u.)
D
600
650
Wavelength (nm)
650
700
Wavelength (nm)
5
550
Fig. 1
C
Fluorescence intensity (a.u.)
5
E
Fluorescence intensity (a.u.)
550
B
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
A 5
700
5
550
600
650
Wavelength (nm)
700
Fig. 2 Fluorescence peak area per cell (a.u.)
ALA mALA bALA
P1 P2
100
Survival rate (%)
80
60
40
20
0 ALA
Fig. 3
mALA
oALA
bALA Without additives
S1572-1000(17)30306-X http://dx.doi.org/doi:10.1016/j.pdpdt.2017.06.004 PDPDT 975
To appear in:
Photodiagnosis and Photodynamic Therapy
Received date: Accepted date:
14-5-2017 7-6-2017
Please cite this article as: Ogata Arisa, Hasunuma Yuya, Kikuchi Emii, Ishii Takuya, Ishizuka Masahiro, Tokuoka Yoshikazu.Accumulation of Porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters.Photodiagnosis and Photodynamic Therapy http://dx.doi.org/10.1016/j.pdpdt.2017.06.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short Communication
Accumulation of Porphyrins in Propionibacterium acnes by 5-aminolevulinic acid and its esters. Arisa Ogata1, Yuya Hasunuma1, 2, Emii Kikuchi2, Takuya Ishii3, Masahiro Ishizuka3, and Yoshikazu Tokuoka1,2
1:
Graduate School of Engineering, Toin University of Yokohama,
1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan.
2:
Faculty of Biomedical Engineering, Toin University of Yokohama,
1614 Kurogane-cho, Aoba-ku, Yokohama 225-8502, Japan.
3:
SBI Pharmaceuticals Co., Ltd.
Izumi Garden Tower 20F, 1-6-1, Roppongi, Minato-ku, Tokyo 106-6020, Japan.
Corresponding author: Yoshikazu Tokuoka
E-mail: [email protected]
Highlights
ALA is superior to ALA esters in accumulating porphyrins in P.acnes.
PDT efficacy for P.acnes is greater than by ALA than by ALA esters.
Peptidoglycans in cell wall of P.acnes could make uptake of ALA esters difficult.
1
Abstract We have investigated the accumulation of porphyrins in Propionibacterium acnes (P.acnes) by 5-aminolevulinic acid hydrochloride (ALA) and its esters, ALA methyl ester hydrochloride (mALA), ALA octyl ester hydrochloride (oALA), and ALA benzyl ester hydrochloride (bALA). From the fluorescence spectra of porphyrins accumulated in P.acnes, the order of porphyrin accumulation is as follows: ALA >> mALA ≈ bALA > oALA (≈ 0). Moreover, the PDT efficacy is reduced in the order of ALA > mALA ≈ bALA > oALA (≈ without additives). These results confirm that ALA is superior to ALA esters in accumulating porphyrins in P.acnes.
Keywords: 5-aminolevulinic acid; 5-aminolevulinic acid esters; P.acnes; porphyrin accumulation; and PDT efficacy
2
Introduction Propionibacterium acnes (P.acnes) is one of the major causes of the pathogenesis of acne vulgaris. Photodynamic therapy (PDT) with 5-aminolevulinic acid hydrochloride (ALA) has attracted attention to treat acne vulgaris. When ALA is incorporated into P.acnes, porphyrins are produced, and the photoactivated porphyrin forms cytotoxic active oxygen species, leading to photochemical and photobiological processes that cause irreversible damage to P.acnes. An ALA ester is a well-known method to enhance penetrability of ALA through plasma membranes and accumulation of porphyrins, protoporphyrin IX (PpIX), in cancer cells [1]. Thus, there are many biochemical and clinical studies on ALA-based PDT for cancer treatments using ALA esters [2-6]. In the present study, we investigate the accumulation of porphyrins in P.acnes by ALA and its esters, ALA methyl ester hydrochloride (mALA), ALA octyl ester hydrochloride (oALA), and ALA benzyl ester hydrochloride (bALA).
3
Materials and Methods Materials
ALA was provided from SBI Pharmaceuticals Co., Ltd., and mALA was purchased
from Cosmo Bio Co., Ltd. We synthesized oALA and bALA according to previous paper [7].
Accumulation of porphyrins A control strain, P.acnes ATCC 6919, was allowed to grow anaerobically in modified GAM agar with 6x10-4 mol/L of ALA and its esters for 48 hours at 37 °C. After being grown, P.acnes was dispersed in a physiological saline solution, and the fluorescence spectrum of the dispersion was measured using a fluorescence spectrometer (FP-6500/6000, JASCO). Here, the excitation wavelength was 410 nm, and the emission wavelength ranged from 550 to 700 nm. Also, the P.acnes dispersion was diluted and was cultured in modified GAM agar for 72 hours to measure cell count in the dispersion. We then evaluated accumulation of porphyrins in P.acnes by the fluorescence peak area per cell.
PDT efficacy
P.acnes grown anaerobically in modified GAM agar with ALA and its esters, as
mentioned above, was dispersed and was diluted with a physiological saline solution at McFarland No. between 0.5 and 0.8. The P.acnes dispersion was inoculated into modified GAM agar and was irradiated aerobically for an hour by LED light of which the wavelength ranged from 400 to 800 nm. Its total fluence rate was 6.54 mW/cm2. After the irradiation, the medium with P.acnes was incubated anaerobically at 37 °C for 72 hours, and the viable cell count was measured. P.acnes dispersion without light irradiation was also set as a control group. The PDT efficacy is defined as the survival rate of viable cell count with and without LED light irradiation; the smaller the survival rate is, the greater the PDT efficacy is.
4
Results Figures 1 are fluorescence spectra of P.acnes cultured with ALA and its esters. No fluorescence peak is observed in P.acnes cultured without additives (Fig. 1 A). In P.acnes cultured with ALA, two fluorescence peaks are recognized at 580 and 620 nm (Fig. 1 B). We suggest that the peak at 580 nm is derived from metalloporphyrins and that the peak at 620 nm is ascribed to coproporphyrin III and/or uroporphyrin III [8]. Moreover, the fluorescence peak of porphyrins in P.acnes is observed in the case with mALA and bALA (Fig. 1 C and D), but not in the case with oALA (Fig. 1 E). Figure 2 shows the fluorescence peak area per cell. Since there is no peak in the case with oALA, we neglect its fluorescence peak area per cell. Figure 2 provides that the fluorescence peak area per cell is as follows: ALA >> mALA ≈ bALA.
The survival rate of P.acnes by PDT with ALA and its esters is depicted in Fig. 3. This figure indicates that the PDT efficacy without additives is almost equal to zero. Also, the PDT efficacy is the greatest in the case with ALA and is reduced as follows: ALA > mALA ≈ bALA > oALA (≈ without additives). The tendency of PDT efficacy is consistent with that of porphyrin accumulation in P.acnes.
5
Discussion Lipophilicity by esterification is one of the most effective methods for pharmacologically active agents to enhance their uptake into animal cells, because of increment in passive permeability of the agent through plasma membranes. In fact, ALA esters are more lipophilic than ALA and penetrate plasma membranes more readily, thus leading to the biosynthesis of porphyrins, PpIX, in a large quantity in animal cells. However, surprisingly, Figs. 2 and 3 indicate that ALA is superior to ALA esters in accumulating porphyrins in P.acnes. In contrast to animal cells, P.accnes is a gram-positive bacterium, having a thick cell wall of which the skeleton is composed of a peptidoglycan [9]. Since peptidoglycan is hydrophilic, the cell wall makes uptake of lipophilic agents difficult. This may be the
reason why porphyrin accumulation is reduced by ALA esters.
According to our result of porphyrin accumulation in P.acnes, we expect that clinical PDT treatment for acne vulgaris is more effective by ALA than by ALA esters. Unfortunately, Wiegell et al demonstrated that there were no significant differences in the PDT efficacy for inflammatory acne vulgaris between ALA and mALA that were applied topically [10]. We can infer that mALA permeates through stratum corneum more readily than ALA because of its lipophilicity, resulting in similar efficacy to ALA on the PDT treatment [11]. We suggest therefore that enhancement techniques for improving ALA skin permeation promote PDT efficacy on acne vulgaris treatment. This interest warrants further investigation.
6
References [1]
V. Zenzen, H. Zankl, In vitro evaluation of the cytotoxic and mutagenic potential of the 5aminolevulinic acid hexylester-mediated photodynamic therapy, Mutat. Res., 561 (2004) 91-100.
[2]
O.A. Gederaas, A. Holroyd, S.B. Brown, D. Vernon, J. Moan, K. Berg, 5-Aminolaevulinic acid methyl ester transport on amino acid carriers in a human colon adenocarcinoma cell line, Photochem. Photobiol., 73 (2001) 164-9.
[3]
S. El Khatib, J. Didelon, A. Leroux, L. Bezdetnaya, D. Notter, M. D'Hallewin, Kinetics, biodistribution and therapeutic efficacy of hexylester 5-aminolevulinate induced photodynamic therapy in an orthotopic rat bladder tumor model, J. Urol., 172 (2004) 2013-2017.
[4]
T. Smits, A.C. Moor, New aspects in photodynamic therapy of actinic keratoses, J. Photochem. Photobiol. B., 96 (2009) 159-69.
[5]
M. Wakui, Y. Yokoyama, H. Wang, T. Shigeto, M. Futagami, H. Mizunuma, Efficacy of a methyl ester of 5-aminolevulinic acid in photodynamic therapy for ovarian cancers, J Cancer Res. Clin. Oncol., 136 (2010) 1143-50.
[6]
M.J. Bader, H. Stepp, W. Beyer, T. Pongratz, R. Sroka, M. Kriegmair, D. Zaak, M. Welschof, D. Tilki, C.G. Stief, R. Waidelich, Photodynamic therapy of bladder cancer - a phase I study using hexaminolevulinate (HAL), Urol Oncol., 31 (2013) 1178-83.
[7]
N. Miyachi, T. Tanaka, S. Nishikawa, H. Takeya, Y. Hotta, Preparation and chemical properties of 5-aminolevulinic acid and its derivatives, Porphyrins, 7 (1998) 342-347.
[8]
S. Ramstad, N.L. Anh-Vu, A. Johnsson, The temperature dependence of porphyrin production in Propionibacterium acnes after incubation with 5-aminolevulinic acid (ALA) and its methyl ester (m-ALA), Photochem. Photobiol. Sci., 5 (2006) 66-72.
[9]
W.W. Navarre, O. Schneewind, Surface Proteins of Gram-Positive Bacteria and Mechanisms of Their Targeting to the Cell Wall Envelope, Microbiol. Mol. Biol. Rev., 63 (1999) 174-229.
7
[10] S.R. Wiegell, H.C. Wulf, Photodynamic therapy of acne vulgaris using 5-aminolevulinic acid versus methyl aminolevulinate, J. Am. Acad. Dermatol., 54 (2006) 647-51. [11] L.W Zhang, Y.P. Fang, J.Y. Fang, Enhancement techniques for improving 5-aminolevulinic acid delivery through the skin, Dermatol. Sin., 29 (2011) 1-7.
8
Figure captions Figure 1
Fluorescence spectra of porphyrins induced by ALA and its esters. A: non-additives, B: ALA, C: mALA, D: oALA, and E: bALA.
Figure 2
Fluorescence peak area per cell. P1: at 580 nm and P2: at 620 nm. Values are mean ± SD (n=3).
Figure 3
Survival rate of P.acnes by PDT with ALA and its esters. Values are mean ± SD (n=3).
9
600
650
700
550
600
Wavelength (nm)
650
700
5
550
600
Wavelength (nm)
Fluorescence intensity (a.u.)
D
600
650
Wavelength (nm)
650
700
Wavelength (nm)
5
550
Fig. 1
C
Fluorescence intensity (a.u.)
5
E
Fluorescence intensity (a.u.)
550
B
Fluorescence intensity (a.u.)
Fluorescence intensity (a.u.)
A 5
700
5
550
600
650
Wavelength (nm)
700
Fig. 2 Fluorescence peak area per cell (a.u.)
ALA mALA bALA
P1 P2
100
Survival rate (%)
80
60
40
20
0 ALA
Fig. 3
mALA
oALA
bALA Without additives