Photodiagnosis and Photodynamic Therapy (2006) 3, 162—167
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis Carrie-Anne Bristow a, Robert Hudson a, Timothy A. Paget PhD b,∗∗, Ross W. Boyle PhD a,∗ a
Photobiology and Photomedicine Group, Department of Chemistry & Clinical Biosciences Institute, University of Hull, Kingston-upon-Hull HU6 7RX, United Kingdom b Medway School of Pharmacy, The Universities of Kent and Greenwich at Medway, Kent ME4 4TB, United Kingdom Available online 5 July 2006 KEYWORDS Leishmaniasis; Photodynamic therapy; Porphyrins
Summary Four tetracationic porphyrins have been investigated for their ability to photo-inactivate Leishmania major promastigotes. Parallel photocytotoxicity assays against keratinocytes and macrophages show significant differences in activity between the microorganism and mammalian cells. Results suggest that it may be possible to photodynamically inactivate macrophages infected with Leishmania and the promastigote form of the microorganism, while minimising damage to surrounding tissue. © 2006 Elsevier B.V. All rights reserved.
Introduction Leishmaniasis affects 12 million people worldwide with an estimated 350 million people at risk of infection and 2 million new cases each year. The disease can be found in 66 old world countries and 22 new world countries in tropical and sub tropical areas, human infection is found in 16 countries in Europe [1,2]. Leishmaniasis is the term used to describe a group of diseases caused by species of the genus Leishmania. These parasitic protozoa are transmitted to mammalian hosts by the bite of infected female sandflies ∗
Corresponding author. Tel.: +44 1482 466353; fax: +44 1482 466410. ∗∗ Corresponding author. E-mail address: [email protected] (R.W. Boyle).
of the genera Phlebotomus or Lutzomyia. The life cycle of this organism involves two distinct forms, the promastigote, which is found in the insect and involved in transmission, and the amastigote, which is an intracellular form found in the mammalian host. There are many clinical variations of the disease, but the three most common forms are cutaneous, mucocutaneous and visceral leishmaniasis. The lesions of cutaneous and mucocutaneous leishmaniasis are localized to the skin and mucous membranes, whereas visceral leishmaniasis involves the reticulo-endothelial system. [3] The need for alternative methods of treatment is great, as current chemotherapy is costly and toxic with increasing reports of drug resistance. ‘‘Photodynamic therapy (PDT) is based on the dye-sensitized photo-oxidation of biological matter
1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2006.04.004
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis in target tissue’’ [4]. PDT is a promising treatment modality for superficial cancers either on its own or in combination with surgery. The dye or photosensitiser (PS) is introduced into the tissues, which are then irradiated with light of suitable wavelengths to photo-activate the PS. This leads to the production of cytotoxic species, such as singlet molecular oxygen and other reactive oxygen species, which kill cells in the immediate vicinity [5]. PDT has also proved to be effective at killing both grampositive and gram-negative bacteria, this up and coming area is known as photodynamic antimicro-
163
bial chemotherapy (PACT). Recently, two reports on the treatment of cutaneous leishmaniasis with PDT have been published [6,7], however these studies utilised a derivative of 5-aminolaevulinic acid (ALA), a biochemical precursor to the photosensitiser protoporphyrin IX (PPIX). Interestingly, as Leishmania are known to lack the haem biosynthetic pathway [8], required for conversion of ALA into PPIX, the clinical PDT effect must in this case be indirect, most likely by PPIX formation within infected macrophages, rather than within Leishmania itself.
Figure 1 Structures of cationic photosensitisers 1—4.
164 We report here, for the first time, structure activity relationships for a small group of cationic porphyrin-based PDT sensitisers on cells representing Leishmania promastigotes, macrophages, and keratinocytes, and show significant differences in activity and selectivity which will be important in anti-leishmanial PDT. Cationic PSs have been shown to photoinactivate gram-positive and gram-negative bacteria and parasitic protozoa such as Acanthamoeba palestinensis and cysts of Calpada inflata [9,10]. These cationic PSs are thought to be attracted to the net negative charge of the lipopolysaccharides on the surface of gram-negative bacteria. There is substantial literature [11—15] proving the efficacy of photo-inactivation of gram-negative bacteria using cationic porphyrins; this, and similarities in negatively charged liposaccharide character of membranes of Leishmania spp. and gramnegative bacteria, led us to investigate a small series of cationic photosensitisers as potential photodynamic anti-Leishmania agents. Two phosphorous centred cationic porphyrins and two nitrogen centred cationic porphyrins (Fig. 1) were tested for their ability to photoinactivate macrophages, keratinocytes and L. major promastigotes. All the cationic photosensitisers were chosen as they had previously shown good PDT activity against carcinoma cells [16]. Keratinocytes were used to model the effect of the PSs on healthy tissue surrounding infected lesions, and macrophages were used as the intracellular amastigote stage of the parasite resides primarily within macrophage cells.
Materials and methods Preparation of tetra cationic porphyrins Phosphoniumyl tetra cationic porphyrins 1 and 2 were synthesised by a previously described method [16] while compounds 3 and 4 were a gift from Frontier Scientific Inc.
Leishmania, keratinocytes and macrophages L. major (J118) was cultivated in Schneiders drosophila media supplemented with 10% (v/v) foetal bovine serum (FBS) and stored between 24 and 26 ◦ C. The promastigotes were kindly donated by Dr. V. Yardley (London School of Hygiene and Tropical Medicine) and passaged every 4—7 days. Macrophages (U937) were cultivated in RPMI media supplemented with 10% (v/v) FBS and 1% (v/v)
C.-A. Bristow et al. l-glutamine and stored at 37 ◦ C, 5% CO2 . The macrophage cells were kindly donated by the Medical Research Laboratory (Hull) and were passaged every fourth day. Keratinocytes (NCTC 2554) were cultivated in M119 media supplemented with 10% (v/v) FBS and 1% (v/v) l-glutamine and stored at 37 ◦ C, 5% CO2 . Kratinocytes were kindly donated by Dr. S. MacFarlane (University of Strathclyde) and were passaged when they became confluent, usually after 3—4 days.
Photodynamic inactivation studies Assays were performed using the cationic compounds 1—4 (Fig. 1). For each assay cells were washed and re-suspended in fresh Schneiders media with no FBS (FBS non-specifically binds porphyrins) [17] to give a concentration of 3.4 × 106 cells per ml. Compounds 1, 2, 3, or 4 dissolved in dimethylsulphoxide (DMSO) were added to cells in fresh medium and then incubated in the dark at 25 ◦ C for 3 h. Controls used were solvent (DMSO) control and a 100% kill using 50 M of menadione. All assays were performed in quadruplicate. After incubation excess unbound compound was removed by dilution and centrifugation at 1000 × g. Cells were re-suspended in 1.5 ml of fresh Schneiders media containing no FBS. Cells (200 l), either treated with drug or controls, were then transferred into duplicate flat bottomed 96-well plates. One of the plates was kept in the dark as a control and the other was irradiated with red light (633 ± 3 nm) for 16 min and 30 s using an Omnilux diode array PDT lamp (EL10000AG) delivering a fluence of 80 J/cm2 . One row of wells contained no drug and acted as a light/no drug control. After illumination, 10 l of FBS was added to every well of both the dark plate and the illuminated plates and these were then wrapped in foil and incubated at 25 ◦ C for 24 h before growth was assessed. Cell viability was assessed by MTS reduction [18] briefly 20 l of MTS was added per 100 l of media and cells and incubated in the dark at 25 ◦ C for 4 h. After incubation MTS reduction was measured at 490 nm in a microtitre plate reader (Dynex MRX2). All assays were performed in quadruplicate. After incubation, wells were also assayed microscopically to determine if any whole cells were present or if cells were motile after treatment. The killing of macrophages and keratinocytes was determined as for the Leishmania except that the cell pellet was re-suspended in fresh media to give a concentration of 1 × 106 cells per ml. The cells were then incubated with the compounds in the dark at 37 ◦ C for 1 h.
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis
165
Table 1 Comparison of the LC 50 values for the photoinactivation of L. major promastigotes, keratinocytes and macrophages using 1—4 Compound no.
Name
L. major LC50 (M)
Macrophage LC50 (M)
Keratinocytes LC50 (M)
1
meso-Tetra(4-(tributyl phosphoniumyl-methyl)phenyl)porphyrin tetrabromide meso-Tetra(4-(dicyclohexylphenylphophoniumylmethyl)phenyl)porphyrin tetrabromide meso-Tetra (4-N,N,N,N-trimethylanilinium)porphyrin tetrachloride meso-Tetra(N-methyl-4-pyridyl)porphyrin tetratosylate
0.85
>25
0.45
2 3 4
3.35
3.95
0.7
0.74
0.91
7.77
No activity
3.76
7.20
All values are means of three separate experiments each carried out in quadruplicate.
Results The four tetracationic compounds showed significantly differing activities between L. major, macrophages and keratinocytes. LC50 data for each compound is summarised in Table 1 and full photoinactivation curves are shown in Fig. 2. Compounds 1 and 3 showed the most effective antileishmanial activity with similar LC50 values of
0.85 and 0.74 M, respectively, however their photodynamic activity against macrophages and keratinocytes was strikingly different. 1 showed no activity against macrophages, even at the highest dose of 25 M while 3 inactivated 50% of macrophages at 0.91 M, a concentration close to the LD50 for L. major. Conversely, while 1 inactivated 50% of keratinocytes at a concentration approximately half
Figure 2 Concentration dependent photodynamic inactivation of macrophages, keratinocytes and L. major promastigotes using compounds 1—4. All values are expressed as percentage viability compared to untreated controls and are the means of three separate experiments carried out in quadruplicate. Bars show standard deviation. Empty squares, dark control; filled squares, macrophage cells; empty triangles, keratinocytes; filled triangles, Leishmania major.
166
C.-A. Bristow et al.
that required to elicit the same effect on L. major, 3 required a concentration more than ten times that of the LD50 for L. major to achieve the same end point. Compound 2, the second of the phosphorous centred cationic molecules showed similarities to 1, in the respect that it was more photocytotoxic against keratinocytes than against either L. major or macrophages. Compound 4 was the only compound tested which showed no activity against L. major, however the same compound also showed relatively low levels of photocytotoxicity against macrophages and keratinocytes.
by employing cationic photosensitisers, due to the specific anionic character that has been reported for regions of the cell membrane of Leishmania, and this may well be the factor responsible for the striking differences in photoinactivation seen for the four cationic porphyrins and three cell lines studied here. Work is now underway in our laboratory to synthesise and test a wider range of cationic photosensitisers to identify structural motifs which give optimal photodynamic activity and selectivity against Leishmania.
Discussion
Acknowledgement
The results clearly show that there are relatively complex structure activity relationships between each of the cationic porphyrins and the three different cell lines. Compound 1 shows good inhibition of L. major promastigotes but is more phototoxic to keratinocytes at all concentrations tested, suggesting this compound could potentially cause major damage to skin surrounding infected lesions. The differentiation in photocytotoxicity between the three cell lines with compound 3 is however of particular importance, and offers the possibility that a dose of PS could be administered that would kill Leishmania and infected macrophages, but would not cause significant damage to the surrounding healthy tissue, hence decreasing the risk of scarring. A therapeutic window exists for this compound from approximately 2 to 7 M where 60% inactivation of L. major is achieved with between 90 and 100% inactivation of macrophages, while this may be insufficient to completely cure leishmania lesions in one treatment, multiple treatments could be performed, this is a major advantage of PDT. During the course of this study the photodynamic inactivation of Leishmania amazonensis has been reported using aluminium phthalocyanine as the photosensitising agent [19], however this report considered a single neutral photosensitiser. Aluminium phthalocyanine is also insoluble in water, thus hindering its use clinically. In contrast all four compounds described here are soluble in polar solvents such as water and alcohol, which would allow easy formulation and application to lesions. It was also noteworthy that, while both promastigote and amastigote forms of L. amazonensis were found to be almost equally susceptible to photodynamic inactivation, and these were compared with activity against macrophages, no comparison was made with cells representing skin tissue of the infected lesions. Finally, we believe that the potential for selectivity is considerably enhanced
The authors would like to thank the Sir Halley Stewart Trust for funding.
References [1] The world health organisation website viewed 11/10/05 http://www.who.int/mediacentre/factsheets/fs116/en/. [2] Desjeux P. Leishmaniasis—–public health aspects and control. Clin Dermatol 1996;14(5):417—23. [3] Herwaldt B. Leishmaniasis. The Lancet 1999;354:1191—9. [4] Henderson B, Dougherty T. How does photodynamic therapy work? Photochem Photobiol 1992;55(1):145—57. [5] MacDonald I, Dougherty T. Basic principles of photodynamic therapy. J Porphyrins Phthalocyanines 2001;5:105—29. [6] Enk CD, Fritsch C, Jonas F, et al. Treatment of cutaneous leishmaniasis with photodynamic therapy. Arch Dermatol 2003;139:432—4. [7] Gardlo K, Horska Z, Enk CD, et al. Treatment of cutaneous leishmaniasis by photodynamic therapy. J Am Acad Dermatol 2003;48:893—6. [8] Sah JF, Ito H, Kolli BK, Peterson DA, Sassal S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002;277:14902—9. [9] Kassab K, Amor T, Jori G, Coppellotti O. Photosensitization of Colpdoda inflata cysts by meso-substituted cationic compounds. Photochem Photobiol Sci 2002;1:560—4. [10] Kassab K, Dei D, Roncucci G, Jori G, Coppellotti O. Phthalocyanine-photosensitized inactivation of a pathogenic protozoan, Acanthamoeba palestinensis. Photochem Photobiol Sci 2003;2:668—72. [11] Merchat M, Spikes J, Bertoloni G, Jori G. Studies on the mechanism of bacteria photosensitization by mesosubstituted cationic compounds. J Photochem Photobiol B: Biol 1996;35:149—57. [12] Segalla A, Boasarelli C, Braslavsky S, et al. Photophysical, photochemical and antibacterial photosensitizing properties of a novel octacationic Zn(II)-phthalocyanine. Photochem Photobiol Sci 2002;1:641—8. [13] Hamblin M, O’Donnell D, Murthy N, Contag C, Hasan T. Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol 2002;75:51—7. [14] Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 1998;42:13—28.
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis [15] Hamblin M, Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci 2004;3:436—50. [16] Hudson R, Savoie H, Boyle RW. Lipophilic cationic compounds as photodynamic sensitisers, synthesis and structure-activity relationships. J Photodiagn Photodyn Therapy 2005;2(3):193—6. [17] Obochi M, Boyle R, van Lier J. Biological activities of phthalocyanines. XIII. The effects of human serum compo-
167
nents on the in vitro uptake of and photodynamic activity of zinc phthalocyanine. Photochem Photobiol 1993;37:4. [18] Deyes R. Cell viability assays. Promega Notes 2002;81: 32—3. [19] Dutta S, Ray D, Kolli BK, Chang K-P. Photodynamic sensitisation of Leishmania amazonensis in both extracellular and intracellular stages with aluminium phthalocyanine chloride for photolysis in vitro. Antimicrob Agents Chemother 2005;49:4474—84.
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis Carrie-Anne Bristow a, Robert Hudson a, Timothy A. Paget PhD b,∗∗, Ross W. Boyle PhD a,∗ a
Photobiology and Photomedicine Group, Department of Chemistry & Clinical Biosciences Institute, University of Hull, Kingston-upon-Hull HU6 7RX, United Kingdom b Medway School of Pharmacy, The Universities of Kent and Greenwich at Medway, Kent ME4 4TB, United Kingdom Available online 5 July 2006 KEYWORDS Leishmaniasis; Photodynamic therapy; Porphyrins
Summary Four tetracationic porphyrins have been investigated for their ability to photo-inactivate Leishmania major promastigotes. Parallel photocytotoxicity assays against keratinocytes and macrophages show significant differences in activity between the microorganism and mammalian cells. Results suggest that it may be possible to photodynamically inactivate macrophages infected with Leishmania and the promastigote form of the microorganism, while minimising damage to surrounding tissue. © 2006 Elsevier B.V. All rights reserved.
Introduction Leishmaniasis affects 12 million people worldwide with an estimated 350 million people at risk of infection and 2 million new cases each year. The disease can be found in 66 old world countries and 22 new world countries in tropical and sub tropical areas, human infection is found in 16 countries in Europe [1,2]. Leishmaniasis is the term used to describe a group of diseases caused by species of the genus Leishmania. These parasitic protozoa are transmitted to mammalian hosts by the bite of infected female sandflies ∗
Corresponding author. Tel.: +44 1482 466353; fax: +44 1482 466410. ∗∗ Corresponding author. E-mail address: [email protected] (R.W. Boyle).
of the genera Phlebotomus or Lutzomyia. The life cycle of this organism involves two distinct forms, the promastigote, which is found in the insect and involved in transmission, and the amastigote, which is an intracellular form found in the mammalian host. There are many clinical variations of the disease, but the three most common forms are cutaneous, mucocutaneous and visceral leishmaniasis. The lesions of cutaneous and mucocutaneous leishmaniasis are localized to the skin and mucous membranes, whereas visceral leishmaniasis involves the reticulo-endothelial system. [3] The need for alternative methods of treatment is great, as current chemotherapy is costly and toxic with increasing reports of drug resistance. ‘‘Photodynamic therapy (PDT) is based on the dye-sensitized photo-oxidation of biological matter
1572-1000/$ — see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.pdpdt.2006.04.004
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis in target tissue’’ [4]. PDT is a promising treatment modality for superficial cancers either on its own or in combination with surgery. The dye or photosensitiser (PS) is introduced into the tissues, which are then irradiated with light of suitable wavelengths to photo-activate the PS. This leads to the production of cytotoxic species, such as singlet molecular oxygen and other reactive oxygen species, which kill cells in the immediate vicinity [5]. PDT has also proved to be effective at killing both grampositive and gram-negative bacteria, this up and coming area is known as photodynamic antimicro-
163
bial chemotherapy (PACT). Recently, two reports on the treatment of cutaneous leishmaniasis with PDT have been published [6,7], however these studies utilised a derivative of 5-aminolaevulinic acid (ALA), a biochemical precursor to the photosensitiser protoporphyrin IX (PPIX). Interestingly, as Leishmania are known to lack the haem biosynthetic pathway [8], required for conversion of ALA into PPIX, the clinical PDT effect must in this case be indirect, most likely by PPIX formation within infected macrophages, rather than within Leishmania itself.
Figure 1 Structures of cationic photosensitisers 1—4.
164 We report here, for the first time, structure activity relationships for a small group of cationic porphyrin-based PDT sensitisers on cells representing Leishmania promastigotes, macrophages, and keratinocytes, and show significant differences in activity and selectivity which will be important in anti-leishmanial PDT. Cationic PSs have been shown to photoinactivate gram-positive and gram-negative bacteria and parasitic protozoa such as Acanthamoeba palestinensis and cysts of Calpada inflata [9,10]. These cationic PSs are thought to be attracted to the net negative charge of the lipopolysaccharides on the surface of gram-negative bacteria. There is substantial literature [11—15] proving the efficacy of photo-inactivation of gram-negative bacteria using cationic porphyrins; this, and similarities in negatively charged liposaccharide character of membranes of Leishmania spp. and gramnegative bacteria, led us to investigate a small series of cationic photosensitisers as potential photodynamic anti-Leishmania agents. Two phosphorous centred cationic porphyrins and two nitrogen centred cationic porphyrins (Fig. 1) were tested for their ability to photoinactivate macrophages, keratinocytes and L. major promastigotes. All the cationic photosensitisers were chosen as they had previously shown good PDT activity against carcinoma cells [16]. Keratinocytes were used to model the effect of the PSs on healthy tissue surrounding infected lesions, and macrophages were used as the intracellular amastigote stage of the parasite resides primarily within macrophage cells.
Materials and methods Preparation of tetra cationic porphyrins Phosphoniumyl tetra cationic porphyrins 1 and 2 were synthesised by a previously described method [16] while compounds 3 and 4 were a gift from Frontier Scientific Inc.
Leishmania, keratinocytes and macrophages L. major (J118) was cultivated in Schneiders drosophila media supplemented with 10% (v/v) foetal bovine serum (FBS) and stored between 24 and 26 ◦ C. The promastigotes were kindly donated by Dr. V. Yardley (London School of Hygiene and Tropical Medicine) and passaged every 4—7 days. Macrophages (U937) were cultivated in RPMI media supplemented with 10% (v/v) FBS and 1% (v/v)
C.-A. Bristow et al. l-glutamine and stored at 37 ◦ C, 5% CO2 . The macrophage cells were kindly donated by the Medical Research Laboratory (Hull) and were passaged every fourth day. Keratinocytes (NCTC 2554) were cultivated in M119 media supplemented with 10% (v/v) FBS and 1% (v/v) l-glutamine and stored at 37 ◦ C, 5% CO2 . Kratinocytes were kindly donated by Dr. S. MacFarlane (University of Strathclyde) and were passaged when they became confluent, usually after 3—4 days.
Photodynamic inactivation studies Assays were performed using the cationic compounds 1—4 (Fig. 1). For each assay cells were washed and re-suspended in fresh Schneiders media with no FBS (FBS non-specifically binds porphyrins) [17] to give a concentration of 3.4 × 106 cells per ml. Compounds 1, 2, 3, or 4 dissolved in dimethylsulphoxide (DMSO) were added to cells in fresh medium and then incubated in the dark at 25 ◦ C for 3 h. Controls used were solvent (DMSO) control and a 100% kill using 50 M of menadione. All assays were performed in quadruplicate. After incubation excess unbound compound was removed by dilution and centrifugation at 1000 × g. Cells were re-suspended in 1.5 ml of fresh Schneiders media containing no FBS. Cells (200 l), either treated with drug or controls, were then transferred into duplicate flat bottomed 96-well plates. One of the plates was kept in the dark as a control and the other was irradiated with red light (633 ± 3 nm) for 16 min and 30 s using an Omnilux diode array PDT lamp (EL10000AG) delivering a fluence of 80 J/cm2 . One row of wells contained no drug and acted as a light/no drug control. After illumination, 10 l of FBS was added to every well of both the dark plate and the illuminated plates and these were then wrapped in foil and incubated at 25 ◦ C for 24 h before growth was assessed. Cell viability was assessed by MTS reduction [18] briefly 20 l of MTS was added per 100 l of media and cells and incubated in the dark at 25 ◦ C for 4 h. After incubation MTS reduction was measured at 490 nm in a microtitre plate reader (Dynex MRX2). All assays were performed in quadruplicate. After incubation, wells were also assayed microscopically to determine if any whole cells were present or if cells were motile after treatment. The killing of macrophages and keratinocytes was determined as for the Leishmania except that the cell pellet was re-suspended in fresh media to give a concentration of 1 × 106 cells per ml. The cells were then incubated with the compounds in the dark at 37 ◦ C for 1 h.
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis
165
Table 1 Comparison of the LC 50 values for the photoinactivation of L. major promastigotes, keratinocytes and macrophages using 1—4 Compound no.
Name
L. major LC50 (M)
Macrophage LC50 (M)
Keratinocytes LC50 (M)
1
meso-Tetra(4-(tributyl phosphoniumyl-methyl)phenyl)porphyrin tetrabromide meso-Tetra(4-(dicyclohexylphenylphophoniumylmethyl)phenyl)porphyrin tetrabromide meso-Tetra (4-N,N,N,N-trimethylanilinium)porphyrin tetrachloride meso-Tetra(N-methyl-4-pyridyl)porphyrin tetratosylate
0.85
>25
0.45
2 3 4
3.35
3.95
0.7
0.74
0.91
7.77
No activity
3.76
7.20
All values are means of three separate experiments each carried out in quadruplicate.
Results The four tetracationic compounds showed significantly differing activities between L. major, macrophages and keratinocytes. LC50 data for each compound is summarised in Table 1 and full photoinactivation curves are shown in Fig. 2. Compounds 1 and 3 showed the most effective antileishmanial activity with similar LC50 values of
0.85 and 0.74 M, respectively, however their photodynamic activity against macrophages and keratinocytes was strikingly different. 1 showed no activity against macrophages, even at the highest dose of 25 M while 3 inactivated 50% of macrophages at 0.91 M, a concentration close to the LD50 for L. major. Conversely, while 1 inactivated 50% of keratinocytes at a concentration approximately half
Figure 2 Concentration dependent photodynamic inactivation of macrophages, keratinocytes and L. major promastigotes using compounds 1—4. All values are expressed as percentage viability compared to untreated controls and are the means of three separate experiments carried out in quadruplicate. Bars show standard deviation. Empty squares, dark control; filled squares, macrophage cells; empty triangles, keratinocytes; filled triangles, Leishmania major.
166
C.-A. Bristow et al.
that required to elicit the same effect on L. major, 3 required a concentration more than ten times that of the LD50 for L. major to achieve the same end point. Compound 2, the second of the phosphorous centred cationic molecules showed similarities to 1, in the respect that it was more photocytotoxic against keratinocytes than against either L. major or macrophages. Compound 4 was the only compound tested which showed no activity against L. major, however the same compound also showed relatively low levels of photocytotoxicity against macrophages and keratinocytes.
by employing cationic photosensitisers, due to the specific anionic character that has been reported for regions of the cell membrane of Leishmania, and this may well be the factor responsible for the striking differences in photoinactivation seen for the four cationic porphyrins and three cell lines studied here. Work is now underway in our laboratory to synthesise and test a wider range of cationic photosensitisers to identify structural motifs which give optimal photodynamic activity and selectivity against Leishmania.
Discussion
Acknowledgement
The results clearly show that there are relatively complex structure activity relationships between each of the cationic porphyrins and the three different cell lines. Compound 1 shows good inhibition of L. major promastigotes but is more phototoxic to keratinocytes at all concentrations tested, suggesting this compound could potentially cause major damage to skin surrounding infected lesions. The differentiation in photocytotoxicity between the three cell lines with compound 3 is however of particular importance, and offers the possibility that a dose of PS could be administered that would kill Leishmania and infected macrophages, but would not cause significant damage to the surrounding healthy tissue, hence decreasing the risk of scarring. A therapeutic window exists for this compound from approximately 2 to 7 M where 60% inactivation of L. major is achieved with between 90 and 100% inactivation of macrophages, while this may be insufficient to completely cure leishmania lesions in one treatment, multiple treatments could be performed, this is a major advantage of PDT. During the course of this study the photodynamic inactivation of Leishmania amazonensis has been reported using aluminium phthalocyanine as the photosensitising agent [19], however this report considered a single neutral photosensitiser. Aluminium phthalocyanine is also insoluble in water, thus hindering its use clinically. In contrast all four compounds described here are soluble in polar solvents such as water and alcohol, which would allow easy formulation and application to lesions. It was also noteworthy that, while both promastigote and amastigote forms of L. amazonensis were found to be almost equally susceptible to photodynamic inactivation, and these were compared with activity against macrophages, no comparison was made with cells representing skin tissue of the infected lesions. Finally, we believe that the potential for selectivity is considerably enhanced
The authors would like to thank the Sir Halley Stewart Trust for funding.
References [1] The world health organisation website viewed 11/10/05 http://www.who.int/mediacentre/factsheets/fs116/en/. [2] Desjeux P. Leishmaniasis—–public health aspects and control. Clin Dermatol 1996;14(5):417—23. [3] Herwaldt B. Leishmaniasis. The Lancet 1999;354:1191—9. [4] Henderson B, Dougherty T. How does photodynamic therapy work? Photochem Photobiol 1992;55(1):145—57. [5] MacDonald I, Dougherty T. Basic principles of photodynamic therapy. J Porphyrins Phthalocyanines 2001;5:105—29. [6] Enk CD, Fritsch C, Jonas F, et al. Treatment of cutaneous leishmaniasis with photodynamic therapy. Arch Dermatol 2003;139:432—4. [7] Gardlo K, Horska Z, Enk CD, et al. Treatment of cutaneous leishmaniasis by photodynamic therapy. J Am Acad Dermatol 2003;48:893—6. [8] Sah JF, Ito H, Kolli BK, Peterson DA, Sassal S, Chang KP. Genetic rescue of Leishmania deficiency in porphyrin biosynthesis creates mutants suitable for analysis of cellular events in uroporphyria and for photodynamic therapy. J Biol Chem 2002;277:14902—9. [9] Kassab K, Amor T, Jori G, Coppellotti O. Photosensitization of Colpdoda inflata cysts by meso-substituted cationic compounds. Photochem Photobiol Sci 2002;1:560—4. [10] Kassab K, Dei D, Roncucci G, Jori G, Coppellotti O. Phthalocyanine-photosensitized inactivation of a pathogenic protozoan, Acanthamoeba palestinensis. Photochem Photobiol Sci 2003;2:668—72. [11] Merchat M, Spikes J, Bertoloni G, Jori G. Studies on the mechanism of bacteria photosensitization by mesosubstituted cationic compounds. J Photochem Photobiol B: Biol 1996;35:149—57. [12] Segalla A, Boasarelli C, Braslavsky S, et al. Photophysical, photochemical and antibacterial photosensitizing properties of a novel octacationic Zn(II)-phthalocyanine. Photochem Photobiol Sci 2002;1:641—8. [13] Hamblin M, O’Donnell D, Murthy N, Contag C, Hasan T. Rapid control of wound infections by targeted photodynamic therapy monitored by in vivo bioluminescence imaging. Photochem Photobiol 2002;75:51—7. [14] Wainwright M. Photodynamic antimicrobial chemotherapy (PACT). J Antimicrob Chemother 1998;42:13—28.
Potential of cationic porphyrins for photodynamic treatment of cutaneous Leishmaniasis [15] Hamblin M, Hasan T. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem Photobiol Sci 2004;3:436—50. [16] Hudson R, Savoie H, Boyle RW. Lipophilic cationic compounds as photodynamic sensitisers, synthesis and structure-activity relationships. J Photodiagn Photodyn Therapy 2005;2(3):193—6. [17] Obochi M, Boyle R, van Lier J. Biological activities of phthalocyanines. XIII. The effects of human serum compo-
167
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