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Photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP) and the corresponding chlorin (m-THPC) and bacteriochlorin(m-THPBC). A comparative study
www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 53 (1999) 136–143
Photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP) and the corresponding chlorin (m-THPC) and bacteriochlorin (m-THPBC). A comparative study Raymond Bonnett *, Birgul D. Djelal, Peter A. Hamilton, Gabriel Martinez, Franz Wierrani Department of Chemistry, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK Received 8 July 1999; accepted 1 November 1999
Abstract The photobleaching of compounds of the 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin series at different reduction levels (m-THPP 1, m-THPC 2, m-THPBC 3) has been studied in methanol and in methanol–water (3:2, v/v) using an argon laser (514 nm) by observing the diminution of absorbance of band I (for 2 and 3) and of band IV (for 1) with time. Under the conditions studied here, true photobleaching only occurs for m-THPC (2) and m-THPBC (3), with photomodification being the major process for m-THPP (1). The rates for the photobleaching of 2 and 3 are presented in different solvents. The photobleaching rate of the bacteriochlorin 3 is found to be 90 times higher than that of the chlorin 2 in methanol–water (3:2, v/v). Singlet oxygen appears to be the reactive species responsible for the photobleaching of 2 and 3 and the photomodification of 1. q1999 Elsevier Science S.A. All rights reserved. Keywords: Photodynamic therapy; Photobleaching; m-THPP; m-THPC; m-THPBC
1. Introduction Photobleaching (otherwise referred to as photofading, or in terms of the absence of light fastness) is a well-established study in the science of dyes and pigments [1]. In this context, photobleaching is defined as the loss of absorption of the chromophore caused by visible radiation. Generally it is light fastness that is the desirable property in that area (the indigo of blue denim being a fashionable exception). However, in photochemistry and photobiology the custom has grown up to define photobleaching as the loss of absorption or emission intensity [2] caused by light. Because fluorescence is very sensitive to a variety of quenching effects, the loss of fluorescence does not usually parallel loss of absorption, and it is important to state which mode of observation is being employed. The present paper refers to loss of absorption of photosensitizers that are candidates for applications in photodynamic therapy (PDT). Photodynamic therapy may be defined as the use of light and a sensitizer in the presence of oxygen to treat disease: currently the most intensively studied application is in cancer phototherapy. The process involves some modest cancer tar* Corresponding author. Fax: q44-20-7882-7794; e-mail: R.Bonnett@ qmw.ac.uk
getting on the part of the photosensitizer; the excellent targetting properties of the light (frequently, laser) beam; and the generation within the tumour of singlet oxygen and other reactive oxygen species, which lead to the degradation of the tumour [3]. Photobleaching of photosensitizers during PDT has been observed both in cells in vitro and skin in vivo using the fluorescence mode of observation, both directly and after pigment extraction [4,5]. While photobleaching might be thought to be a disadvantageous property in a tumour photosensitizer (in that the source of the toxic species is being destroyed), it is possible to envisage potential benefits [4– 6]. These would arise if, while the dosage was adjusted to keep tumour levels of photosensitizer at effective levels, the amounts of photosensitizer in surrounding tissue during the irradiation, and in skin and muscle subsequently exposed to ambient light, were below the threshold levels for tissue photosensitization, or, if not, quickly fell below such levels because of photobleaching. Photobleaching of the porphyrin analogues most commonly used in PDT can occur by photo-oxidation or photoreduction processes, photo-oxidation being the more common. Two types of irreversible photobleaching process leading to chemical change in the chromophore are encountered:
1011-1344/99/$ - see front matter q1999 Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 9 9 ) 0 0 1 3 9 - 6
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(i) Photomodification, where loss of absorbance or fluorescence occurs at some wavelengths, but the chromophore is retained in modified form. The formation of the isomeric photoprotoporphyrins A and B [7] and the photo-oxidation of the etiopurpurin series of PDT photosensitizers [8] are examples here. A reductive photomodification is to be found in the photoreactions of haematoporphyrin, where there is evidence for chlorin formation [9]. (ii) True photobleaching, where chemical changes are deep seated, and result in small fragments that no longer have appreciable absorption in the visible region. These fragments have commonly not been identified: the photo-oxidation of bilirubin (which occurs as one of the pathways in the phototherapy of neonatal jaundice) provides an example where the structures of the colourless products are known (maleimides, propentdyopent adducts, dipyrrylmethanes) [10]. It seems that where photomodification occurs, true photobleaching often occurs concomitantly. Early studies [11] showed that 5,10,15,20-tetrakis(mhydroxyphenyl)porphyrin (m-THPP, 1, Fig. 1) was a potent PDT photosensitizer. It was subsequently overshadowed by the corresponding chlorin (m-THPC, 2, Fig. 1), which was even more active [12]. This compound is now being developed under the proprietary name ‘Foscan’ (international nonproprietary name: temoporfin) by Scotia Pharmaceuticals Ltd., Stirling, Scotland. The recent emergence of 2 as a potentially valuable PDT sensitizer has stimulated a number of studies on its photobleaching. Mild photo-oxidation of 2 in methanol in ambient laboratory light for seven days led to the identification of hydroxylated and dehydrogenated derivatives by HPLC– electrospray ionization tandem mass spectrometry. The products were formulated as hydroxy m-THPC (three isomers) and hydroxy m-THPP derivatives: some m-THPP (1) was also detected [13]. The photobleaching of 2 has been compared with that of 1 and with Photofrin in in vitro experiments with Chinese hamster lung fibroblasts [14]. Studies with 2 in protein-containing solutions (foetal calf serum) have shown that the rate of loss of fluorescence is about 15 times the rate of loss of absorbance [15] and that a new product
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with lmaxf320 nm is formed [15,16]. Preliminary studies have appeared on the photobleaching of m-THPBC (3, Fig. 1), which occurs more rapidly than that of the corresponding chlorin [17]. Although the photophysical parameters of 1, 2 and 3 (Fig. 1) have recently been reported [18], there is no comparative kinetic study of the photobleaching of these three substances under defined chemical conditions, and this is now provided.
2. Materials and methods 2.1. Chemicals m-THPP (1) and m-THPBC (3) were synthesized as described [11,12]. m-THPBC (3) contained about 5% of the chlorin (2). m-THPC (2) was a gift from Scotia Pharmaceuticals Ltd. Solvents used were methanol Analar (Merck, Poole, UK), formamide 99.5% (Aldrich, Gillingham, UK) and glass-distilled water. 2.2. Photobleaching kinetics The sensitizer solution (3 ml) was irradiated in a 10 mm=10 mm stoppered quartz cuvette with an argon laser (Spectraphysics 164) tuned to 514 nm. This wavelength approximately corresponds to band IV in the three substrates: the solutions were optically matched (As0.5"10%) at 514 nm at ts0 (1, 0.03 mM; 2, 0.04 mM; 3, 0.02 mM). The laser power was set at 0.78 W and measured using a power meter (Coherent, model 210). At intervals the cuvette was removed and the spectrum in the range 450–800 nm was recorded (Perkin–Elmer Lambda 2 spectrometer) in order to follow the course of photomodification (1, absorbance decay at 512 nm) or photobleaching (absorbance decay at 649 nm for 2, and 734 nm for 3). Preliminary experiments (data not shown) indicated that, under the conditions employed here, the observed rates were insensitive to (i) stirring the solution (Teflon flea, magnetic stirring), (ii) temperature changes within the range 20–368C, and (iii) power density (degree of focusing of the laser within
Fig. 1. Structural formulae for m-THPP (1), m-THPC (2) and m-THPBC (3).
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the window of the cuvette). The experiments described below were carried out with stirring at 20"1oC.
3. Results and discussion 3.1. Effect of structural variation of the substrate In Figs. 2–4, a comparison is made of the photobleaching of 1, 2 and 3, respectively, in methanol–water (3:2 v/v) solutions at the same initial optical density at 514 nm. Fig. 2 (photobleaching of 1) shows that photobleaching does occur slowly at band IV but that band II actually increases in absorbance over the course of 60 min, and the rate of photochange then diminishes. In fact, this Figure demonstrates that the major process occurring here is photomodification and not true photobleaching. Visual observation under a Woods lamp (366 nm) of the starting solution, and of the solution of photoproducts after 60 min, showed that the latter was much less fluorescent than the starting solution, so that a study based on fluorescence diminution would give a quite different apparent result (i.e., it would suggest true photobleaching). Figs. 3 and 4 show the corresponding results for the chlorin 2 and the bacteriochlorin 3, which are in strong contrast to the result for the porphyrin 1. True photobleaching in the visible region occurs in both cases, although in the case of
the chlorin, a new absorbance band appears to be developing below 480 nm. The photobleaching is rapid in both cases, but particularly rapid with the bacteriochlorin 3. Since the reaction is carried out in equilibrium with air, it is presumed that a photo-oxidation process is occurring, and the relative rates thus accord with the known redox potentials (bacteriochlorin-chlorin [19]). The photobleaching of band I (diminution of absorbance at lmax) for the chlorin 2 and bacteriochlorin 3 follows firstorder kinetics in both cases (Fig. 5). The first-order rate constant is 6.3=10y4 sy1 for the chlorin and 5.7=10y2 sy1 for the bacteriochlorin. Thus, under the conditions of our experiment the bacteriochlorin is photobleached about 90 times faster than the chlorin in methanol–water (3:2, v/v). 3.2. Effect of solvent Spikes has shown [20] that the rates of photobleaching of haematoporphyrin, Photofrin, tetraphenylporphyrin tetrasulfonic acid and uroporphyrin are sensitive to solvent changes. Similar effects are found in the present series. As shown in Table 1, the rates of photobleaching of 2 and 3 are lower in pure methanol than in methanol–water (3:2, v/v). The effect is shown graphically for the chlorin 2 in Fig. 6, which should be compared with Fig. 3. In methanol–formamide (1:1, v/ v) and formamide the initial rate for the chlorin 2 was markedly increased, and in each case the reaction kinetics become
Fig. 2. Changes in the visible spectrum of m-THPP (1) at an initial concentration of 0.03 mM in methanol–water (3:2, v/v) in equilibrium with air, on irradiation at 514 nm with an argon laser (Ps0.78 W).
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Fig. 3. Changes in the visible spectrum of m-THPC (2) at an initial concentration of 0.04 mM in methanol–water (3:2, v/v) under the same conditions as described for Fig. 2, except for the time scale.
biphasic, possibly due to oxygen depletion. Because of the rapidity of the reaction, the rate with the bacteriochlorin in these two solvents could not be measured using our methodology. The origins of these solvent effects on rate are not really understood, but it is apparent that the rate depends on the polar nature of the solvent. Aggregation does not appear to play a part, because in the solvent systems employed the spectra suggest that the substrates are monomeric. Spikes [20] has pointed out an apparent correlation of photobleaching with the dielectric constant of the solvent (so k is in the sequence formamide)aqueous methanol)methanol). Possibly the more polar solvents are stabilizing polar or dipolar transition states involved in the reaction with singlet oxygen or other reactive oxygen species. 3.3. Mechanistic considerations In order to assess the role of oxygen in the photobleaching of m-THPC (2) and m-THPBC (3), and in the photomodification of m-THPP (1), solutions of 1, 2 and 3 in methanol were deoxygenated by bubbling nitrogen for at least 1 h; the UV cell was sealed and then irradiated. The photobleaching of the bacteriochlorin 3 was almost suppressed. However, photoreaction of the porphyrin 1 and the chlorin 2 still
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occurred (albeit more slowly), although the changes in the visible spectra were different from those observed in aerated solutions (data not shown). The mechanisms of these photo-oxygenation processes are expected to be complex. It is known that in air-saturated methanol all three of the photosensitizers studied here have significant singlet oxygen quantum yields (FD: for 1, 0.46; for 2, 0.43; for 3, 0.43)[18]. Evidence to support a type I (radical) mechanism and/or a type II (singlet oxygen) mechanism is frequently sought using additives that physically or chemically quench the putative intermediate species [21]. For example, sodium azide physically quenches singlet oxygen, while bilirubin and 1,3-diphenylisobenzofuran rapidly react with it. As type I probes, superoxide dismutase (SOD), catalase and mannitol react rapidly with superoxide radical anion, hydrogen peroxide and hydroxyl radicals, respectively: if these species influence the rate-determining step, the relevant additions will lower the rate of reaction. Singlet oxygen has a longer lifetime in a deuterated solvent compared with that in the corresponding protiated solvent, and so, provided the substrate is of modest reactivity and deactivation by solvent molecules determines singlet oxygen concentration, the rate will be enhanced in the deuterated solvent.
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Fig. 4. Changes in the visible spectrum of m-THPBC (3) at an initial concentration of 0.02 mM in methanol–water (3:2, v/v) under the same conditions as Fig. 2, except for the time scale. Table 1 First-order rate constants (k, sy1) for photobleaching in various solvents MeOH
m-THPC
1.7=10y4 6.3=10y4
m-THPBC
1.0=10y2 5.7=10y2
a b
Fig. 5. First-order plots of the photobleaching of m-THPC (2) and mTHPBC (3) in methanol–water (3:2, v/v).
It has already been shown that, for m-THPC 2 in aqueous medium containing 10% foetal calf serum, the photobleaching process involves singlet oxygen on the evidence of rate diminution in the presence of singlet oxygen quenchers (DABCO, 25% decrease observed; histidine, 35% decrease observed), whereas evidence for a type I process was not found (no appreciable effect of SOD, catalase or deferoxamine) [16]. In ethanol, the rate of photobleaching of 1,3-
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MeOH–H2O MeOH–formamide Formamide (3:2) (1:1) 1.0=10y3 a 2.2=10y4 b
2.8=10y3 a 1.2=10y4 b
ts0–7.5 min. ts10–30 min.
diphenylisobenzofuran in the presence of 2 was enhanced on deuteration of the solvent [16]. In the present study, evidence has been found that supports a contribution from a singlet oxygen mechanism in the photomodification of 1 and in the photobleaching of 2 and 3 in methanol and in methanol–water (3:2, v/v). The results are presented in Table 2 for the porphyrin 1, Table 3 for the chlorin 2, and Table 4 for the bacteriochlorin 3. When sodium azide (cs8 mM) was incorporated into methanolic solutions, the reaction rates were reduced in each case (by 51% for 1, by 60% for 2, and by 29% for 3). Addition of Rose Bengal (3=10y6 M), a well-known and effective photogenerator of singlet oxygen [21], increased the rate of reaction for 1 (by 100%) and for 2 (by 47%).
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Fig. 6. Example of the solvent effect on photobleaching: course of photobleaching of m-THPC in methanol using the same conditions as in Fig. 3.
Table 2 Apparent first-order rate constants for the diminution of band IV of m-THPP 1 (cs3=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
None NaN3 (8=10y3 M) Rose Bengal (3=10y6 M)
MeOH MeOH MeOH
None None SOD (0.25 mg mly1) Catalase (0.15 mg mly1) Mannitol (cs10y2 M)
MeOH–H2O (3:2) CD3OD–D2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
k (sy1) 3.3=10y5 1.6=10y5 6.8=10y5 9.6=10y5 8.4=10-4 10.0=10y5 10.2=10y5 10.2=10y5
Table 3 First-order rate constants for the diminution of band I of m-THPC 2 (cs4=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
k (sy1)
None NaN3 (8=10y3 M) Rose Bengal (3=10y6 M)
MeOH MeOH MeOH
1.7=10y4 6.7=10y5 2.5=10y4
None SOD (0.25 mg mly1) Catalase (0.15 mg mly1) Mannitol (10y2 M)
MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
6.3=10y4 6.6=10y4 7.7=10y4 7.8=10y4
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Table 4 First-order rate constants for the diminution of band I of m-THPBC 3 (cs2=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
k (sy1)
None NaN3 (8=10y3 M) Mannitol (10y2 M)
MeOH MeOH MeOH
1.0=10y2 7.1=10y3 1.1=10y2
None SOD (0.25 mg mly1) Catalase (0.15 mg mly1)
MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
5.7=10y2 5.3=10y2 4.6=10y2
(This additive was not studied with the bacteriochlorin, since the rate was already as fast as we could reliably measure using our methodology.) In one case, that of the porphyrin in methanol–water (3:2, v/v), we have observed the effect of using a completely deuterated solvent under otherwise identical reaction conditions. The observed first-order rate constant rose from ks9.6=10y5 to 8.4=10y4 sy1, consistent with the involvement of singlet oxygen in the reaction. On the other hand, additives designed to test for a type I pathway (SOD, catalase) in methanol–water (3:2, v/v) were essentially without effect on the reaction rate (Tables 2–4). Mannitol (cs10 mM), which reacts rapidly with hydroxyl radicals, was added to the bacteriochlorin 3 in methanol
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(Table 4) and to the porphyrin 1 and the chlorin 2 in methanol–water (3:2, v/v) (Tables 2 and 3). The rates of reaction were essentially unaffected. Overall these results do not reveal evidence for a type I pathway, but do provide some support for a type II pathway involving singlet oxygen for the reactions studied in methanol and in methanol–water (3:2, v/v). 4. Conclusions In conclusion, we have shown that the photoreactions of porphyrin (1), chlorin (2) and bacteriochlorin (3) in aqueous methanol in air, using 514 nm laser irradiation under the same conditions in each case, proceed at very different rates and by distinct pathways, even though these compounds have the same peripheral substitution pattern. The porphyrin (1) resists photobleaching and undergoes photomodification to give a new chromophore, whereas the chlorin (2) and the bacteriochlorin (3) are photobleached rapidly. We have evidence to support the view that singlet oxygen is a key reactive species in all three cases. This behaviour is considered to be relevant to the development of these compounds as tumour photosensitizers. Especially is this true for the bacteriochlorin, where the rate of photobleaching is very rapid. In the presence of biomolecules in vitro or in vivo, the rate would be expected to increase still further [17,22], and careful consideration of drug dosage would be expected to produce effective tumour damage with minimal, and rapidly diminishing, generalized photosensitization. The laser experiment is not suitable for product structure studies, but we have observed similar kinetic data using noncoherent sources (tungsten and mercury discharge lamps) in larger-scale reactions. These are now being studied with the aim of separating and identifying the products of photomodification and photobleaching. 5. Abbreviations DABCO HPLC m-THPBC m-THPC m-THPP PDT SOD
1,4-diazabicyclo[2.2.2]octane high-pressure liquid chromatography 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin photodynamic therapy superoxide dismutase
Acknowledgements We thank Scott Loftus for some preliminary experiments and Scotia Pharmaceuticals Ltd. for a gift of m-THPC. This work was supported by EPSRC (CASE Award with Scotia
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Pharmaceuticals Ltd. to B.D.D.) and by the University of London (Laura de Saliceto Cancer Research Postgraduate Studentship to G.M.).
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Photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin (m-THPP) and the corresponding chlorin (m-THPC) and bacteriochlorin (m-THPBC). A comparative study Raymond Bonnett *, Birgul D. Djelal, Peter A. Hamilton, Gabriel Martinez, Franz Wierrani Department of Chemistry, Queen Mary and Westfield College, Mile End Road, London E1 4NS, UK Received 8 July 1999; accepted 1 November 1999
Abstract The photobleaching of compounds of the 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin series at different reduction levels (m-THPP 1, m-THPC 2, m-THPBC 3) has been studied in methanol and in methanol–water (3:2, v/v) using an argon laser (514 nm) by observing the diminution of absorbance of band I (for 2 and 3) and of band IV (for 1) with time. Under the conditions studied here, true photobleaching only occurs for m-THPC (2) and m-THPBC (3), with photomodification being the major process for m-THPP (1). The rates for the photobleaching of 2 and 3 are presented in different solvents. The photobleaching rate of the bacteriochlorin 3 is found to be 90 times higher than that of the chlorin 2 in methanol–water (3:2, v/v). Singlet oxygen appears to be the reactive species responsible for the photobleaching of 2 and 3 and the photomodification of 1. q1999 Elsevier Science S.A. All rights reserved. Keywords: Photodynamic therapy; Photobleaching; m-THPP; m-THPC; m-THPBC
1. Introduction Photobleaching (otherwise referred to as photofading, or in terms of the absence of light fastness) is a well-established study in the science of dyes and pigments [1]. In this context, photobleaching is defined as the loss of absorption of the chromophore caused by visible radiation. Generally it is light fastness that is the desirable property in that area (the indigo of blue denim being a fashionable exception). However, in photochemistry and photobiology the custom has grown up to define photobleaching as the loss of absorption or emission intensity [2] caused by light. Because fluorescence is very sensitive to a variety of quenching effects, the loss of fluorescence does not usually parallel loss of absorption, and it is important to state which mode of observation is being employed. The present paper refers to loss of absorption of photosensitizers that are candidates for applications in photodynamic therapy (PDT). Photodynamic therapy may be defined as the use of light and a sensitizer in the presence of oxygen to treat disease: currently the most intensively studied application is in cancer phototherapy. The process involves some modest cancer tar* Corresponding author. Fax: q44-20-7882-7794; e-mail: R.Bonnett@ qmw.ac.uk
getting on the part of the photosensitizer; the excellent targetting properties of the light (frequently, laser) beam; and the generation within the tumour of singlet oxygen and other reactive oxygen species, which lead to the degradation of the tumour [3]. Photobleaching of photosensitizers during PDT has been observed both in cells in vitro and skin in vivo using the fluorescence mode of observation, both directly and after pigment extraction [4,5]. While photobleaching might be thought to be a disadvantageous property in a tumour photosensitizer (in that the source of the toxic species is being destroyed), it is possible to envisage potential benefits [4– 6]. These would arise if, while the dosage was adjusted to keep tumour levels of photosensitizer at effective levels, the amounts of photosensitizer in surrounding tissue during the irradiation, and in skin and muscle subsequently exposed to ambient light, were below the threshold levels for tissue photosensitization, or, if not, quickly fell below such levels because of photobleaching. Photobleaching of the porphyrin analogues most commonly used in PDT can occur by photo-oxidation or photoreduction processes, photo-oxidation being the more common. Two types of irreversible photobleaching process leading to chemical change in the chromophore are encountered:
1011-1344/99/$ - see front matter q1999 Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 9 9 ) 0 0 1 3 9 - 6
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(i) Photomodification, where loss of absorbance or fluorescence occurs at some wavelengths, but the chromophore is retained in modified form. The formation of the isomeric photoprotoporphyrins A and B [7] and the photo-oxidation of the etiopurpurin series of PDT photosensitizers [8] are examples here. A reductive photomodification is to be found in the photoreactions of haematoporphyrin, where there is evidence for chlorin formation [9]. (ii) True photobleaching, where chemical changes are deep seated, and result in small fragments that no longer have appreciable absorption in the visible region. These fragments have commonly not been identified: the photo-oxidation of bilirubin (which occurs as one of the pathways in the phototherapy of neonatal jaundice) provides an example where the structures of the colourless products are known (maleimides, propentdyopent adducts, dipyrrylmethanes) [10]. It seems that where photomodification occurs, true photobleaching often occurs concomitantly. Early studies [11] showed that 5,10,15,20-tetrakis(mhydroxyphenyl)porphyrin (m-THPP, 1, Fig. 1) was a potent PDT photosensitizer. It was subsequently overshadowed by the corresponding chlorin (m-THPC, 2, Fig. 1), which was even more active [12]. This compound is now being developed under the proprietary name ‘Foscan’ (international nonproprietary name: temoporfin) by Scotia Pharmaceuticals Ltd., Stirling, Scotland. The recent emergence of 2 as a potentially valuable PDT sensitizer has stimulated a number of studies on its photobleaching. Mild photo-oxidation of 2 in methanol in ambient laboratory light for seven days led to the identification of hydroxylated and dehydrogenated derivatives by HPLC– electrospray ionization tandem mass spectrometry. The products were formulated as hydroxy m-THPC (three isomers) and hydroxy m-THPP derivatives: some m-THPP (1) was also detected [13]. The photobleaching of 2 has been compared with that of 1 and with Photofrin in in vitro experiments with Chinese hamster lung fibroblasts [14]. Studies with 2 in protein-containing solutions (foetal calf serum) have shown that the rate of loss of fluorescence is about 15 times the rate of loss of absorbance [15] and that a new product
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with lmaxf320 nm is formed [15,16]. Preliminary studies have appeared on the photobleaching of m-THPBC (3, Fig. 1), which occurs more rapidly than that of the corresponding chlorin [17]. Although the photophysical parameters of 1, 2 and 3 (Fig. 1) have recently been reported [18], there is no comparative kinetic study of the photobleaching of these three substances under defined chemical conditions, and this is now provided.
2. Materials and methods 2.1. Chemicals m-THPP (1) and m-THPBC (3) were synthesized as described [11,12]. m-THPBC (3) contained about 5% of the chlorin (2). m-THPC (2) was a gift from Scotia Pharmaceuticals Ltd. Solvents used were methanol Analar (Merck, Poole, UK), formamide 99.5% (Aldrich, Gillingham, UK) and glass-distilled water. 2.2. Photobleaching kinetics The sensitizer solution (3 ml) was irradiated in a 10 mm=10 mm stoppered quartz cuvette with an argon laser (Spectraphysics 164) tuned to 514 nm. This wavelength approximately corresponds to band IV in the three substrates: the solutions were optically matched (As0.5"10%) at 514 nm at ts0 (1, 0.03 mM; 2, 0.04 mM; 3, 0.02 mM). The laser power was set at 0.78 W and measured using a power meter (Coherent, model 210). At intervals the cuvette was removed and the spectrum in the range 450–800 nm was recorded (Perkin–Elmer Lambda 2 spectrometer) in order to follow the course of photomodification (1, absorbance decay at 512 nm) or photobleaching (absorbance decay at 649 nm for 2, and 734 nm for 3). Preliminary experiments (data not shown) indicated that, under the conditions employed here, the observed rates were insensitive to (i) stirring the solution (Teflon flea, magnetic stirring), (ii) temperature changes within the range 20–368C, and (iii) power density (degree of focusing of the laser within
Fig. 1. Structural formulae for m-THPP (1), m-THPC (2) and m-THPBC (3).
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the window of the cuvette). The experiments described below were carried out with stirring at 20"1oC.
3. Results and discussion 3.1. Effect of structural variation of the substrate In Figs. 2–4, a comparison is made of the photobleaching of 1, 2 and 3, respectively, in methanol–water (3:2 v/v) solutions at the same initial optical density at 514 nm. Fig. 2 (photobleaching of 1) shows that photobleaching does occur slowly at band IV but that band II actually increases in absorbance over the course of 60 min, and the rate of photochange then diminishes. In fact, this Figure demonstrates that the major process occurring here is photomodification and not true photobleaching. Visual observation under a Woods lamp (366 nm) of the starting solution, and of the solution of photoproducts after 60 min, showed that the latter was much less fluorescent than the starting solution, so that a study based on fluorescence diminution would give a quite different apparent result (i.e., it would suggest true photobleaching). Figs. 3 and 4 show the corresponding results for the chlorin 2 and the bacteriochlorin 3, which are in strong contrast to the result for the porphyrin 1. True photobleaching in the visible region occurs in both cases, although in the case of
the chlorin, a new absorbance band appears to be developing below 480 nm. The photobleaching is rapid in both cases, but particularly rapid with the bacteriochlorin 3. Since the reaction is carried out in equilibrium with air, it is presumed that a photo-oxidation process is occurring, and the relative rates thus accord with the known redox potentials (bacteriochlorin-chlorin [19]). The photobleaching of band I (diminution of absorbance at lmax) for the chlorin 2 and bacteriochlorin 3 follows firstorder kinetics in both cases (Fig. 5). The first-order rate constant is 6.3=10y4 sy1 for the chlorin and 5.7=10y2 sy1 for the bacteriochlorin. Thus, under the conditions of our experiment the bacteriochlorin is photobleached about 90 times faster than the chlorin in methanol–water (3:2, v/v). 3.2. Effect of solvent Spikes has shown [20] that the rates of photobleaching of haematoporphyrin, Photofrin, tetraphenylporphyrin tetrasulfonic acid and uroporphyrin are sensitive to solvent changes. Similar effects are found in the present series. As shown in Table 1, the rates of photobleaching of 2 and 3 are lower in pure methanol than in methanol–water (3:2, v/v). The effect is shown graphically for the chlorin 2 in Fig. 6, which should be compared with Fig. 3. In methanol–formamide (1:1, v/ v) and formamide the initial rate for the chlorin 2 was markedly increased, and in each case the reaction kinetics become
Fig. 2. Changes in the visible spectrum of m-THPP (1) at an initial concentration of 0.03 mM in methanol–water (3:2, v/v) in equilibrium with air, on irradiation at 514 nm with an argon laser (Ps0.78 W).
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Fig. 3. Changes in the visible spectrum of m-THPC (2) at an initial concentration of 0.04 mM in methanol–water (3:2, v/v) under the same conditions as described for Fig. 2, except for the time scale.
biphasic, possibly due to oxygen depletion. Because of the rapidity of the reaction, the rate with the bacteriochlorin in these two solvents could not be measured using our methodology. The origins of these solvent effects on rate are not really understood, but it is apparent that the rate depends on the polar nature of the solvent. Aggregation does not appear to play a part, because in the solvent systems employed the spectra suggest that the substrates are monomeric. Spikes [20] has pointed out an apparent correlation of photobleaching with the dielectric constant of the solvent (so k is in the sequence formamide)aqueous methanol)methanol). Possibly the more polar solvents are stabilizing polar or dipolar transition states involved in the reaction with singlet oxygen or other reactive oxygen species. 3.3. Mechanistic considerations In order to assess the role of oxygen in the photobleaching of m-THPC (2) and m-THPBC (3), and in the photomodification of m-THPP (1), solutions of 1, 2 and 3 in methanol were deoxygenated by bubbling nitrogen for at least 1 h; the UV cell was sealed and then irradiated. The photobleaching of the bacteriochlorin 3 was almost suppressed. However, photoreaction of the porphyrin 1 and the chlorin 2 still
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occurred (albeit more slowly), although the changes in the visible spectra were different from those observed in aerated solutions (data not shown). The mechanisms of these photo-oxygenation processes are expected to be complex. It is known that in air-saturated methanol all three of the photosensitizers studied here have significant singlet oxygen quantum yields (FD: for 1, 0.46; for 2, 0.43; for 3, 0.43)[18]. Evidence to support a type I (radical) mechanism and/or a type II (singlet oxygen) mechanism is frequently sought using additives that physically or chemically quench the putative intermediate species [21]. For example, sodium azide physically quenches singlet oxygen, while bilirubin and 1,3-diphenylisobenzofuran rapidly react with it. As type I probes, superoxide dismutase (SOD), catalase and mannitol react rapidly with superoxide radical anion, hydrogen peroxide and hydroxyl radicals, respectively: if these species influence the rate-determining step, the relevant additions will lower the rate of reaction. Singlet oxygen has a longer lifetime in a deuterated solvent compared with that in the corresponding protiated solvent, and so, provided the substrate is of modest reactivity and deactivation by solvent molecules determines singlet oxygen concentration, the rate will be enhanced in the deuterated solvent.
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Fig. 4. Changes in the visible spectrum of m-THPBC (3) at an initial concentration of 0.02 mM in methanol–water (3:2, v/v) under the same conditions as Fig. 2, except for the time scale. Table 1 First-order rate constants (k, sy1) for photobleaching in various solvents MeOH
m-THPC
1.7=10y4 6.3=10y4
m-THPBC
1.0=10y2 5.7=10y2
a b
Fig. 5. First-order plots of the photobleaching of m-THPC (2) and mTHPBC (3) in methanol–water (3:2, v/v).
It has already been shown that, for m-THPC 2 in aqueous medium containing 10% foetal calf serum, the photobleaching process involves singlet oxygen on the evidence of rate diminution in the presence of singlet oxygen quenchers (DABCO, 25% decrease observed; histidine, 35% decrease observed), whereas evidence for a type I process was not found (no appreciable effect of SOD, catalase or deferoxamine) [16]. In ethanol, the rate of photobleaching of 1,3-
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MeOH–H2O MeOH–formamide Formamide (3:2) (1:1) 1.0=10y3 a 2.2=10y4 b
2.8=10y3 a 1.2=10y4 b
ts0–7.5 min. ts10–30 min.
diphenylisobenzofuran in the presence of 2 was enhanced on deuteration of the solvent [16]. In the present study, evidence has been found that supports a contribution from a singlet oxygen mechanism in the photomodification of 1 and in the photobleaching of 2 and 3 in methanol and in methanol–water (3:2, v/v). The results are presented in Table 2 for the porphyrin 1, Table 3 for the chlorin 2, and Table 4 for the bacteriochlorin 3. When sodium azide (cs8 mM) was incorporated into methanolic solutions, the reaction rates were reduced in each case (by 51% for 1, by 60% for 2, and by 29% for 3). Addition of Rose Bengal (3=10y6 M), a well-known and effective photogenerator of singlet oxygen [21], increased the rate of reaction for 1 (by 100%) and for 2 (by 47%).
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Fig. 6. Example of the solvent effect on photobleaching: course of photobleaching of m-THPC in methanol using the same conditions as in Fig. 3.
Table 2 Apparent first-order rate constants for the diminution of band IV of m-THPP 1 (cs3=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
None NaN3 (8=10y3 M) Rose Bengal (3=10y6 M)
MeOH MeOH MeOH
None None SOD (0.25 mg mly1) Catalase (0.15 mg mly1) Mannitol (cs10y2 M)
MeOH–H2O (3:2) CD3OD–D2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
k (sy1) 3.3=10y5 1.6=10y5 6.8=10y5 9.6=10y5 8.4=10-4 10.0=10y5 10.2=10y5 10.2=10y5
Table 3 First-order rate constants for the diminution of band I of m-THPC 2 (cs4=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
k (sy1)
None NaN3 (8=10y3 M) Rose Bengal (3=10y6 M)
MeOH MeOH MeOH
1.7=10y4 6.7=10y5 2.5=10y4
None SOD (0.25 mg mly1) Catalase (0.15 mg mly1) Mannitol (10y2 M)
MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
6.3=10y4 6.6=10y4 7.7=10y4 7.8=10y4
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Table 4 First-order rate constants for the diminution of band I of m-THPBC 3 (cs2=10y5 M) with irradiation at 514 nm in air under various conditions Additive
Solvent
k (sy1)
None NaN3 (8=10y3 M) Mannitol (10y2 M)
MeOH MeOH MeOH
1.0=10y2 7.1=10y3 1.1=10y2
None SOD (0.25 mg mly1) Catalase (0.15 mg mly1)
MeOH–H2O (3:2) MeOH–H2O (3:2) MeOH–H2O (3:2)
5.7=10y2 5.3=10y2 4.6=10y2
(This additive was not studied with the bacteriochlorin, since the rate was already as fast as we could reliably measure using our methodology.) In one case, that of the porphyrin in methanol–water (3:2, v/v), we have observed the effect of using a completely deuterated solvent under otherwise identical reaction conditions. The observed first-order rate constant rose from ks9.6=10y5 to 8.4=10y4 sy1, consistent with the involvement of singlet oxygen in the reaction. On the other hand, additives designed to test for a type I pathway (SOD, catalase) in methanol–water (3:2, v/v) were essentially without effect on the reaction rate (Tables 2–4). Mannitol (cs10 mM), which reacts rapidly with hydroxyl radicals, was added to the bacteriochlorin 3 in methanol
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(Table 4) and to the porphyrin 1 and the chlorin 2 in methanol–water (3:2, v/v) (Tables 2 and 3). The rates of reaction were essentially unaffected. Overall these results do not reveal evidence for a type I pathway, but do provide some support for a type II pathway involving singlet oxygen for the reactions studied in methanol and in methanol–water (3:2, v/v). 4. Conclusions In conclusion, we have shown that the photoreactions of porphyrin (1), chlorin (2) and bacteriochlorin (3) in aqueous methanol in air, using 514 nm laser irradiation under the same conditions in each case, proceed at very different rates and by distinct pathways, even though these compounds have the same peripheral substitution pattern. The porphyrin (1) resists photobleaching and undergoes photomodification to give a new chromophore, whereas the chlorin (2) and the bacteriochlorin (3) are photobleached rapidly. We have evidence to support the view that singlet oxygen is a key reactive species in all three cases. This behaviour is considered to be relevant to the development of these compounds as tumour photosensitizers. Especially is this true for the bacteriochlorin, where the rate of photobleaching is very rapid. In the presence of biomolecules in vitro or in vivo, the rate would be expected to increase still further [17,22], and careful consideration of drug dosage would be expected to produce effective tumour damage with minimal, and rapidly diminishing, generalized photosensitization. The laser experiment is not suitable for product structure studies, but we have observed similar kinetic data using noncoherent sources (tungsten and mercury discharge lamps) in larger-scale reactions. These are now being studied with the aim of separating and identifying the products of photomodification and photobleaching. 5. Abbreviations DABCO HPLC m-THPBC m-THPC m-THPP PDT SOD
1,4-diazabicyclo[2.2.2]octane high-pressure liquid chromatography 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin 5,10,15,20-tetrakis(m-hydroxyphenyl)porphyrin photodynamic therapy superoxide dismutase
Acknowledgements We thank Scott Loftus for some preliminary experiments and Scotia Pharmaceuticals Ltd. for a gift of m-THPC. This work was supported by EPSRC (CASE Award with Scotia
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Pharmaceuticals Ltd. to B.D.D.) and by the University of London (Laura de Saliceto Cancer Research Postgraduate Studentship to G.M.).
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