Volume 4, Issue 1, Pages 3-11 (March 2007)

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The history of PDT in Norway: Part one: Identification of basic mechanisms of general PDT

published online 28 December 2006.

Summary

Photodynamic therapy (PDT) is now an established treatment of malignant and premalignant dysplasias. A number of first and second generation photosensitizers have been studied in Norway. The aim has been to improve PDT efficiency and applicability. Many critical details regarding the mechanisms of PDT were elucidated by researchers in Norway. In this review we focus on the most important findings related to these basic mechanisms, such as generation of singlet oxygen, estimations of its lifetime, the oxygen effect itself, the subcellular localization of photosensitizers with different properties, their photodegradation during PDT and their tumour selectivity.

Abbreviations: ALA, 5-aminolevulinic acid, AlPcS2, disulfonated aluminum phthalocyanine, AlPcSn, sulfonated aluminum phthalocyanines, AlPcTs, aluminium phthalocyanine tetrasulfonate, BPD-MA, benzoporphyrin derivative monoacid ring A, CLSM, confocal laser scanning microscopy, DHE, dihematoporphyrin ether, ESR, electron spin resonance, Hp, hematoporphyrin, HpD, hematoporphyrin derivative, MBD, methylene blue derivative, MC 540, merocyanine 540, m-THPBC, meso-tetrahydroxyphenyl-bacter-iochlorin, m-THPC, meso-tetra(hydroxyphenyl)chlorin, m-THPP, meso-tetra(3-hydroxyphenyl)porphine, PDT, photodynamic therapy, PpIX, protoporphyrin IX, 1O2, singlet oxygen, TAN, 2,2,6,6-tetramethyl-4-piperidone-N-oxyl, TMPyPH2, meso-tetra-(N-methyl-4-pyridyl)porphine, TMPD, 2,2,6,6-tetramethyl-4-piperidone, TPPSn, sulfonated tetraphenylporphines, ZnPc, zinc phthalocyanine

Keywords: Photodynamic therapy, Photosensitizers, Singlet oxygen, Photodegradation

Article Outline

• Summary

• Introduction

• First generation photosensitizers

• Second generation photosensitizers

• Oxygen

• Action spectra for photosensitizers in vitro and in vivo and choice of optimal wavelength for PDT

• The subcellular localization of photosensitizers and the target sites in cells and tissues

• Photodegradation of photosensitizers during PDT

• Effects of PDT on DNA and chromosomes

• References

• Copyright

Introduction

Clinical applications of photodynamic therapy (PDT) using a derivative of hematoporphyrin (HpD) were pioneered by Thomas J. Dougherty (USA) in the 1970s. At that time, in 1976, Johan Moan at the Norwegian Radium Hospital in Oslo (Norway) became interested in PDT and from then on his group contributed continuously to the development of PDT (Fig. 1). After 17 years of intense research, PDT was approved for the treatment of bladder cancer first in Canada and later in other countries. Chemical and cellular processes leading to photodynamic cell inactivation were poorly known. Large contributions in the field of PDT have been made in the Norwegian Radium Hospital. Many local and visiting scientists from more than 20 countries have participated in these studies in Norway. Some of them just got Ph.D. degrees in the PDT field: Terje Christensen, Stein Sommer, Jon Folkvard Evensen, Magne Kongshaug, Egil Kvam, Helle Anholt, Barbara Noodt, Jostein Dahle, Øystein Bech Gadmar, Beata Čunderlíková, Petras Juzenas and others. Kristian Berg, Qian Peng, Li-Wei Ma, Ana Soler, Trond Warloe, Odrun Arna Gederaas and others continued to work in the field.

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Figure 1. Prof. Johan Moan. Johan Moan is a Professor of Biophysics at the University of Oslo and the leader of the group of ‘Biophysics and PDT’ at the Norwegian Radium Hospital in Oslo (Norway). He obtained his master degree at Trondheim University in 1970, with the highest marks, and consequently was reported to His Majesty the King of Norway. He received his Ph.D. in 1975 at the University of Oslo in the fields of radiation chemistry and physics. Since 1976 the main research field has been PDT. This research has led to the start of clinical PDT at the Norwegian Radium Hospital in 1991. His main research fields are PDT, photocarcinogenesis, skin cancer epidemiology and Vitamin D photobiophysics. He is a co-author of 425 scientific papers and co-editor of 6 books.

First generation photosensitizers

In the 1970s hematoporphyrin (Hp) and HpD were the most frequently used photosensitizers. They were later called the first generation photosensitizers. HpD is a mixture of porphyrins, and its main chemical composition, photochemical and photosensitizing properties were thoroughly analysed by Norwegian scientists [1], [2], [3], [4], [5], [6]. It was shown that HpD contained at least seven components, both lipophilic and hydrophilic. These components have different intracellular and tumour localization properties, and photosensitivities. As an important task it was to identify the component that contributed most to an optimal therapeutic outcome when tumours and cells were exposed to light in the presence of HpD. A world-famous biochemist Professor Claude Rimington joined Moan's group in 1984. He soon realized that in order to find the most active component of HpD one needed to study pure porphyrins with different hydrophobicities. He developed methods of preparing and purifying a series of new photosensitizers, including hematoporphyrin ethers [7], [8], [9]. Their biochemical properties were tested, and those most likely to be effective in treating cancers were selected for further work [10], [11], [12], [13], [14]. Prof. Rimington's research laid the ground for the development of better photosensitizers, and for his work he received a prestigious national award, the Kings medal of honour.

Second generation photosensitizers

The major side effect of HpD is cutaneous photosensitivity. A number of second generation photosensitizers of different chemical families were synthesized in the 1980s. Sulfonated tetraphenylporphines (TPPSn) [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], aluminum and zinc phthalocyanines [34], [35], [36], [37], [38], [39], [40], [41], meso-tetra(hydroxyphenyl)chlorin (m-THPC) [31], [33], [42], [43], [44], [45], [46], [47], [48], chlorin e6 [16], [31], [49], meso-tetra(3-hydroxyphenyl)porphine (m-THPP) [31], [33], [43], [50], [51], merocyanine 540 (MC 540) [16], [52], hypericin [53], [54], [55], meso-tetra-(N-methyl-4-pyridyl)porphine (TMPyPH2) [27], [30], methylene blue derivative (MBD) [56], [57], etc., were tested in our laboratories. Scientists in Norway evaluated their physicochemical properties, intracellular/tissue uptake, localization, photodegradation and photodynamic efficiencies both in vitro and in vivo.

For example, Ma et al. [42] and Peng [58] found in 1994 that m-THPC was an extremely potent photosensitizer for tumour eradication. Disulfonated aluminum phthalocyanine (AlPcS2) was shown to be another efficient photosensitizer [58]. In 2001, PDT using m-THPC (Foscan, Biolitec, The Netherlands) was approved in the European Union, Norway and Iceland as a local therapy for the palliative treatment of patients with advanced head and neck cancer who had failed prior therapies or were unsuitable for radiotherapy, surgery or systemic chemotherapy.

The properties of an ideal photosensitizer should include [59]: (1) minimal dark toxicity, (2) selective accumulation in target tissue, (3) a short time interval between administration and maximal accumulation in target tissue, (4) rapid excretion from the body to give minimal systemic toxicity, (5) a high quantum yield of cell inactivation, which is often mediated by singlet oxygen (1O2) and (6) a high extinction coefficient in the 600–800nm range, where light penetration into tissue is maximal, and where the photons still have enough energy to produce 1O2. However, such an ideal photosensitizer has not been found yet. Design of strategies for new photosensitizers are directed towards specificity, i.e. greater tumour selectivity with reduced skin phototoxicity.

Oxygen

Already in 1978 the researchers in Moan's group recognized the importance of 1O2 in a variety of chemical and biological processes, for example, dye-sensitized photooxidation of lipids, proteins and nucleic acids, photodynamic inactivation of viruses and cells, and phototherapy of cancer. Highly reactive 1O2, formed during PDT, was found to be the main toxic reagent. Evidence for the production of 1O2, its role in cell photoinactivation during PDT and the importance of oxygenation status in tumour tissues were extensively studied by the group [60], [61], [62], [63], [64], [65].

The methods used to detect 1O2 at that time were unspecific, of low sensitivity or laborious. A new, specific electron spin resonance (ESR) method of detecting 1O2 production from Hp exposed to light was proposed by Moan and Wold in 1979 [60]. The detection was based on the reaction of 1O2 with sterically hindered amines 2,2,6,6-tetramethyl-4-piperidone (TMPD) leading to a stable free nitroxide radical adduct 2,2,6,6-tetramethyl-4-piperidone-N-oxyl (TAN) [66], which can be measured by ESR [60]. ESR employing TMPD was capable of determining a concentration of 10−8M of TAN. The ESR signal of TAN can be used for highly selective monitoring of 1O2 [60].

The most widely used and studied photosensitizers until 1990 were Hp, HpD (Photofrin I) and dihematoporphyrin ether (DHE, Photofrin II). It was known that Hp, as well as other porphyrins, aggregate when their concentration is increased. HpD and DHE are mixtures of porphyrins in different states of aggregation. The photochemical yield of 1O2 from porphyrins in different states of aggregation was investigated by Moan in 1984 [62]. He found that photoexcitation of the clinically used Photofrin I and Photofrin II produces 1O2 with significantly lower yields than photoexcitation of Hp. It was concluded that the fluorescence quantum yield and the singlet oxygen quantum yield of an aggregated sensitizer are remarkably lower than those of the monomeric form [62].

In 1985 Moan and Sommer studied the oxygen dependence of the PDT effect of HpD in cancer cells [63]. They found that the efficiency of photoinactivation of the photosensitized cells decreased with decreasing oxygen concentration. In anoxic suspensions no photosensitivity was observed. The quantum yield for photoinactivation was reduced by 50% when was reduced from 100 to 1% O2 (1% O2∼=7.6mmHg). They found that the inactivation efficiency is strongly dependent on the O2 concentration, that hypoxic tumour cells are resistant to PDT, and suggested to increase the tumour oxygenation status during the treatment by adding O2 to the air breathed by patients during the therapy [63].

Moan and Boye observed that 1O2 generated outside Escherichia coli bacteria or human cells does not introduce DNA strand breaks as long as the photosensitizer is outside the bacteria or the cells [67], [68]. These experiments indicated that 1O2 generated outside the cell wall cannot penetrate this wall, which thickness is approximately 0.025–0.050μm [64]. In another series of experiments Moan et al. incubated NHIK 3025 cells with Photofrin II and/or tetra (3-hydroxyphenyl)porphyrin (3-THPP) and exposed to light at either 400 or 420nm, and studied the kinetics of the photodegradation of the dyes. The diffusing agent, which caused the photodegradation, was supposedly 1O2 [12]. The distance diffused by 1O2 was estimated to be of the order of 0.01–0.02μm, which corresponds to a lifetime of 0.01–0.04μs of the intermediate in the cells [65]. Thus, 1O2 can diffuse less than 0.05μm during its short lifetime from its site of origin before reacting with, or being quenched by, a variety of cellular targets [64], [65]. Therefore, photodynamic damage occurs close to the localization of photosensitizing molecules during light exposure. The subcellular localization of the photosensitizer is of crucial importance, since it determines the localization of the primary damage and its impact.

Action spectra for photosensitizers in vitro and in vivo and choice of optimal wavelength for PDT

Lasers as well as non-coherent light sources are being used for PDT [69]. An advantage of using lasers is that their light can be focused into fibre systems and led to otherwise inaccessible locations, such as urinary bladder, digestive tract or brain. For dermatology, however, non-laser sources are superior to laser systems because of their large illumination field, lower cost, smaller size, reliability and easy setup.

Choice of light source depends primary on the depth, which must be reached by light [70], [71], [72]. Because of the shape of the absorption spectrum of the main chromophores in tissue, hemoglobin and melanin, the penetration depth increases with the wavelength in the visible and near infrared spectral regions [70], [71], [72]. The chosen wavelength has to match an absorption band of the photosensitizer. Aggregated and monomeric porphyrins have different absorption spectra and fluorescence quantum yields [1], [62]. The optimal wavelength should give maximal yield of 1O2 at maximal depth. Such spectrum of the photosensitizer with respect to cell photoinactivation has to be considered. The action spectrum describes the relative effectiveness of different wavelengths in producing the desired biological response. Moan [62] found in 1984 that the action spectra indicated that aggregated porphyrins do not contribute significantly to the photosensitivity of cells. Furthermore, they found that only cell bound photosensitizers, and not those present in the fluid around the cell, were efficient [73]. The action spectra for HpD, Photofrin II, 3-THPP, chlorin e6, aluminium phthalocyanine tetrasulfonate (AlPcTs), benzoporphyrin derivative monoacid ring A (BPD-MA), zinc phthalocyanine tetrasulfonate (ZnPcS4), AlPcS2, ZnPc, etiopurpurin were determined in vitro or in vivo [37], [74], [75], [76]. These spectra were found to have the same shape as the fluorescence excitation spectrum of the photosensitizer, indicating that primarily nonaggregated molecules generate 1O2 [37], [74], [75], [76]. Thus, only the fluorescent fraction of a photosensitizer is efficient.

Action spectra, adjusted for penetration spectra through tissue, show the optimal wavelength for killing cells at a given depth below the tissue surface, as demonstrated by Moan et al. [77]. Down to about 2mm blue light in the Soret band (∼410nm) is more efficient with porphyrin photosensitizers than red light [77]. This is due to the high extinction coefficient of porphyrins in the blue region, being about 20 times larger than that in the red.

The subcellular localization of photosensitizers and the target sites in cells and tissues

As mentioned, the highly reactive 1O2 has a short radius of action of only about 20nm in cells [64], [65]. Consequently, 1O2-mediated oxidative damage will occur in the immediate vicinity of the subcellular site of photosensitizing molecule localization during light exposure. Therefore, the availability of oxygen and the subcellular localization of the photosensitizer determine which organelles are primarily damaged by PDT. In order to increase the PDT efficiency, and reduce side effects, the subcellular/intratumoural localization was continuously investigated with the aim to find photosensitizers with high and selective uptake in sensitive subcellular sites.

Intracellular distribution patterns of photosensitizing dyes were thoroughly studied in vitro by Moan's group [78], [79], [80] and others [81], [82], [83], [84], [85], using conventional fluorescence microscopy. This method shows the intracellular localization of fluorescing photosensitizers. Unfortunately, photobleaching and photodamage are serious limitations of fluorescence microscopy in the study of living cells and tissues. Many photosensitizers are rapidly photodegraded by the strong light of the microscope, or they have too weak fluorescence. By confocal laser scanning microscopy (CLSM) it is possible to performe detailed studies of weakly fluorescencing fluorochromes with high accuracy, even under conditions when they are rapidly photobleached. CLSM was for the first time used in our hospital by Peng et al. in 1990 [21], [35].

HpD and Photofrin are lipophilic compounds, and are concentrated in plasma membranes, mitochondria, endoplasmic reticulum, nuclear membranes and perinuclear regions in cells in vitro [78]. HpD and Photofrin contain a number of porphyrins with different hydrophobicities with different retention in a variety of intracellular compartments. Following light exposure, membranes are preferentially damaged [78]. However, damage can also be seen at other sites, including mitochondria, endoplasmic reticulum and Golgi complex. It was noticed by Peng that pure photosensitizers are better suited than HpD for studying subcellular distributions and PDT effects [58]. Photosensitizers vary widely in PDT activity, even within series of molecules with similar basic structure (e.g. sulfonated aluminum phthalocyanines (AlPcSn) and TPPSn) [19], [32], [40]. Small differences in the physicochemical characteristics of the compounds alter uptake and subcellular distribution [32]. Even when different photosensitizers appear to target the same organelle, their effectiveness can differ significantly [32]. Protoporphyrin IX (PpIX), induced by 5-aminolevulinic acid (ALA), is initially localized in mitochondria, whereas exogenous PpIX is mainly distributed in plasma membranes [86]. At similar intracellular concentration of PpIX, PDT with ALA is significantly more efficient than PDT with exogenously added PpIX [86]. Factors such as charge, aggregation and lipophilicity, mode of drug delivery, time interval between drug administration and light exposure influence the subcellular localization [32]. The differences between photosensitizers depend on cell/tumour type [87], [88]. Lipophilic dyes generally localize in membrane structures [78] and hydrophilic ones usually in lysosomes [21], [39].

The PDT efficacy may be related to the intratumoural localization of the photosensitizer. It was shown by Peng [58], that photosensitizers which were localized in the vasculature and in the tumour cells (as m-THPC and AlPcS2) were the strongest photosensitizers.

In our hospital it was found that cell-bound photosensitizers are much more efficient in cell inactivation than unbound photosensitizers [3]. The efficiency of a photosensitizer is strongly dependent on its localization within a cell (Table 1): hydrophilic sensitizers are far less efficient than lipophilic ones with comparable singlet oxygen yields [12], [40]. During exposure to light the intracellular localization may alter, giving rise to a change in the quantum yield of cell inactivation [89]. The intracellular localization of photosensitizers is also related to the mechanism by which the photosensitizers are taken up or produced by the cells. For therapeutic applications it may be of interest to modify the uptake of a photosensitizer or its production from a precursor [79] in the cell. The intracellular localization of a photosensitizer determines the PDT efficiency and the mechanism of cell death [39].

Table 1.

Sites of photosensitizer localization in cells


Photosensitizer	Cell linesa	Localization/targets	References
HpD, Photofrin	NHIK 3025, LOX, V79, THX	Mainly on the membranes, some in the cytoplasm, some in mitochondria	Moan et al. [78], Peng et al. [21], Noodt et al. [27], Prasmickaite et al. [90]
3-THPP	LOX, V79, MDCK II, THX	Golgi apparatus, endoplasmic reticulum, plasma membranes	Peng et al. [21], Noodt et al. [27], Dahle et al. [91], Prasmickaite et al. [90]
TPPS1	NHIK 3025	Membranes	Moan et al. [79], Berg et al. [18]
TPPS2a	NHIK 3025, THX	Lysosomes, endosomes, membranes	Berg et al. [18], Prasmickaite et al. [90]
TPPS4	NHIK 3025, V79, THX	Lysosomes, endosomes, membranes, nucleus	Moan et al. [79], Berg et al. [18], Noodt et al. [27], Prasmickaite et al. [90]
TPPS2o	NHIK 3025	Lysosomes	Moan et al. [79], Berg et al. [18]
AlPcS1, AlPcS2	LOX	Membranes	Peng et al. [21], Peng et al. [58]
AlPcS3, AlPcS4	LOX	Lysosomes	Peng et al. [58]
AlPcS2a	THX	Lysosomes, endosomes	Prasmickaite et al. [90]
p-TMPyPH2	THX	Cytosol, lysosomes, endosomes	Noodt et al. [27], Gaullier et al. [92], Prasmickaite et al. [90]
ZnPc	NHIK 3025	Mitochondria, Golgi apparatus	Rodal et al. [93], Rodal et al., 1998 [94]
MBD	V79	Mitochondria	Noodt et al. [57]
Nile blue A	THX	Lysosomes, endosomes, perinuclear region	Prasmickaite et al. [90]
ALA-PpIX	WiDr, NHIK 3025, V79, THX, KYSE-450, KYSE-70, Het-1A	Mitochondria, endoplasmic reticulum and plasma membrane	Gaullier et al. [88], Prasmickaite et al. [90], Ji et al. [86]
ALA-esters-PpIX	WiDr, NHIK 3025, V79	Cytoplasm, plasma membrane	Gaullier et al. [88]
PpIX	KYSE-450, KYSE-70, Het-1A	Membranes	Ji et al. [86]
Hypericin	WiDr, NHIK 3025, D54Mg	Endoplasmic reticulum, Golgi apparatus, nuclear	Uzdensky et al. [53]

Human cervix carcinoma cell line NHIK 3025, human adenocarcinoma cell line WiDr, human melanoma cell line LOX, Chinese hamster lung fibroblast cell line V79, human malignant melanoma cell line THX, Madison–Darby canine kidney cell line MDCK II, human oesophageal cell lines KYSE-450, KYSE-70 and Het-1A and human glioma cell line D54Mg.

Photodegradation of photosensitizers during PDT

Until 1984–1986, it was assumed that porphyrins were rather stable during PDT and could be activated indefinitely to produce the desired therapeutic effect. However, Moan reported the first relevant observations of loss of fluorescence (photobleaching) of HpD [95] and of DHE (Photofrin II) incubated in NHIK 3025 cells [96] in 1984–1986. Shortly afterwards, in collaboration with Moan, photobleaching was observed by Mang et al. in patients receiving treatment with DHE in USA [97]. The crucial point of photobleaching was already noticed by Moan in 1984 [95], who pointed out that a tumour should contain enough photosensitizer in order not to be completely bleached during PDT. At the same time the potential benefit of photobleaching was recognized: he proposed to use a low dose of the photosensitizer in combination with high dose of light in order to reduce the damaging effects to normal tissue [96]. Since the porphyrin level in normal tissue is usually much lower than that in tumours, it may be possible to choose the porphyrin dose at a level where normal tissue is little damaged (due to photobleaching) while tumour tissue is destroyed.

Photostability is a therefore fundamental factor to consider in the evaluation of new photosensitizers for PDT. Moan's group has investigated the photodegradation of a number of photosensitizers including AlPcSn (n=1–4) in LOX cells [36], m-THPC and m-THPP in V79 cells [42], m-THPC and meso-tetrahydroxyphenyl-bacteriochlorin (m-THPBC) in mice [42], [44], ALA-induced PpIX in WiDr cells [98], [99], [100] and in mice [101], [102], [103], PpIX induced by methyl 5-aminolevulinate in WiDr cells [69], [104] and in mice [69], Photofrin (PII), benzoporphyrin derivative mono acid ring A (BPD), chlorin e6, m-THPC, m-THPP, tetraphenylporphine tetrasulfonated (TPPS4), AlPcS2, AlPcS4 and zinc phthalocyanine (ZnPc) in mice [31], hypericin in WiDr cells and in mice [54]. The main results are described systematically in a review [105]. Almost all photosensitizers are photolabile, but have widely different photostabilities. Water-soluble dyes seem to be more stable than lipophilic ones [31], [105]. This may be related to the intracellular localization [21] and to the diffusion length of 1O2 [65]. Photobleaching and phototransformation of photosensitizers, mediated by 1O2, result in formation of fluorescing photoproducts and/or breakdown of the porphyrin macrocycle [106]. Photobleaching also requires 1O2, just as tumour destruction does. Thus, photodegradation rates can be used for clinical dosimetry.

In their review Bonnett and Martinez recognized Moan and his group as not just the first to observe the photobleaching, but also as the most prominent workers in the field [107].

Effects of PDT on DNA and chromosomes

In the early 1980s it was well known that DNA and chromosome damage were produced by chemotherapy and radiotherapy. Evaluation of the carcinogenic and mutagenic risk of PDT became necessary. As a first stage of such an evaluation, the PDT effect of Hp on DNA in vitro was studied in 1980. Hp does not bind to DNA but still PDT with this dye gave rise to 1O2 mediated alkali-labile sites in DNA [67]. At a given level of survival the frequency of single-strand breaks and the induction of sister chromatid exchanges were higher after X-radiation than after PDT [68], [108], [109]. This indicated that the carcinogenic risk was smaller after PDT than after ionizing radiation [108]. PDT with HpD also induced DNA and chromosome damage, but significantly more damage were generated with X-rays [73], [110]. Even though the extent of damage to DNA induced by PDT seemed to be small when compared with the effects on DNA induced by other forms of cancer therapy, they must be considered whenever new PDT photosensitizers are introduced [111]. Later a variety of photosensitizers was investigated in our hospital to study the mechanisms of induction of DNA and chromosomes damage, to evaluate the genotoxic and mutagenic potential of PDT [27], [29], [30], [112], [113], [114], [115], [116], [117]. It was concluded that PDT can directly or indirectly damage DNA. Photodynamic damage, due to a short action radius of 1O2, occurs close to the location of a photosensitizer during light absorption. In agreement with this, it was found that only a small fraction of DNA in cells can be damaged by PDT, since the most porphyrins do not enter the nucleus. In fact PDT could be exploited to study the chromosome organization in interphase cells [110]. On the contrary, we and others have shown that PpIX in mouse skin has an antiphotocarcinogenic action when the skin is exposed to otherwise carcinogenic UV radiation [118], [119], [120], [121].

References

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a Department of Radiation Biology, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

b Institute of Physics, Oslo University, Blindern, 0316 Oslo, Norway

Corresponding author. Tel.: +47 22935113; fax: +47 22934270.

PII: S1572-1000(06)00150-5

doi:10.1016/j.pdpdt.2006.11.002

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