Practica: Modulación de FM

The use of light in the treatment of various diseases can be dated back to about three thousand years ago, used by both Egyptians and Chinese civilisations.110 Phototherapy involves the use of light in combination with a chemical agent to induce a cytotoxic effect in the biological region of interest. The concept of phototherapy can be dated back to ca. 1900 from reports by Oscar Raab, effectively reporting on the cytotoxic effect of acridine in combination with light on infusoria.111

1.7.1 Photodynamic therapy

Studies performed by Dougherty led to the development of photodynamic therapy, PDT.112,113 PDT involves the administration of a non-toxic complex, a photo- sensitiser (PS), into a patient’s bloodstream. After an incubation period (to allow sufficient accumulation of the PS into the target tissue) the PS is subject to

36 irradiation at a specific wavelength. Longer wavelengths of light (>600 nm) are used in PDT owing to its ability to penetrate into deeper tissues (Figure 1.23).114

PDT is often used in cancer treatment115 but extends into treatment of both infectious diseases and disorders.116

Figure 1.23Percentage and depth penetration by various wavelengths of light (figure adapted from ref 114).

Often, PS are porphyins consisting of four pyrrole rings fused together by methine bridges and possess an absorption in the region of 600-800 nm.117 The first generation photo-sensitiser synthesised by Dougherty, Photofrin® (Figure 1.24A), was approved clinically for the treatment of lung, esophageal and bladder cancer.118 In contrast, Foscan® _{(Figure 1.24B), a second generation PS compound, has been}

clinically approved for head and neck cancer. Interestingly, this compound accumulates more specifically in the cancerous tissue and is irradiated with a slightly longer wavelength of light (652 nm) compared to Photofrin at 630 nm.119

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Figure 1.24 Structures of the clinically approved photo-sensitisers Photofrin (30) and Foscan (31).

1.7.1.1 Mechanism of PDT

PDT has three essential requirements: (a) a photo-sensitiser, (b) light and (c) molecular oxygen (O2) necessary to induce a PDT effect in the cancerous tissue.

The PS should have minimal dark toxicity in both human and animal models.115 The process involves the irradiation of the PS in the ground (S0) state with a specific wavelength of light (generally matched to the absorption of the administered compound). Photon excitation promotes the PS into a single excited (S1) state. Once absorption occurs, the excited state can dissipate its energy through photo-physical pathways, as previously described (Figure 1.25).113,117 The longer lifetime (ca. µs – ms) of the triplet excited (T1) state compared to the singlet excited (S1) state suggests that the T1 excited state is involved in the photodynamic effect. The energy of the T1 state can be dissipated through phosphorescence (T1S0).

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Figure 1.25 Absorption of light by the singlet ground state (S0), generating a singlet excited (S1) and the triplet excited (T1) states, the latter formed through intersystem crossing (ISC). Quenching of the T1 state by molecular oxygen (3O2) via type I ()

or type II () processes induces the PDT effect through the formation of various reactive oxygen species (figure adapted from ref 117).

Quenching mechanisms for the T1 excited state have been reported to induce the observed PDT effect.120 Two quenching processes exist, type I and type II. The type I process involves either an electron or hydrogen transfer between the T1 state and a molecular substrate i.e. cell membrane, generating free radical species. These formed radical-based species interact with molecular oxygen (3_O

2) present in the

cell leading to the formation of ROS. In contrast, a direct interaction between the T1 excited state and 3O2 occurs during type II. Through an energy transfer

mechanism, singlet oxygen (1O2, 1g) is generated from this interaction (Figure

1.25). As mentioned earlier, 1O2 is a well-known cytotoxic species and is believed

39 reported to proceed during PDT. The ratio of type I over type II is dependent on the concentration of the substrate, molecular oxygen and the administered photosensitiser.121

PDT remains a highly used cancer therapy treatment as opposed to surgery due to its non-invasive nature. Additionally, the formation of 1O2 has been attributed as the

main cytotoxic species inducing the PDT effect. Formed at the site of irradiation,

1_O

2 has a short life-time and more importantly possesses a very short diffusion

distance of ca. 0.02 µm.122 Thereby, healthy nearby cells remain undamaged. However, numerous disadvantages are associated with current PDT therapy. Firstly, the requirement of molecular oxygen inside the cell is a limiting factor of PDT treatment, due to the common hypoxic (3O2-deficient) environment of cancerous

tissue.123 Secondly, despite the reported non-invasive nature, the long clearance times (4-8 weeks) of the PS from the patient’s body limit the patient’s exposure to daylight up to periods of a couple of months.117 Finally, resistance mechanisms similar to those previously reported for conventional chemotherapy and radiotherapy treatments have emerged.124 PDT resistance has been reported to be dependent on both the concentration of the PS and the light dose administered.125 Antioxidant enzymes have been reported to inactivate the toxic reactive oxygen species (●OH, O2●- and 1O2) during first stage PDT treatment.125 Another mode of

resistance has been noted through heat shock proteins (HSPs) which possess the ability to prevent unwanted protein aggregation and stabilise unfolded proteins, resulting in the repair of the damage induced by PDT.126 Therefore, another mode of cancer therapy is needed to overcome current resistance mechanisms.

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