Silicon Phthalocyanine 4 Phototoxicity in Trichophyton rubrum

Authors: Minh Lam, Matthew L. Dimaano, Patricia Oyetakin-White, Mauricio A. Retuerto, Jyotsna Chandra, Pranab K. Mukherjee, Mahmoud A. Ghannoum, Kevin D. Cooper, and Elma D. Baron

Publication Year: 2014

PMID: 24614382

Utilizing mainly keratin for their source of energy, dermatophytes can cause dermatophytosis or tinea of the skin, hair, and nails that impact the quality of life of infected patients, especially in immunocompromised individuals. Trichophyton rubrum is the main culprit of superficial mycosis of the nail (onychomycosis), which is often long lasting and has a high incidence of recurrence. Onychomycosis affects 10% of the general population, 20% of the population older than 60 years, 50% of people older than 70 years, and 30% of diabetic patients, and it can often result in pain, disability, and psychosocial stress, therefore significantly reducing quality of life (1,–3). The conventional treatments involve excruciating surgical nail avulsion and toxic systemic antifungal drugs. Topical therapy is a less invasive and hence more attractive treatment for the eradication of fungal infection. However, dismal patient compliance often contributes to the high rate of unsuccessful treatment (4). It is for these reasons that alternative therapeutic agents need to be developed that can effectively target T. rubrum without significantly harming the host.

In vitro studies have demonstrated that yeast-like fungi, such as Candida albicans and T. rubrum, are highly susceptible to photodynamic therapy (PDT) using Photofrin (porfimer sodium) (4), the protoporphyrin IX (PpIX) precursor 5-aminolevulinic acid (ALA), or other photosensitizers that are not yet approved by the FDA (5,–9). However, before PDT can become a mainstream therapeutic strategy or be used in conjunction with an FDA-approved antifungal agent to combat dermatophytes, a comprehensive understanding of the cellular and molecular PDT pathways is necessary.

Allylamines, such as terbinafine and naftifine, are a relatively new class of ergosterol biosynthetic inhibitors. As an essential component of fungal membranes, ergosterol is also known to be involved in the modulation of membrane fluidity and permeability (10). Specifically, terbinafine acts by inhibiting the early steps of ergosterol biosynthesis, which leads to the accumulation of squalene, the sterol precursor, and the absence of other sterol intermediates (11). In other words, terbinafine inhibition of sterol synthesis possibly occurs during the epoxidation of squalene, which is catalyzed by the enzyme squalene epoxidase (12). This enzyme is reportedly encoded by ERG1 (13), and mutations in this particular gene have been demonstrated to confer resistance to terbinafine in fungi (12, 14,–16).

Conversely, PDT with the combination of a photosensitizer, light, and molecular oxygen in producing cytotoxic reactive oxygen species (ROS) is generally known to target intracellular organelles, such as mitochondria, lysosomes, and endoplasmic reticulum (ER) (17, 18). In essence, upon absorption of a photon generated by light of an appropriate wavelength, the photosensitizer would undergo at least one energy transition to go into an excited triplet state. It then can either (i) react with cellular molecules to produce free radical intermediates which subsequently form ROS within the presence of molecular oxygen (photochemical type I) or (ii) directly transfer energy to ground state molecular oxygen (photochemical type II) to generate singlet oxygen, which can cause irrevocable damage to lipids, proteins, and other biological molecules that are vital for the cells (19, 20). Most studied photosensitizers for PDT are known to produce type I and II photochemistry (21), and for most microbial systems, both type I and II photochemical reactions are typically produced by photosensitizers following irradiation and can result in cell death due to the oxidative damage caused by the formation of ROS (22).

PDT sensitized by silicon phthalocyanine (Pc) 4, a second-generation photosensitizer (Fig. 1) developed at Case Western Reserve University, induces biological responses (17). Pc 4-PDT may be effective in targeting T cell-mediated malignant and nonmalignant dermatological diseases, such as cutaneous T cell lymphoma and psoriasis, respectively, as multiple clinical trials have been undertaken here at the University Hospitals Case Medical Center. Unlike current FDA-approved PDT agents (e.g., 5-aminolevulinic acid or ALA, which is principally a metabolic precursor of the protoporphyrin IX [PpIX] photosensitizer), Pc 4 is a chemically pure photosensitizer which requires no bioconversion. Pharmacokinetic data in swine (M. Lam, K. D. Cooper, and E. D. Baron, unpublished data) and mice indicate that Pc 4 clears much more rapidly than other photosensitizers, such as Photofrin, when delivered systemically, thereby minimizing the possibility of prolonged generalized photosensitivity. In addition, with the absorbance maximum occurring at a wavelength of 672 nm (Fig. 1), Pc 4 is ideal for tissue penetration with minimal cutaneous photosensitivity (23, 24). Pc 4-PDT also demonstrated an excellent safety profile in a phase 1 trial on cutaneous neoplasms (25). Although the susceptibility of T. rubrum to PDT using other photosensitizers has been extensively investigated in vitro, the mechanism of phototoxicity has not been clearly defined (2, 9, 26,–28). In this study, we showed that a common dose of Pc 4-PDT can induce cell death in microconidia (terbinafine-sensitive and -resistant strains of T. rubrum). Our data also showed specific intracellular localizations of the photosensitizer and diminished metabolic activity immediately following Pc 4 irradiation and subsequently cell death, suggesting that the mechanism of cell death in the two strains involves the mitochondrial pathway.

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