Review
Photodynamic therapy with the phthalocyanine photosensitizer Pc 4: The case experience with preclinical mechanistic and early clinical–translational studies

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Abstract

Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers. PDT involves either local or systemic administration of a photosensitizing drug, which preferentially localizes within the tumor, followed by illumination of the involved organ with light, usually from a laser source. Here, we provide a selective overview of our experience with PDT at Case Western Reserve University, specifically with the silicon phthalocyanine photosensitizer Pc 4. We first review our in vitro studies evaluating the mechanism of cell killing by Pc 4-PDT. Then we briefly describe our clinical experience in a Phase I trial of Pc 4-PDT and our preliminary translational studies evaluating the mechanisms behind tumor responses. Preclinical work identified (a) cardiolipin and the anti-apoptotic proteins Bcl-2 and Bcl-xL as targets of Pc 4-PDT, (b) the intrinsic pathway of apoptosis, with the key participation of caspase-3, as a central response of many human cancer cells to Pc 4-PDT, (c) signaling pathways that could modify apoptosis, and (d) a formulation by which Pc 4 could be applied topically to human skin and penetrate at least through the basal layer of the epidermis. Clinical–translational studies enabled us to develop an immunohistochemical assay for caspase-3 activation, using biopsies from patients treated with topical Pc 4 in a Phase I PDT trial for cutaneous T-cell lymphoma. Results suggest that this assay may be used as an early biomarker of clinical response.

Section snippets

Background: photodynamic therapy

Photodynamic therapy (PDT) is emerging as a promising non-invasive treatment for cancers (Babilas et al., 2005, Dougherty et al., 1998). It has been shown to induce favorable responses in the treatment of cutaneous squamous cell and basal cell carcinomas, as well as cancers of the head, neck, lung, esophagus, and bladder (Wolfsen, 2005, Sibata et al., 2001). This treatment involves either local or systemic administration of a photosensitizing drug, which preferentially localizes within the

Overview of the case experience with PDT

Research on PDT at Case Western Reserve University began in the mid-1980s with collaborations among faculty in several departments, most notably Chemistry, Dermatology, and Radiation Oncology. These early studies led to the synthesis and characterization of novel silicon phthalocyanines that proved to be strong, active photosensitizers in cell cultures and model tumors. Phthalocyanines are structurally related to porphyrins but with a larger macrocycle ring system, and hence they absorb longer

From DNA damage to caspases

Our earliest exploration into the mechanism of PDT with phthalocyanine photosensitizers focused on DNA damage, in particular, the formation of DNA single-strand breaks and DNA–protein crosslinks. In one study, we exposed L929 mouse fibroblasts to an LD90 dose of ionizing radiation (i.e., a dose causing 90% reduction of clonogenic survival) and/or a sub-lethal dose of PDT. Radiation-killed cells remained attached to the culture dish and repaired the initially produced strand breaks in their DNA.

The role of caspases in PDT-induced apoptosis

Addressing part a, we tested the relevance of the caspase-3-dependent steps in the killing of human breast cancer MCF-7 cells (Xue et al., 2001a, Whitacre et al., 2002). MCF-7 cells have a deletion in the CASP-3 gene and do not express procaspase-3 at all. We obtained two derivative cell lines from Dr. C. Froehlich (Northwestern University) that were stably transfected with human procaspase-3 (MCF-7c3 cells) or the empty vector (MCF-7v cells). These lines were exposed to various doses of Pc 4

Bcl-2 family proteins in PDT-induced apoptosis

Addressing part b, we explored the role of Bcl-2 and its homologues in PDT-induced apoptosis to evaluate the earliest identifiable biochemical changes post-PDT. Bcl-2 is a 26-kDa protein that resides in the outer mitochondrial membrane and the endoplasmic reticulum (ER), anchored via a C-terminal transmembrane (TM) domain. Bcl-2 contains four BH (Bcl-2 homology) domains (BH1-BH4). The other members of the family share one or more of these domains. We (Xue et al., 2001b) and the Kessel

Signaling pathways and apoptosis in response to Pc 4-PDT

PDT has been found to upregulate numerous signaling pathways (Oleinick and Evans, 1998, Moor, 2000). For example, stress kinases such as SAPK/JNK and p38/HOG are strongly activated by PDT and may help to promote apoptosis (Xue et al., 1999). When Hasan Mukhtar was studying Pc 4-PDT at Case, he made several notable contributions to our understanding, including demonstrating the presence of apoptosis within PDT-treated murine tumors (Agarwal et al., 1996, Whitacre et al., 2000, Colussi et al.,

The possible role of cardiolipin in cell killing by Pc 4-PDT

Returning to our earlier question concerning the immediate critical reaction following PDT that commits cells to apoptosis (or cell death by another mechanism), we now describe some recent observations implicating mitochondrial lipids as key mediators of the initial photodynamic damage. In an attempt to use the high-affinity ligand of cardiolipin, nonyl-acridine orange (NAO), as a probe of cardiolipin oxidation, we were surprised to observe that NAO could undergo fluorescence resonance energy

Clinical development of Pc 4 in photodynamic therapy at Case Western Reserve University and our translational mechanistic studies

While the mechanistic details of cell killing by Pc 4-PDT were being elucidated, steps were taken to bring Pc 4 into clinical studies. With the help of the National Cancer Institute's (NCI) Drug Decision Network, pre-clinical pharmacokinetic, toxicity, and efficacy studies were carried out. Following these studies, the NCI made available formulated GMP (Good Manufacturing Practices)-grade Pc 4 for use in phase I clinical trials. Subsequently an Investigational New Drug Application was approved

Discussion and conclusions

Topical application is a convenient and safe method of specifically directing photosensitizers to accessible lesions, avoiding the widespread distribution that occurs following intravenous administration. This concept has been well studied for the administration of the porphyrin prodrugs, ALA and its esters. However, until now, little attention has been given to the delivery of active photosensitizers after the apparent failure of studies with porphyrins (Brown et al., 2004). Our clinical study

Acknowledgments

Research on PDT in our laboratories has been supported by the following NIH grants: R01 CA83917 (to NLO), R01 CA-106491 (to NLO), P01 CA48735 (to NLO), P30 CA43703, AR-39750 (to KDC), T32 AR-007569 (to KDC), R01 AR-051498 (to KDC).

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