Allan R. Oseroff

Mitochondria-based photodynamic anti-cancer therapy

As photodynamic therapy (PDT) becomes established as a treatment for cancer, there is increasing interest in identifying critical mechanisms of cell killing and understanding the bases for effective photosensitizers. The existence of multiple cellular targets makes it difficult to distinguish the critical events leading to cell death from PDT. However, with more sensitive techniques to detect photosensitizer localization, the isolation of PDT-resistant and -sensitive mutants and the use of innovative molecular and biochemical strategies to map cellular events occurring during and after photosensitization, some order is emerging from the chaos. The subcellular localization of many photosensitizers and the early responses to light activation indicate that mitochondria play a major role in photodynamic cell death. PDT with many agents which damage or inhibit different or multiple mitochondrial targets has many of the desirable characteristics for an effective anti-cancer therapy.

Irradiance-Dependent Photobleaching and Pain in δ-Aminolevulinic Acid-Photodynamic Therapy of Superficial Basal Cell Carcinomas

Purpose: In superficial basal cell carcinomas treated with photodynamic therapy with topical δ-aminolevulinic acid, we examined effects of light irradiance on photodynamic efficiency and pain. The rate of singlet-oxygen production depends on the product of irradiance and photosensitizer and oxygen concentrations. High irradiance and/or photosensitizer levels cause inefficient treatment from oxygen depletion in preclinical models. Experimental Design: Self-sensitized photobleaching of protoporphyrin IX (PpIX) fluorescence was used as a surrogate metric for photodynamic dose. We developed instrumentation measuring fluorescence and reflectance from lesions and margins during treatment at 633 nm with various irradiances. When PpIX was 90% bleached, irradiance was increased to 150 mW/cm2 until 200 J/cm2 were delivered. Pain was monitored. Results: In 33 superficial basal cell carcinomas in 26 patients, photobleaching efficiency decreased with increasing irradiance above 20 mW/cm2, consistent with oxygen depletion. Fluences bleaching PpIX fluorescence 80% (D80) were 5.7 ± 1.6, 4.5 ± 0.3, 7.5 ± 0.8, 7.4 ± 0.3, 12.4 ± 0.3, and 28.7 ± 7.1 J/cm2, respectively, at 10, 20, 40, 50, 60 and 150 mW/cm2. At 20-150 mW/cm2, D80 doses required 2.5-3.5 min; times for the total 200 J/cm2 were 22.2-25.3 min. No significant pain occurred up to 50 mW/cm2; pain was not significant when irradiance then increased. Clinical responses were comparable to continuous 150 mW/cm2 treatment. Conclusions: Photodynamic therapy with topical δ-aminolevulinic acid using ∼40 mW/cm2 at 633 nm is photodynamically efficient with minimum pain. Once PpIX is largely photobleached, higher irradiances allow efficient, rapid delivery of additional light. Optimal fluence at a single low irradiance is yet to be determined.

Photodynamic therapy for nonmelanoma skin cancers: Current review and update

Photodynamic therapy (PDT) is a therapeutic modality involving the use of a photosensitizing agent activated by light to destroy tumor cells. Over the past 25 years, PDT has been shown useful in the treatment of actinic keratoses and certain nonmelanoma skin cancers, such as Bowens disease and basal cell carcinoma. We review the current data available for PDT with systemic photofrin and topical 5-aminolevulinic acid (ALA). PDT offers many advantages including its non-invasiveness and its ability to treat multiple lesions simultaneously and is, therefore, an interesting alternative for treating certain skin malignancies.

Photodynamic therapy for the treatment of extramammary Paget's disease

Summary Background Surgical and ablative treatment modalities for extramammary Pagets disease (EMPD) have high recurrence rates and can be associated with significant morbidity.

THE ROLE OF TRANSFERRIN RECEPTOR (CD71) IN PHOTODYNAMIC THERAPY OF ACTIVATED AND MALIGNANT LYMPHOCYTES USING THE HEME PRECURSOR DELTA -AMINOLEVULINIC ACID (ALA)

Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71 +) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA‐induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71 + T lymphocytes from a patient with cutaneous T‐cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to norma] lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA‐PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.

The Tyrosine Kinase Inhibitor Imatinib Mesylate Enhances the Efficacy of Photodynamic Therapy by Inhibiting ABCG2

Purpose: The ATP-binding cassette protein ABCG2 (breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) and, thus, may confer resistance to this treatment modality. Tyrosine kinase inhibitors (TKI) can block the function of ABCG2. Therefore, we tested the effects of the TKI imatinib mesylate (Gleevec) on photosensitizer accumulation and in vitro and in vivo PDT efficacy. Experimental Design: Energy-dependent photosensitizer efflux and imatinib mesylates effects on intracellular accumulation of clinically used second- and first-generation photosensitizers were studied by flow cytometry in murine and human cells with and without ABCG2 expression. Effects of ABCG2 inhibition on PDT were examined in vitro using cell viability assays and in vivo measuring photosensitizer accumulation and time to regrowth in a RIF-1 tumor model. Results: Energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX (PpIX) synthesized from 5-aminolevulenic acid, and the benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) was shown in ABCG2+ cell lines, but the first-generation multimeric photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were minimally transported. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2+ cells, but not in ABCG2− cells, and enhanced PDT efficacy both in vitro and in vivo. Conclusions: Second-generation clinical photosensitizers are transported out of cells by ABCG2, and this effect can be abrogated by coadministration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other ABCG2 transport inhibitors may enhance efficacy and selectivity of clinical PDT.

Enhanced Systemic Immune Reactivity to a Basal Cell Carcinoma Associated Antigen Following Photodynamic Therapy

Purpose: Numerous preclinical studies have shown that local photodynamic therapy (PDT) of tumors enhances systemic antitumor immunity. However, other than single-case and anecdotal reports, this phenomenon has not been examined following clinical PDT. To determine whether PDT in a clinical setting enhances systemic recognition of tumor cells, we examined whether PDT of basal cell carcinoma resulted in an increased systemic immune response to Hip1, a tumor antigen associated with basal cell carcinoma. Experimental Design: Basal cell carcinoma lesions were either treated with PDT or surgically removed. Blood was collected from patients immediately before or 7 to 10 days following treatment. Peripheral blood leukocytes were isolated from HLA-A2–expressing patients and reactivity to a HLA-A2–restricted Hip1 peptide was measured by INF-γ ELISpot assay. Results: Immune recognition of Hip1 increased in patients whose basal cell carcinoma lesions were treated with PDT. This increase in reactivity was significantly greater than reactivity observed in patients whose lesions were surgically removed. Patients with superficial lesions exhibited greater enhancement of reactivity compared with patients with nodular lesions. Immune reactivity following PDT was inversely correlated with treatment area and light dose. Conclusions: These findings show for the first time that local tumor PDT can enhance systemic immune responses to tumors in patients, and validate previous preclinical findings.

PHOTOSENSITIZATION OF MURINE TUMOR, VASCULATURE and SKIN BY 5‐AMINOLEVULINIC ACID‐INDUCED PORPHYRIN

Abstract— The effects of topical and systemic administration of 5‐aminolevulinic acid (ALA) were examined in several murine tumor systems with regard to porphyrin accumulation kinetics in tumor, skin and blood, vascular and tumor cell photosensitization and tumor response after light exposure. Marked, transient increases in porphyrin levels were observed in tumor and skin after systemic and topical ALA. Rapid, transient, dose‐dependent porphyrin increases were also observed in blood; these were pronounced after systemic ALA injection and mild after topical application. They were highest within 1 h after ALA injection, thereafter declining rapidly. This matched the clearing kinetics of injected exogenous protoporphyrin IX (PpIX). Initially, vascular photosensitivity changed inversely to blood porphyrin levels, increasing gradually up to 5 h post‐ALA, as porphyrin was clearing from the bloodstream. This pattern was again matched by injected, exogenous PpIX. After therapeutic tumor treatment vascular disruption of the tumor bed, while observed, was incomplete, especially at the tumor base. Minimal direct tumor cell kill was found at low photodynamic therapy (PDT) doses (250 mg/kg ALA, 135 J/cm2 light). Significant, but limited (<1 log) direct photodynamic tumor cell kill was obtained when the PDT dose was raised to 500 mg/kg systemic ALA, followed 3 h later by 270 J/cm2, a dose that was however toxic to the animals. The further reduction of clonogenic tumor cells over 24 h following treatment was moderate and probably limited by the incomplete disruption of the vasculature. Tumor responses were highest when light treatment was carried out at the time of highest tumor porphyrin content rather than at the time of highest vascular photosensitivity. Tumor destruction did not reach the tumor base, regardless of treatment conditions.

Tumor Vascular Response to Photodynamic Therapy and the Antivascular Agent 5,6-Dimethylxanthenone-4-Acetic Acid: Implications for Combination Therapy

Purpose: Photodynamic therapy (PDT) is a clinically approved treatment for a variety of solid malignancies. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) is a potent vascular targeting agent that has been shown to be effective against a variety of experimental rodent tumors and xenografts and is currently undergoing clinical evaluation. We have previously reported that the activity of PDT against transplanted mouse tumors is selectively enhanced by DMXAA. In the present study, we investigated the in vivo tumor vascular responses to the two treatments given alone and in combination. Experimental Design: Vascular responses to (i) four different PDT regimens using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) at two different fluences (128 and 48 J/cm2) and fluence rates (112 and 14 mW/cm2), (ii) 5-aminolevulinic acid (ALA)–sensitized PDT (135 J/cm2 at 75 mW/cm2), (iii) DMXAA at a high (30 mg/kg) and low dose (25 mg/kg), and (iv) the combination of HPPH-PDT (48 J/cm2 at 112 mW/cm2) and low-dose DMXAA were studied in BALB/c mice bearing Colon-26 tumors. Results: PDT-induced changes in vascular permeability, determined using noninvasive magnetic resonance imaging with a macromolecular contrast agent, were regimen dependent and did not predict tumor curability. However, a pattern of increasing (4 hours after treatment) and then decreasing (24 hours after) contrast agent concentrations in tumors, seen after high-dose DMXAA or the combination of PDT and low-dose DMXAA, was associated with long-term cure rates of >70%. This pattern was attributed to an initial increase in vessel permeability followed by substantial endothelial cell damage (CD31 immunohistochemistry) and loss of blood flow (fluorescein exclusion assay). Low dose–rate PDT, regardless of the delivered dose, increased the level of magnetic resonance contrast agent in peritumoral tissue, whereas treatment with either DMXAA alone, or PDT and DMXAA in combination resulted in a more selective tumor vascular response. Conclusions: The observed temporal and spatial differences in the response of tumor vessels to PDT and DMXAA treatments could provide valuable assistance in the optimization of scheduling when combining these therapies. The combination of PDT and DMXAA provides therapeutically synergistic and selective antitumor activity. Clinical evaluation of this combination is warranted.

Characterization of electric-pulse-induced permeabilization of porcine skin using surface electrodes

We measured the transient and long-term changes of permeability of full-thickness porcine skin after the application of a single or a train of electric pulses, as the basis for optimization of the electrical parameters for enhancing transdermal drug or gene delivery by electroporation. Two electrodes were attached to the stratum corneum of excised skin for transdermal electric pulse delivery and impedance measurement. Both transient and long-term permeabilization were found to be dependent on the electrical exposure dose, i.e., the product of pulse voltage and cumulative pulsing (exposure) time. Skin resistance dropped to about 20% of its prepulsing value when pulsed beyond a critical dosage of 0.4 V-s (with 20-40 V across each skin path), but recovered rapidly within seconds after the pulse. Long-term permeabilization of the skin required repeated pulsing with a minimum potential of 160 V (80 V across each skin path). The maximum long-term resistance drop, to 35% of the initial value, required a dose greater than 200 V-s, recovering slowly and seldom completely in tens of minutes to hours. The decrease and recovery of the resistance were dependent on the frequency and pulse length only for low-dose electrical exposure.