Lurine A. Vaughan

Potentiation of photodynamic therapy antitumor activity in mice by nitric oxide synthase inhibition is fluence rate dependent.

Abstract— The effects of systemic administration of the nitric oxide synthase (NOS) inhibitor NG‐nitro‐L‐arginine (L‐NNA) in combination with photodynamic therapy (PDT) on tumor response, tumor oxygenation and tumor and normal skin perfusion were studied in C3H mice bearing subcutaneous radiation‐induced fibrosarcoma tumors. Photodynamic therapy was carried out using the photosensitizer Photofrin®(5 mg/kg) in conjunction with a low fluence rate (30 mW/cm2) and a high fluence rate (150 raW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced –15% tumor cures, a response not significantly altered by administration of 20 mg/kg L‐NNA either 5 min before or after PDT. In contrast, high fluence rate PDT produced no tumor cures by itself, but addition of L‐NNA either pre‐ or post‐PDT resulted in –30% and ‐10% tumor cures, respectively. The L‐NNA by itself tended to decrease tumor pO2 levels and perfusion, but statistically significant differences were reached only at one time point (1 h) with one of the oxygenation parameters measured (% values < 2 mm Hg). Photodynamic therapy by itself decreased tumor oxygenation and perfusion more significantly. Addition of L‐NNA before PDT further potentiated this effect. The L‐NNA exerted its most striking effects on the PDT response of the normal skin microvasculature. Low fluence rate PDT caused severe and lasting shut‐down of skin microvascular perfusion. With high fluence rate PDT, skin perfusion was initially decreased but recovered to persistent normal levels within 1 h of treatment. Administration of L‐NNA reversed this response, converting it to complete and lasting vascular shut‐down identical to that achieved with low fluence rate PDT. This effect was somewhat L‐NNA dose dependent but was still marked at a dose of 1 mg/ kg. It occurred whether L‐NNA was given before or after PDT. The L‐NNA did not alter the long‐term vascular response of skin to low fluence rate PDT. The ability of L‐NNA to correspondingly improve tumor response and severely limit skin vascular perfusion following high fluence rate PDT, while providing no benefit for the low fluence rate protocol, suggests that vascular changes in the tumor surrounding normal tissue contribute to the enhanced tumor curability with adjuvant L‐NNA treatment.

Light Delivery Over Extended Time Periods Enhances the Effectiveness of Photodynamic Therapy

Purpose: The rate of energy delivery is a principal factor determining the biological consequences of photodynamic therapy (PDT). In contrast to conventional high-irradiance treatments, recent preclinical and clinical studies have focused on low-irradiance schemes. The objective of this study was to investigate the relationship between irradiance, photosensitizer dose, and PDT dose with regard to treatment outcome and tumor oxygenation in a rat tumor model. Experimental Design: Using the photosensitizer HPPH (2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide), a wide range of PDT doses that included clinically relevant photosensitizer concentrations was evaluated. Magnetic resonance imaging and oxygen tension measurements were done along with the Evans blue exclusion assay to assess vascular response, oxygenation status, and tumor necrosis. Results: In contrast to high-incident laser power (150 mW), low-power regimens (7 mW) yielded effective tumor destruction. This was largely independent of PDT dose (drug-light product), with up to 30-fold differences in photosensitizer dose and 15-fold differences in drug-light product. For all drug-light products, the duration of light treatment positively influenced tumor response. Regimens using treatment times of 120 to 240 min showed marked reduction in signal intensity in T2-weighted magnetic resonance images at both low (0.1 mg/kg) and high (3 mg/kg) drug doses compared with short-duration (6-11 min) regimens. Significantly greater reductions in pO2 were observed with extended exposures, which persisted after completion of treatment. Conclusions: These results confirm the benefit of prolonged light exposure, identify vascular response as a major contributor, and suggest that duration of light treatment (time) may be an important new treatment variable.

Cross-Linking of Signal Transducer and Activator of Transcription 3—A Molecular Marker for the Photodynamic Reaction in Cells and Tumors

Purpose: Photodynamic therapy (PDT) depends on the delivery of a photosensitizer to the target tissue that, under light exposure, produces singlet oxygen and other reactive oxygen species, which in turn cause the death of the treated cell. This study establishes a quantitative marker for the photoreaction that will predict the outcome of PDT. Experimental Design: Cells in tissue culture, murine s.c. tumors, and endobronchial carcinomas in patients were treated with PDT, and the noncleavable cross-linking of the latent signal transducer and activator of transcription 3 (STAT3) was determined. Results: Murine and human cancer cell lines reacted to PDT by an immediate covalent cross-linking of STAT3 to homodimeric and other complexes. The magnitude of this effect was strictly a function of the PDT reaction that is determined by the photosensitizer concentration and light dose. The cross-link reaction of STAT3 was proportional to the subsequent cytotoxic outcome of PDT. An equivalent photoreaction as detected in vitro occurred in tumors treated in situ with PDT. The light dose-dependent STAT3 cross-linking indicated the relative effectiveness of PDT as a function of the distance of the tissue to the treating laser light source. Absence of cross-links correlated with treatment failure. Conclusions: The data suggest that the relative amount of cross-linked STAT3 predicts the probability for beneficial outcome, whereas absence of cross-links predicts treatment failure. Determination of STAT3 cross-links after PDT might be clinically useful for early assessment of PDT response.

Parabolic Quantitative Structure‐Activity Relationships and Photodynamic Therapy: Application of a Three‐Compartment Model with Clearance to the In Vivo Quantitative Structure‐Activity Relationships of a Congeneric Series of Pyropheophorbide Derivatives Used as Photosensitizers for Photodynamic Therapy

An open three‐compartment pharmacokinetic model was applied to the in vivo quantitative structure‐activity relationship (QSAR) data of a homologous series of pyropheophorbide photosensitizers for photodynamic therapy (PDT). The physical model was a lipid compartment sandwiched between two identical aqueous compartments. The first compartment was assumed to clear irreversibly at a rate K0+. The measured octanol‐water partition coefficients, P1 (where i is the number of carbons in the alkyl chain) and the clearance rate K0 determined the clearance kinetics of the drugs. Solving the coupled differential equations of the three‐compartment model produced clearance kinetics for each of the sensitizers in each of the compartments. The third compartment was found to contain the target of PDT. This series of compounds is quite lipophilic. Therefore these drugs are found mainly in the second compartment. The drug level in the third compartment represents a small fraction of the tissue level and is thus not accessible to direct measurement by extraction. The second compartment of the model accurately predicted the clearance from the serum of mice of the hexyl ether of pyropheophorbide a, one member of this series of compounds. The diffusion and clearance rate constants were those found by fitting the pharmacokinetics of the third compartment to the QSAR data. This result validated the magnitude and mechanistic significance of the rate constants used to model the QSAR data. The PDT response to dose theory was applied to the kinetic behavior of the target compartment drug concentration. This produced a pharmacokinetic‐based function connecting PDT response to dose as a function of time postinjection. This mechanistic dose‐response function was fitted to published, single time point QSAR data for the pheophorbides. As a result, the PDT target threshold dose together with the predicted QSAR as a function of time postinjection was found.

IL-10 does not play a role in cutaneous Photofrin photodynamic therapy-induced suppression of the contact hypersensitivity response.

Abstract Photodynamic therapy (PDT) treatment of both malignant and benign skin diseases has proven to be effective, and its use is increasing worldwide. However, preclinical studies using murine models have shown that PDT of the skin inhibits cell-mediated immune reactions, as measured by the suppression of the contact hypersensitivity (CHS) reaction. We have previously demonstrated that PDT enhances IL-10 expression in treated skin, and that the kinetics of induction of IL-10 is similar to the kinetics of suppression of systemic CHS reactions by cutaneous PDT. In the following report we have expanded upon these studies to demonstrate that cutaneous PDT, using Photofrin®, induces elevated levels of systemic IL-10 that persist for at least 28 days following treatment. The increase in systemic IL-10 correlates to a prolonged suppression of CHS of at least 28 days following cutaneous PDT. IL-10 has been implicated as the causative agent in the suppression of cell-mediated immune reactions by UVB and transdermal PDT. However, in the studies reported here we demonstrate that the suppression of CHS by cutaneous PDT occurs via an IL-10 independent mechanism, as administration of anti–IL-10 antibodies had no effect on the ability of PDT to induce CHS suppression. These results were further confirmed using IL-10 knockout (KO) mice. Cutaneous PDT of IL-10 KO mice resulted in CHS suppression that was not significantly different from suppression induced in wild-type mice. Thus, it appears as though IL-10 does not play a role in CHS suppression by cutaneous PDT. Suppression of cell-mediated immune reactions by UVB and transdermal PDT is reversible by IL-12, which is critical for the development of these reactions. We show that administration of exogenous IL-12 is also able to reverse CHS suppression induced by cutaneous PDT, suggesting that whereas suppression of cell-mediated immune reactions by UVB, transdermal PDT and cutaneous PDT occurs via different mechanisms, a common regulatory point exists.

Activation of the IL-10 Gene Promoter Following Photodynamic Therapy of Murine Keratinocytes

Abstract Photodynamic therapy (PDT), an anticancer treatment modality, has recently been shown to be an effective treatment for several autoimmune disease models including antigen-induced arthritis. PDT was found to induce the expression of IL-10 messenger RNA (mRNA) and protein in the skin, and this expression has similar kinetics to the appearance of PDT-induced suppression of skin-mediated immune responses such as the contract hypersensitivity (CHS) response. Some aspects of the UVB-induced suppression of the immune response have been linked to the induction of IL-10. IL-10 has been shown to inhibit the development and activation of Th1 cells, which are critical for many cell-mediated immune responses, including CHS. We have examined the effect of PDT and UVB irradiation on the activity of the IL-10 gene promoter and on IL-10 mRNA stability using the murine keratinocyte line, PAM 212. In vitro PDT induces IL-10 mRNA and protein expression from PAM 212 cells, which can be correlated with an increase in AP-1 DNA binding activity and activation of the IL-10 gene promoter by PDT. Deletion of an AP-1 response element from the IL-10 gene promoter was shown to abrogate the PDT-induced promoter activity indicating that the AP-1 response element is critical to IL-10 induction by PDT. In addition, PDT results in an increase in IL-10 mRNA stability, which may also contribute to the increased IL-10 expression in PAM 212 cells following PDT. In vitro UVB irradiation also results in activation of the IL-10 promoter. However, in contrast to PDT, UVB-induced activation of the IL-10 promoter is not AP-1 dependent and did not increase IL-10 mRNA stability.

The Role of the Peripheral Benzodiazepine Receptor in Photodynamic Activity of Certain Pyropheophorbide Ether Photosensitizers: Albumin Site II as a Surrogate Marker for Activity¶

A study has been carried out to define the importance of the peripheral benzodiazepine receptor (PBR) as a binding site for a series of chlorin‐type photosensitizers, pyropheophorbide‐a ethers, the subject of a previous quantitative structure–activity relationship study by us. The effects of the PBR ligand PK11195 on the photodynamic activity have been determined in vivo for certain members of this series of alkyl‐substituted ethers: two of the most active derivatives (hexyl and heptyl), the least active derivative (dodecyl [C12]) and one of intermediate activity (octyl [C8]). The photodynamic therapy (PDT) effect was inhibited by PK11195 for both of the most active derivatives, but no effect on PDT activity was found for the less active C12 or C8 ethers. The inhibitory effects of PK11195 were predicted by the binding of only the active derivatives to the benzodiazepine site on albumin, i.e. human serum albumin (HSA)‐Site II. Thus, as with certain other types of photosensitizers, it has been demonstrated with this series of pyropheophorbide ethers that in vitro binding to HSA‐Site II is a predictor of both optimal in vivo activity and binding to the PBR in vivo.

THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS

Abstract— The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase‐locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators.

Lipid-associated methylpheophorbide-a (hexyl-ether) as a photodynamic agent in tumor-bearing mice.

Abstract Liposomes are a potential system for more selective delivery of photosensitizers (PS) to tumors. Pheo‐phorbides are one series of new PS under investigation for use in photodynamic therapy. The pharmacokinetics, anti‐tumor response and normal tissue effects of methylpheophorbide‐a‐(hexyl‐ether) (MPH) associated with negatively charged phospholipid vesicles composed of high and low transition temperature lipids were determined in mice. In some preparations monosialoganglioside, which is known to impart long circulation time to liposomes was also included. Normally water‐insoluble MPH could be quantitatively incorporated in multilamellar liposomes up to at least 20 mol MPH/mol lipid% for most liposome compositions and sonicated to form clear suspensions. Evidence from electron microscopy and entrapment of aqueous space markers indicated that the particles formed by sonication were not standard liposomes. Anti‐tumor responses to light treatment (135 J/cm2, 665 nm argon‐dye laser) 24 h after MPH (0.4 μmol/kg) administration were slightly but significantly greater (P < 0.05) for lipid associated MPH compared to MPH solubilized in Tween 80. There were no major differences in tumor uptake and tumor cell photosensitization between lipid or Tween 80 formulations of MPH, whereas, dependent on lipid composition and time after MPH administration, the doses of light required to cause occlusive vascular damage were increased for the lipid formulations. Pharmacokinetic studies showed rapid dissociation between lipids and MPH in vivo. Lipid formulations are useful for solubilizing MPH and may improve the therapeutic effects of this PS.

Photodynamic therapy (PDT) treatment enhances tumor cell antigenicity

Several groups, including our own, have reported that PDT enhances the host anti-tumor immune response and it is known that the enhanced immune response plays a role in the overall tumor response to PDT. The mechanism behind this enhancement is unknown, however it has been shown that the initiation of an inflammatory response and the infiltration of neutrophils into the tumor bed is critical to the tumor response. We have shown that PDT induces the expression of chemokines that play a critical role in neutrophil infiltration. Recent studies in our laboratory have shown that in addition to affecting the inflammatory and chemokine/cytokine response, PDT also alters the immunogenicity of the tumor, either through changes in antigen structure or enhancement of presentation of tumor associated antigens by host antigen presenting cells to tumor specific T cells. These recent studies and the underlying mechanisms will be discussed.