Barbara W. Henderson

HOW DOES PHOTODYNAMIC THERAPY WORK

Those readers already familiar with the field of photodynamic therapy (PDT)t will consider this title somewhat presumptuous since it implies that the answer to the posed question is known. Indeed, answers to many questions regarding PDT have been found over the past decade, but a comprehensive understanding of all mechanisms involved in PDT tumor destruction has not yet emerged. This paper will attempt to deal with this complex subject by giving a sequential account of the effects occurring during PDT tissue treatment on a cellular and tissue level. Photodynamic therapy is based on the dye-sensitized photooxidation of biological matter in the target tissue (Foote, 1990). This requires the presence of a dye (sensitizer) in the tissue to be treated. Although such sensitizers can be naturally occurring constituents of cells and tissues, in the case of PDT they are introduced into the organism as the first step of treatment. In the second step, the tissuelocalized sensitizer is exposed to light of wavelength appropriate for absorption by the sensitizer. Through various photophysical pathways, also involving molecular oxygen, oxygenated products harmful to cell function arise and eventual tissue destruction results. In keeping with the chronological nature of this review, the subject matter will be divided into the

Role of cytokines in photodynamic therapy-induced local and systemic inflammation.

Photodynamic therapy (PDT) of tumour results in the rapid induction of an inflammatory response that is considered important for the activation of antitumour immunity, but may be detrimental if excessive. The response is characterised by the infiltration of leucocytes, predominantly neutrophils, into the treated tumour. Several preclinical studies have suggested that suppression of long-term tumour growth following PDT using Photofrin® is dependent upon the presence of neutrophils. The inflammatory pathways leading to the PDT-induced neutrophil migration into the treated tumour are unknown. In the following study, we examined, in mice, the ability of PDT using the second-generation photosensitiser 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) to induce proinflammatory cytokines and chemokines, as well as adhesion molecules, known to be involved in neutrophil migration. We also examined the role that these mediators play in PDT-induced neutrophil migration. Our studies show that HPPH-PDT induced neutrophil migration into the treated tumour, which was associated with a transient, local increase in the expression of the chemokines macrophage inflammatory protein (MIP)-2 and KC. A similar increase was detected in functional expression of adhesion molecules, that is, E-selectin and intracellular adhesion molecule (ICAM)-1, and both local and systemic expression of interleukin (IL)-6 was detected. The kinetics of neutrophil immigration mirrored those observed for the enhanced production of chemokines, IL-6 and adhesion molecules. Subsequent studies showed that PDT-induced neutrophil recruitment is dependent upon the presence of MIP-2 and E-selectin, but not on IL-6 or KC. These results demonstrate a PDT-induced inflammatory response similar to, but less severe than obtained with Photofrin® PDT. They also lay the mechanistic groundwork for further ongoing studies that attempt to optimise PDT through the modulation of the critical inflammatory mediators.

Choice of Oxygen-Conserving Treatment Regimen Determines the Inflammatory Response and Outcome of Photodynamic Therapy of Tumors

The rate of light delivery (fluence rate) plays a critical role in photodynamic therapy (PDT) through its control of tumor oxygenation. This study tests the hypothesis that fluence rate also influences the inflammatory responses associated with PDT. PDT regimens of two different fluences (48 and 128 J/cm2) were designed for the Colo 26 murine tumor that either conserved or depleted tissue oxygen during PDT using two fluence rates (14 and 112 mW/cm2). Tumor oxygenation, extent and regional distribution of tumor damage, and vascular damage were correlated with induction of inflammation as measured by interleukin 6, macrophage inflammatory protein 1 and 2 expression, presence of inflammatory cells, and treatment outcome. Oxygen-conserving low fluence rate PDT of 14 mW/cm2 at a fluence of 128 J/cm2 yielded ∼70–80% tumor cures, whereas the same fluence at the oxygen-depleting fluence rate of 112 mW/cm2 yielded ∼10–15% tumor cures. Low fluence rate induced higher levels of apoptosis than high fluence rate PDT as indicated by caspase-3 activity and terminal deoxynucleotidyl transferase-mediated nick end labeling analysis. The latter revealed PDT-protected tumor regions distant from vessels in the high fluence rate conditions, confirming regional tumor hypoxia shown by 2-(2-nitroimidazol-1[H]-yl)-N-(3,3,3-trifluoropropyl) acetamide staining. High fluence at a low fluence rate led to ablation of CD31-stained endothelium, whereas the same fluence at a high fluence rate maintained vessel endothelium. The highest levels of inflammatory cytokines and chemokines and neutrophilic infiltrates were measured with 48 J/cm2 delivered at 14 mW/cm2 (∼10–20% cures). The optimally curative PDT regimen (128 J/cm2 at 14 mW/cm2) produced minimal inflammation. Depletion of neutrophils did not significantly change the high cure rates of that regimen but abolished curability in the maximally inflammatory regimen. The data show that a strong inflammatory response can contribute substantially to local tumor control when the PDT regimen is suboptimal. Local inflammation is not a critical factor for tumor control under optimal PDT treatment conditions.

Photodynamic Therapy Enhancement of Antitumor Immunity Is Regulated by Neutrophils

Photodynamic therapy (PDT) is a Food and Drug Administration-approved local cancer treatment that can be curative of early disease and palliative in advanced disease. PDT of murine tumors results in regimen-dependent induction of an acute local inflammatory reaction, characterized in part by rapid neutrophil infiltration into the treated tumor bed. In this study, we show that a PDT regimen that induced a high level of neutrophilic infiltrate generated tumor-specific primary and memory CD8(+) T-cell responses. In contrast, immune cells isolated from mice treated with a PDT regimen that induced little or no neutrophilic infiltrate exhibited minimal antitumor immunity. Mice defective in neutrophil homing to peripheral tissues (CXCR2(-/-) mice) or mice depleted of neutrophils were unable to mount strong antitumor CD8(+) T-cell responses following PDT. Neutrophils seemed to be directly affecting T-cell proliferation and/or survival rather than dendritic cell maturation or T-cell migration. These novel findings indicate that by augmenting T-cell proliferation and/or survival, tumor-infiltrating neutrophils play an essential role in establishment of antitumor immunity following PDT. Furthermore, our results may suggest a mechanism by which neutrophils might affect antitumor immunity following other inflammation-inducing cancer therapies. Our findings lay the foundation for the rational design of PDT regimens that lead to optimal enhancement of antitumor immunity in a clinical setting. Immune-enhancing PDT regimens may then be combined with treatments that result in optimal ablation of primary tumors, thus inhibiting growth of primary tumor and controlling disseminated disease.

Alkyl ether analogs of chlorophyll-a derivatives: Part 1. Synthesis, photophysical properties and photodynamic efficacy.

The synthesis, preliminary in vivo biological activity, singlet oxygen and fluorescence yields of a series of alkyl ether derivatives of chlorophyll‐a analogs are described. For short‐chain carbon ethers (1–7carbon units), it was observed that the biological activity increased by increasing the length of the carbon chain, being maximum in compounds with n‐hexyl and n‐heptyl chains. Related sensitizers prepared by reacting 2‐(1‐bromoethyl)‐2‐devinylpyropheophorbide‐a with (sec)alcohols were found to be less effective. Under similar treatment conditions, photosensitizers containing cis‐ and trans‐ 3‐hexenyl side chains were ineffective. Thus, both stereochemical and steric factors caused differences in sensitizing activity. In general, pyropheophorbide‐a analogs were found to be more active than related chlorin e6 derivatives, in which the isocyclic ring (ring “E”) was cleaved. Related photosensitizers in the 9‐deoxy‐ series were found to be as effective as the corresponding pyropheophorbide‐a analogs. The photosensitizers prepared from pyropheophorbide‐a methyl ester and chlorin e6 trimethyl ester have long wavelength absorption at 660 nm (ε 45000 to 50000). Reduction of the carbonyl group in the pyropheophorbide‐a to methylene (ring E) resulted in a blue shift to 648 nm (ε 38000).

DRUG and LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR and NORMAL TISSUE RESPONSE

Abstract It is clinically relevant to determine drug and light dose combinations where complete tumor response is accompanied by little or no photosensitivity, and minimal damage to normal tissues. Although reciprocity of RIF tumor cell clonogenicity has been established within a range of drug and light doses, no quantitative data exist for reciprocity of tumor response. This study has examined reciprocity of drug and light doses for tumor response and normal tissue damage in two experimental mouse models. Representative tumors were examined for vascular damage after treatment. Reciprocity of drug and light doses for tumor response was observed over a range of drug/light combinations in both tumor models. Reciprocity failed when drug dose was reduced below a threshold value. For reciprocal drug/light combinations, complete vascular stasis occurred in the tumor and surrounding skin which was followed by necrosis of those tissues. In non‐reciprocal PDT combinations, there was vascular damage to the tumor but no damage to the surrounding normal tissues. Tumors responded initially, but no cure was obtained. Tumor cure was only observed under conditions where a considerable margin of normal tissue surrounding the tumor was damaged. This conclusion was supported by shielding experiments done to assess the contribution of normal tissue damage to tumor response. Reciprocity of drug and light doses for tumor response was therefore shown to exist only at high drug doses, which were not low enough to reduce skin photosensitivity in our models.

Murine pharmacokinetics and antitumor efficacy of the photodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a.

The combination of the new photodynamic sensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (HPPH) and laser light of wavelength 665 nm showed antitumor activity against two s.c.-implanted murine tumors. HPPH also sensitized normal mouse foot tissue to light but photosensitivity decreased rapidly with time after HPPH administration. Mechanistic studies revealed that HPPH induced little direct tumor cell toxicity but was an effective mediator of vascular photodamage. Pharmacokinetic studies following intravenous injection of 1 mg [14C]HPPH per kilogram revealed a biexponential decay with time, with plasma alpha and beta half-lives of 0.69 and 21 h respectively. Fecal excretion was the primary route of elimination. The highest levels of [14C]HPPH were found in the liver, which also showed the greatest long-term retention. The sequence of decreasing uptake levels was the liver, adrenals, lung, spleen, kidney, urinary bladder, heart, eye, skin, pancreas, muscle, testes, fat and brain. This distribution correlated with the relative blood perfusion rates in the tissues.

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

Abstract The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation‐induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C‐labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin‐free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only. These results indicate that the prerequisites for tumor destruction via direct photodynamic tumor cell inactivation in vivo are high photosensitizer content in tumor cells and relative insensitivity of the tissue microvasculature. Under conditions where the tumor bed is sensitive to photodynamic damage, tumor cell kill can occur at a wider range of photosensitizer levels as a consequence of PDT induced ischemia.

Anti-Angiogenic Activity of Selected Receptor Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implications for Combination Treatment with Photodynamic Therapy

Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR1TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR-β-, EGFR-, and FGFR1TKs and c-src TK by 50% (IC50) at concentrations between 7−85nM. PD173074 displayed selective inhibitory activity towards FGFR1TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1−25 mg/kg) or PD173074 (25−100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5−10 mg/kg) or PD173074 (30−60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR1TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.

The Effect of Fluence Rate on Tumor and Normal Tissue Responses to Photodynamic Therapy

Photodynamic therapy (PDT), carried out at low fluence rates, may enhance tumor response as well as affect treatment selectivity. We have studied the effects of fluence rate on the response of the murine radiation‐induced fibrosarcoma (RIF) to PDT using Photofrin® (5 mg/kg). Tumor response was tested over a large range of fluence rates (10‐200 mW/cm2) and fluences (25‐378 J/ cm2). Low fluence rates were more efficient; ‐60 J/cm2 at 10 mW/cm2 was needed to achieve the same tumor growth delay as ‐100 J/cm2 at 150 mW/cm2 and ‐150 J/cm2 at 200 mW/cm2. Despite this increased efficiency, lower fluence rates still required longer treatment times for equivalent anti‐tumor effects: 95 min for 57 J/cm2 at 10 mW/cm2versus 11 min for 100 J/cm2 at 150 mW/cm2. Effects of fluence rate on the PDT toxicity to normal tissue were examined through the response of the murine (C311) foot to Photofrin® PDT. Treatment with conditions that produced equivalent tumor responses, i.e. 57 J/cm2 at 10 mW/cm2 and 100 J/cm2 at 150 mW/cm2, resulted in a more severe foot response at the higher fluence rate (median peak response: 0.9 at 10 mW/cm2, 1.5 at 150 mW/cm2) with more time required for tissue to return to normal (8 days at 10 mW/cm2, at least 30 days at 150 mW/cm2). However, when feet were treated with an equal fluence of 100 J/cm2 at various fluence rates, longer healing times accompanied the lower fluence rate treatments. Overall, this paper demonstrates that lower PDT fluence rates are associated with increased efficiency of tumor response. If this increased efficiency is accounted for by lowering treatment fluence, lower fluence rates also may result in a more favorable normal tissue response to treatment.