Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols

Effective photodynamic therapy (PDT) depends on the optimization of factors such as drug dose, drug-light interval, fluence rate and total light dose (or fluence). In addition sufficient oxygen has to be present for the photochemical reaction to occur. Oxygen deficits may arise during PDT if the photochemical reaction consumes oxygen more rapidly than it can be replenished, and this could limit the efficacy of PDT. In this study we investigated the influence of the drug-light interval, illumination-fluence rate and total fluence on PDT efficacy for the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). The effect of increasing the oxygenation status of tumors during PDT was also investigated. PDT response was assessed from tumor-growth delay and from cures for human malignant mesothelioma xenografts grown in nude mice. Tumor-bearing mice were injected intravenously with 0.15 or 0.3 mg.kg-1 mTHPC, and after intervals of 24-120 h, the subcutaneous tumors were illuminated with laser light (652 nm) at fluence rates of 20, 100 or 200 mW.cm-2. Tumor response was strongly dependent on the drug-light interval. Illumination at 24 h after photosensitization was always significantly more effective than illumination at 72 or 120 h. For a drug-light interval of 24 h the tumor response increased with total fluence, but for longer drug-light intervals even high total fluences failed to produce a significant delay in tumor regrowth. No fluence-rate dependence of PDT response was demonstrated in these studies. Nicotinamide injection and carbogen breathing significantly increased tumor oxygenation and increased the tumor response for PDT schedules with illumination at 24 h after photosensitizer injection.     

Outcome of mTHPC mediated photodynamic therapy is primarily determined by the vascular response

We have previously shown that the efficacy of photodynamic therapy (PDT) using the photosensitizer meso-tetra-hydroxyphenyl-chlorin (mTHPC) correlated with plasma drug levels at the time of illumination rather than drug levels in human tumor xenografts or mouse skin. These results suggested that vascular-mediated effects could be important determinants of PDT response in vivo. In the present study we further investigated the relationship between PDT response, mTHPC pharmacokinetics and the localization and extent of vascular damage induced in human squamous cell carcinoma xenografts (HNXOE). Plasma levels of mTHPC decreased exponentially with time after injection, whereas tumor drug levels remained maximal for at least 48 h. At 3 h after administration mTHPC was localized in the blood vessels, whereas at later times it was distributed throughout the whole tumor. Illumination at 3 h after mTHPC, which resulted in 100% long-term tumor cure, led to a marked reduction of vascular perfusion and increased tumor hypoxia at 1 h after treatment. Illumination at 48 h resulted in rapid regrowth of most tumors and only 10% cure. This protocol did not affect a significant decrease in vascular perfusion or increase in tumor hypoxia. These data show that optimal responses to mTHPC-mediated PDT were primarily dependent on the early vascular response, and that plasma drug levels at the time of illumination could predict this relationship.     

Potentiation of photodynamic therapy with hypericin by mitomycin C in the radiation-induced fibrosarcoma-1 mouse tumor model

Hypericin, a polycyclic quinone obtained from plants of the genus Hypericum, has been shown to be a promising photosensitizer. We investigated the combination of hypericin-photodynamic therapy (PDT) and a bioreductive drug mitomycin C (MMC) in the present study. The radiation-induced fibrosarcoma-1 tumors were exposed to laser light (120 J/cm2 at 595 nm) 24 h after an intravenous injection of hypericin (1 mg/kg). Hypericin-PDT alone significantly decreased tumor perfusion and oxygen tension as demonstrated by India ink staining technique and OxyLite pO2 measurement, respectively. The in vivo-in vitro cell-survival assay revealed about 60% direct tumor cell killing immediately after PDT. No significant delayed tumor cell death was observed after PDT, which suggests that vascular damage does not contribute significantly to the overall tumor cell death. Injection of a 2.5 mg/kg dose of MMC 20 min before light application significantly decreased tumor cell survival and delayed tumor growth compared with PDT or MMC alone. No greater skin reaction was observed after the combination of MMC and PDT than after PDT alone. Our study demonstrates that combining hypericin-PDT with MMC can be effective in enhancing tumor response with little side effect.     

Antivascular treatment of solid melanoma tumors with bacteriochlorophyll-serine-based photodynamic therapy

We describe here a strategy for photodynamic eradication of solid melanoma tumors that is based on photo-induced vascular destruction. The suggested protocol relies on synchronizing illumination with maximal circulating drug concentration in the tumor vasculature attained within the first minute after administrating the sensitizer. This differs from conventional photodynamic therapy (PDT) of tumors where illumination coincides with a maximal concentration differential of sensitizer in favor of the tumor, relative to the normal surrounding tissue. This time window is often achieved after a delay (3-48 h) following sensitizer administration. We used a novel photosensitizer, bacteriochlorophyll-serine (Bchl-Ser), which is water soluble, highly toxic upon illumination in the near-infrared (lambda max 765-780 nm) and clears from the circulation in less than 24 h. Nude CD1 mice bearing malignant M2R melanotic melanoma xenografts (76-212 mm3) received a single complete treatment session. Massive vascular damage was already apparent 1 h after treatment. Changes in vascular permeability were observed in vivo using contrast-enhanced magnetic resonance imaging (MRI), with the contrast reagent Gd-DTPA, by shortening spin-spin relaxation time because of hemorrhage formation and by determination of vascular macromolecular leakage. Twenty-four hours after treatment a complete arrest of vascular perfusion was observed by Gd-DTPA-enhanced MRI. Histopathology performed at the same time confirmed primary vascular damage with occlusive thrombi, hemorrhage and tumor necrosis. The success rate of cure of over 80% with Bchl-Ser indicates the benefits of the short and effective treatment protocol. Combining the sensitizer administration and illumination steps into one treatment session (30 min) suggests a clear advantage for future PDT of solid tumors.     

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.     

Potentiation of Photodynamic Therapy Antitumor Activity in Mice by Nitric Oxide Synthase Inhibition Is Fluence Rate Dependent

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 mW/cm2) protocol at a total fluence of 100 J/cm2. Low fluence rate PDT produced approximately 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 approximately 30% and approximately 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.     

Wavelength-dependent properties of photodynamic therapy using hypericin in vitro and in an animal model

Wavelength effects in photodynamic therapy (PDT) with hypericin (HY) were examined in a C26 colon carcinoma model both in vitro and in vivo. Irradiation of HY-sensitized cells in vitro with either 550 or 590 nm caused the loss of cell viability in a drug- and light-dose-dependent manner. The calculated ratio of HY-based PDT (HY-PDT) efficiencies at these two wavelengths was found to correlate with the numerical ratio of absorbed photons at each wavelength. In vivo irradiation of C26-derived tumors, 6 h after intraperitoneal administration of HY (5 mg/kg), caused extensive vascular damage and tumor necrosis. The depth of tumor necrosis (d) was more pronounced at 590 than at 550 nm and increased when the light dose was raised from 60 to 120 J/cm2. The maximal depths of tumor necrosis (at 120 J/cm2) were 7.5+/-1.5 mm at 550 nm and 9.9+/-0.8 mm at 590 nm. Both values are rather high in view of the limited penetration of green-yellow light into the tissue. Moreover, the depth ratio, d590/d550 = 1.3 (P < 0.001), is smaller than expected considering the 2.2-fold lower HY absorbance and the 1.7-fold lower tissue penetration of radiation at 550 than at 590 nm. This finding indicates that in vivo the depth at which HY-PDT elicits tumor necrosis is not only determined by photophysical considerations (light penetration, number of absorbed photons) but is also influenced significantly by other mechanisms such as vascular effects. Therefore, despite the relatively short-wavelength peaks of absorption, our observations suggest that HY is an effective photodynamic agent that can be useful in the treatment of tumors with depths in the range of 1 cm.     

Measurement of Tumor Vascular Damage in Mice with 99m Tc-MIBI Following Photodynamic Therapy

Abstract— The clinical perfusion agent ^99mTc-MIBI was used to monitor changes in tumor vascular perfusion (TVP) induced by Photofrin® (Pll)-mediated photodynamic therapy (PDT). BALB/c mice bearing an EMT-6 tumor on each hind thigh were given an intravenous injection of 1, 2 or 5 mg kg⁻¹ PII. Twenty-four hours later, one tumor was illuminated (600–650 run, 200 mW cm⁻² 400 J cm⁻²) while the other served as a control. At various time intervals after PDT (0, 2 and 24 h) mice received an intravenous injection of ^99mTc-hexakismethoxy(sobutyusonitri-le (MIBI) (0.18 MBq g⁻¹) and were sacrificed 2 min later. The light-treated and the untreated tumors were then dissected, the radioactivity was counted and the percentage of the injected dose per gram of tumor (%ID g⁻¹) was calculated as a measure of TVP. We observed that TVP is drug dose dependent, develops progressively with time post-PDT and is inversely related to PDT efficacy. Our data show that early tumor retention of ^99mMIBI is a simple method to assess TVP and vascular damage induced by PDT.     

THE EFFECTS OF THROMBOXANE INHIBITORS ON THE MICROVASCULAR AND TUMOR RESPONSE TO PHOTODYNAMIC THERAPY

Abstract— Vascular stasis and tissue ischemia are known to cause tumor cell death in several experimental models after photodynamic therapy (PDT); however, the mechanisms leading to this damage remain unclear. Because previous studies indicated that thromboxane release is implicated in vessel damage, we further examined the role of throm-boxane in PDT. Rats bearing chondrosarcoma were injected with 25 mg/kg Photofrin® (intravenously) 24 h before treatment. Light (135 J/cm ² , 630 nm) was delivered to thc tumor area after injection of one of the following inhibitors: (1) R68070: a thromboxane synthetase inhibitor; (2) SQ-29548: a thromboxane receptor antagonist; and (3) Flunarizine: an inhibitor of platelet shape change. Systemic thromboxane levels were determined. Vessel constriction and leakage were evaluated by intravital microscopy. Tumor response was assessed after treatment. Thromboxane levels were decreased more than 50% with SQ-29548 as compared to controls. Thromboxane levels in animals given R68070 and Flunarizine remained at baseline levels. SQ-29548 and R68070 reduced vessel constriction compared to controls, while Flunarizine totally prevented vessel constriction. R68070 and SQ-29548 inhibited vessel permeability compared to PDT controls; Flunarizine did not. Animals given these inhibitors showed markedly reduced tumor cure. These results indicate that the release of thromboxane is linked to the vascular response in PDT.     

DH-I-180-3-mediated photodynamic therapy: biodistribution and tumor vascular damage

An important goal of photodynamic therapy (PDT) for treatment of various cancers is to shorten PDT-performing time and simultaneously enhance PDT efficacy. Here, we investigated the nontumor tissue distribution of and the tumor vascular damage caused by a new photosensitizer, DH-I-180-3, in mice with implanted EMT6 mammary tumor cells. In addition, we performed cell-based assays to evaluate the basic antitumor effect of DH-I-180-3/PDT in EMT6 cells. After administration of PDT, the type of cell death was characterized to be apoptosis, and a change in the mitochondrial membrane potential was also observed within minutes. On the other hand, tumor growth was remarkably retarded in vivo in mice that received DH-I-180-3/PDT, compared with mice in the control group, which were exposed to light irradiation alone. Finally, tumors in some mice nearly healed. The antitumor drug reached a maximum concentration approximately 3 h after administration. However, PDT was most effective when there was substantial accumulation of DH-I-180-3 in the tumor vasculature and in healthy tissue. The histological demonstration provided further evidence of tumor vascular damage. On the basis of these findings, we suggest that PDT with the photosensitizer DH-I-180-3 induces vascular damage with blood vessel shutdown, in addition to direct killing of tumor cells, in mice.     

Fluorescence imaging and phototoxicity effects of new formulation of chlorin e6-polyvinylpyrrolidone

Evaluations of the efficiency of a new formulation of chlorin consisting of a complex of trisodium salt chlorin e6 (Ce6) and polyvinylpyrrolidone (PVP) in photodynamic therapy (PDT) and fluorescence diagnosis was performed on poorly differentiated human bladder carcinoma murine model with the following specific aims: (i) to qualitatively evaluate the fluorescence accumulation in human bladder tumor, (ii) to determine fluorescence distribution of Ce6-PVP using the tissue extraction technique and fluorescence imaging technique, (iii) to compare the fluorescence distribution of Ce6, Ce6-PVP and Photofrin in skin of nude mice, and (iv) to investigate phototoxicity caused by different parameters (drug-light interval, drug dose, irradiation fluence rate and total light fluence) in PDT. The fluorescence of the Ce6-PVP formulation was determined either by fluorescence imaging measurements or by chemical extraction from the tissues displaying similar trends of distribution. Our results demonstrated that the Ce6-PVP formulation possesses less in vivo phototoxic effect compared to Ce6 alone. The phototoxicity revealed a strong dependence on the drug and light dosimetry as well as on the drug-light interval. In PDT, the Ce6-PVP compound was most toxic at the 1h drug-light interval at 200J/cm(2), while Ce6 alone was most toxic at a light dose of more that 50J/cm(2) at the 1 and 3h drug-light interval. We also confirmed that Ce6-PVP has a faster clearance compared to Ce6 alone or Photofrin. This eliminates the need for long-term photosensitivity precautions. In conclusion, the Ce6-PVP formulation seems to be a promising photosensitizer for fluorescence imaging as well as for photodynamic treatment.     

Hyperoxygenation enhances the tumor cell killing of photofrin-mediated photodynamic therapy

Tumor hypoxia, either preexisting or as a result of oxygen depletion during photodynamic therapy (PDT) light irradiation, can significantly reduce the effectiveness of PDT-induced cell killing. To overcome tumor hypoxia and improve tumor cell killing, we propose using supplemental hyperoxygenation during Photofrin-PDT. The mechanism for the tumor cure enhancement of the hyperoxygenation-PDT combination is investigated using an in vivo-in vitro technique. A hypoxic tumor model was established by implanting mammary adenocarcinoma in the hind legs of mice. Light irradiation (200 J/cm2 at either 75 or 150 mW/cm2), under various oxygen supplemental conditions (room air, carbogen, 100% normobaric or hyperbaric oxygen), was delivered to animals that received 12.5 mg/kg Photofrin 24 h before light irradiation. Tumors were harvested at various time points after PDT and grown in vitro for colony formation analysis. Treated tumors were also analyzed histologically. The results show that when PDT is combined with hyperoxygenation, the hypoxic condition could be improved and the cell killing rate at various time points after PDT could be significantly enhanced over that without hyperoxygenation, suggesting an enhanced direct and indirect cell killing associated with high-concentration oxygen breathing. This study further confirms our earlier observation that when a PDT treatment is combined with hyperoxygenation it can be more effective in controlling hypoxic tumors.     

SITES OF PHOTODAMAGE in vivo and in vitro BY A CATIONIC PORPHYRIN

Abstract— Localization and photodynamic efficacy of a monocationic porphyrin (MCP) were assessed using murine leukemia cells in culture. This sensitizer localized at surface membrane loci and catalyzed selective photodamage to membrane structures. Although both cationic and hydrophobic, this porphyrin was not recognized by the multidrug transporter, which excludes many cationic agents from cells that express multidrug resistance. Photodynamic studies with the murine radiation-induced fibrosarcoma tumor model indicated moderate photosensitization of neoplastic lesions in vivo at 3 h, but not at 24 h after sensitizer administration. Pharmacokinetic studies indicate that plasma levels, not tissue levels were the major determinant of photodynamic therapy (PDT) response. Consistent with this observation, vascular damage and disturbances of tissue perfusion followed PDT. These effects were more pronounced in tumor-bearing skin than in normal skin. The therapeutic response to MCP appeared to be related mainly to secondary, probably vascular, effects.     

Angiogenesis induced by photodynamic therapy in normal rat brains

Angiogenesis promotes tumor growth and invasiveness in brain. Because brain injury often induces expression of angiogenic-promoting molecules, we hypothesize that oxidative insult induced by photodynamic therapy (PDT) could lead to an endogenous angiogenic response, possibly diminishing the efficacy of PDT treatment of tumors. Therefore, we sought to establish whether PDT induced an angiogenic response within the nontumored brain. PDT using Photofrin as a sensitizer at an optical dose of 140 J/cm2 was performed on normal rat brain (n = 30). Animals were sacrificed at 24 h, and 1, 2, 3 and 6 weeks after PDT treatment. Fluorescein isothiocyanatedextran perfusion was performed, and brains were fixed for immunohistological study. Immunostaining revealed that vascular endothelial growth factor (VEGF) expression increased within the PDT-treated hemisphere 1 week after treatment and remained elevated for 6 weeks. Three-dimensional morphologic analysis of vasculature within PDT-treated and contralateral brain demonstrated PDT-induced angiogenesis, as indicated by a significant increase in vessel connectivity (P < 0.001) concomitant with decreased (P < 0.05) mean segment length compared with vessels within the contralateral hemisphere. Volumetric measurement of angiogenic regions indicate that neovascular expansion continued for 4 weeks after PDT. These data demonstrate that PDT induces VEGF expression and neovascularization within normal brain. Because angiogenesis promotes growth and invasiveness of tumor, antagonizing this endogenous angiogenic response to PDT may present a practical means to enhance the efficacy of PDT.     

The vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid improves the antitumor efficacy and shortens treatment time associated with Photochlor-sensitized photodynamic therapy in vivo

In this report, we examined the antitumor activity of photodynamic therapy (PDT) in combination with 5,6-dimethylxanthenone-4-acetic acid (DMXAA), a vascular disrupting agent currently undergoing clinical evaluation. BALB/c mice bearing subcutaneous CT-26 colon carcinomas were treated with PDT using the second-generation chlorin-based sensitizer, 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a (Photochlor) with or without DMXAA. Long-term (60-days) treatment outcome, induction of tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), vascular damage (microvessel density, MVD) were evaluated as endpoints. In addition, treatment selectivity was evaluated using magnetic resonance imaging (MRI) and the foot response assay. A highly synergistic interaction was observed with the combination of low-dose DMXAA and PDT (48 J cm⁻² at 112 mW cm⁻²) resulting in ∼60% long-term cures. The duration of the PDT session for this combination therapy protocol was only 7 min, while the duration of a monotherapy PDT session, selected to yield the equivalent cure rate, was 152 min. MRI showed markedly less peritumoral edema after DMXAA + short-duration PDT compared with long-duration PDT monotherapy. Similarly, DMXAA + PDT caused significantly less phototoxicity to normal mouse foot tissue than PDT alone. Increased induction of cytokines TNF-α and IL-6 ( P  < 0.001) was observed at 4 h followed by extensive vascular damage, demonstrated by a significant reduction in MVD at 24 h after combination treatment. In conclusion, Photochlor-sensitized PDT in combination with DMXAA exhibits superior efficacy and improved selectivity with clinically feasible illumination schemes. Clinical evaluation of this novel combination strategy is currently being planned.     

Interaction between photodynamic therapy and BCG immunotherapy responsible for the reduced recurrence of treated mouse tumors

Subcutaneous mouse EMT6 tumors were treated by individual or combined regimens of a single Bacillus Calmette-Guérin (BCG) vaccine administration and photodynamic therapy (PDT). Six clinically relevant photosensitizers characterized by different action mechanisms were used: Photofrin, benzoporphyrin derivative, tetra(m-hydroxyphenyl)chlorin (foscan), mono-L-aspartylchlorin e6, lutetium texaphyrin or zinc phthalocyanine. Irrespective of the type of photosensitizer used, the optimized BCG protocols improved the cure rate of PDT-treated tumors. This indicates that the interaction does not take place during the early phase of tumor ablation but at later events involved in preventing tumor recurrence. Beneficial effects on tumor cure were observed even when the BCG injection was delayed to 7 days after PDT. The accumulation of activated myeloid cells that markedly increases in tumors treated by Photofrin-based PDT was not additionally affected by BCG treatment. However, the incidence of immune memory T cells in tumor-draining lymph nodes that almost doubled at 6 days after Photofrin-PDT further increased close to three-fold with adjuvant BCG. This suggests that BCG immunotherapy amplifies the T-lymphocyte-mediated immune response against PDT-treated tumors. Since both these modalities are established for the treatment of superficial bladder carcinomas, use of their combination for this condition should be clinically tested.     

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.     

Antitumor efficacy of photodynamic therapy using novel nanoformulations of hypocrellin photosensitizer SL052

Recent preclinical and clinical testing of hypocrellin-based photosensitizer SL052 for use in photodynamic therapy (PDT) of cancer has shown encouraging results. Further optimization of its formulation for delivery could considerably extend the therapeutic efficiency of this drug. A nanoformulation encapsulating SL052 into biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) was developed using a single-emulsion solvent evaporation technique and characterized in terms of particle size and loading of the photosensitizing agent. This nanoformulation, SL052-PLGA-nanoparticles (NPs), was compared with recently created nanoformulation based on polyvinylpyrrolidone (SL052-PVP-NPs) and standard liposomal SL052 preparation in terms of efficacy when used for PDT treatment of squamous cell carcinomas SCCVII growing subcutaneously in syngeneic mice. The therapeutic effect of PDT using these three different SL052 formulations was tested for both 1 and 4 h intervals between drug injection and tumor light exposure. The longer time interval produced higher tumor cure rates with all SL052 preparations. With both drug-light intervals, PDT based on SL052-PLGA-NPs produced superior therapeutic benefit compared with the other two SL052 formulations.     

In vitro fungicidal photodynamic effect of hypericin on Candida species

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 μm, respectively, at a fluence of 18 J cm(-2) (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 μM, C. parapsilosis 320 μM and C. krusei 320 μM; light dose 37 J cm(-2)). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 μm and the light dose below 37 J cm(-2). C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.     

Improvement of tumor response by manipulation of tumor oxygenation during photodynamic therapy

Photodynamic therapy (PDT) requires molecular oxygen during light irradiation to generate reactive oxygen species. Tumor hypoxia, either preexisting or induced by PDT, can severely hamper the effectiveness of PDT. Lowering the light irradiation dose rate or fractionating a light dose may improve cell kill of PDT-induced hypoxic cells but will have no effect on preexisting hypoxic cells. In this study hyperoxygenation technique was used during PDT to overcome hypoxia. C3H mice with transplanted mammary carcinoma tumors were injected with 12.5 mg/kg Photofrin and irradiated with 630 nm laser light 24 h later. Tumor oxygenation was manipulated by subjecting the animals to 3 atp (atmospheric pressure) hyperbaric oxygen or normobaric oxygen during PDT light irradiation. The results show a significant improvement in tumor response when PDT was delivered during hyperoxygenation. With hyperoxygenation up to 80% of treated tumors showed no regrowth after 60 days. In comparison, when animals breathed room air, only 20% of treated tumors did not regrow. To explore the effect of hyperoxygenation on tumor oxygenation, tumor partial oxygen pressure was measured with microelectrodes positioned in preexisting hypoxic regions before and during the PDT. The results show that hyperoxygenation may oxygenate preexisting hypoxic cells and compensate for oxygen depletion induced by PDT light irradiation. In conclusion, hyperoxygenation may provide effective ways to improve PDT efficiency by oxygenating both preexisting and treatment-induced cell hypoxia.