Photodynamic Therapy Regimens Research Articles

Rationale of Combined PDT and SDT Modalities for Treating Cancer Patients in Terminal Stage: The Proper Use of Photosensitizer

We read with interest the recent article by Wang et al, reporting the clinical application of SF1 (Sonoflora 1)—a sensitizer that can be activated by light and ultrasound. SF1 is an analog of chlorophyll in that its macrocycle backbone is porphyrin-based and the center of the porphyrin ring consists of a metal ion. The chlorophyll derivative has light absorption peaks at 402 and 636 nm. A very unusual combination of this investigational agent and various prototype light sources and ultrasound devices was used in the treatment of three patients who suffered from late stages of breast cancer with systemic metastases to multiple organs. The combination of photosensitizer and light illumination is commonly known as photodynamic therapy (PDT) and the combination of drug or sonosensitizer and acoustic activation as sonodynamic therapy (SDT). In theory, the direct tumoricidal effects of these modalities are mediated by cytotoxic agents generated through photochemical or sonochemical reactions inside tumor tissue. The narrow gap between potential benefit and potential risk is determined by intrinsic cellular sensitivity and the sensitizer ratio between tumor and normal tissue. The synergistic effects of sensitizer and low-power ultrasound have been examined in many in vitro studies and to a lesser extent in in vivo models. Although the sonosensitization-induced cytotoxicity might be attributed to the sonochemical reaction, such as sensitizer-mediated energy transfer and free radical generation, data suggest that the mechanism of sonosensitization is probably not governed by a universal mechanism, but may be influenced by multiple factors including the nature of the biological model, sensitizer distribution, ultrasound parameters and acoustic energy distribution. Nevertheless, to date, the correlation between ultrasound parameters and biological effects has not been fully established in preclinical investigation (in vitro or in vivo). There is no convincing data that shows that ultrasound used in this way is effective in the treatment of primary tumor and multiple metastases. Therefore, this article raises a few basic questions. First of all, without those critical safety and efficacy information, it is unjustifiable to test the unproven prototypes in humans, particularly those in terminal stages for any purposes. The authors state that acoustic activation was delivered after intravenous administration of the chlorophyll derivative in two different modes—local and whole body ultrasound activation. A handheld ultrasound transducer was used for two patients, which was associated with a side effect of pain. Another patient was placed in a water tub to receive whole body ultrasound treatment. It was expected that ultrasound could reach deep-seated tumor(s) and sensitizers that are beyond the reach of external light of 630 nm. Although no details were provided in terms of the number of transducers in the water tub, the geometric distribution of acoustic energy within the body, and its accessibility to the multiple tumor sites (e.g., breast, lung, bones, liver, neck, axillary lymph nodes, and abdominal lymph nodes), the authors indicated that high acoustic power (2 W/cm at 1 MHz for 20 minutes) was used in both settings without providing justifications on the selection of those parameters. It is also questionable that sufficient acoustic energy will reach the deep-seated metastases in lung and bone tissues. Second, single high-dose PDT and metronomic PDT (i.e., to deliver photosensitizer and/or light doses at low rates over an extended period) have distinct and complementary therapeutic goals. In contrast to the conventional PDT regimen of single dose drug administration (intravenous or oral), the authors used a repeated lingual delivery of the chlorophyll derivative (a total dose of 30 or 60 mg) for 2 to 3 consecutive days followed by light plus ultrasound treatment daily for 3 days starting at day 3 or day 4 after the onset

Monitoring the efficacy of antimicrobial photodynamic therapy in a murine model of cutaneous leishmaniasis using L. major expressing GFP.

A murine model of cutaneous leishmaniasis with green fluorescent protein positive (GFP+) L. major enables the monitoring of parasitic load via measurements of GFP fluorescence intensity, allowing for a faster and more efficient way of monitoring the clinical outcome of photodynamic therapy (PDT). This model may provide new insights on the phototoxic aspects in PDT. Although PDT regimens may be somewhat different in humans, it is expected that the developed model will facilitate the optimization and clinical translation of PDT as a therapy for cutaneous leishmaniasis and the eventual development of topical PDT treatments for other granulomatous infections.

Photodynamic therapy of cottontail rabbit papillomavirus‐induced papillomas in a severe combined immunodeficient mouse xenograft system

To evaluate the efficacy of photodynamic therapy (PDT) with the phthalocyanine photosensitizer Pc 4 for treating an animal model of recurrent respiratory papillomatosis (RRP). Rabbit skin was grafted onto the dorsum of severe combined immunodeficient mice, two xenografts per animal. After the graft healed, it was inoculated with cottontail rabbit papillomavirus (CRPV). When papillomas developed, Pc 4 (0.6 or 1.0 mg/kg) was administered systemically, and 48 hours later, one papilloma of the two on each animal was exposed to 675-nm photoactivating light at either 100 or 150 J/cm(2). In addition to the contralateral tumors, which received Pc 4 but no light, other controls included animals receiving light only or neither agent. Response was assessed by measuring papilloma size with a caliper. Some papillomas and residual skin were harvested for histological assessment. For the lower-dose PDT regimens, papilloma growth rates were not significantly different from the controls. In contrast, 13 of 15 papillomas receiving the higher Pc 4 dose (1.0 mg/kg) and the higher light fluence (150 J/cm(2)) regressed completely and did not regrow within the observation period of up to 79 days. The response of these papillomas was significantly different from the controls (P < .001). Histological analysis confirmed the absence of residual tumor following complete response and replacement with near-normal epithelium. Pc 4-PDT is highly effective in treating virally induced (CRPV) papillomas in a murine model of RRP, and thus warrants further study as a treatment for HPV-induced papillomas.

Combination of photodynamic therapy and immunomodulation: Current status and future trends

Photodynamic therapy (PDT) has been used for the treatment of nonmalignant and malignant diseases from head to toe. Over the last decade its clinical application has gained increasing acceptance around the world. Pre-clinical studies demonstrate that, in addition to the direct local cytotoxicity and vascular effects, PDT can induce various host immune responses. Recent clinical data also show that improved clinical outcomes are obtained through the combination of PDT and immunomodulation. This review will summarize and discuss recent progress in developing innovative regimen of PDT combined with immunomodulation for the treatment of both nonmalignant and malignant diseases.

Photodynamic therapy for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation

Photodynamic therapy (PDT) is emerging as a therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). The efficacy of PDT against CL has been demonstrated previously with aminolevulinic acid, although the prolonged terms of therapy were less than ideal, and the search for new photosensitizers (PS) is ongoing. However, phenothiaziniums have demonstrated high parasiticidal effects in vitro. The subject of our investigation is the in vivo activity of two PS, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride (EtNBSe) and (3,7-Bis(N,N-dibutylamino) phenothiazinium bromide (PPA904). The results of our comparative analysis of the efficacy of these two phenothiazinium analogues demonstrated a high antiparasitic activity of EtNBSe in vitro, and the higher efficacy of PPA904 in a mouse model of CL. The kinetics of photodestruction are different in parasite and mammalian cells, and with both dyes, the macrophages are more susceptible to photodynamic effects than L. major parasites. As the number of parasites in the lesions undergoes a biphasic change, temporarily increasing on days 2-4 and decreasing on days 5-7, more than one treatment is required within an interval of 5 to 7 days. We have also shown that PPA904-PDT can provide an immunomodulating, dose-dependent efflux on IL-12p70 production. This mechanism could be responsible for promoting a more rapid healing in PPA904-PDT treated mice. Our initial data indicate that phenothiaziniums exhibit a high parasiticidal effect in vivo against CL; this finding may be of use in establishing curative PDT regimens for future clinical trials.

TWOFOLD ILLUMINATION PHOTODYNAMIC THERAPY SCHEME FOR SUBFOVEAL CHOROIDAL NEOVASCULARIZATION IN PATHOLOGIC MYOPIA

To compare the short-term efficacy and safety of standard verteporfin photodynamic therapy (PDT) to modified PDT regimen using a twofold illumination scheme for treating choroidal neovascularization in pathologic myopia. Randomized active-controlled clinical trial including 16 patients with subfoveal choroidal neovascularization caused by pathologic myopia and best-corrected visual acuity (BCVA) of 20/320 or better. Patients were randomly assigned (1:1) to standard PDT regimen (50 J/cm; n=8) or twofold illumination PDT scheme (50+50 J/cm; n=8). Comprehensive ophthalmic evaluation including fluorescein angiography was performed at baseline and at weeks 1, 12+/-2, and 24+/-2 following treatment. At week 12 follow-up examination, an additional treatment session with either standard or modified PDT regimens (as assigned at baseline) was applied to areas of fluorescein leakage if present. Changes in BCVA, retreatment rate, and incidence of adverse events were evaluated. All patients completed the 24-week study period. Baseline median BCVA was 20/100 and 20/100 for patients assigned to standard and modified PDT regimens, respectively. At week 24, median improvement in BCVA was significantly greater in patients submitted to twofold illumination PDT scheme (P=0.005; Mann-Whitney U test). Median change from baseline in BCVA (ETDRS lines) was +1.8 (P<0.05; Wilcoxon signed-rank test), +0.4, and +0.5 lines for patients submitted to standard PDT regimen, and +1.8 (P<0.05; Wilcoxon signed-rank test), +1.9 (P<0.05; Wilcoxon signed-rank test), and +2.4 (P<0.05; Wilcoxon signed-rank test) lines for patients submitted to twofold illumination PDT scheme at week 1, 12, and 24 follow-up examinations, respectively. An additional PDT session was performed in 7/8 patients assigned to standard PDT regimen, and in 4/8 patients assigned to modified PDT (P=0.28; Fisher exact test). No PDT-related complications were observed. Although the number of patients and length of follow-up in this study was limited, a better visual outcome and diminished retreatment rate were observed in patients assigned to twofold illumination PDT scheme in comparison to those submitted to standard PDT regimen. Larger studies with longer follow-up to look at twofold illumination PDT scheme versus standard one to confirm our preliminary results are warranted.

Traitement des néo-vaisseaux choroïdiens rétrofovéolaires du myope fort par photothérapie dynamique : résultats à 1 an et à 2 ans

To evaluate the visual outcome of photodynamic therapy with verteporfin in patients with subfoveal choroidal neovascularization (CNV) caused by pathologic myopia. Retrospective and noncomparative analysis of 42 patients with CNV secondary to pathologic myopia recruited between September 2001 and 2003, who were treated with a standard regimen of photodynamic therapy with verteporfin. Forty-two patients were followed up for 12 months; 15 out of 42 completed 24 months of follow-up. Initial visual acuity ranged from 1/10 to 5/10 with a mean of 1.6/10. Twenty-five (56.7%) eyes had stable or improved visual acuity at 12 months with a mean of 2.5/10. In the group of patients who completed 24 months of follow-up, six eyes (39.9%) had stable or improved visual acuity with a mean of 1.6/10 at 1 year and 1.25/10 at 2 years. The average photodynamic therapy treatment required in 1 and 2 years were 1.5 and 2.1, respectively, and they are less than those reported by the Verteporfin in Photodynamic Therapy (VIP) study. Photodynamic therapy with verteporfin for subfoveal choroidal neovascularization caused by pathologic myopia maintained a visual benefit at 1 and 2 years.

Minimally Invasive Photodynamic Therapy (PDT) for Ablation of Experimental Rat Glioma

The feasibility of using ALA-mediated photodynamic therapy (PDT) tumor ablation as a minimally invasive treatment alternative for malignant brain tumors was evaluated in a rodent model. Treatment efficacy and side effects were evaluated with MRI, histopathology and survival rates. BT (4)C orthotopic brain tumors were induced in BD-IX rats. At various time intervals following tumor induction the animals were given 5-aminolevulinic acid (ALA) and 4 hours later optical fibers were inserted directly into the tumor without mechanical debulking or cranial decompression. A 3-day course of steroid treatment was initiated immediately prior to PDT. All untreated animals inevitably died within one month after tumor implantation (28.5 +/- 2.5 days). Complete tumor eradication was achieved in only 1/17 rats, but a significant increase in survival was obtained in the group of animals receiving 125 mg/kg ALA and 26 Joules of light fluence. Histopathology revealed large areas of central tumor necrosis, although clusters of viable tumor cells were often found at the tumor periphery. Pronounced edema in the necrotic tumor center as well as in the surrounding brain, and along white matter tracts was evident in all the brains studied from PDT-treated animal. This study suggests that ALA-mediated PDT may become a promising alternative therapy for the minimally invasive treatment of brain tumors. A judicious choice of PDT regimens that minimizes inflammatory responses through the use multiple fractionated long-term treatment protocols would likely be required.

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

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. 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/cm(2)) and fluence rates (112 and 14 mW/cm(2)), (ii) 5-aminolevulinic acid (ALA)-sensitized PDT (135 J/cm(2) at 75 mW/cm(2)), (iii) DMXAA at a high (30 mg/kg) and low dose (25 mg/kg), and (iv) the combination of HPPH-PDT (48 J/cm(2) at 112 mW/cm(2)) and low-dose DMXAA were studied in BALB/c mice bearing Colon-26 tumors. 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. 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.

5‐aminolaevulinic acid photodynamic therapy in a transgenic mouse model of skin melanoma

Photodynamic therapy (PDT) is widely used to treat preneoplastic skin lesions and non-melanoma skin tumours. Studies analyzing the effects of PDT on malignant melanoma have yielded conflicting results. On the one hand, melanoma cell lines in culture as well as cell lines transplanted into experimental animals were sensitive to PDT. On the other hand, spontaneous melanomas of human patients responded poorly to most PDT regimens tested so far. Here, we analyzed effects of 5-aminolaevulinic acid (5-ALA)-based PDT on melanoma cell lines and on experimental melanomas. To mimic the clinical situation as closely as possible, metallothionein-I/ret (MT-ret) mice, a transgenic model of skin melanoma development, were used. Optimal doses of 5-ALA as well as energy doses and power densities were determined in vitro using a cell line (Mel25) established by us from a melanoma of an MT-ret transgenic mouse as well as commercially available human and mouse melanoma cell lines. Treatment with light irradiation alone had no effect. In combination with 5-ALA, however, this illumination readily induced the death of all mouse and human melanoma cell lines examined. Still, 5-ALA PDT caused only minor focal regressive changes including haemorrhages and fibrosis of MT-ret melanomas in vivo and did not significantly delay tumour growth. These results show that, even though MT-ret melanoma cells are vulnerable to 5-ALA PDT in vitro, malignant MT-ret melanomas in vivo are quite resistant to this type of therapy at doses which are highly effective in vitro.

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/cm(2)) 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/cm(2)). 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/cm(2) at a fluence of 128 J/cm(2) yielded approximately 70-80% tumor cures, whereas the same fluence at the oxygen-depleting fluence rate of 112 mW/cm(2) yielded approximately 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/cm(2) delivered at 14 mW/cm(2) ( approximately 10-20% cures). The optimally curative PDT regimen (128 J/cm(2) at 14 mW/cm(2)) 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.

Treatment of choroidal neovascularization in central serous chorioretinopathy by photodynamic therapy with verteporfin

Purpose To evaluate the safety and efficacy of photodynamic therapy (PDT) with verteporfin in the treatment of patients with choroidal neovascularization (CNV) secondary to central serous chorioretinopathy (CSC). Design Open-label, two-center, noncomparative, prospective interventional case series. Methods Consecutive patients with subfoveal or juxtafoveal CNV secondary to CSC were recruited and treated with a standard regimen of PDT with verteporfin. At regular 3-month follow-up examinations, re-treatment was considered if fluorescein angiography showed evidence of leakage. Outcome measures included the proportion of patients who had improvement (gained 2 more lines), stable, or loss (dropped in 2 or more lines) in vision at the final follow-up and the changes in best-corrected visual acuity (BCVA) from baseline. Results Ten eyes of 10 patients were recruited into the study. The mean age of the patients was 57.3 years with a mean follow-up duration of 12.6 months. At the last follow-up, six (60%) eyes gained 2 or more lines of BCVA with four (40%) patients having final BCVA of within 1 line. No patient lost 2 or more lines of BCVA. The mean logarithm of the minimal angle of resolution BCVA improvement after PDT was 2.4 lines (Wilcoxon signed-rank test, P = .013). No patient suffered serious ocular or systemic complications from PDT. Conclusions Photodynamic therapy with verteporfin therapy is a safe and well-tolerated treatment in patients with CNV associated with CSC. A randomized, controlled trial with a longer follow-up period is warranted to further study the efficacy of PDT in the management of CNV secondary to CSC.

Association between the photodynamic loss of Bcl-2 and the sensitivity to apoptosis caused by phthalocyanine photodynamic therapy.

We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT-induced apoptosis, whether overall cell killing is also sensitized or how up-regulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2delta33-54 and Bcl-2delta37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2delta210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2delta33-54 or Bcl-2delta37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells.

Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy

Background and Objective A human glioma spheroid model is used to investigate the efficacy of different light delivery schemes in 5-aminolevulinic acid (ALA)—mediated photodynamic therapy (PDT). The results provide the rationale for the development of an indwelling balloon applicator for optimizing light delivery. Study Design/Materials and Methods Human glioma spheroids were incubated in ALA (100 or 1000 μg ml−1) for 4 hours and subjected to various light irradiation schemes. In one set of experiments, spheroid survival was monitored as a function of light fluence rate (5–200 mW cm−2). In all cases, spheroids were exposed to fluences of either 25 or 50 J cm−2. In a second study, the effects of repeated weekly PDT treatments, using sub-threshold fluences, were investigated. One group of spheroids was subjected to three treatments using fluences of 12, 12, and 25 J cm−2. Results were compared to spheroids receiving single treatments of either 12 or 25 J cm−2. A fluence rate of 25 mW cm−2 was used for all three groups of spheroids. In all cases, the effect of a given irradiation scheme was evaluated by monitoring spheroid growth. Results Low fluence rates produce greater cell kill than high fluence rates. The minimum effective fluence rate in human glioma spheroids is approximately 10 mW cm−2. Repeated weekly PDT treatments with sub-threshold fluences result in significant cell kill. In spheroids surviving the PDT treatments, growth is suppressed for the duration of the treatment period. Conclusion The results of the in vitro studies support the development of an indwelling balloon applicator for the delivery of light doses in long term multi-fractionated PDT regimens. Lasers Surg. Med. 29:406–412, 2001. © 2001 Wiley-Liss, Inc.

Development of a novel indwelling balloon applicator for optimizing light delivery in photodynamic therapy.

A human glioma spheroid model is used to investigate the efficacy of different light delivery schemes in 5-aminolevulinic acid (ALA)--mediated photodynamic therapy (PDT). The results provide the rationale for the development of an indwelling balloon applicator for optimizing light delivery. Human glioma spheroids were incubated in ALA (100 or 1000 microg /ml-1) for 4 hours and subjected to various light irradiation schemes. In one set of experiments, spheroid survival was monitored as a function of light fluence rate (5-200 mW cm-2). In all cases, spheroids were exposed to fluences of either 25 or 50 J cm-2. In a second study, the effects of repeated weekly PDT treatments, using sub-threshold fluences, were investigated. One group of spheroids was subjected to three treatments using fluences of 12, 12, and 25 J cm-2. Results were compared to spheroids receiving single treatments of either 12 or 25 J cm-2. A fluence rate of 25 mW cm-2 was used for all three groups of spheroids. In all cases, the effect of a given irradiation scheme was evaluated by monitoring spheroid growth. Low fluence rates produce greater cell kill than high fluence rates. The minimum effective fluence rate in human glioma spheroids is approximately 10 mW cm-2. Repeated weekly PDT treatments with sub-threshold fluences result in significant cell kill. In spheroids surviving the PDT treatments, growth is suppressed for the duration of the treatment period. The results of the in vitro studies support the development of an indwelling balloon applicator for the delivery of light doses in long term multi-fractionated PDT regimens.

Photodynamic therapy targeted to tumor-induced angiogenic vessels

Cancer photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA, verteporfin) may be effective not only by being directly cytotoxic to tumor cells, but also by being cytotoxic to the endothelium of tumor neovasculature. In the present study, we investigated the effect of PDT with an experimental liposomal formulation of BPD-MA on tumor-induced angiogenic vessels using a murine dorsal air sac model. First, hemostasis of neovasculature was examined by varying the regimen of PDT. Laser irradiation at 15 min after injection of 2 mg/kg liposomal BPD-MA (15 min PDT) caused complete blocking of blood flow in neovasculature. In contrast, PDT did not inhibit blood flow when the irradiation occurred 3 h after the injection of liposomal BPD-MA (3 h PDT). Next, the antitumor effect of PDT on Meth A sarcoma-bearing mice was investigated by using the hemostasis-inducing regimen. Tumor growth was strongly inhibited after the 15 min PDT with BPD-MA at a dose of 0.5–2 mg/kg. In contrast, 3 h PDT with BPD-MA at a dose of 2 mg/kg suppressed tumor growth only partially. The current study indicates that 15 min PDT causes strong suppression of tumor growth, perhaps through damaging endothelial cells in the tumor neovasculature rather than through a direct cytotoxic effect on tumor cells.

Systems Approach to Optimization of the Regimen of Photodynamic Therapy of Solid Sarcoma M-1 Using the “Fotosens” Preparation as Example

A systems analysis of the interaction between objects (tumor, healthy tissue, radiation, and photosensitizer of the singlet state of oxygen) involved in photodynamic therapy was carried out. A schematic diagram of the biologically significative processes proceeding under these circumstances was proposed. This schematic diagram can serve as a basis for a systems approach to the optimization of photodynamic therapy. The efficiency of the systems approach in photodynamic therapy was demonstrated using “Fotosens” and “Khlorin S” preparations, β‐carotene, and dodecaprene β‐-carotene in biological experiments on Wistar rats and mouses inoculated for the solid sarcoma M-1.

Nuclear factor-κB activation by the photochemotherapeutic agent verteporfin

The nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκB levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκB levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκB is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

Nuclear factor-κB activation by the photochemotherapeutic agent verteporfin

AbstractThe nuclear factor-kappa B (NF-κB) gene transactivator serves in the formation of immune, inflammatory, and stress responses. In quiescent cells, NF-κB principally resides within the cytoplasm in association with inhibitory κ (IκB) proteins. The status of IκB and NF-κB proteins was evaluated for promyelocytic leukemia HL-60 cells treated at different intensities of photodynamic therapy (PDT). The action of the potent photosensitizer, benzoporphyrin derivative monoacid ring A (verteporfin), and visible light irradiation were assessed. At a verteporfin concentration that produced the death of a high proportion of cells after light irradiation, evidence of caspase-3 and caspase-9 processing and of poly(ADP-ribose) polymerase cleavage was present within whole cell lysates. The general caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD.fmk) effectively blocked these apoptosis-related changes. Recent studies indicate that IκB proteins may be caspase substrates during apoptosis. However, the level of IκBβ was unchanged for HL-60 cells undergoing PDT-induced apoptosis. IκBα levels decreased during PDT-induced apoptosis, though ZVAD.fmk did not affect this change. At a less intensive level of photosensitization, cellular IκBα levels were transiently depressed after PDT. At these times, p50 and RelA NF-κB species were increased within nuclear extracts, as revealed by electrophoretic mobility supershift assays. HL-60 cells transiently transfected with a κB-luciferase reporter construct exhibited elevated luciferase activity after PDT or treatment with tumor necrosis factor-α, a well-characterized NF-κB activator. Productive NF-κB activation and associated gene transcription may influence the phenotype and behavior of cells exposed to less intensive PDT regimens. However, IκBα is not subject to caspase-mediated degradation as a component of PDT-induced apoptosis. (Blood. 2000;95:256-262)

Photodynamic therapy of epithelial skin tumours using delta-aminolaevulinic acid and desferrioxamine.

Photodynamic therapy (PDT) uses photosensitizing drugs, such as porphyrins, and light for cancer treatment. In the present clinical study we employed topical application of the porphyrin precursor delta-aminolaevulinic acid (ALA) in combination with desferrioxamine (df) for the induction of endogenous porphyrin synthesis. Irradiation was performed with a light source consisting of a halogen lamp with a red filter and a fibreoptic device. Irradiances ranged from 50 to 300 mW/cm2. We treated 49 patients with this PDT regimen. In 32 patients we treated a total of 34 superficial basal cell carcinomas (BCCs) and 22 nodular BCCs, in nine patients 43 solar keratoses, and in eight patients 10 lesions of Bowen's disease. After a single treatment, 30 (88.2%) of the superficial and seven (31.8%) of the nodular BCCs, 35 of 43 (81.4%) solar keratoses, and three (30%) of the Bowen's disease lesions showed complete remission. The post-treatment observation period was up to 20 months. Repeat therapy was required in six nodular and four superficial BCCs. Biopsies were obtained before treatment, and at variable intervals after treatment. Topical PDT of skin tumours with ALA-df-induced porphyrins is effective in the management of epithelial skin tumours.