TARGETING ACTIVATED LYMPHOCYTES WITH PHOTODYNAMIC THERAPY: SUSCEPTIBILITY OF MITOGEN-STIMULATED SPLENIC LYMPHOCYTES TO BENZOPORPHYRIN DERIVATIVE (BPD) PHOTOSENSITIZATION

Abstract— Benzoporphyrin derivative monoacid ring A (BPD), a hydrophobic chlorin-like porphyrin derivative, which fluoresces strongly at 690 nm, may have potential for both oncologic and nononcologic applications in photodynamic therapy (PDT). To study the influence of cellular characteristics on the uptake of BPD, the murine tumor cell line (P815), and in vitro and in vivo concanavalin A (Con A)-stimulated and unstimulated murine splenic lymphocytes were incubated with 2 µg/mL BPD at 37°C for 0–60 min. At various times, cells were lysed and the amount of BPD taken up by the cells was quantified by fluorescence measurements. The subsets of cells taking up BPD were analyzed using a panel of monoclonal antibodies and the Coulter XL* fluorescence-activated cell sorter. Furthermore, Con A-stimulated and unstimulated spleen cells were incubated with 0–50 ng/mL of BPD for 1 h prior to exposure to red light (7.2 J/cm²). Cell survival 24 h post-PDT was measured by the MTT assay. We found that the rapidly dividing tumor cell line and mitogen-stimulated murine T cells (mainly CD4V IL-2R⁺) took up significantly more BPD (5–10-fold) than do unstimulated splenic lymphocytes. Increased BPD uptake correlated with greater photoinactivation when these cells were exposed to light at a wavelength of 690 nm. These findings suggest that activated cells of the immune system may be a target for photoinactivation by BPD.     

Photodynamic Synovectomy Using Benzoporphyrin Derivative in an Antigen-induced Arthritis Model for Rheumatoid Arthritis

Experimental photodynamic therapy (PDT) has recently been adapted for the treatment of inflammatory and rheumatoid arthritis. The biodistribution of benzoporphyrin derivative monoacid ring A (BPD-MA) and the effect of percutaneous light activation via intra-articular bare cleaved optical fibers was investigated using a rabbit-antigen-induced arthritis model. Qualitative evaluation of intra-articular photosensitizer clearance was performed with laser-induced fluorescence from 0 to 6 h following intravenous injection. The compound was rapidly taken up within the joint and then cleared steadily over the 6 h interval. Biodistribution was determined by fluorescence microscopy and spectrofluoroscopic extraction techniques 3 h following intravenous injection of 2 mg/kg BPD-MA. The biodistribution study demonstrated elevated levels of BPD-MA in synovium (0.35 μg/g) and muscle (0.35μg/g). Fluorescence microscopy demonstrated presence of the compound within pathologic synovium but absence of the photosensitizer within meniscus, ligament, bone and articular cartilage. Tissue effects were evaluated histologically at 2 and 4 weeks posttreatment. BPD-MA-mediated PDT caused synovial necrosis in the region of light activation in 50% of treatment knees at 2 weeks and 43% at 4 weeks. No damage to nonpathologic tissues was observed. These studies indicate that selective destruction of synovium can be achieved by the light-activated photosensitizing agent BPD-MA without damage to articular cartilage or periarticular soft tissues. PDT needs to be further evaluated to optimize treatment parameters to provide for a new minimally invasive synovectomy technique.     

Calculation of singlet oxygen dose using explicit and implicit dose metrics during benzoporphyrin derivative monoacid ring A (BPD-MA)-PDT in vitro and correlation with MLL cell survival

Photodynamic therapy (PDT) oxygen consumption, clonogenic cell survival, fluorescence photobleaching and photoproduct formation were investigated during benzoporphyrin derivative monoacid (BPD-MA)-PDT of MAT-LyLu cells in vitro. Cells were incubated with BPD-MA concentrations of 0.1, 0.5 or 2.5 μg mL(-1) for 2 h and then treated with 405 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment, and photobleaching and photoproduct generation were quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony-forming assay. The amount of oxygen consumed by PDT per photon absorbed decreased with BPD-MA intracellular concentration. Survival was correlated with the total amount of oxygen consumed by PDT per unit volume, which is assumed to be equivalent to the amount of singlet oxygen that reacted. A photobleaching-based singlet oxygen dose metric was also found to predict survival independent of intracellular BPD-MA concentration. The BPD-MA photoproduct was bleached during the treatment. Two singlet oxygen dose metrics based on photoproduct kinetics could not be correlated with cell survival over the full range of intracellular BPD-MA concentrations used.     

Effects of photodynamic therapy on adhesion molecules and metastasis

Photodynamic therapy (PDT) induces among numerous cell targets membrane damage and alteration in cancer cell adhesiveness, an important parameter in cancer metastasis. We have previously shown that hematoporphyrin derivative (HPD)-PDT decreases cancer cell adhesiveness to endothelial cells in vitro and that it reduces the metastatic potential of cells injected into rats. The present study analyzes the influence of PDT in vivo on the metastatic potential of cancers cells and in vitro on the expression of molecules involved in adhesion and in the metastatic process. Photofrin and benzoporphyrin derivative monoacid ring A (BPD) have been evaluated on two colon cancer cell lines obtained from the same cancer [progressive (PROb) and regressive (REGb)] with different metastatic properties. Studies of BPD and Photofrin toxicity and phototoxicity are performed by colorimetric MTT assay on PROb and REGb cells to determine the PDT doses inducing around 25% cell death. Flow cytometry is then used to determine adhesion-molecule expression at the cell surface. ICAM-I, MHC-I, CD44V6 and its lectins (àHt1.3, PNA, SNA and UEA) are studied using cells treated either with BPD (50 ng/ml, 457 nm light, 10 J/cm2) or Photofrin (0.5 microgram/ml, 514 nm light, 25 J/cm2). Changes of metastatic patterns of PROb cells have been assessed by the subcutaneous injection of non-lethally treated BPD or Photofrin cells and counting lung metastases. First, we confirm the metastatic potential reduction induced by PDT with respectively a 71 or 96% decrease of the mean number of metastases (as compared with controls) for PROb cells treated with 50 ng/ml BPD and 10 or 20 J/cm2 irradiation. Concerning Photofrin-PDT-treated cells, we find respectively a 90 or 97% decrease (as compared with controls) of the mean number of metastases for PROb cells treated with 0.5 microgram/ml Photofrin and 25 or 50 J/cm2 irradiation. Then, we observe that CD44V6, its lectins (àHt1.3, PNA, SNA) and MHC-I are significantly decreased (compared with the other molecules tested) in PROb and REGb cells after both BPD and Photofrin PDT treatment. These modifications in adhesion-molecule expression, particularly of CD44V6, can thus account only for part of the decrease in the metastatic potential of PDT-treated cancer cells. Changes in adhesion-molecule expression induced by PDT are only transient, implying that the rate of metastatic reduction is probably not linked simply to these changes.     

LIPOSOMAL DELIVERY OF A PHOTOSENSITIZER, BENZOPORPHYRIN DERIVATIVE MONOACID RING A (BPD), TO TUMOR TISSUE IN A MOUSE TUMOR MODEL

Abstract— Biodistribution studies were carried out on ¹⁴C-labeled benzoporphyrin derivative monoacid ring A (BPD), which had been formulated as a unilamellar liposome or taken from a stock solution in dimethyl sulfoxide diluted into phosphate-buffered saline immediately before intravenous injection into DBA/2 mice. By and large the general distribution of BPD to various organs and tissues was comparable for both formulations. It was noted, however, that liposomal material appeared to enter tissues more rapidly and to be cleared more rapidly, as demonstrated by shorter half-lives for a number of tissues including skin, lung and fat, and generally lower levels in most tissues 24 h following administration. Accumulation in tumor tissue was slightly higher with liposomal BPD, and clearance rates for this tissue were equivalent (half-lives 16.1 h for liposomal BPD and 16.9 h for aqueous BPD). When the two preparations were tested in a bioassay in tumor-bearing mice, photodynamic therapy (PDT) with liposomal BPD proved to be superior to the aqueous preparation when PDT was administered 3 h following intravenous administration of BPD. Plasma distribution studies in vitro demonstrated that 91.1 ± 0.3% of the liposomal BPD distributed to the lipoprotein fraction within the first hour of mixing, whereas only 49.1 ± 2.6% of nonliposomal BPD was associated with lipoprotein under the same conditions. Furthermore, while lipoprotein-associated liposomal BPD distributed evenly between all three types of lipoprotein (high, low and very low density), a majority of nonliposomal BPD associated with the high-density lipoprotein fraction.     

Benzophenothiazine and Benzoporphyrin Derivative Combination Phototherapy Effectively Eradicates Large Murine Sarcomas

Abstract— The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[ a ] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (∼20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm² at 652 nm and then by 100 J/cm² at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.     

Photodynamic Therapy of 9L Gliosarcoma with Liposome-Delivered Photofrin

Abstract— The effect of Photofrin encapsulated in a liposome delivery vehicle for photodynamic therapy (PDT) of the 9L gliosarcoma and normal rat brain was tested. We hypothesized that the liposome vehicle enhances therapeutic efficacy, possibly by increasing tumor tissue concentration of Photofrin. Male Fisher rats bearing a 9L gliosarcoma were treated 16 days after intracerebral tumor implantation with either Photofrin in dextrose (n = 5) or Photofrin in liposome (n = 6). Nontumor-bearing animals were treated with Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle. Tissue concentrations of Photofrin delivered either in dextrose (n = 4) or liposome (n = 4) vehicle were measured in tumor, brain adjacent to tumor and in normal brain tissue. Photofrin was administered (intraperitoneally) at a dose of 12.5 mg/kg and PDT (17 J/cm₂ of 632 nm light at 100 mW/cm₂) was performed 24 h after Photofrin administration. Brains were removed 24 h after PDT and stained with hematoxylin and eosin for analysis of cellular damage. The PDT using Photofrin in the liposome vehicle caused significantly more damage to the tumor ( P < 0.001) than did PDT with Photofrin in dextrose. The PDT of tumor with Photofrin delivered in liposomes caused a 22% volume of cellular necrosis, while PDT of tumor with Photofrin delivered in dextrose caused only scattered cellular damage. Photofrin concentration in tumors was significantly higher ( P = 0.021) using liposome (33.8 ± 18.9 μg/g) compared to dextrose delivery (5.5 ± 1.5 μg/g). Normal brain was affected similarly in both groups, with only scattered cellular necrosis. Our data suggest that the liposome vehicle enhances the therapeutic efficacy of PDT treatment of 9L tumors.     

Significance of Dosimetry in Photodynamic Therapy of Injured Arteries: Classification of Biological Responses

With conflicting results in the literature on the ability of photodynamic therapy (PDT) to inhibit intimal hyperplasia (IH), the present study systematically investigated the effects of drug and light dosimetry on the biologic responses in the artery wall. The rat common carotid artery was balloon-injured and pressurized with benzoporphyrin-derivative monoacid ring (BPD). Then, PDT was performed with an external laser at different fluences and the biologic responses of the artery wall were histologically examined at 24 h and at 2 weeks. Photodynamic therapy effects on injured arteries can be classified into four stages: low-dose PDT using 0.5 microgram/mL BPD at 50 J/cm2 (stage I) resulted in incomplete cell eradication and significant IH at 2 weeks. Irradiation with 100 J/cm2 at the same BPD concentration (stage II) completely eradicated the cells in the artery wall at 24 h but still led to IH at 2 weeks. However, 25 micrograms/mL BPD at 100 J/cm2 (stage III) resulted in total cell eradication at 24 h and inhibition of IH at 2 weeks. In contrast, high-dose PDT with 25 micrograms/mL BPD and 200 J/cm2 (stage IV) led to thrombus development and vascular occlusion at 24 h. These data, demonstrating the different stages of PDT effects on injured arteries, emphasize the critical importance of appropriate PDT dosimetry for the effective inhibition of IH.     

Monitoring tumor response during photodynamic therapy using near-infrared photon-migration spectroscopy.

Benzoporphyrin-derivative (BPD)-monoacid-ring A photodynamic therapy (PDT) was performed on subcutaneous tumor implants in a rat ovarian cancer model. In order to assess PDT efficacy the tumor and normal tissue optical properties were measured noninvasively prior to and during PDT using frequency-domain photon migration (FDPM). FDPM data were used to quantify tissue absorption and reduced scattering properties (given by the parameters mu a and mu's, respectively) at four near-infrared (NIR) wavelengths (674, 811, 849 and 956 nm). Tissue physiologic properties, including the in vivo concentration of BPD, deoxy-hemoglobin (Hb), oxy-hemoglobin (HbO2), total hemoglobin (TotHb), water (H2O) and percent tissue hemoglobin oxygen saturation (%StO2), were calculated from optical property data. PDT efficacy was also determined from morphometric analysis of tumor necrosis in histologic specimens. All the measured tumor properties changed significantly during PDT. [Hb] increased by 9%, while [HbO2], [TotHb] and %StO2 decreased by 18, 7 and 12%, respectively. Using histologic data we show that long-term PDT efficacy is highly correlated to mean BPD concentration in tumor and PDT-induced acute changes in [HbO2], [TotHb] and %StO2 (correlation coefficients of 0.829, 0.817 and 0.953, respectively). Overall, our results indicate that NIR FDPM spectroscopy is able to quantify noninvasively and dynamically the PDT-induced physiological effects in vivo that are highly correlated with therapeutic efficacy.     

Delivery of Benzoporphyrin Derivative, a Photosensitizer, into Atherosclerotic Plaque of Watanabe Heritable Hyperlipidemic Rabbits and Balloon-Injured New Zealand Rabbits

Abstract— In this study we compared the plasma distribution and arterial accumulation of a photosensitizer, benzoporphyrin derivative (BPD), in two models of atherosclerosis: the spontaneous lesions of the Watanabe heritable hyperlipidemic (WHHL) rabbit and induced lesions of the balloon-injured, cholesterol-fed New Zealand white (NZW) rabbit. Selective uptake and retention of a photosensitizer by the abnormal portion of a vessel is a necessity in order for photodynamic therapy to become a successful modality for inhibition of intimal hyperplasia, selective removal of atherosclerotic tissue or imaging of diseased arteries. Liposome-based formulations were compared to freshly isolated native low density lipoprotein (LDL) and acetylated-LDL (Ac-LDL) as delivery vehicles for BPD. Plasma distribution of the photosensitizer was analyzed by KBr density gradient ultracentrifuga-tion. Although the delivery vehicle influenced plasma distribution immediately postinjection, BPD subsequently partitioned according to the plasma concentration of the lipoproteins. Photosensitizer level in plaque and normal artery specimens was determined by ethyl acetate extraction and spectrofluorometric measurement. The measurement of BPD in normal and atherosclerotic arterial tissue demonstrated a selective accumulation in atherosclerotic tissue. Preassociation with LDL and Ac-LDL enhanced accumulation of BPD in atherosclerotic tissue when compared with normal artery (mean ratios of 2.8 and 4.1 were achieved, respectively). These results indicate that the preferential uptake of BPD by atherosclerotic plaque can be enhanced by preassociation with plasma lipoproteins, suggesting that light activation could lead to a highly selective destruction of diseased vascular tissue.     

The Selective Uptake of Benzoporphyrin Derivative Mono-Acid Ring A Results in Differential Cell Kill of Multiple Myeloma Cells in vitro

High-dose chemotherapy (HDCT) and autologous bone marrow/blood stem cell transplantation are an effective combination for treating a number of malignant disorders. The contamination of the autograft by malignant cells may be a reason for recurrences in spite of this treatment, for instance, in multiple myeloma. Therefore, we evaluated the use of photodynamic therapy (PDT) using the photosensitizer benzoporphyrin derivative mono-acid ring A (BPD-MA) on multiple myeloma cells in comparison to the components of the normal bone marrow (NBM) and peripheral blood apheresis product. Flow cytometry was used to measure differential BPDMA uptake of NBM components: namely lymphocytes, monocytes, granulocytes and enriched hematopoietic stem cell (CD34⁺) populations and also the multiple myeloma cell lines OCI-MY7 and OCI-MY4. When each population was measured individually, the order of uptake was [OCI-MY7/MY4] > [CD34⁺] > [granulocytes] = [monocytes] ? [lymphocytes]. Further, clonogenic assay was used to demonstrate surviving fractions for OCI-MY7, OCI-MY4 and NBM in vitro. The LD₉₀ for OCI-MY7 and OCI-MY4 was between 10 and 20 ng/mL BPD-MA whereas this concentration did not show any significant cell kill for the colony-forming units-granulocyte/macrophage (CFU-GM) and burst-forming units—erythrocyte (BFU-E). When the NBM was “contaminated” with multiple myeloma cells in vitro, the LD₉₀ for OCI-MY7 in this cell mixture was shifted to between 40 and 80 ng/mL BPD-MA. However, at 40 ng/ mL BPD-MA at least 50% of normal CFU-GM and BFU-E colonies survived. For CFU-GM and BFU-E derived from the enriched CD34⁺ cell population, BPDMA up to a concentration of 80 ng/mL did not significantly reduce the surviving fractions. We have observed a 3–4 log therapeutic window with differential cell kill when comparing multiple myeloma cell lines to the components of the NBM and apheresis product in vitro. We conclude, that BPD-MA is a molecule potentially useful as an ex vivo purging agent.     

Skin Necrosis due to Photodynamic Action of Benzoporphyrin Depends on Circulating Rather than Tissue Drug Levels: Implications for Control of Photodynamic Therapy

In an ideal world, photodynamic therapy (PDT) of abnormal tissue would reliably spare the surrounding normal tissue. Normal tissue responses set the limits for light and drug dosimetry. The threshold fluence for necrosis (TFN) was measured in normal skin following intravenous infusion with a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) Verteporfin as a function of drug dose (0.25-2.0 mg/kg), wavelength of irradiation (458 and 690 nm) and time interval (0–5h) between drug administration and irradiation. The BPD-MA levels were measured in plasma and skin tissue to elucidate the relationship between TFN, drug kinetics and biodistribution. The PDT response of normal skin was highly reproducible. The TFN for 458 and 690 nm wavelengths was nearly identical and the estimated quantum efficiency for skin response was equal at these two wavelengths. Skin phototoxicity, quantified in terms of 1/ TFN, closely correlated with the plasma pharmacokinetics rather than the tissue pharmacokinetics and was quadratically dependent on the plasma drug concentration regardless of the administered drug dose or time interval between drug and light exposure. This study strongly suggests that noninvasive measurements of the circulating drug level at the time of light treatment will be important for setting optimal light dosimetry for PDT with liposomal BPD-MA, a vascular photosensitizer.     

THYMIDINE UPTAKE, INCORPORATION AND DNA POLYMERASE ACTIVITY IN MURINE BLADDER TUMOR CELL,MBT–2, EXPOSED TO UV ACTIVATED DIHEMATOPORPHYRIN ETHER

Abstract— Photodynamic therapy (PDT) is a new modality for treatment of malignancy. In this paper, we reported the effect of UV activated dihematoporphyrin ether (DHE) on [³H] thymidine uptake and DNA synthesis in murine bladder tumor cells,MBT–2. Exponentially growing cells were pretreated with 0.05–5 μg/ml of DHE for 30 min in complete darkness prior to irradiation with 0.15-0.90 J/cm² of UV light (265 nm). The rates of thymidine uptake and DNA synthesis were suppressed in a DHE concentration and photic energy dependent manner. Double reciprocal analysis on the kinetics of the thymidine uptake and DNA synthesis indicated that the inhibition was non-competitive, i.e. decrease in both the apparent K^m value and maximum velocity in DHE plus UV light treated cells. The activities of DNA polymerase a and (3 were determined by [*H] dATP incorporation into DNA of permeabilizedMBT–2 cells. DNA polymerase a activity was approximately 60% of the control after 0.45 J/cm² of UV light exposure; a further inhibition of DNA polymerase a was observed when 0.5–5ng/W of DHE and UV photoradiation were combined. In contrast, a slight stimulation of DNA polymerase fJ was noted after a similar treatment. This study demonstrates that photodynamic therapy-induced suppression of DNA synthesis inMBT–2 cells is a complex process involving in reduction of thymidine transport as well as the perturbation of the enzymes involved in DNA synthesis.     

In vitro survival of nonsmall cell lung cancer cells following combined treatment with ionizing radiation and photofrin-mediated photodynamic therapy

It has been suggested that combination high dose rate (HDR) intraluminal brachytherapy and photodynamic therapy (PDT) in nonsmall cell lung cancer (NSCLC) may improve efficacy of treatment, reduce toxicity and enhance quality of life for patients. To provide a cellular basis for this we examined the in vitro sensitivity of MRC5 normal lung fibroblasts and four NSCLC cell lines following HDR radiation, PDT and combined HDR radiation and PDT. HDR radiation was cobalt-60 gamma rays (1.5–1.9 Gy min⁻¹). For PDT treatment, cells were exposed to 2.5 μg mL⁻¹ Photofrin for 18–24 h followed by light exposure (20 mW cm⁻²). For combined treatment cells were exposed to Photofrin and then either exposed to light and 15–30 min later exposed to HDR radiation or exposed to HDR radiation and 15–30 min later exposed to light. D₃₇ values calculated from clonogenic survival curves indicated a six-fold difference in HDR radiation sensitivity and an eight-fold difference in PDT sensitivity. The effect of combined treatment was not significantly different from an additive effect of the individual treatment modalities for the NSCLC cells, but was significantly less than additive for the MRC5 cells. These results suggest an equivalent tumor cell kill may be possible at reduced systemic effects to patients.     

Amino acid derivatives of pyropheophorbide-α ethers as photosensitizer: Synthesis and photodynamic activity

Ten new water-soluble amino acid conjugates of pyropheophorbide-α ethers 4a-4j were synthesized and investigated for their in vitro photodynamic antitumor activity. The results showed that all compounds exhibited higher phototoxicity and lower dark toxicity against three kinds of tumor cell lines than BPD-MA. In particular, themost phototoxic compound 4d and 4j individually showed IC₅₀ values of 41 nmol/L and 33 nmol/L against HCT116 cell, which represented 7.8- and 9.7-fold increase of antitumor potency compared to BPD-MA, respectively, suggesting that they were promising photosensitizers for PDT applications because of their strong absorption at long wavelength (λ_max>650 nm), high phototoxicity, low dark cytotoxicity and good water-solubility.     

PHOTODYNAMIC THERAPY OF A MURINE TUMOR FOLLOWING SENSITISATION WITH CHLORO ALUMINUM SULFONATED PHTHALOCYANINE

Abstract Syngeneic mice bearing a colorectal carcinoma (Colo 26) growing subcutaneously in the flank region received photodynamic therapy (PDT) when the tumor was 7-12 mm diameter. Light (emission at 675 nm from an argon ion pumped dye laser, laser energy 100 J, power 50 mW) was delivered to the tumor 24-28 h after the i.v. injection of a single dose of chloro aluminum sulfonated phthalocyanine (ClAlSPc) at 5 mg kg¹ body weight. Control tumor-bearing animals received (1) phosphate buffered saline (PBS) but not PDT, (2) ClAlSPc but not PDT, or (3) PBS injection plus PDT. Five days later PDT tumors of the ClAlSPc + PDT group were markedly reduced in size (mean weight 0.075 ± 0.027 g) as compared with those from control groups 1 (0.408 ± 0.167 g), 2 (0.475 ± 0.143 g) or 3 (0.376 ± 0.153 g). Histological examination revealed that ClAlSPc + PDT induced severe necrosis and cytotoxicity of neoplastic cells with viable tumor limited to a small peripheral margin. Animals in the ClAlSPc + PDT group in a repeat experiment survived significantly longer than animals in the three control groups suggesting that ClAlSPc may be a useful photosensitiser for PDT of cancer.     

In situ COMPARISON OF 665 nm and 633 nm WAVELENGTH LIGHT PENETRATION IN THE HUMAN PROSTATE GLAND

Abstract— The depth of treatment in photodynamic therapy (PDT) of tumors varies with the wavelength of light activating the photosensitizer. New generation photosensitizers that are excited at longer wavelengths have the potential for increasing treatment depths. Tin ethyl etiopurpurin (SnET2), a promising second-generation photosensitizer is maximally activated at 665 nm, which may be significantly more penetrating than 633 nm light currently used with porphyrins in PDT. The penetration of 665 nm and 633 nm wavelength red light in the prostate gland was compared in 11 patients undergoing prostatic biopsies for suspected prostatic cancer. Interstitial optical fibers determined the light attenuation within the prostate gland. Of the 11 patients, 7 had dual wavelength and 4 had single wavelength studies. The mean attenuation coefficients, μ_eff, for 665 nm and 633 nm wavelength light were 0.32 ± 0.05 mm^-1 and 0.39 ± 0.05 mm^-1, respectively, showing a statistically significant difference (P = 0.0003). This represented a 22% increase in the mean penetration depth and at 10 mm from the delivery fiber there was 1.8 times as much 665 nm light fluence than 633 nm. The mean μ_eff at 665 nm for benign and malignant prostate tissue were similar ( P = 0.42), however, there was significant interpatient variation (μ_eff ranging from 0.24 to 0.42 mm^-1) reflecting biological differences of therapeutic importance. The enhanced light fluence and penetration depth with 665 nm light should allow significantly larger volumes of prostatic tissue to be treated with SnET2-mediated PDT.     

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.     

THE IN VITRO PHOTOCHEMISTRY OF BIOLOGICAL MOLECULES—II. THE TRIPLET STATES OF β-CAROTENE AND LYCOPENE EXCITED BY PULSE RADIOLYSIS

Abstract— –Pulse radiolysis has been used to excite the triplet states of β-carotene (τ# 9μ sec) and lycopene (τ= 8μsec) in hexane solution, both in the presence and absence of naphthalene as a triplet sensitiser. The absorption spectra of both triplets have been measured in the range 430–550 nm and have thus been extended into the region of the corresponding singlet absorptions. The overlap of the triplet and singlet spectra is discussed in relation to in vivo studies. Extinction coefficients of 1.3±0.1 × 10⁵ l/mole cm for β-carotene triplet 515 nm and 3.9±0.2 × 10⁵ l/mole cm for lycopene triplet at 525 nm were obtained. Isomerisation of the all- trans polyenes used was detected and preliminary measurements indicate that the yield of isomerisation was greater than the triplet yield. The rate of triplet energy transfer from naphthalene to β-carotene was estimated to be 1.5 × 10¹⁰ l/mole sec. The corresponding value for lycopene was 1.4× 10¹⁰ l/mole sec. The measured efficient quenching of triplet β-carotene by oxygen may occur by an energy transfer mechanism, leading to the formation of singlet oxygen (¹Δ_g. This would suggest that the triplet energy level of β-carotene lies between 121 and 94 kJ mole^-1.     

MOUSE SKIN PHOTOSENSITIZATION WITH BENZOPORPHYRIN DERIVATIVES AND PHOTOFRIN®: MACROSCOPIC AND MICROSCOPIC EVALUATION

A comparative study, at both the macroscopic and microscopic level, of skin photosensitivity caused by four isomeric forms of benzoporphyrin derivative (BPD) has been carried out, and compared to effects of Photofrin. Animals injected intravenously with BPD analogues and exposed to light 3 h later showed extensive photosensitivity. Animals receiving the monoacid derivatives of BPD (BPD-MA and BPD-MB) showed markedly more photosensitivity than those receiving the diacid derivatives (BPD-DA and BPD-DB). Animals receiving BPD analogues which were exposed to light 24 h or more later showed only minimal reactivity. Histological examination of biopsies taken after photosensitizer injection and light exposure showed extensive changes in epidermis and dermis, including epidermal erosion, degranulation of the stratum granulosum, spongiosis, depletion in cellularity and mast cell degranulation. These changes were noted to be similar to changes caused by Photofrin.