Preferential light absorption in atheromas in vitro. Implications for laser angioplasty.

Laser angioplasty, the in situ ablation of arterial obstructions with laser radiation, has been demonstrated in animal models and early clinical trials. A problem with this technique, however, is the possibility of thermal damage to adjacent or underlying normal tissues that also absorb the radiation. Using a spectrophotometer with an integrating sphere and a specially constructed tunable-dye laser-based spectrophotometer, we evaluated the transmittance and remittance of human cadaveric atheromas and adjacent normal aorta from 250 to 1,300 nm to identify wavebands where there is preferential light absorption by atheromas. Data were analyzed by both the Kubelka-Munk formalism and a Beer's law model. Both methods indicate that atheromas absorb more than normal aorta between 420 and 530 nm. At 470 nm the average Kubelka-Munk absorption coefficient of atheromas from 10 cadavers was 54 +/- 9 cm-1 compared with 26 +/- 6 cm-1 for normal aortic specimens from seven cadavers. Yellow chromophores responsible for the atheroma absorbance were extractable with xylenes. Thin-layer chromatography and absorption spectra identified the extracted chromophores as predominantly consisting of a mix of carotenoids, which are known constituents of atheromatous lesions. Preferential absorption of blue light by carotenoids in atheromas may permit selective ablation of atheromatous obstructions with appropriate pulses of laser radiation.

Photosensitizing dyes for the selection of nontumorigenic revertants from human lung cancer cell lines.

Certain positively charged, lipophilic dyes have been noted by various authors to localize selectively in the mitochondria of carcinoma cells. Oseroff et al. (Proc. Natl. Acad. Sci. USA, 83:9729-9733, 1986) studied 10 carcinoma-specific mitochondrial photosensitizers and judged N,N'-bis(2-ethyl-1,3-dioxolane)kryptocyanine (EDKC) to be the most effective in selective carcinoma cell photolysis, a system where light-absorbing molecules accumulate only in carcinoma cells and on illumination initiate a reaction that kills or damages those cells. The present study duplicated the published EDKC retention result for the normal monkey kidney epithelial cell line CV-1. A series of nontumorigenic and tumorigenic human bronchial epithelial and human pleural mesothelial cells were assayed for EDKC uptake and retention, with the intent of using selective carcinoma cell photolysis to isolate nontumorigenic revertants of the tumorigenic lung cell lines. In addition, the uptake and retention of the fluorescent, mitochondria- and carcinoma-specific dye rhodamine-123 were surveyed in a series of hybrids between tumorigenic and nontumorigenic human bronchial epithelial cells. The half-life of dye retention ranged from 6 to 12 h in all the bronchial epithelial and mesothelial cells studied, with little or no dye selectivity for tumorigenic cells. When EDKC-retaining bronchial epithelial cells were illuminated with red light, significant reductions in short term viability and colony-forming efficiency were seen, which became more pronounced as light and dye doses were increased. However, these effects did not correlate with tumorigenicity within the cell series. The method, therefore, does not appear generally useful for the selection of nontumorigenic variants of human bronchial epithelial or pleural mesothelial cancers of the lung.

Cytotoxicity and mutagenicity of low intensity, 248 and 193 nm excimer laser radiation in mammalian cells.

The cytotoxicity of 193 and 248 nm excimer laser radiation was compared to that produced by a germicidal lamp (predominantly 254 nm) using Chinese hamster ovary cells (CHO), and a human diploid fibroblast line, AG-1522A. Excimer laser radiation at 248 nm (3.5 X 10(2) w/m2) and germicidal radiation (5.3 X 10(-5) w/m2) caused toxicity in both cell lines, with the AG-1522A cells (D37 = 7-8 J/m2) being slightly more sensitive than the CHO cells (D37 = 11 J/m2). Incident 193 nm radiation was less cytotoxic than 248 nm to AG-1522A and CHO cells with D37 values of 18 and 85 J/m2, respectively. The mutagenic potential of UV excimer radiation at 193 and 248 nm was evaluated using the hypoxanthine guanine phosphoribosyl transfer assay system with CHO cells. Excimer laser radiation at 248 nm induced mutation in proportion to dose (1.7 X 10(-5) resistant colonies per survivor per J/m2 incident radiation) up to 14 J/m2, similar to results reported for 254 nm light. However, excimer laser radiation at 193 nm did not cause mutation greater than the dark control. The decreased cytotoxicity and mutagenicity of 193 nm radiation may be due to the shielding of the nucleus by cytoplasmic and membrane components or to the formation of different DNA photoproducts. These differences between 193 and 248 nm radiation may be important in choosing an excimer wavelength for ablation in biological systems.

Mitochondrial toxicity of cationic photosensitizers for photochemotherapy.

The triarylmethane derivative Victoria Blue-BO (VB-BO) and the chalcogenapyrylium (CP) dyes have potential for use in photochemotherapy, because they are taken up by the mitochondria of malignant cells and cause cell death. To clarify the mechanism of cell killing we examined the phototoxic effects of VB-BO and a series of three CP dyes on bioenergetic function in isolated rat liver mitochondria. Without photoirradiation, and irrespective of the respiratory substrate used, each of the compounds tested induced some uncoupling of oxidative phosphorylation. Visible irradiation of VB-BO produced an inhibition of mitochondrial respiration when glutamate plus malate, but not succinate, was used as the respiratory substrate. With photoirradiation VB-BO was also shown to inhibit rotenone-sensitive NADH-cytochrome c reductase activity, but it had no effect on succinate-cytochrome c reductase activity. These data indicate that photoactivation of VB-BO produces selective inhibition of mitochondrial respiratory complex I. Photoirradiation of the CP dyes inhibited both complex I and complex II initiated respiratory activity. With photoirradiation, the CP dyes also inhibited both NADH- and succinate-cytochrome c reductase activities, as well as other membrane-bound enzymes, cytochrome c oxidase and succinate dehydrogenase, but not the mitochondrial matrix enzyme, citrate synthetase, or the cytosolic enzyme, lactate dehydrogenase. alpha-Tocopherol protected bioenergetic activities against CP dye photodamage. These results suggest that mitochondrial photosensitization by CP compounds is mediated by the production of membrane-damaging singlet oxygen which causes nonspecific damage to membranes and membrane-bound enzymes.

Mechanisms of mitochondrial photosensitization by the cationic dye, N,N-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC): preferential inactivation of complex I in the electron transport chain.

We investigated mechanisms of mitochondrial phototoxicity caused by the cationic cyanine dye N,N'-bis(2-ethyl-1,3-dioxylene)kryptocyanine (EDKC), examining the role of the mitochondrial membrane potential on the dye uptake by carcinoma cells in vitro, and both the dark and photosensitizing effects of the dye on the function of isolated mouse liver mitochondria. When human bladder carcinoma cells (EJ) were pretreated with 2,4-dinitrophenol or nigericin, cellular uptake of EDKC decreased or increased, respectively, consistent with dye uptake that is dependent on membrane potentials. In isolated liver mitochondria, during NADH linked substrate oxidation (using glutamate plus malate or beta-hydroxybutyrate as substrates), low concentrations of the dye (0.25-0.5 microM) sensitized mitochondria to illumination with long wavelength light and inhibited both basal and ADP-stimulated respiration. Similar effects were observed during succinate oxidation, but only at higher concentrations of EDKC (greater than 5 microM) and at 10-fold greater light doses. NADH coenzyme Q reductase (Complex I) activity was inhibited by dye with or without light to an extent comparable to the inhibition of glutamate plus malate oxidation. Activity of cytochrome c oxidase, the terminal enzyme in the electron transport chain, was photosensitized with high dye doses (greater than 5 microM) and light, but the extent of inhibition was much less than the inhibition of respiration with succinate as substrate. ATP synthetase (F0F1 ATPase) activity was minimally affected by 4.0 microM EDKC with or without 24 J/cm2 light. We conclude that at low concentrations of dye, respiratory Complex I is a primary target for EDKC dark and light-induced toxicities. If Complex I is bypassed by using succinate as a respiratory substrate, the mitochondria can tolerate much higher dye concentrations and light doses.

Photofrin photodynamic therapy can significantly deplete or preserve oxygenation in human basal cell carcinomas during treatment, depending on fluence rate.

At high fluence rates in animal models, photodynamic therapy (PDT) can photochemically deplete ambient tumor oxygen through the generation of singlet oxygen, causing acute hypoxia and limiting treatment effectiveness. We report that standard clinical treatment conditions (1 mg/kg Photofrin, light at 630 nm and 150 mW/cm2), which are highly effective for treating human basal cell carcinomas, significantly diminished tumor oxygen levels during initial light delivery in a majority of carcinomas. Oxygen depletion could be found during at least 40% of the total light dose, but tumors appeared well oxygenated toward the end of treatment. In contrast, initial light delivery at a lower fluence rate of 30 mW/cm2 increased tumor oxygenation in a majority of carcinomas. Laser treatment caused an intensity- and treatment time-dependent increase in tumor temperature. The data suggest that high fluence rate treatment, although effective, may be inefficient.

Pivotal phase III trial of two dose levels of denileukin diftitox for the treatment of cutaneous T-cell lymphoma.

PURPOSE: The objective of this phase III study was to determine the efficacy, safety, and pharmacokinetics of denileukin diftitox (DAB389IL-2, Ontak [Ligand Pharmaceuticals Inc, San Diego, CA]) in patients with stage Ib to IVa cutaneous T-cell lymphoma (CTCL) who have previously received other therapeutic interventions. PATIENTS AND METHODS: Patients with biopsy-proven CTCL that expressed CD25 on > or = 20% of lymphocytes were assigned to one of two dose levels (9 or 18 microg/kg/d) of denileukin diftitox administered 5 consecutive days every 3 weeks for up to 8 cycles. Patients were monitored for toxicity and clinical efficacy, the latter assessed by changes in disease burden and quality of life measurements. Antibody levels of antidenileukin diftitox and anti-interleukin-2 and serum concentrations of denileukin diftitox were also measured. RESULTS: Overall, 30% of the 71 patients with CTCL treated with denileukin diftitox had an objective response (20% partial response; 10% complete response). The response rate and duration of response based on the time of the first dose of study drug for all responders (median of 6.9 months with a range of 2.7 to more than 46.1 months) were not statistically different between the two doses. Adverse events consisted of flu-like symptoms (fever/chills, nausea/vomiting, and myalgias/arthralgias), acute infusion-related events (hypotension, dyspnea, chest pain, and back pain), and a vascular leak syndrome (hypotension, hypoalbuminemia, edema). In addition, 61% of the patients experienced transient elevations of hepatic transaminase levels with 17% grade 3 or 4. Hypoalbuminemia occurred in 79%, including 15% with grade 3 or 4 changes. Tolerability at 9 and 18 microg/kg/d was similar, and there was no evidence of cumulative toxicity. CONCLUSION: Denileukin diftitox has been shown to be a useful and important agent in the treatment of patients whose CTCL is persistent or recurrent despite other therapeutic interventions.

Pleiotropic expression of heterologous cytokine/receptor genes in HTLV-1 associated diseases: candidate TRS for chimeric gene therapy.

DNA motifs that encode for specific transcriptional regulatory sequences (TRS) when engineered adjacent to the structural protein coding domain of a suicide enzyme can provide cell-lineage specific protein expression. The disparate up-regulation of several genes in adult T-cell leukemia (ATL) versus HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), seropositive carriers (SPC) and uninfected normals may reflect events at the molecular level related to leukemogenesis or to processes maintaining the heme-oncologic phenotype. Further, the genetic transduction of cytokine and receptor genes uniquely associated with ATL may provide targets for the development of leukemia-specific gene therapies aimed at exploiting differences in the production of certain growth factors and growth factor receptors. Comparisons of the transcriptional and translational levels of interleukin-2 receptor alpha chain (IL-2R alpha), transforming growth factor-beta 1 (TGF-beta 1) and intracellular adhesion molecule-1 (ICAM-1) in ATL, HAM/TSP, and SPC and in several control populations revealed selectively up-regulated expression in ATL. We evaluated the feasibility of using lymphoid-specific TRS to activate herpes simplex virus thymidine kinase (HSVtk) to achieve selective cytotoxicity in leukemias expressing terminal deoxynucleotidyl transferase (TdT). Selective and efficient leukemic cell killing was produced and suggests that similar chimeric gene constructs containing TRS elements for IL-2R alpha, TGF-beta 1, or ICAM-1 may prove useful in designing gene therapies to treat ATL.

Immune responses in patients with T-cell lymphoma treated with an anti-idiotype antibody mimicking a highly restricted T-cell antigen.

We generated an IgG1 murine monoclonal anti-idiotype antibody (Ab2) to a highly restricted T-cell antigen designated glycoprotein (gp) 37 that is found on T-cell malignancies but not on normal cells. gp37 is identified by the murine monoclonal antibody SN2 (Ab1) against which the Ab2 was raised. Each of four patients with T-cell lymphoma predominantly confined to the skin received a minimum of four intracutaneous injections of aluminum hydroxide precipitated anti-idiotype murine monoclonal antibody (1 mg/injection) given every 2 weeks. For responding patients, injections were continued on a monthly basis. All tumors were measured along orthogonal major and minor axes, using a ruler and/or calipers, by the same observer. Tumor sizes were documented photographically. Three of the four patients developed specific idiotypic humoral immune responses, and two of the four patients also demonstrated idiotypic cell-mediated responses. Humoral responses included binding of the patients' sera to the anti-idiotype antibody as well as specific inhibition of binding of the SN2 antibody (Ab1) to the anti-idiotype antibody (Ab2). Anti-anti-idiotypic (Ab3) antibody from one patient's serum bound specifically to the gp37-positive cell line MOLT-4 and also to semipurified gp37 antigen. Cell-mediated responses were demonstrated by specific proliferative response to the aluminum hydroxide precipitated anti-idiotype antibody by patients' peripheral blood mononuclear cells. While three of the four patients had extensive disease and did not have clinical responses, one of the patients who had nine discrete skin tumors and peripheral blood involvement without other detectable disease had virtually complete disappearance of the tumors lasting over 11 months. Our results demonstrate that this particular anti-idiotype antibody can induce humoral and cellular immune responses, and at least in one patient led to a meaningful therapeutic response. Future trials should focus on immunocompetent patients with minimal disease.

Photofrin photodynamic therapy for treatment of AIDS-related cutaneous Kaposi's sarcoma.

OBJECTIVE: Kaposi's sarcoma, the most common malignancy in AIDS patients, often presents with painful cutaneous lesions that are difficult to treat effectively despite a wide variety of therapeutic approaches. We used photodynamic therapy in an attempt to provide effective palliative treatment for this disease. METHODS: Photodynamic therapy utilizes the activation by light of a photosensitizing drug that preferentially accumulates in tumor tissue such as Kaposi's sarcoma. We enrolled 25 patients who received 1.0 mg/kg of Photofrin 48 h before exposure to 100-400 J/cm2 of 630 nm light. RESULTS: Of the 348 lesions treated, 289 were evaluable: 32.5% had complete clinical response, 63.3% had partial clinical response and 4.2% were clinical failures. There was a strong correlation between response and light dose: 54% of lesions achieved a complete clinical response at optimum light dose (> 250 J/cm2). There was no correlation of response with CD4 cell count nor was there a change in CD4 cell count post-treatment. At 400 J/cm2 full field scabbing and necrosis occurred in 90% of the treated fields. Thus, the maximum tolerated dose was determined to be 300 J/cm2. At light doses of 250 J/cm2 and below the toxicities were limited to erythema and edema in the treatment field. Forty-three biopsies were taken 0.5 h to 4 months post-treatment. These showed little change in the B and T cell infiltrates identified. Kaposi's sarcoma cells disappeared post-treatment in certain lesions. CONCLUSION: Photofrin is effective palliative treatment for HIV-associated Kaposi's sarcoma.

Mitochondria-based photodynamic anti-cancer therapy.

As photodynamic therapy (PDT) becomes established as a treatment for cancer, there is increasing interest in identifying critical mechanisms of cell killing and understanding the bases for effective photosensitizers. The existence of multiple cellular targets makes it difficult to distinguish the critical events leading to cell death from PDT. However, with more sensitive techniques to detect photosensitizer localization, the isolation of PDT-resistant and -sensitive mutants and the use of innovative molecular and biochemical strategies to map cellular events occurring during and after photosensitization, some order is emerging from the chaos. The subcellular localization of many photosensitizers and the early responses to light activation indicate that mitochondria play a major role in photodynamic cell death. PDT with many agents which damage or inhibit different or multiple mitochondrial targets has many of the desirable characteristics for an effective anti-cancer therapy.

A murine monoclonal antibody (VM-1) against human basal cells inhibits the growth of human keratinocytes in culture.

Using epidermal cells from psoriatic plaques as the immunogen, an IgG1 murine monoclonal antibody, VM-1, has been produced which stains basal keratinocytes on frozen sections of skin obtained from normal individuals and from psoriatic plaques. In some areas of both normal and psoriatic epidermis, the cell layer immediately above the basal cells is also stained. Cells in the external root sheath of the hair follicles also bind VM-1. The antibody binding site is trypsin-resistant, and is not blocked by bullous pemphigoid serum. If dispersed epidermal cells are preincubated with VM-1 for 1 h or more before plating, the majority of the cells do not attach and spread out on a collagen-coated Petri dish surface or on a fibroblast feeder layer. When added to attached, preconfluent cultures of keratinocytes, VM-1 inhibits growth and alters cell morphology. The growth inhibition is specific for keratinocytes, and viability studies show that it is not due to an immediate toxic effect of the antibody. The VM-1-induced inhibition of keratinocyte growth is not reversed by soy bean or lima bean trypsin inhibitors added at the time of cell plating or at the time of addition of antibody.

Unscheduled DNA synthesis in human skin after in vitro ultraviolet-excimer laser ablation.

DNA damage repaired by the excision repair system and measured as unscheduled DNA synthesis (UDS) was assessed in freshly excised human skin after 193 and 248 nm ultraviolet (UV)-excimer laser ablative incisions. Laser irradiation at 248 nm induced DNA damage throughout a zone of cells surrounding the ablated and heat-damaged area. In contrast, with 193 nm irradiation UDS was not detected in cells adjacent to the ablated area, even though DNA strongly absorbs this wavelength. Our results suggest that the lack of UDS after 193 nm irradiation is due to: "shielding" of DNA by the cellular interstitium, membrane, and cytoplasm, DNA damage that is not repaired by excision repair, or thermal effects that either temporarily or permanently inhibit the excision repair processes.

Selective enrichment of human epidermal cell subpopulations using monoclonal antibodies.

In studying the mechanisms that regulate the growth and differentiation of the human epidermis, it would be helpful to obtain relatively pure populations of the different epidermal cell types. We have used a solid-phase immunoabsorption method termed "panning" to positively select two types of epidermal cells: Langerhans cells and the keratinocytes found in the basal cell layer (basal cells). To attach basal cells to a goat anti-mouse IgG-coated plastic surface, we used murine monoclonal antibodies (VM-1 or VM-2), which were recently produced in our laboratory and bind specifically to antigens on human basal cells. Using antibodies VM-1 or VM-2, we panned for basal cells and obtained a yield of about 40 percent (an enrichment of about 2.5-fold). The cells enriched for basal cells demonstrated much better growth and DNA synthesis than did the cell fraction depleted of basal cells. For positive selection of Langerhans cells, we used OKT6, a murine monoclonal antibody that binds specifically to Langerhans cells in the epidermis. We determined that of those cells preincubated with OKT6 and adherent to an antibody-coated petri dish surface, about 70 percent demonstrated OKT6 binding by fluorescence microscopy. This represents a 15- to 20-fold enrichment for Langerhans cells. The nonadherent cell fraction contained less than 1 percent OKT6-positive cells. Ultrastructural studies showed that the cells thus separated were Langerhans cells. The OKT6-positive but not the OKT6-negative cells were capable of stimulating allogeneic lymphocytes in the skin-cell-lymphocyte reaction. Thus the panning technique is an effective method for obtaining greatly enriched subpopulations of viable epidermal cells.

A pulsed electric field enhances cutaneous delivery of methylene blue in excised full-thickness porcine skin.

We used electric pulses to permeabilize porcine stratum corneum and demonstrate enhanced epidermal transport of methylene blue, a water-soluble cationic dye. Electrodes were placed on the outer surface of excised full-thickness porcine skin, and methylene blue was applied to the skin beneath the positive electrode; 1 ms pulses of up to 240 V were delivered at frequencies of 20-100 Hz for up to 30 min. The amount of dye in a skin sample was determined from absorbance spectra of dissolved punch biopsy sections. Penetration depth and concentration of the dye were measured with light and fluorescence microscopy of cryosections. At an electric exposure dose VT (applied voltage x frequency x pulse width x treatment duration) of about 4700 Vs, there is a threshold for efficient drug delivery. Increasing the applied voltage or field application time resulted in increased dye penetration. Transport induced by electric pulses was more than an order of magnitude greater than that seen following iontophoresis. We believe that the enhanced cutaneous delivery of methylene blue is due to a combination of de novo permeabilization of the stratum corneum by electric pulses, passive diffusion through the permeabilization sites, and electrophoretic and electroosmotic transport by the electric pulses. Pulsed electric fields may have important applications for drug delivery in a variety of fields where topical drug delivery is a goal.

Water-soluble, core-modified porphyrins as novel, longer-wavelength-absorbing sensitizers for photodynamic therapy.

Water-soluble, core-modified 5,10,15, 20-tetrakis(4-sulfonatophenyl)-21,23-dithiaporphyrin (1) and 5,10,15, 20-tetrakis(4-sulfonatophenyl)-21,23-diselenaporphyrin (2) were prepared as the tetrasodium salts by the sulfonation of 5,10,15, 20-tetraphenyl-21,23-dithiaporphyrin (3) and -21, 23-diselenaporphyrin (4), respectively, with sulfuric acid. Compounds 3 and 4 were prepared by the condensation of pyrrole with either 2,5-bis(phenylhydroxymethyl)thiophene (5) or 2, 5-bis(phenylhydroxymethyl)selenophene (6) in propionic acid. The addition of benzaldehyde to 2,5-dilithiothiophene or 2, 5-dilithioselenophene gives 5 or 6, respectively, as a nearly equimolar mixture of meso- and d,l-diastereomers. Careful crystallization of 5 gives a single diastereomer by removing the crystalline product from the equilibrating mixture of diastereomers in solution. Photodynamic therapy (PDT) with 1 has an LD(50) of less than 25 microg/mL against Colo-26 cells in culture and exhibits a lethal dose for 90% or more at concentrations greater than 50 microg/mL. In contrast, PDT with 5,10,15, 20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS(4)) requires concentrations of greater than 100 microg/mL to achieve LD(50). Neither 1 nor TPPS(4) shows significant photoactivity against the murine T-cell line, MOLT-4, above the dark toxicity. Sensitizer 1 shows no toxicity or side effects in BALB/c mice observed for 30 days following a single intravenous injection of 10 mg (9.1 micromol)/kg. Distribution studies show that sensitizer 1 accumulates in the tumors of BALB/c mice bearing Colo-26 or EMT-6 tumors with sensitizer concentration roughly doubling as the dosage of 1 increased from 5 to 10 mg/kg. In vivo studies show that PDT with sensitizer 1 at both 3.25 and 10 mg/kg with 135 J cm(-2) of 694-nm light is effective against Colo-26 tumors in BALB/c mice.

Synthesis and evaluation of chalcogenopyrylium dyes as potential sensitizers for the photodynamic therapy of cancer.

A series of thiopyrylium (2), selenopyrylium (3), and telluropyrylium dyes (4) was prepared via the addition of Grignard reagents to either 2, 6-di(4-dimethylamino)phenylchalcogenopyran-4-ones (5a) or 2-[4-(dimethylamino)phenyl]-6-phenylchalcogenopyran-4-ones (5b) followed by elimination and ion exchange to give the chloride salts. The absorption spectra and quantum yields for singlet oxygen generation of these dyes suggested that the dyes would have utility as sensitizers for PDT. Selenopyrylium dyes 3a and 3d with quantum yields for singlet oxygen generation of 0.040 and 0.045, respectively, were phototoxic to Colo-26 cells in culture. The toxicity of the dyes 2-4 was evaluated in clonogenic assays of human carcinoma cell lines. Importantly, the presence of a sulfur, selenium, or tellurium heteroatom in the molecules had no predictable impact on the toxicity of any particular dye set. Substituents at the 2-, 4-, and 6-positions of the dye had a much greater impact on cytotoxicity. The IC(50) values determined in the clonogenic assays did not correlate with chemical properties in the dye molecules such as reduction potential or lipophilicity. Initial in vivo toxicity studies showed no toxicity for these dyes at dosages between 7.2 and 38 micromol/kg in BALB/c mice.

A selenopyrylium photosensitizer for photodynamic therapy related in structure to the antitumor agent AA1 with potent in vivo activity and no long-term skin photosensitization.

Cationic chalcogenopyrylium dyes 5 were synthesized in six steps from p-aminophenylacetylene (9), have absorption maxima in methanol of 623, 654, and 680 nm for thio-, seleno-, and telluropyrylium dyes, respectively, and generate singlet oxygen with quantum yields [Phi((1)O(2))] of 0.013, 0.029, and 0.030, respectively. Selenopyrylium dye 5-Se was phototoxic to cultured murine Colo-26 and Molt-4 cells. Initial acute toxicity studies in vivo demonstrate that, at 29 mg (62 micromol)/kg, no toxicity was observed with 5-Se in animals followed for 90 days under normal vivarium conditions. In animals given 10 mg/kg of 5-Se via intravenous injection, 2-8 nmol of 5-Se/g of tumor was found at 3, 6, and 24 h postinjection. Animals bearing R3230AC rat mammary adenocarcinomas were treated with 10 mg/kg of 5-Se via tail-vein injection and with 720 J cm(-2) of 570-750-nm light from a filtered tungsten lamp at 200 mW cm(-2) (24 h postinjection of 5-Se). Treated animals gave a tumor-doubling time of 9 +/- 4 days, which is a 300% increase in tumor-doubling time relative to the 3 +/- 2 days for untreated dark controls. Mechanistically, the mitochondria appear to be a target. In cultured R3230AC rat mammary adenocarcinoma cells treated with 0.1 and 1.0 microM 5-Se and light, mitochondrial cytochrome c oxidase activity was inhibited relative to cytochrome c oxidase activity in untreated cells. Irradiation of isolated mitochondrial suspensions treated with 10 microM dye 5-Se inhibited cytochrome c oxidase activity. The degree of enzyme inhibition was abated in a reduced oxygen environment. Superoxide dismutase, at a final concentration of 30 U, did not alter the photosensitized inhibition of mitochondrial cytochrome c oxidase by dye 5-Se. The data suggest that singlet oxygen may play a major role in the photosensitized inhibition of mitochondrial cytochrome c oxidase.

Design and construction of a light-delivery system for photodynamic therapy.

We have developed a device to divide the output from a dye laser into as many as eight beams of equal power with negligible total power loss. In this system, 630-nm s-plane polarized laser light was split by a series of highly polarization-sensitive plate beamsplitters. Each of the beams was coupled to a 200, 400, or 600 microm diameter optical fiber. Brewster-window-type attenuators allowed the power of each beam to be individually set. It was possible to reconfigure the device to produce four, two, or one output(s). We discuss the design requirements of the beamsplitter device and describe its construction from mostly commercially available components. An apparatus for positioning and stabilizing each optical fiber relative to the skin surface of a patient is also described. The illumination from the fiberoptic supported by such an apparatus strikes a defined surface area and is independent of patient movement. Both the beamsplitter device and the optical fiber positioner are used routinely in photodynamic therapy (PDT) of malignant tumors in the clinic and in the laboratory.

The use of tetrazolium salts to determine sites of damage to the mitochondrial electron transport chain in intact cells following in vitro photodynamic therapy with Photofrin II.

A method is described utilizing the tetrazolium salts neotetrazolium chloride (NTC), triphenyltetrazolium chloride (TTC), C,N-diphenyl-N'-4,5-dimethylthiazol-2-yltetrazolium bromide (MTT) and various substrates to elucidate damage to the mitochondrial electron transport chain of intact cells following in vitro photodynamic therapy (PDT). Using this methodology, a portion of the dark toxicity manifested by Photofrin II (PII) was found to occur prior to entry of electrons into the transport chain through Complex I, as evidenced by the fact that the inhibition of MTT reduction was reversible by the addition of malic acid to the culture media. A second site of dark toxicity was found to be Complex IV (cytochrome oxidase). After photoirradiation of the cells, Complex I was found to be affected since malic acid could no longer reverse the inhibition of MTT reduction but it could be reversed by the addition of succinic acid, whose electrons enter the transport chain at Complex II. A second and more sensitive site of photoirradiation damage was found to be Complex IV. A region near cytochrome C was also affected by photoirradiation but appreciably less so than noted for Complexes I and IV. A kinetic analysis of MTT and TTC reduction following photoirradiation indicated that MTT reduction was sustained at a normal rate for 1 h after which it slowed down and eventually plateaued. In contrast, TTC reduction was found to be inhibited almost immediately indicating Complex IV is extremely susceptible to photoirradiation damage. Compared to other assays of mitochondrial function requiring subcellular fractionation, the use of tetrazolium salts is simpler to perform and can be done using physiologically relevant conditions.