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Treatment with benozopophyrin derivative monoacid ring A (BPD-MA, verteporfin) and broad-spectrum fluorescent light rapidly produced apoptosis in murine P815 mastocytoma cells. Fragmentation of DNA, a fundamental characteristic of cells undergoing apoptosis, was evident within 3 h following the photodynamic treatment. Western immunoblot analysis using the specific antiphosphotyrosine monoclonal antibody 4G10 indicated that molecular species of > 200 kDa were phosphorylated on tyrosine residues during or immediately following the irradiation of cells loaded with BPD-MA. Increased tyrosine phosphorylation of a 15 kDa protein was evident by 15 min postirradiation. In the absence of light, BPD-MA did not affect the status of cellular protein tyrosine phosphorylation or cause DNA fragmentation. The protein kinase inhibitor staurosporine prevented tyrosine phosphorylation of the > 200 kDa species but did not affect tyrosine phosphorylation of the 15 kDa protein or the level of DNA fragmentation produced by the photodynamic treatment. The protein tyrosine phosphorylation events observed for P815 cells treated with cytotoxic levels of BPD-MA and light may not be directly related to the induction of the apoptotic cell death pathway.

Transcutaneous photodynamic therapy (PDT), utilizing benzoporphyrin derivative monoacid ring A (BPD, verteporfin) and whole-body light exposure, was assessed for its capacity to modify the course of adoptively transferred experimental autoimmune encephalomyelitis (EAE) in PL mice. Using a novel cell culture technique to facilitate the induction of this neurodegenerative condition, disease signs commenced 3-4 weeks after the transfer of myelin basic protein (MBP)-reactive lymph node or spleen cells to naive syngeneic recipients. Mice administered MBP-sensitized lymph node cells preincubated with BPD followed by whole-body 690 nm light irradiation (15 J/cm2) did not display symptoms of EAE. Although almost all animals given MBP-sensitized spleen cells developed EAE, mice given BPD (1 mg/kg) and the light treatment 24, 48 or 120 h after spleen cell transfer exhibited significantly less severe disease symptoms than control animals. Mice given the photodynamic treatment 24 h after spleen cell transfer also exhibited a significantly later disease onset than the control animals. Treatment of mice with PDT 24 h prior to spleen cell transfer did not influence subsequent disease severity but modestly delayed its onset. In the absence of directed light, BPD did not influence the development of EAE. Spinal cord tissues were evaluated for the presence of T cell receptor (TCR) V alpha 4 mRNA transcripts that specifically encode for the TCR alpha-chain of MBP-reactive T cells of PL mice. Using the polymerase chain reaction, V alpha 4 TCR mRNA transcripts were present in spinal cord samples prepared from almost all control mice but in only about one-half of spinal cord samples prepared from mice treated with PDT 24 h after spleen cell transfer. These observations indicated that PDT had limited the expansion of MBP-specific V alpha 4+ T cells within the central nervous system. Transcutaneous PDT represents a new technique with which to approach the treatment of autoimmune disease.

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 micrograms/mL BPD at 37 degrees C for 0-60 min. At various times, cells were lysed and the amount of BPD taken up by 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/mliter 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 CD4+/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.

— This study compared the ability of highly purified resting and activated DBA/2 mouse peritoneal macrophages to survive treatment with the photosensitizer benzoporphyrin derivative (BPD, verteporfin) and light. Culture of macrophages with recombinant murine interferon-γ (rIFN-γ, 100 U/mL) for 72 h imparted a phenotypic and functional activation by dramatically increasing cell surface expression of major histocompatibility complex Class II (Ia) molecules and the formation of nitric oxide. The rIFN-γ-activated macrophages were significantly (P < 0.05) more sensitive (lethal dose to cause a 50% reduction in cell survival, LD50= 14.4 ± 1.1 ng/mL) to photodynamic killing with BPD and light (10 J/cm2) than cells (LD50= 18.2 ± 2.0 ng/mL) cultured in medium alone. In contrast, macrophages treated with different concentrations of bacterial lipopolysaccharide (LPS) were as resistant or more resistant to photodynamic killing than cells cultured in medium alone. No cytotoxic effect of BPD was detected in cultures containing the drug but protected from light. Comparable amounts of BPD were taken up in vitro by unactivated and rIFN-γ-activated macrophages, as detected by flow cytometric analysis. However, cells cultured with LPS (10 μg/mL) took up more BPD than macrophages cultured in medium alone or with rIFN-γ. The DBA/2 P815 mastocytoma cells took up greater amounts of the drug and were subsequently more vulnerable to treatment with BPD and light (LD50= 6.9 ng/mL) than macrophages cultured under any condition. The explanation for the increased vulnerability of rIFN-γ-activated macrophages and the greater resistance of LPS-activated macrophages, relative to medium-cultured macrophages, to photodynamic killing with BPD is uncertain. However, the increased susceptibility of macrophages, activated with the immunomodulatory cytokine IFN-γ, to treatment with BPD and light might indicate how photodynamic therapy could interfere with the development of experimental autoimmune disease, conditions in which activated macrophages are known to be involved.

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.

Four structural analogs of benzoporphyrin derivative (BPD) have been studied and compared for photosensitizing activity in vitro. All analogs have an identical reduced tetrapyrrol porphyrin ring, and differ by the position of a cyclohexadiene ring (fused at either ring A or ring B of the porphyrin) and the presence of either two acid groups or one acid and one ester group at rings C and D of the porphyrin. Photosensitizer activity was tested with the M1 tumor cell line using an assay (the MTT assay) which detects mitochondrial hydrogenases as a measure of cell viability. This assay was shown to be equivalent to the standard clonogenicity or [3H]thymidine uptake assay. Comparative studies with the BPD analogs showed that the monoacid derivatives had equivalent cytotoxicity and were about five-fold more active than the diacid forms. This was the case whether the assays were performed in the presence or absence of fetal calf serum.

Photodynamic therapy is now recognized as a legitimate therapy for both palliative and potentially curative treatment of solid tumors. This represents a major achievement for investigators who have committed their careers to PDT. Scientific research characteristically runs well ahead of accepted views and technologies. This Symposium-in-Print perhaps provides a snapshot of the potential for this technology during the next two decades.

Inhibition of the growth of a transplantable methylcholanthrene-induced tumor was achieved in mice by administering indomethacin or aspirin in the drinking water, these drugs having in common the ability to inhibit prostaglandin synthesis. Indomethacin was able to reduce the levels of prostaglandin E (PGE) in the tumor tissue. When tumors from these animals were cultured in vitro, the culture supernatant fluid from drug-treated animals showed lower levels of PGE released into the media, but after several days in culture, tumor cells from indomethacin-treated animals fully recovered their ability to produce PGE. The relative size of tumors in untreated animals was directly related to the amount of PGE activity present in these tumors.

A monoclonal antibody (Fd-B2) to ferredoxin, which bears an idiotype scarcely expressed in any of a wide variety of mouse strains, is able to markedly enhance the response to ferredoxin of both high-responder and intermediate-responder strains. A rabbit anti-idiotype serum to Fd-B2 also specifically enhances the response to ferredoxin in such mice. Most remarkably, the treatment of nonresponder T cells by either the idiotype (Fd-B2) plus complement or anti-idiotype antiserum plus complement causes them to be responsive in adoptive transfer experiments. The two responding populations (idiotype-treated and anti-idiotype-treated) can then be combined to reconstitute the nonresponsive state. When the nonresponders are treated with either Fd-B2 idiotype plus complement or anti-idiotype plus complement and subsequently respond, the idiotype of the anti-ferredoxin antibody produced does not bear the Fd-B2 idiotype. We interpret the results as being consistent with a model in which the unresponsive state for ferredoxin is a state of high network connectivity of the ferredoxin-specific T cells.