Evaluation of the immunotoxicity of benzoporphyrin derivative (BPD-MA) in mice.

Photodynamic therapy has been shown to selectively eliminate activated lymphocytes in a number of experimental situations. These findings have important implications in therapies involving selective immunomodulation. In this study we report the effects of intravenous dosing with the photosensitizer benzoporphyrin derivative-monoacid A(BPD) on normal immunological function. Therapeutic doses of BPD and light had no effect on natural killer cell activity, the mixed lymphocyte reaction, cell-mediated lympholysis, the primary immune response to sheep red blood cells, or the secondary memory response to T cell-dependent antigens. In non-light treated controls, BPD at concentrations up to 10-fold higher had a limited effect on cell-mediated lympholysis. We conclude that the primary effect of BPD in several therapeutic modalities in not due to a generalized suppression of the immune system.

Accelerated myelopoietic recovery in irradiated mice treated with Photofrin.

The porphyrin photosensitizer, Photofrin porfimer sodium (Photofrin), has been widely studied for its capacity to evoke destruction of malignant tissues. In addition to its photosensitizing properties, Photofrin may exert myelostimulatory effects in normal and immunosuppressed mice in the absence of activating light. In the present set of experiments, we examined the effect of Photofrin upon the immunohematopoietic axis of sublethally irradiated DBA/2 mice. Administration of Photofrin (10 mg/kg) 1 and 4 days following irradiation (4 Gy) significantly enhanced the recovery of spleen cellularity, spleen and bone granulocyte/macrophage progenitors (colony-forming units, CFU-GM) and peripheral blood leukocytes levels. Proliferative responses to the T-cell mitogen concanavalin A by spleen cells prepared from Photofrin-treated mice were significantly less than those of cells from irradiated control mice 8 and 15 days post-irradiation. Photofrin given 1, 4 and 7 days following irradiation elevated splenic CFU-GM 3 to 4-fold 10 days post-irradiation relative to the irradiated controls and mice given only two injections of Photofrin. In contrast to the effect of two injections, multiple (three or four) injections of Photofrin did not elevate bone marrow CFU-GM above control levels beyond 8 days post-irradiation. In addition, splenic CFU-GM levels in animals receiving three or four injections of Photofrin were no different than those of the irradiated controls later than 10 days post-irradiation. These findings indicate that prolonged exposure to Photofrin in sublethally irradiated mice may induce regulatory factors which dampen the enhanced myelopoietic recovery stimulated by only two injections of the drug.

Wavelength-dependent effects of benzoporphyrin derivative monoacid ring A in vivo and in vitro.

Benzoporphyrin derivative monoacid ring A (BPD-MA) is a chlorin-like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P815 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon-ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBA/2 mice injected with 2 mg/kg of BPD-MA and exposed to light at 125 J/cm2, between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630-640 nm and 680-690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680-690 nm caused slightly more damage to normal skin than light delivered at 630-640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 680-695 nm. Antitumor efficacy in tumor-bearing DBA/2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor-free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of porphyrins on the hematopoietic recovery of mice treated with gamma-radiation.

Porphyrins are a group of organic compounds involved in a wide spectrum of fundamental biological processes. Non-metallic, naturally occurring and synthetic porphyrin derivatives may produce cytotoxic effects in malignant or normal tissues exposed to visible light. Supra-clinical doses of the photosensitizing porphyrin, Photofrin are hematostimulatory when administered to normal and immunosuppressed inbred mice. To determine if a non-photosensitizing metalloporphyrin has similar hematostimulatory activity, we have synthesized iron (III) hematoporphyrin chloride (FeHp) and administered it to sub-lethally irradiated mice. FeHp (10 mg/kg) given 1 and 4 days or 1, 4 and 7 days following sub-lethal (7 Gy) whole body irradiation significantly increased spleen colony forming units of progenitor cells of the granulocyte-macrophage lineage (CFU-GM) 14 days post-irradiation, relative to irradiated controls. In addition, total splenocyte numbers were significantly increased 17 days post-irradiation in mice that had received FeHp 1 and 4 days post-irradiation. When FeHp was given 24 hours prior to irradiation and again 48 hours or 48 and 96 hours post-irradiation, significant increases in splenic CFU-GM and spleen cell numbers, relative to control mice, were observed 15 days post-irradiation. A non-metallic photosensitizing monomeric fraction of Photofrin, deuteroporphyrin IX, 2,4 (4,2) hydroxyethyl vinyl (HVD) was compared to Photofrin for its ability to influence the hematopoietic recovery of irradiated mice. Only Photofrin but not HVD given in 3 doses (10 mg/kg) 1, 4 and 7 days following irradiation (4.8 Gy) significantly enhanced the recovery of spleen cellularity and splenic CFU-GM. In addition, Photofrin significantly increased bone marrow CFU-GM 7 and 10 days following the sub-lethal dose of gamma-radiation. The mechanism by which certain porphyrins augment hematopoiesis in the mouse is unknown. However, the identification of FeHp as a non-photosensitizing monomeric porphyrin with hematostimulatory activity in vivo, indicates that further study of metalloporphyrins is warranted and may reveal their clinical potential within the context of therapeutically-induced immunosuppression.

Liposomal delivery of a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD), to tumor tissue in a mouse tumor model.

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.

Photosensitizing efficiency of two regioisomers of the benzoporphyrin derivative monoacid ring A (BPD-MA).

Benzoporphyrin derivative, monoacid ring A (BPD-MA), currently in clinical trials as a photosensitizer for photodynamic therapy for cancer, consists of two regioisomers (A1 and A2) present in equal proportions. The contribution of the regioisomers to the overall photosensitizing potency of BPD-MA was tested in vitro and in vivo. The in vitro photosensitizing potencies of BPD-MA-A1 and -A2 were tested in a standard cytotoxicity assay using M1 (rhabdomyosarcoma of DBA/2 mice) tumor cells and were found to be equivalent. The in vivo photosensitizing efficacies of the regioisomers were tested in the M1 tumor model in DBA/2 mice and were also found to be equivalent. Biodistribution of the regioisomers in mouse plasma, tumor and liver was studied in M1 tumor-bearing DBA/2 mice at 15 min and 3 hr post intravenous injection of [14C]BPD-MA-A1/A2 at 4 mg/kg body weight. Plasma and extracts from tumor and liver were analysed by HPLC and tested for radioactivity. The two regioisomers were eliminated from plasma and liver at different rates, which resulted in A1:A2 ratios of 1:0.28 in plasma and 1:0.75 in liver at 3 hr post injection. The differential elimination was not observed to any significant degree in the tumor, where even at 3 hr post injection the A1:A2 ratio was 1:1.15. Therefore, we concluded that in tumor tissue, at 3 hr post injection, the time at which laser photodynamic therapy is carried out, both regioisomers were present in about equal proportions. Further, both regioisomers were fully active as determined by an in vitro cytotoxicity assay following extraction.

The effects of plasma lipoproteins on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative.

The influence of lipoprotein association on in vitro tumor cell killing and in vivo tumor photosensitization with benzoporphyrin derivative (BPD) has been investigated in M-1 tumor bearing mice. The association of benzoporphyrin mono acid ring A with either low or high density lipoprotein increased tumor cell killing in an in vivo/in vitro cytotoxicity assay performed 3 h post intravenous drug administration. Eight hours following photosensitizer injection only low density lipoprotein (LDL) mixtures produced significant (P less than or equal to 0.005) increases in tumor cell killing compared to BPD in unfractionated plasma. The efficacy of in vivo photosensitization in the presence of lipoproteins correlated with the in vivo/in vitro cytotoxicity. Association of BPD with low or high density lipoproteins resulted in delayed tumor regrowth and higher cure rates when light exposure (125J/cm2) was performed 3 h post drug administration. When light exposure was performed 8 h post-injection only LDL-BPD mixtures led to enhanced tumor eradication compared to BPD administered in aqueous solution or unfractionated plasma.

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.

Photosensitising potency of structural analogues of benzoporphyrin derivative (BPD) in a mouse tumour model.

The in vivo characteristics of four analogues of benzoporphyrin derivative (BPD) have been investigated. Biodistribution data obtained in DBA/2J mice with BPD-MA (monoacid ring A analogue) which had been tritiated or internally labelled with 14C showed that both labelled materials acted in an essentially identical manner during the period of study. Biodistribution and clearance studies showed that relative distribution in a variety of mouse tissues was similar for all BPD analogues. M1 tumour cells (rhabdomyosarcoma in DBA/2J mice) taken from tumours excised from animals treated 3 h earlier with BPD, and tested in vitro for photosensitivity provided evidence that significant levels of photosensitiser detected in tumour was both active and associated with tumour cells. The monoacid forms of BPD were found to be much more photodynamically active in this test than were the diacid analogues. The ability of the analogues to ablate tumours in mice by photodynamic therapy was also tested. Again, BPD-MA and BPD-MB proved to be measurably better than the diacid analogues. These findings are discussed in reference to structural and physical differences between the analogues.

In vitro evaluation of phototoxic properties of four structurally related benzoporphyrin derivatives.

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.

Restriction specificities, alloreactivity, and allotolerance expressed by T cells from nude mice reconstituted with H-2-compatible or -incompatible thymus grafts.

Congenitally thymusless nude mice that lacked functional T cells were reconstituted with H-2-compatible or -incompatible thymus grafts taken from either fetal, newborn, or adult mice and transplanted under the kidney capsule or subcutaneously. Transplantation with unirradiated fetal (15--17 d) or newborn thymus grafts reconstituted the nude mice as assessed by their subsequent generation of virus-specific cytotoxic T cells in vivo or alloreactive T cells in vitro. The restriction specificity of T cells from homozygous mice was exclusively for the nude host H-2, as shown by direct cytolysis or by cold target competitive inhibition assays. irrespective of whether nude mice were reconstituted with H-2-compatible, semiallogeneic, or H-2-incompatible, unirradiated newborn or fetal thymus grafts (in order of decreasing efficiency of reconstitution). The restriction specificity for the nonhost H-2 of the thymus could not be demonstrated even after primary or secondary sensitization in an infected appropriate F1 environment. These nude mice reconstituted with fetal or newborn grafts were tolerant to the H-2 of the thymus donors. Nude mice transplanted with irradiated adult thymus grafts were reconstituted functionally with syngeneic or semisyngeneic but not with allogeneic thymus grafts. In homozygous nu/nu irradiated heterozygous recipients of F1 thymus grafts, the restriction specificity for the nonhost thymic H-2 could not be elicited upon adoptive sensitization in irradiated and infected F1 heterozygote stimulator mice; in fact, these chimeras' lymphocytes were not tolerant to the nonhost H-2. The discrepancy between the restorative capacity of unirradiated vs. irradiated thymus grafts suggests that precursors of T cells in nude mice can acquire restriction specificity and immunocompetence independently of a conventional, functioning H-2-compatible thymus if exposed to an allogeneic fetal or a newborn thymus that contains functioning thymocytes of donor type but not if reconstituted with an irradiated adult allogeneic thymus.

Lymphocyte activation by Con A.

Con A can activate cytotoxic effector cells, helper cells and suppressor cells. In all cases Con A activates polyclonally and thus the entire T cell repertoir can be revealed by Con A activation. Nylon purified T cells cannot be activated by Con A. however, in the presence of helper cells, such as adherent cells, or in the presence of serum from several species, Con A activates purified T cells. The serum factors that are responsible for Con A activation of purified T cells are present in the albumin fraction. It is suggested that Ia molecules may be responsible for the ability of Con A to activate purified T cells and a general hypothesis for T cell activation is outlined.

Characterization of human adenoid cells using surface and functional markers for lymphocyte subpopulations.

Adenoid lymphocytes from children undergoing adenoidectomy were compared with blood cells from the same children using techniques for identifying T cells and B cells. A high proportion of adenoid lymphocytes were immunoglobulin positive cells. Of these only a minor fraction carried receptors for the Fc part of IgG. Adenoid B lymphocytes respond poorly if at all to polyclonal B-cell activators, such as LPS or PPD, which show a different reactivity compared to human splenic cells. The response to anti B2-microglobulin was also different; blood cells responded better than adenoid cells. Thus distinct subpopulations of B lymphocytes reside in different lymphoid organs. The adenoid lymphocyte reactivity might reflect their function in the defence mechanism against infections.

Polyclonal antibody secretion in human lymphocytes induced by killed staphylococcal bacteria and by lipopolysaccharide.

Preparations of Staphylococcus aureus strains Cowan 1 and Wood 46 and of lipopolysaccharide (LPS) were found to act as polyclonal B-cell-activating substances for human splenic and blood lymphocytes. All three substances induced polyclonal antibody secretion in blood and spleen cell cultures, as tested against fluorescein isothiocyanate-coupled sheep erythrocytes by a modification of the local hemolysis-in-gel assay. Antibodies were of IgM class, as shown by inhibition of plaque formation by anti-IgM but not by anti-IgG or anti-IgA antisera. All these substances also consistently induced the formation of intracellular immunoglobulin and increased DNA synthesis in stimulated spleen cells. In blood lymphocytes Staph. aureus Cowan 1 induced a consistent increase in DNA synthesis, whereas Staph, aureus Wood and LPS often gave low or no increase in DNA synthesis. Peak antibody formation was observed on day 3 in spleen cells and on day 6 in blood lymphocyte cultures. Stimulation into high-rate immunoglobulin secretion occurred with all PBAs also in B-cell-enriched cell suspensions but not in T-cell-enriched cells. Optimal responses were, however, always noted in unseparated cell suspensions. It is concluded that preparations of killed bacteria can be useful tools for the clinical evaluation of both specific and nonspecific antibody-forming ability in cells from different groups of patients.

Lymphocyte-mediated cytotoxicity against tumor cells II. Characteristics and tissue distribution of concanavalin A-activated cytotoxic effector cells.

In vivo or in vitro activation of cytotoxic effector lymphocytes by concanavalin A (Con A) has been shown to result in a population of effector cells exhibiting a degree of immunological specificity when Con A was present during the cytotoxic assay (Waterfield, J.D. et al., Cell. Immunol. 1975. 17:392). In this communication we have characterized the Con A-activated effector cell by physical criteria and tried to assign it to a given subpopulation of thymus-derived lymphocytes. It was shown that macrophages played no role in target cell lysis in this system, nor was their presence required for Con A activation of the effector cell. The effector cell proved insensitive to anti-theta serum, but was classified as a T cell by the enrichment of cytotoxicity when a purified population of T cells was activated by Con A in vitro and by the failure to activate the effector cell in nude mice. Precursors of the effector cell were shown to reside primarily in the spleen, to be radioresistant up to 400 R, and to be short-lived after adult thymectomy. Thus, the effector cell can be classified with the T1 population of T cells and has similar characteristics as a cytolytically active cell described by Stobo et al. (J. Immunol. 1973. 110: 362, 652).

Lymphocyte-mediated cytotoxicity against tumor cells. III Differentiation of concanavalin A-activated cytotoxic effector cells.

The maturation of selected T cell responses in the lymphoid organs of irradiated CBA mice was followed after adoptive transfer of syngeneic fetal liver cells. Mitogenic responsiveness to phytohemagglutinin (PHA) and concanavalin A (Con A) was found to reach control values 3 weeks after reconstitution in the thymus, spleen, and lymph node, of fetal liver repopulated animals. Spleen and lymph node cell reactivity in mixed lymphocyte culture reactions required 6 weeks to reach significant values. However, the ability of spleen cell suspensions to be activated by Con A into cytotoxic effector lymphocytes appeared after only 2 to 3 weeks. It is concluded that two functionally distinct T cell subpopulations exist in the spleen, one which can be activated into cytotoxic effector lymphocytes by Con A, and one which responds to alloantigens by DNA synthesis.

Lymphocyte-mediated cytotoxicity against tumor cells specificity and characterization of concanavalin A-activated cytotoxic effector lymphocytes.

The selective T cell mitogen Con A was found to induce cytotoxic effectorlymphocytes after in vivo or in vitro treatment. The effector cells exhibited immunologic specificity when tested against tumor cells, killing targets only across H-2 histocompatibility barriers. Thus, Con A caused a polyclonal T cell activation, resulting in the appearance of effector cells capable of recognizing all H-2 antigens, except those coded for by the major histocompatibility loci of the lymphocyte donor. These experiments demonstrate that Con A is capable of activating pre-existing T cells to reveal their genetically determined immunological specificity, and furthermore suggest that there is a deletion of T cell clones with specificity for self. The activated effector cells were characterized as T blasts being relatively insensitive to treatment with anti-O serum plus complement. The precursors of the Con A-activated effector cells were shown to reside primarily in the spleen, to be radio-resistant up to 400 R, and to be short-lived after adult thymectomy. Thus, the population of T cells capable of activation to cytotoxic effector lymphocytes by Con A has similar physical characteristics to the splenic subset(s) of T cells mediating both GVH and cytotoxicity responses upon alloimmunization.