Various different photosensitizers were compared to Photofrin® II with respect to their potential for causing direct tumor cell inactivation and/or vascular damage. GaCl-phthalocyanine of mixed sulfonation, while being 10 x more efficient than Photofrin® II in tumor cell inactivation, caused equal vascular photosensitization. Monosulfonated AlC1-phthalocyanine, on the other hand, was sparing to the vasculature, while still exhibiting good tumor cell effects. Large differences in vascular photosensitization were also observed with tetra- and monosulfonated tetraphenylporphines.

Tumor destruction in photodynamic therapy is the result of the combination of direct cellular toxicity and damage to tumor microvasculature. These phenomena appear to be caused by tissue interactions with toxic oxygen compounds which are formed when light interacts with photosensitizing agents. Although injury to cell membranes, mitochondria and the nucleus have been noted, such injuries by themselves tend to be sublethal and cannot totally account for the effectiveness of PDT. The mechanism of effect of PDT on the vasculature has not been fully investigated. The vascular effects are believed to involve both intravascular and perivascular phenomena. Platelet aggregation appears to be an early event. Changes to the endothelium, and smooth muscle contraction as well as increased capillary permeability have also been observed during therapy. Initial experiments using'cyclooxygenase inhibitors indicate that arachidonic acid metabolites are active elements in producing the vascular phase of the therapeutic response and that these microvasculature effects appear to be critical to permanent tumor destruction.

The effects of porphyrins and radiation on inflammation include activation of the complement system, generation eicosanoids from mast cells, macrophages, and endothelial cells, alteration in cutaneaous eicosanoid metabolism, activation of factor XII-dependent pathways, release of mediators from mast cells, and dermal accumulation of neutrophils. These effects contribute to the development of porphyrin-induced cutaneous inflammation, which can be seen in patients with porphyrias and as a consequence of photodynamic therapy.

Changes in blood flow to transplantable bladder tumors growing in Fischer rats were measured after photodynamic therapy with the photosensitizer tin (II) etiopurpurin dichloride (SnET2) using the radioactive microsphere technique. As with the other photosensitizers, hematoporphyrin derivative and chloroaluminum tetrasulfophthalocyanine, SnET2 and light caused a rapid decrease in tumor blood flow. This occurred when the vehicle for photosensitizer delivery was either an emulsion or a liposome. Systemic heparinization of animals did not alter changes in tumor blood flow.

We have been investigating the dose dependent modification of p02, pH, and phosphate metabolism within tumors subjected to PDT. The system employed in all studies has been the C3H mouse mammary adenocarcinoma tumor model. All tumors were treated at a size of 7.0 ± 0.5 mm (maximum diameter) with biologically defined "doses" ranging from the TCD10 to the TCD90 response level (i.e. from 200 J/cm2 to 600 J/cm2 in this system). The dose of Photofrin II employed in all studies was 12.5 mg/kg (IV) with light treatment 24 hours later (drug kindly supplied by QLT Inc.). All light treatments were at 632 nm (Coherent). Both microelectrodes and 31P NMR spectroscopy (Varian VXR 400) were employed to measure the intralesional values. Results indicate a "dose" dependent reduction in intracellular ATP occurring within 4 hours post treatment. At the TCD90 level, no detectible high energy phosphates are seen within 4 hours following treatment. A similar trend is observed when intralesional p02 is measured with significant decreases seen within 4 hours post treatment. Microelectrode measurements of pH (predominantly extracellular) showed no significant change for up to 72 hours post treatment, independent of "dose". However, some "dose" dependent changes in pH gradient [pH (intracellular) - pH (extracellular)] have been observed.

Porphyrin photodynamic therapy directed specifically to the hind leg of various strains of mice was found to induce a high percentage of lethality at dosages which would be required to achieve cures in tumor bearing mice. Toxicity was observed in both pigmented and albino mouse strains. An inverse relationship between light dose rate and lethality was documented. Anti-coagulant drugs and anti-inflammatory agents which inhibit cyclo-oxygenase had protective effects. The response induced by localized PDT appears to mimic that of a classical traumatic shock syndrome and may be limited to PDT in small animals such as mice.

One of the side effects of peritoneal photodynamic treatment (PDT) of mice is a systemic suppression of contact hypersensitivity (CH) responses. Treatment with either laser alone or the photosensitizer, Photofrin II (PFII), alone does not cause suppression of CH responses. Immunosuppression of CH responses is an active process that is adoptively transferable using viable cells, but not serum, from PDT-treated mice. The induction of adoptively transferable suppressor cells in PDT-treated mice requires exposure to an antigenic stimulus, yet the suppressor cells are antigen non-specific in their function. T cell function in PDT-treated mice, as measured by the ability of splenic lymphoid cells to generate allogeneic cytotoxic T lymphocyte responses, is comparable to that detected in normal mice. However, the ability of spleen cells from PDT-treated mice to act as stimulators in a mixed lymhocyte reaction is dramatically impaired, suggesting that the major cell type affected by peritoneal PDT is of the macrophage lineage. Support for this concept is provided by experiments in which spleen cells from PDT-treated mice were chromatographically separated into populations of T cells, B cells and macrophages prior to adoptive transfer into naive recipients. The results indicate that the cell type mediating adoptively transferable suppression of CH responsiveness is of the macrophage lineage. Analysis of hematologic parameters revealed that induction of suppression by PDT-treatment was associated with a marked neutrophilia and lymphocytosis, and was also accompanied by a 5-fold increase in concentration of the acute phase protein, Serum Amyloid P. Finally, attempts to ameliorate PDT-induced immunosuppression by pharmacologic intervention have proved successful using implants of pellets that release indomethacin at a rate of 1.25µg/day. Thus, the data suggest that PDT-treatment induces macrophages to produce factors (e.g., prostaglandins) that are known to be potently immunosuppressive.

This study was undertaken to determine if photodynamic therapy (PDT) produces an immunologic response in patients treated for bladder cancer. Gamma interferon, interleukin 1-beta, interleukin 2 and tumor necrosis factor-alpha were assayed in the urine of four patients treated with photodynamic therapy for bladder cancer, in seven patients undergoing transurethral procedures, and in five healthy control subjects. Quantifiable concentrations of all cytokines, except gamma interferon, were measured in urine samples from the PDT patients treated with the highest light energies, while no urinary cytokines were found in the PDT patient who received the lowest light energy or in the control subjects. These findings suggest that a local immunologic response may occur following PDT for bladder cancer. Such an immunologic response activated by PDT may be an additional mechanism involved in bladder tumor destruction.

Monoclonal antibodies with reactivity to T suppressor cells and T suppressor factors, conjugated to hematoporphyrin, when injected intravenously into mice bearing either the P815 mastocytoma or L1210 thymoma were able to reject a significant number of palpable tumors, in comparison to appropriate controls. This effect is attributed to immune modulation by selective elimination of T suppressor cells. Investigation of novel methodology for reliable linkage of photosensitizers to antibodies indicate that derivatives of polyvinyl alcohol provide suitable carriers for photosensitizers. Such carriers can relatively easily be conjugated to monoclonal antibodies without jeopardizing the biological activity of the antibody or the photosensitizer, or the solubility of the complex.

A number of laboratories have demonstrated that immunoconjugates may have potential in phototherapy of cancer. The phototoxic effectiveness of these conjugates may be increased by manipulation of irradiation parameters and by biochemical and immunologic modulation. Results from experiments aimed at potentiation of immunoconjugate photosensitization are presented.

Determination of the dependence of Photofrin II concentration on the time integrated space irradiance in tissue allows the definition of Photodynamic dose to be formulated as an integral of sensitizer concentration over the time integrated space irradiance at each point in tissue. This integral, together with a measurement of the depth of necrosis produced by the clinically measurable sensitizer and light doses, and the tissue optical properties allows the calculation of the expected necrotic depth for any similar tissue. These predictive calculations are carried out for various dosimetric conditions and a variety of clinically interesting sources e.g. long cylinders or spherically symmetric sources imbedded in the tissue, uniform surface illumination over a wide field or a spherical source at the center of a water filled bladder. The results are presented in graphical form and are based on published clinical data.

Dosimetry of laser fluence rates within a tissue are required for proper planning of photodynamic therapy at a specific site on a given individual. A simple one-dimensional theory of light penetration into tissue is presented. A device for measurement of the total reflectance and the lateral diffusion of light provides a simple means for specifying the tissue optical parameters that govern laser dosimetry. Rules of thumb for complicating factors such as narrow laser beams and optical fiber delivery are discussed.

Effective treatment of non-superficial tumors using HpD-PDT requires the development of interstitial light application. In order to determine the efficacy of interstitial HpD-PDT, a dose effect relationship has been established in a rat rhabdomyosarcoma. Because of the lack of data about the light distribution in tissue a miniature isotropic light detector was used throughout this study for in vitro and in vivo light dosimetry, permitting evaluation of tumor response versus "light dose" (i.e. light energy fluence) distribution. Effects of increased light absorption due to the photosensitizer concentration in the tumor tissue were observed and taken into account in reporting the light dose throughout the tumor volumes. Light dosimetry during HpD-PDT treatments was performed as a check on the prescribed calculated light dose levels. Measurements demonstrate a gradual but drastic decrease in light penetration, beginning almost immediately after the start of the treatment. This phenomenon, which is also observed in clinical HpD-PDT treatments, complicated the assessment of the required light energy fluence for tumor control. The dose-effect experiments show that a high tumor control rate is achievable in a single HpD-FDT treatment. Failures were strongly correlated with inadequate light dose or light dose distribution. Successful treatment appeared to be highly dependent on the light dose at the tumor periphery. Vascular damage in a small margin of the surrounding normal tissue seems to be required. This implies that tumor treatment selectivity is mainly determined by restricted light penetration in tissues irradiated by local light application.

When light is diffusely reflected from tissue containing a dye, such as a photosensitizer, with a known absorption spectrum, the changes in the reflectance spectrum caused by the presence of the dye can be identified and correlated with the dye concentration. A feasibility study' of this method for the noninvasive determination of the concentration of photosensitizers in tissue found, however, that the changes in reflectance also depended on the optical properties of the tissue. In this study we propose a simple model of light propagation which allows quantitative prediction of the sensitivity of the method. The optical absorption and scattering coefficients required as input for the model are obtained from two ancillary noninvasive measurements: the total diffuse reflectance and the spatial variation of the local diffuse reflectance. Experiments performed using tissue-simulating phantoms suggest that the simple model, when combined with the ancillary measurements, allows absolute calibration of the method to within 20%.

Photochemical reactions are used in photodynamic therapy of cancer and other disease. The cytotoxic agent in photochemotherapy is usually singlet oxygen. Thus measurements of singlet oxygen production or concentration may allow prediction of the biological response. The decrease in fluorescence of L-tryptophan because of reaction with singlet oxygen, the decrease in absorbance of a dye such as RNO subject to secondary oxidation by singlet oxygen, and the decrease in fluorescence of the most common photosensitizer, dihematoporphyrin ether/ester (DHE) because of photobleaching, have been investigated in solutions in vitro. The most promising method for dosimetry and prediction of biological response appears to be the photobleaching of DHE.

Methods for the pre-clinical evaluation of new photosensitizing dyes are described. The resulting information can provide useful leads concerning likely modes and sites of localization. But correlations between results obtained in cell culture and in animal tumor models are sufficiently weak to indicate the need for caution in extrapolation of any in vitro result.

Sulfonated metallo phthalocyanines (M-SPC) are extensively studied as sensitizers for photodynamic therapy of cancer. They strongly absorb clinically useful red light with absorption maxima between 670-680 nm. Their photodynamic properties depend on the nature of the central metal ion as well as the degree of substitution on the macrocycle. The zinc, aluminum and gallium complexes are efficient photo-generators of singlet oxygen, the species most likely responsible for their phototoxicity and tumoricidal action. Tissue distribution pattern, cell penetration and dye aggregation are strongly affected by the degree of sulfonation of the dyes. Mono- and disulfonated M-SPC have the highest tendency to form photo-inactive aggregates. However, these lower sulfonated dyes more readily cross cell membranes resulting, in vitro, in enhanced phototoxicity. In vivo, the highly sulfonated hydrophilic M-SPC show the best tumor localization properties but the lower sulfonated dyes still exhibit the best photo-activity. Variations in activities between the differently sulfonated M-SPC are far less pronounced in vivo as compared to in vitro conditions. Such discrepancies are explained by the combined action of numerous vectors including interaction of M-SPC with plasma proteins, vascular versus cellular photo-damage, tumor retention, cell penetration as well as the degree of aggregation of the dye.

A series of benzochlorins have been prepared and tested for tumoricidal activity using the FANFT-induced rat bladder tumor model. Results indicate that these chlorin derivatives, in combination with red light, can cause significant tumor regression at doses as low as 0.5 - 1.0 mg/kg body weight. This compares favorably with other sensitizers tested in the same model and suggests that further studies on the effectiveness of benzochlorins in photodynamic therapy be performed.

Spectrofluorometric and FACS (Fluorescence Activated Cell Sorting) analyses were employed to determine 1) the maximal fluorescence excitation and emission peaks characteristic of BPD, benzoporphyrin derivative, 2) which structural analogue of BPD, BPD-monoacid ring A (BPD-MA), BPD-monoacid ring B (BPD-MB), BPD-diacid ring A (BPD-DA) or BPD-diacid ring B (BPD-DB) fluoresced to the greatest extent in the presence of leukemic cells and 3) to determine whether substantive differences existed in the uptake of BPD by human or murine leukemic versus normal human or murine mononuclear cells. Spectrofluorometric analysis revealed that the maximal fluorescence excitation peak of BPD (BPD-diacid ring A) was situated at 420 nm with a less prominent peak at 356 nm. Fluorescence emission scans, in which 420 nm was used as the excitation wavelength, revealed a single prominent fluorescence peak at 690 nm. FACS analysis revealed that negligible differences in fluorescence existed between leukemic cells incubated with BPD-MA, BPD-MB, BPD-DA, or BPD-DB upon excitation with visible light (488nm). However, subsequent to uv excitation cells incubated with BPD-MA fluoresced to the greatest extent followed by BPD-MB, BPD-DA, and BPD-DB respectively. Pronounced differences in red fluorescence were consistently observed between leukemic cells (HL60, K562, and L1210) and normal human or murine bone marrow cells incubated with BPD-MA. These observed differences in BPD-mediated fluorescence provide the rationale for sorting leukemic from normal cells via FACS and may constitute a novel method for extra-corporeal purging of remission marrow in autologous bone marrow transplantation.

The biological activity of a series of porphyrin dimers and trimers with ester and ether linkages, related to Photofrin II and some new photosensitizers with better optical properties are discussed. The structure and biological activity of a large number of photosensitizers, recently reported in the literature, are also reviewed.

The cellular characteristics of three photodynamic sensitizers are described. The mechanism of uptake by cultured cells was determined for Photofrin II, Mono L-aspartyl Chlorin e6, and Chloroaluminum Sulfonated Phthalocyanine. In addition, the subcellular localization, sensitizer photostability and photochemistry were studied. Also, the integral relationship between the biological characteristics and the photochemistry of the sensitizers is discussed. Photofrin II enters the cells through passive diffusion across the plasma membrane and primarily localizes in the mitochondria. Both the chlorin and phthalocyanine are transported into the cell through constitutive pinocytosis and thus localize in the lysosomes.

Chloroaluminum sulfonated phthalocyanine (A1PCS) was administered intravenously to clinically normal dogs, and A1PCS levels were determined in tissues using a sensitive assay. A1PCS accumulated to high levels in liver, spleen, bone marrow, kidney, and lung. These tissue levels confirm previous determinations in mice and rats. Only a small amount of dye was retained in skin and very small amounts in muscle and brain. A1PCS was cleared from the blood within 24 h, and excreted primarily by urine. Serum clearance was faster in males than in females. There were also significant tissue distribution differences between the genders, particularly during the first 12 h. The low levels of A1PCS in skin suggest that cutaneous photosensitivity and toxic skin reactions using this photosensitizer in photodynamic therapy of cancer may be eliminated. The difference in tissue distribution between genders is not only intriguing, but indicates that the optimal time window for treatment of various tissue sites may vary by gender.

A Clark-type microelectrode is used to measure oxygen consumption rates in laser-irradiated solutions of photosensitizer and photosensitizer-containing cells. The presence of a singlet oxygen-specific acceptor molecule, furfuryl alcohol, permits indirect determination of relative singlet oxygen generation efficiencies from oxygen consumption data. Solution and cell measurements are performed which compare photosensitizing efficiency of Photofrin-II (PII), tetraphenylporphine tetrasulfonate (TPPS4), mono-L-aspartyl chlorin e6 (MACE), and chloroaluminum sulfonated phthalocyanine (CASPc). Relative singlet oxygen generating efficiency, per-unit-weight and per-absorbed-photon, were determined to be: MACE > CASPc > TPPS4 > PII and TPPS4 > MACE > PII > CASPc, respectively. When these results are compared to oxygen consumption in photosensitizer-containing cells, differences in the order and magnitude of photosensitizing efficiencies are observed. The relative oxygen consumption rate in cells was: PII CASPc > MACE TPPS4. Additional information concerning cell killing efficiency is derived from clongenicity assays. These data indicate that consideration of singlet oxygen generating ability in solution must be considered in conjuntion with cellular assays in order to provide an in vitro estimate of photosensitizer efficacy.

The Increasing use of porphyrin-based compounds for photodynamic therapy raises concerns about the intracellular distribution of these substances in target cells. We are utilizing chlorophyll derivatives as photosensitizers and exploring their intracellular distribution in in vitro cultivated EJ human bladder tumor cells. The cells are exposed to these compounds delivered within a liposome-based carrier system as well as in the free form. High resolution subcellular fractionation approaches are employed to examine the cellular localization of the photodynamic agents. Free pheophorbide a and hematoporphyrin derivative localize, almost exclusively within the plasma membrane of cells exposed to the agents for 90 min. In contrast the pheophorbide a loaded liposomes are readily taken into lysosomes, presumably by endocytosis, if presented to the cells at 4° and then incubated with the cells at 37°. However cells exposed to the liposomes continuously at 37° accumulate the pheophorbide a in the plasma membrane. These studies indicate that untargeted liposomes exhibiting a decreased fusegenic tendency than employed in these studies may allow for an effective delivery of photodynamic agent to lysosomes.