Oxygen consumption and diffusion effects in photodynamic therapy.

Effects of oxygen consumption in photodynamic therapy (PDT) are considered theoretically and experimentally. A mathematical model of the Type II mechanism of photooxidation is used to compute estimates of the rate of therapy-dependent in vivo oxygen depletion resulting from reactions of singlet oxygen (1O2) with intracellular substrate. Calculations indicate that PDT carried out at incident light intensities of 50 mW/cm2 may consume 3O2 at rates as high as 6-9 microM s-1. An approximate model of oxygen diffusion shows that these consumption rates are large enough to decrease the radius of oxygenated cells around an isolated capillary. Thus, during photoirradiation, cells sufficiently remote from the capillary wall may reside at oxygen tensions that are low enough to preclude or minimize 1O2-mediated damage. This effect is more pronounced at higher power densities and accounts for an enhanced therapeutic response in tumors treated with 360 J/cm2 delivered at 50 mW/cm2 compared to the same light dose delivered at 200 mW/cm2. The analysis further suggests that the oxygen depletion could be partially overcome by fractionating the light delivery. In a transplanted mammary tumor model, a regimen of 30-s exposures followed by 30-s dark periods produced significantly longer delays in tumor growth when compared to the continuous delivery of the same total fluence.

Effects of various photoradiation regimens on the antitumor efficacy of photodynamic therapy for R3230AC mammary carcinomas.

Clinical photodynamic therapy consists of the systemic administration of a derivative of hematoporphyrin (Photofrin II) followed by exposure of malignant lesions to continuous visible laser irradiation. We investigated the effects of various modifications of laser light delivery on the efficacy of photodynamic therapy in controlling R3230AC mammary tumor growth. We observed a significant delay in growth (from initial to 2 times initial volume) of tumors exposed to periodic irradiation (100 mW/cm2/0.25 h, 1-h dark interval, 100 mW/cm2/0.25 h; total fluence, 180 J/cm2), compared to untreated controls or to tumors receiving continuous irradiation at the same total fluence. Other periodic light treatment regimens, consisting of 3-, 6-, or 24-hr dark intervals, delayed tumor growth but not significantly more than continuous irradiation at the same fluence. A biochemical basis was sought by comparing continuous versus periodic irradiation for effects on mitochondrial or cytosolic enzymes in vivo. Although both cytochrome c oxidase and pyruvate kinase activities were reduced dramatically during the first 24 h by continuous or periodic irradiation schemes, recovery of enzyme activity to initial levels took longer after the periodic irradiation protocol (168 h), compared to the continuous irradiation regimen (72 h). We observed a significantly greater delay in the growth of tumors exposed to 50 mW/cm2/2 h continuously, compared to controls or to tumors exposed to the same total fluence but with light delivered at 100 or 200 mW/cm2. The data presented here indicate that the efficacy of photodynamic therapy could be significantly increased by modifications in the delivery of photoradiation.

Increased efficacy of photodynamic therapy of R3230AC mammary adenocarcinoma by intratumoral injection of Photofrin II.

Photodynamic therapy consists of the systemic administration of a derivative of haematoporphyrin (Photofrin II) followed 24-72 h later by exposure of malignant lesions to photoradiation. We investigated the efficacy of this treatment after direct intratumoral injection of Photofrin II. This direct treatment regimen resulted in higher rates of inhibition of mitochondrial cytochrome c oxidase (5.13% J-1 cm-2 x 10(-1) and succinate dehydrogenase (3.14% J-1 cm-2 x 10(-1] in vitro at 2 h after intratumoral injection compared to rates of inhibition obtained after intraperitoneal drug administration: 0.51 and 0.42% J-1 cm-2 x 10(-1), respectively. A significant delay in tumour growth in vivo was observed in animals that received intratumoral injections 2 h before photoradiation compared to animals injected intraperitoneally at either 2 or 24 h before photoradiation. The treatment protocols were compared with control groups, consisting of Photofrin II administration intratumorally or intraperitoneally without photoradiation, or photoradiation in the absence of Photofrin II. These data indicate that the intratumoral injection regimen with Photofrin II enhanced the efficacy of photodynamic therapy. The greater delay in tumour growth observed after intratumoral administration of Photofrin II suggests a mechanism favouring direct cell damage.

In vitro photosensitization of tumour cell enzymes by photofrin II administered in vivo.

The ability of injected Photofrin II, a preparation enriched in hydrophobic dihaematoporphyrin ethers and esters, to photosensitize selected mitochondrial and cytosolic enzymes during illumination in vitro was examined. Preparations of R3230AC mammary tumours, obtained at designated times after a single dose of Photofrin II, displayed a time-dependent photosensitivity. Maximum inhibition of mitochondrial enzymes occurred at 24 hours post-treatment, whereas no inhibition of the cytosolic enzyme, pyruvate kinase, was observed over the 168 hour time course. At the selected 24 hour time point, mitochondrial enzyme photosensitisation was found to be drug dose (5.25 mg kg-1 Photofrin II) and light dose dependent, the rank order of inhibition being cytochrome c oxidase greater than F0F1 ATPase greater than succinate dehydrogenase greater than NADH dehydrogenase. We conclude that porphyrin species contained in Photofrin II accumulate in mitochondria of tumour cells in vivo and produce maximum photosensitisation at 24-72 hours after administration to tumour-bearing animals. The time course observed here with Photofrin II is similar to that seen previously with the more heterogenous haematoporphyrin derivative preparation in this in vivo-in vitro model.

Photosensitizing effects of hematoporphyrin derivative and photofrin II on the plasma membrane enzymes 5'-nucleotidase, Na+K+-ATPase, and Mg2+-ATPase in R3230AC mammary adenocarcinomas.

The sensitivity to photodynamic treatment of three plasma membrane enzymes in R3230AC mammary adenocarcinomas was assessed. The activities of Na+K+-ATPase, Mg2+-ATPase and 5'-nucleotidase in isolated membranes were measured after exposure of membranes to either hematoporphyrin derivative or Photofrin II plus light in vitro or in tumor membranes prepared from animals previously injected with 25 mg/kg Photofrin II and sacrificed at various times prior to exposure to light (in vivo-in vitro protocol). The activities of both Na+K+-ATPase and Mg2+-ATPase were inhibited at equivalent rates by Photofrin II in vitro; inhibition was drug dose and light dose related. For 5'-nucleotidase in vitro, a 10-fold higher porphyrin concentration was required to achieve a similar rate of enzyme inhibition as that for the ion-activated ATPases. Injection of Photofrin II in vivo followed by preparation of tumor plasma membranes, which were subsequently exposed to light in vitro, produced no photosensitization of 5'-nucleotidase activity at any time studied (up to 72 h after Photofrin II administration). Under the same conditions Na+K+-ATPase activity was reduced by 40-60% from 2 to 72 h after drug injection, whereas Mg2+-ATPase activity was inhibited by 10-25% over the same time course. The differential sensitivity of these three enzymes observed in this in vivo-in vitro protocol suggests that each enzyme may possess different characteristics, such as three-dimensional configuration or membrane location, that afford varying susceptibility to porphyrin photosensitization. The data also suggest that photosensitivity-induced damage to these ion-activated plasma membrane ATPases could have deleterious effects on tumor cell survival.

Photosensitizing effects of Photofrin II on the site-selected mitochondrial enzymes adenylate kinase and monoamine oxidase.

The response to Photofrin II-induced photosensitization on the activities of mitochondrial monoamine oxidase (MAO) and adenylate kinase (AK) were studied in order to gain further insight into site specific effects. Utilizing intact mitochondria in vitro, both MAO, located on the cytoplasmic side of the outer mitochondrial membrane, and AK, located in the intermembrane space, were inhibited by exposure to Photofrin II plus light; inhibition was drug-dose and light-dose dependent. However, MAO activity was inhibited to a greater extent than AK; at 35 micrograms/ml of Photofrin II and 160 J/cm2, MAO activity was decreased by 80% whereas AK activity was inhibited by 30%. Higher doses of Photofrin II had no further effect on AK activity. Studies of photosensitization of AK in different mitochondrial preparations demonstrated that inhibition of activity was evident only when mitochondrial membranes containing sequestered porphyrins were present in the reaction mixture. Using an in vivo-in vitro protocol and sampling at 2 to 72 h after administration of 25 mg/kg of Photofrin II, photosensitization of MAO (30% inhibition) was seen at 2 h after drug treatment but inhibition of activity was not observed at later times. AK activity was unchanged over the entire time course. Compared to cytochrome c oxidase, located in the inner mitochondrial membrane and which displayed a sustained inhibition of activity, we suggest that inhibition of MAO or AK activities probably does not contribute to the tumor cytotoxicity under the usual conditions used for photodynamic therapy.

Inhibition of mammalian DNA polymerases by hematoporphyrin derivative and photoradiation.

Hematoporphyrin derivative (HPD) plus photoradiation caused the inactivation of DNA polymerases from calf thymus and R3230AC rat mammary tumor. Photosensitization of purified DNA polymerase-alpha as well as two forms of DNA polymerase-delta (I and II) from calf thymus were evaluated. Although all polymerase enzyme forms were inactivated at 70 micrograms HPD/ml, DNA polymerase-delta II was the most sensitive, displaying a 90% inactivation under conditions that did not cause significant inactivation of the other polymerase forms. Unlike DNA polymerase-alpha, the delta-forms have an associated 3'- to 5'-exonuclease activity. The exonuclease associated with DNA polymerase-delta II was uniquely sensitive to a low level of HPD and light exposure. DNA polymerase-delta II can be distinguished from other polymerase forms in cell extracts by its relative insensitivity to the polymerase inhibitor N2-(p-n-butylphenyl)deoxyadenosine 5'-triphosphate. In cytosols prepared from calf thymus and R3230AC rat mammary tumors, DNA polymerase-delta II was preferentially inhibited by HPD plus light. Furthermore, in experiments in which tumor-bearing rats were administered HPD prior to preparation of tumor cytosols, DNA polymerase-delta II was specifically inactivated by exposure to light. These results are discussed in view of their possible role in cancer therapy, and the potential use of HPD as a specific inhibitory agent of DNA polymerase-delta II is suggested.

Relationship of mitochondrial function and cellular adenosine triphosphate levels to hematoporphyrin derivative-induced photosensitization in R3230AC mammary tumors.

The effects of hematoporphyrin derivative-induced photosensitization on the levels of adenosine triphosphate (ATP) in R3230AC mammary adenocarcinomas were studied. Enzymatically dissociated tumor cells were exposed to various doses of hematoporphyrin derivative (HPD) in vitro plus photoradiation. A drug and light dose-dependent decrease in cellular ATP levels was observed; ATP levels were reduced by 60% after treatment with 7.0 micrograms HPD per ml plus 0.72 J total energy density per cm2. Cell viability, assessed by exclusion of trypan blue, displayed an apparently coordinate behavior to ATP levels. The effects of hematoporphyrin derivative plus photoradiation were examined in the presence of oligomycin, an inhibitor of mitochondrial oxidative phosphorylation, or iodoacetate, an inhibitor of glycolysis, experiments designed to elucidate the site of action leading to reduced ATP levels. The results indicated that HPD-induced photosensitization had little additive effects to oligomycin-sensitive ATP production, whereas significant further reduction in ATP levels was obtained by HPD-induced photosensitization in the presence of iodoacetate. Taken together, along with earlier studies of selected mitochondrial enzymes, we conclude that HPD plus photoradiation inhibits mitochondrial function leading to reduction in cellular ATP levels and loss of viability.

Effects of photosensitization by hematoporphyrin derivative on mitochondrial adenosine triphosphatase-mediated proton transport and membrane integrity of R3230AC mammary adenocarcinoma.

Photodynamic therapy, which consists of treatment with hematoporphyrin derivative (HPD) followed by photoradiation with visible light, is a promising approach to treatment of various cancers. To gain further understanding of the mechanisms whereby this therapy produces cytotoxicity, we undertook a study of the mitochondrial proton-translocating adenosine triphosphatase, an enzyme performing the critical role of coupling electrochemical proton gradients to the formation of adenosine triphosphate. Exposure of submitochondrial particles to HPD and photoradiation in vitro caused a marked inhibition of proton transport; inhibition displayed both drug-dose and light-dose relationships. Inhibition of proton transport was correlated with inhibition of adenosine triphosphate hydrolysis, both demonstrating an inhibition rate of 2% min-1. Investigation of effects of HPD plus light on membrane integrity, measured by [3H]sucrose leakage from submitochondrial particles and by K+ leakage from asolectin phospholipid vesicles, indicated that discrete membrane alterations were not the likely cause of initial loss of pH gradient formation. Likewise, photosensitization by HPD to inhibit adenosine triphosphate hydrolysis and proton transport was not coincident with cross-linking of the major subunits of the enzyme. We conclude that mitochondrial function is severely impaired by photodynamic therapy and offers a potentially important target site leading to cytotoxicity.

Hematoporphyrin derivative-induced photosensitivity of mitochondrial succinate dehydrogenase and selected cytosolic enzymes of R3230AC mammary adenocarcinomas of rats.

The photosensitizing activity of hematoporphyrin derivative (HPD) was investigated by studying selected enzymes localized to mitochondria and cytosol of R3230AC mammary adenocarcinomas. Experiments in vitro demonstrated that mitochondrial succinate dehydrogenase was inhibited in a drug dose- and light exposure time-related manner; at 7.0 micrograms of HPD per ml or higher, enzyme activity was inhibited greater than 50% after 15 min of photoradiation. The three cytosol enzymes studied under the same conditions in vitro demonstrated different photosensitivities. Pyruvate kinase activity was significantly inhibited in a dose- and time-related fashion, whereas lactate dehydrogenase was inhibited to a lesser extent, and glucose phosphate isomerase activity was inhibited only at the highest dose (70 micrograms of HPD per ml) used. The time-course of these responses was examined with an in vivo-in vitro protocol, consisting of photoradiation of mitochondria and cytosol prepared from tumors obtained at various times (up to 1 week) after a single injection of HPD (80 mg/kg). Pyruvate kinase activity was markedly inhibited at early times returning to initial levels by 48 hr; neither lactate dehydrogenase nor glucose phosphate isomerase was inhibited by this treatment. Mitochondrial succinate dehydrogenase and cytochrome c oxidase activities displayed significant photoradiation-induced inhibitions, with greatest inhibition occurring between 24 and 96 hr after injection of HPD; at 1 week, succinate dehydrogenase activity had returned to its initial level, but cytochrome c oxidase activity remained significantly inhibited. These data suggest that HPD-induced photosensitization of mitochondria may be an important site of action contributing to tumor cell cytotoxicity and regression as a result of photoradiation therapy.