Scott L. Gibson

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.

The relative importance of estrogen receptor analysis as a prognostic factor for recurrence or response to chemotherapy in women with breast cancer

The value of estrogen receptor (ER) analysis in primary breast cancer samples as a potential prognostic factor was examined in three clinical situations: time to recurrence in patients with no therapy after mastectomy, failure of patients receiving adjuvant therapy, and response of advanced disease patients to cytotoxic chemotherapy. Other prognostic factors analyzed were menopausal and nodal status. In none of these clinical settings were we able to demonstrate the usefulness of ER status as a prognosticator of the disease course or its response to therapy.

Interdependence of fluence, drug dose and oxygen on hematoporphyrin derivative induced photosensitization of tumor mitochondria.

Abstract— Studies were conducted to assess the interdependence of three discrete parameters known to influence hematoporphyrin derivative (Hpd)‐induced photosensitization. The effects of fiuence, drug dose and oxygen environment were examined for their role in causing an inhibition of the activity of mitochondrial cytochrome c oxidase. Experiments were performed on R3230AC mammary tumor mitochondria in vitro and on mitochondria isolated from tumors of animals pre‐treated with Hpd in vivo. Inhibition of cytochrome c oxidase activity was observed to be directly proportional to total energy density. Photosensitization was dependent on oxygen concentration, with total energy density dependent photosensitization being diminished in environments containing less than 5% oxygen. At 1% oxygen environments, photosensitization was significantly impaired and demonstrated no drug‐dose relationship. These results suggest that tissue oxygen concentration may represent a critical factor for the therapeutic usefulness of Hpd photodynamic therapy in treatment of cancer.

A regulatory role for porphobilinogen deaminase (PBGD) in delta-aminolaevulinic acid (delta-ALA)-induced photosensitization?

As an initial approach to optimize delta-aminolaevulinic acid (delta-ALA)-induced photosensitization of tumours, we examined the response of three enzymes of the haem biosynthetic pathway: delta-ALA dehydratase, porphobilinogen deaminase (PBGD) and ferrochelatase. Only PBGD activity displayed a time- and dose-related increase in tumours after intravenous administration of 300 mg kg(-1) delta-ALA. The time course for porphyrin fluorescence changes, reflecting increased production of the penultimate porphyrin, protoporphyrin IX (PPIX), showed a similar pattern to PBGD. This apparent correlation between PBGD activity and porphyrin fluorescence was also observed in four cultured tumour cell lines exposed to 0.1-2.0 mM delta-ALA in vitro. The increase in PBGD activity and PPIX fluorescence was prevented by the protein synthesis inhibitor cycloheximide. As the apparent Km for PBGD was similar before and after delta-ALA, the increase in PBGD activity was attributed to induction of enzyme de novo. These observations of an associated response of PBGD and PPIX imply that PBGD may be a rate-limiting determinant for the efficacy of delta-ALA-induced photosensitization when used in photodynamic therapy.

Photodynamic inactivation of selected intracellular enzymes by hematoporphyrin derivative and their relationship to tumor cell viability in vitro

The photosensitivity of freshly dissociated R3230AC mammary adenocarcinoma cells was examined by measurement of the activities of selected intracellular enzymes after treatment with hematoporphyrin derivative (Hpd) and exposure to light in vitro. Enzymes selected as representative of the cytosolic cell compartment showed no loss in activity, whereas malate dehydrogenase, located in the mitochondrial matrix, displayed a modest decrease (approximately 15%) in activity. In contrast, cytochrome c oxidase and succinate dehydrogenase, enzymes associated with the mitochondrial membrane, demonstrated a very rapid and more marked inhibition of activities, approximately 45% and 25%, respectively. The time-course of inhibition of these mitochondrial membrane enzymes preceded the loss of cell viability, which displayed slower kinetics as seen by a more gradual and progressive pattern of loss in viability. These data suggest that the mitochondria are an early-affected and important intracellular site for Hpd photosensitization.

The lack of relationship between estrogen receptor status and response to chemotherapy

The possible relationship between estrogen receptors and response of breast cancer patients to chemotherapy was examined in two situations: patients with advanced disease and patients receiving postsurgical adjuvant treatment. In 73 patients with disseminated disease, no relationship between ER status and response to cytotoxic chemotherapy was observed. At this time, 44 of these cases have undergone extramural review; in these 44 cases, we observed no significant relationship between ER status and response to chemotherapy. In a series of 52 patients receiving adjuvant therapy, 27 patients were classified as failures due to recurrence. ER status did not offer prognostic value for failure of patients treated with postsurgical adjuvant therapy. We conclude that the prognostic value of ER data in nonhormonal therapy settings remains to be proven.

31P-NMR spectroscopy demonstrates decreased ATP levels in vivo as an early response to photodynamic therapy

31P-Nuclear magnetic resonance was used to monitor in situ phosphorus containing compounds in mammary tumors after photodynamic therapy, consisting of administration of hematoporphyrin derivative followed by photoradiation of the lesion. A rapid decrease in ATP along with an increase in Pi resonance intensities was observed. The beta-ATP/Pi ratio decreased by 1 hour, dropping in 2 to 8 hours to 0 to 20 percent of that found prior to photoradiation. Disrupted cells and pycnotic nuclei were observed 48 to 72 hours after photoradiation to a depth of approximately 5 mm. Together with previous studies in vitro, reduction in tumor ATP levels appears to be an early biochemical response to photodynamic therapy.

Effects of photodynamic therapy on xenografts of human mesothelioma and rat mammary carcinoma in nude mice.

We have examined the effectiveness of photodynamic therapy against R3230AC rat mammary adenocarcinoma and human mesothelioma as xenografts in the same host. The results demonstrate that the xenografted human tumour is significantly more responsive to photodynamic treatment than the rodent mammary tumour. Studies also showed that the mesothelioma xenograft was fluence rate- and fluence-dependent while the rat tumour exposed to the same conditions demonstrated neither of these dependencies. This disparity in response was not attributable to a difference in either whole-tumour uptake or subcellular distribution of the porphyrin photosensitiser. Analysis of the effects of visible irradiation on cytochrome c oxidase activity, measured in mitochondria prepared from tumours borne on hosts injected with photosensitiser, demonstrated that photoradiation-induced enzyme inhibition was significantly greater in mesothelioma than in R3230AC mammary tumour preparations. However, in parallel studies conducted in vitro, when photosensitiser and light were delivered to previously unperturbed mitochondria, rates of enzyme inhibition were not significantly different. Both tumours were established in long-term cell culture. While the uptake of porphyrin photosensitiser was equivalent in both cell lines, the R3230AC cells displayed a significantly greater photosensitivity than the mesothelioma cells. The data presented here demonstrate that the mechanisms that govern response to photodynamic therapy are complex, but in the case of these two xenografted tumours host response to therapy is not likely to play a significant role.

Evidence against the production of superoxide by photoirradiation of hematoporphyrin derivative

Abstract Experiments were performed to ascertain whether superoxide anion (O2−) was produced by the photodynamic activation of hematoporphyrin derivative (HPD). Three different systems were utilized to detect formation of O2−, oxidation of epinephrine to adrenochrome, reduction of cytochrome c and reduction of nitro blue tetrazolium (NBT). The effects on these detectors under identical conditions for HPD + hν were compared to those obtained with two O2− generating systems, riboflavin + by and xanthine‐xanthine oxidase, and to a singlet oxygen generating system, photoradiation of methylene blue. The results indicated that HPD + hv differed from the two O2− generating systems in failing to reduce cytochrome c or NET, and that HPD + hν was similar to the behavior of methylene blue + hν. In addition, HPD + hν but not the O2− generating systems could inhibit mitochondrial cytochrome c oxidase activity. We conclude that the photodynamic activation of HPD does not produce O2− as a major oxygen radical and that the effects of HPD + hν on mitochondrial cytochrome c oxidase are not caused by O2−.

EARLY BIOCHEMICAL RESPONSES TO PHOTODYNAMIC THERAPY MONITORED BY NMR SPECTROSCOPY

Abstract Studies directed at determining the biochemical events that lead to tumor cytotoxicity following photodynamic therapy, a promising new approach for treatment of neoplasia, have demonstrated that exposure of R3230AC mammary tumors to hematoporphyrin derivative or Photofrin II plus visible light caused marked impairment of mitochondrial enzymes functioning in oxidative phosphorylation and electron transport. 31P‐NMR spectroscopy has now demonstrated that a rapid and striking decrease in NTP (ATP) levels, concomitant with a marked increase in P;, occurs in tumors shortly after photodynamic therapy. These effects appear to be fluence related. Possible changes in tumor vascularity, as detected by 2H‐NMR measurements of the uptake of D20, were not observed under the conditions studied. Taken together with our earlier results, we conclude that the reduction in tumor ATP levels in situ, probably via inhibition of mitochondrial function, is a direct and early response of neoplastic tissue to porphyrin‐induced photosensitization.