Victor H. Fingar

DRUG and LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR and NORMAL TISSUE RESPONSE

Abstract It is clinically relevant to determine drug and light dose combinations where complete tumor response is accompanied by little or no photosensitivity, and minimal damage to normal tissues. Although reciprocity of RIF tumor cell clonogenicity has been established within a range of drug and light doses, no quantitative data exist for reciprocity of tumor response. This study has examined reciprocity of drug and light doses for tumor response and normal tissue damage in two experimental mouse models. Representative tumors were examined for vascular damage after treatment. Reciprocity of drug and light doses for tumor response was observed over a range of drug/light combinations in both tumor models. Reciprocity failed when drug dose was reduced below a threshold value. For reciprocal drug/light combinations, complete vascular stasis occurred in the tumor and surrounding skin which was followed by necrosis of those tissues. In non‐reciprocal PDT combinations, there was vascular damage to the tumor but no damage to the surrounding normal tissues. Tumors responded initially, but no cure was obtained. Tumor cure was only observed under conditions where a considerable margin of normal tissue surrounding the tumor was damaged. This conclusion was supported by shielding experiments done to assess the contribution of normal tissue damage to tumor response. Reciprocity of drug and light doses for tumor response was therefore shown to exist only at high drug doses, which were not low enough to reduce skin photosensitivity in our models.

OXYGEN LIMITATION OF DIRECT TUMOR CELL KILL DURING PHOTODYNAMIC TREATMENT OF A MURINE TUMOR MODEL

Abstract The relationship between levels of in vivo accumulated photosensitizer (Photofrin II), photodynamic cell inactivation upon in vitro or in vivo illumination, and changing tumor oxygenation was studied in the radiation‐induced fibrosarcoma (RIF) mouse tumor model. In vivo porphyrin uptake by tumor cells was assessed by using 14C‐labeled photosensitizer, and found to be linear with injected photosensitizer dose over a range of 10 to 100 mg/kg. Cellular photosensitivity upon exposure in vitro to 630 nm light also varied linearly with in vivo accumulated photosensitizer levels in the range of 25 to 100 mg/kg injected Photofrin II, but was reduced at 10 mg/kg. Insignificant increases in direct photodynamic cell inactivation were observed following in vivo light exposure (135 J/cm2, 630 nm) with increasing cellular porphyrin levels. These data were inconsistent with expected results based on in vitro studies. Assessment of vascular occlusion and hypoxic cell fractions following photodynamic tumor treatment showed the development of significant tumor hypoxia, particularly at 50 and 100 mg/kg of Photofrin II, following very brief light exposures (1 min, 4.5 J/cm2). The mean hyupoxic cell fractions of 25 to 30% in these tumors corresponded closely with the surviving cell fractions found after tumor treatment in vivo, indicating that these hypoxic cells had been protected from PDT damage. Inoculation of tumor cells, isolated from tumors after porphyrin exposure, into porphyrin‐free hosts, followed by in vivo external light treatment, resulted in tumor control in the absence of vascular tumor bed effects at high photosensitizer doses only. These results indicate that the prerequisites for tumor destruction via direct photodynamic tumor cell inactivation in vivo are high photosensitizer content in tumor cells and relative insensitivity of the tissue microvasculature. Under conditions where the tumor bed is sensitive to photodynamic damage, tumor cell kill can occur at a wider range of photosensitizer levels as a consequence of PDT induced ischemia.

DRUG AND LIGHT DOSE DEPENDENCE OF PHOTODYNAMIC THERAPY: A STUDY OF TUMOR CELL CLONOGENICITY AND HISTOLOGIC CHANGES

Abstract— The dependence of photodynamic therapy (PDT) on changes in drug and light doses was determined in C3H/HeJ mice bearing the RIF tumor. Measurements of tumor clonogenicity were determined 24 h after PDT over a range of drug and light doses. Representative histological samples were prepared at each of these doses. Both the drug and light dose dependence experiments showed an exponential decrease in clonogenicity after an initial shoulder region. Reciprocity of drug and light dose was established from those clonogenicity curves. Histological examination of tumors gave information concerning the localization of gross damage within tumors. Increases of light dose in PDT were shown to extend the depth of necrosis within tumors. Increases of drug dose produced enlargements in the area of necrotic spots produced by PDT

THE EFFECTS OF PHOTODYNAMIC THERAPY USING DIFFERENTLY SUBSTITUTED ZINC PHTHALOCYANINES ON VESSEL CONSTRICTION, VESSEL LEAKAGE AND TUMOR RESPONSE

Abstract— The effects of four different zinc phthalocyanines were studied during and after photodynamic therapy (PDT). Measurements of vessel constriction, vessel leakage, tumor interstitial pressure, eicosanoid release, and tumor response of chondrosarcoma were made in Sprague‐Dawley rats. Animals were injected intravenously with 1 μmol/ kg of mono‐, di‐, or tetrasulfonated zinc phthalocyanine, or 1 μmol/kg of a zinc phthalocyanine substituted with four tertiary butyl groups. Tissues were exposed to 400 J/cm2 670 nm light 24 h after photosensitizer injection. An additional group of animals was given indomethacin before treatment. The use of the monosulfonated and tertiary butyl substituted zinc phthalocyanines in PDT caused the release of specific eicosanoids, caused vessel constriction, and induced venule leakage and increases in tumor interstitial pressure. Tumor cures of 27% and 7% were observed. Photodynamic therapy using the disulfonated zinc phthalocyanine did not induce vessel constriction or the release ofeicosanoids, however; tumor cure was 43%. The use of thc tetrasulfonated zinc phthalocyanine caused intermediate effects between the mono‐ and disulfonated compounds. The administration of indornethacin to animals completely inhibited the effects of PDT using the monosulfonated compound but had minimal effects on treatment using the disulfonated compound. This suggests that the monosulfonated and disulfonated compounds act by different mechanisms of destruction.

THE EFFECTS OF THROMBOXANE INHIBITORS ON THE MICROVASCULAR AND TUMOR RESPONSE TO PHOTODYNAMIC THERAPY

Abstract— Vascular stasis and tissue ischemia are known to cause tumor cell death in several experimental models after photodynamic therapy (PDT); however, the mechanisms leading to this damage remain unclear. Because previous studies indicated that thromboxane release is implicated in vessel damage, we further examined the role of throm‐boxane in PDT. Rats bearing chondrosarcoma were injected with 25 mg/kg Photofrin® (intravenously) 24 h before treatment. Light (135 J/cm2, 630 nm) was delivered to thc tumor area after injection of one of the following inhibitors: (1) R68070: a thromboxane synthetase inhibitor; (2) SQ‐29548: a thromboxane receptor antagonist; and (3) Flunarizine: an inhibitor of platelet shape change. Systemic thromboxane levels were determined. Vessel constriction and leakage were evaluated by intravital microscopy. Tumor response was assessed after treatment. Thromboxane levels were decreased more than 50% with SQ‐29548 as compared to controls. Thromboxane levels in animals given R68070 and Flunarizine remained at baseline levels. SQ‐29548 and R68070 reduced vessel constriction compared to controls, while Flunarizine totally prevented vessel constriction. R68070 and SQ‐29548 inhibited vessel permeability compared to PDT controls; Flunarizine did not. Animals given these inhibitors showed markedly reduced tumor cure. These results indicate that the release of thromboxane is linked to the vascular response in PDT.

The effects of thrombocytopenia on vessel stasis and macromolecular leakage after photodynamic therapy using photofrin.

Abstract— Several studies have reported thrombus formation and/or the release of specific vasoactive eicosanoids, suggesting that platelet activation or damage after photodynamic therapy (PDT) may contribute to blood flow stasis. The role of circulating platelets on blood flow stasis and vascular leakage of macromolecules during and after PDT was assessed in an intravital animal model. Sprague‐Daw‐ley rats bearing chondrosarcoma on the right hind limb were injected intravenously (i.v.) with 25 mg/kg Photofrin 24 h before light treatment of 135 J/cm2 at 630 nm. Thrombocytopenia was induced in animals by administration of 3.75 mg/kg of rabbit anti‐rat platelet antibody i.v. 30 min before the initiation of the light treatment. This regimen reduced circulating platelet levels from 300000/mm3 to 20000/mm3. Reductions in the luminal diameter of the microvasculature in normal muscle and tumor were observed in control animals given Photofrin and light. Venule leakage of macromolecules was noted shortly after the start of light treatment and continued throughout the period of observation. Animals made thrombocytopenic showed none of these changes after PDT in either normal tissues or tumor. The lack of vessel response correlated with the absence of thromboxane release in blood during PDT. These data suggest that platelets and eicosanoid release are necessary for vessel constriction and blood flow stasis after PDT using Photofrin.

Implications of a pre-existing tumor hypoxic fraction on photodynamic therapy

The presence of oxygen in tissue is a requirement for photodynamic therapy (PDT)-induced destruction of solid tumors, otherwise no cell death occurs. Since many tumors have been shown to have significant populations of hypoxic cells, it is of clinical interest to determine if pre-existing tumor hypoxia limits phototherapy. This question was examined using RIF tumors where tumor response to PDT of completely oxygenated tumors was compared to tumors with an induced hypoxic fraction. Tumor hypoxia was induced by using vasoactive drugs (epinephrine, chlorpromazine, or isoproterenol), given 30 min prior to PDT, or by a surgical method. PDT consisted of 5 mg/kg Photofrin II ip 24 hr prior to treatment and 135 J/cm2 630-nm light. The administration of the various vasoactive agents induced hypoxic fractions of 2.2 to 10%. The surgical method induced hypoxic fractions of 35%. Tumor response and cure in animals given vasoactive agents did not differ from controls, suggesting that low levels of pre-existing tumor hypoxia do not limit photodynamic therapy in this tumor model. Animals with tumors made hypoxic by a surgical method showed significantly reduced tumor response to PDT. Only 14% of these animals had tumors which became flat and necrotic by the day following PDT, compared to nearly 100% for animals given vasoactive drugs or controls. Furthermore, no tumor cure was observed in animals treated by this method. The higher level of tumor hypoxia in these animals likely represents one point where large proportions of PDT-resistant cells can survive after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

The treatment of malignant endobronchial obstruction with laser ablation

BACKGROUND We compared a new endoscopic treatment for malignant endobronchial obstruction known as photodynamic therapy (PDT) with the more established therapy of neodymium: yttrium-aluminum garnet laser (Nd:YAG) therapy. METHODS A retrospective review was conducted of the medical records at our institution from 1988 to 1999 of patients treated for bronchial obstruction by thermal laser vaporization (Nd:YAG) or by PDT using the tunable dye laser in combination with a light-sensitive dye (PDT). The Nd:YAG procedure vaporized the obstructing neoplasm, whereas the PDT procedure photoablated the obstruction. Thirty-day mortality and morbidity rates were analyzed for both treatment groups using chi-square analysis. RESULTS Of the 102 patients who were suitable for review, 83 received treatment with the Nd:YAG laser and 19 patients received treatment with PDT. Morbidity rates were comparable in both groups (22% for Nd:YAG vs 31% for PDT; P > .05). Equally common complications in both groups were respiratory failure and hypoxemia. Five Nd:YAG patients (6%) died within 30 days after treatment (3 of respiratory failure, 2 of massive hemoptysis), whereas 2 patients (10%) in the PDT group (1 of massive hemoptysis, 1 of acute myocardial infarction) died (P > . 05). CONCLUSIONS PDT and Nd:YAG have similar mortality and morbidity rates. In our experience, PDT is a better choice for the treatment of malignant bronchial obstruction because it is technically easier, potentially safer, and does not require general anesthesia.

Photodynamic therapy for palliation of chest wall recurrence in patients with breast cancer

Background and Objectives: Chest wall recurrence occurs in 5–20% of breast cancer patients. Until recently, the only treatments available were surgical resection or radiotherapy. Photodynamic therapy (PDT) is a new modality that uses a photosensitizer and light to destroy tumor cells selectively. We report here our experience with PDT as a treatment for chest wall recurrence.

In Vivo and In Vitro Photodynamic Studies with Benzochlorin Iminium Salts Delivered by a Lipid Emulsion

Benzochlorin iminium salts (Bis) are hydrophobic photosensitizers based on an octaethylbenzochlorin nucleus that absorb in the near‐IR region of the visible spectrum. In these studies the photodynamic activities of the zinc, copper and metal‐free BI derivatives were compared in vivo in C3H‐HeJ mice bearing a mammary adenocarcinoma tumor line. In vitro studies were also performed with the radiation‐induced fibrosarcoma tumor line. An argon‐pumped Ti‐sapphire laser tuned to deliver light between 710 and 800 nm or an Oriel arc‐lamp filtered to deliver broadband light above 590 nm were used as light source. A lipid emulsion was used as the delivery system for sensitizers in all studies. A pronounced solvent dependence was observed for the Q band for each of all iminium salts examined. As an example, the metal‐free (BI) derivative had an absorption maximum at 798 nm in dichloromethane and at 727 nm in serum. The action spectra showed a greater PDT response at blue‐shifted wavelengths for each of the three iminium salts both in vivo and in vitro. Among the three derivatives, the zinc analog (ZnBI) produced the greatest tumor regression at the low drug/light dose of 0.7 (μ mole/kg and 200 J/cm2. These results indicate that iminium salts have characteristics that may make them promising third‐generation photosensitizers.