A FLUORESCENCE IMAGING DEVICE FOR ENDOSCOPIC DETECTION OF EARLY STAGE CANCER – INSTRUMENTAL and EXPERIMENTAL STUDIES

Abstract Visible detection of early stage cancer labelled with the fluorescing porphyrin mixture of dihematoporphyrin-ether and dihematoporphyrin-ester (DHE) is often limited by a high and inhomogeneously distributed level of autofluorescence. A new imaging method for reducing autofluo-rescence is described. The method uses alternating fluorescence excitation with laser light in the violet and blue spectral ranges. Subtraction of the corresponding fluorescence images results in contrast enhancement due to an effective reduction in the autofluorescence contribution. A prototype version of a highly sensitive fluorescence imaging device including a modified krypton ion laser, an image-intensified solid-state TV-camera, a digital image-processing system, and video recording and monitoring has been developed.
Experiments performed with this fluorescence imaging system on a tumorous dog bladder showed that a dose of 0.2 mg DHE kg⁻¹ body wt, which is far below the dose administered for photodynamic therapy (2-5 mg kg⁻'body wt), is sufficient to give a high-contrast fluorescence image. The corresponding excitation power density was about 1 W m⁻². Photobleaching of DHE was observed during fluorescence detection at excitation power densities exceeding 1 kW m⁻². At these high excitation levels DHE fades out nearly completely within 0.25 min.     

PARTIAL PROTECTION OF PHOTODYNAMIC-INDUCED SKIN REACTIONS IN MICE BY N-ACETYLCYSTEINE: A PRECLINICAL STUDY

Abstract The major side effect of photodynamic therapy (PDT) using Photofrin® is enhanced skin sensitivity for sunlight, which persists for 3-8 weeks after injection. Formation of singlet oxygen and radicals is believed to be involved in the basic mechanism of inducing skin damage. Reducing this side effect would make PDT more widely acceptable, particularly for palliative use. Hairless dorsal skin patches of mice, injected with 10 mg kg⁻¹ photofrin intraperitoneally (i.p.) 24 h before illumination, were used to evaluate the effect of increasing light doses. The light was obtained from a halogen lamp and transmitted via a fiber optic to illuminate a field of 2.5 cm². After establishing a dose-response relationship for single or fractionated light dose illumination of the skin, drugs known to scavenge radicals, quench singlet oxygen or interfere with histamine release were tested for their protective effect. N -acetylcysteine (NAC), a radical scavenger, administered i.p. (1000 and 2000 mg kg⁻¹) 1 h before illumination produced a significant decrease in skin damage at light doses >50 J cm⁻² (protection factor of 1.3-1.8). When NAC was administered in a dose of 500 mg kg⁻¹, no protection was observed. Fractionated illumination experiments in combination with multiple injections of NAC (1000 mg kg⁻¹) also failed to show any protection. The addition of Ranitidine®, a histamine blocking agent (25-100 mg kg⁻¹, given prior to illumination, resulted in a limited protection at higher light doses. From this study we conclude that NAC could be of value in amelioration of the photosensitivity in patients treated with PDT.     

PHOTODYNAMIC THERAPY IN THE TREATMENT OF PANCREATIC CARCINOMA: DIHEMATOPORPHYRIN ETHER UPTAKE and PHOTOBLEACHING KINETICS

Abstract Results of dihematoporphryin ether (DHE) uptake and fluorescence kinetics show that the concentration in the pancreas is on the order of 40-60 μg DHE g⁻¹ of tissue at an injected dose of 40 mg kg⁻¹. Previously concentrations on this order have primarily been found in organs of the reticuloendothelial system. Two intra-pancreatic carcinoma models, one of acinar origin (rat) and one of ductal origin (hamster), were studied. Both showed equal or higher concentrations of DHE as compared with normal pancreas when fluorescence measurements and chemical extraction procedures were performed. Photodynamic therapy (PDT) treatment of the normal pancreas and pancreatic tumors yielded atypical results. When the normal pancreas with DHE present is exposed to 630-nm light from a dye laser (75 mW cm⁻², 30 min), the normal photobleaching measurable by fluorescence decay does not occur. Yet the pancreatic tumor responds with a relatively normal fluorescence decay pattern, with hemorrhaging and a resultant loss of measurable DHE concentration.     

THE INFLUENCE OF SODIUM PENTOBARBITAL ANESTHESIA ON in vivo PHOTODYNAMIC THERAPY

Abstract The use of sodium pentobarbital anesthesia 50 jig gm⁻¹ during localized photodynamic therapy (PDT) was examined in C57BL/6 mice transplanted with the pigmented B-16 melanoma. A 10 mg kg⁻¹ i.p. injection of Photofrin II was administered 24 h prior to light exposure (630 nm, 150 mW, cm⁻², 300-500 J cm⁻²). Separate groups of mice were utilized to monitor tumour temperature and PDT tumor response. Core tumor temperatures decreased by approx. 10^oC following sodium pentobarbital administration. Tumor responses were determined by documenting the percentage of treated animals without tumor recurrences for a period of 50 days following PDT. Superior PDT induced tumor responses were obtained in control (non-anesthetized) mice following light doses of 400 and 500 J cm⁻². The results of this study indicate that sodium pentobarbital can induce a protective effect on B-16 melanomas treated with PDT.     

NECROSIS OF MURINE TAIL SKIN FOLLOWING PHOTODYNAMIC TREATMENT WITH meso-tetra-(p-SULPHOPHENYL) PORPHINE (TPPS)

Abstract. Albino mice were injected intravenously with 2 mg of meso-tefra-(p -sulphophenyl) porphine (TPPS) and subsequently their tails were exposed to a range of doses of full-spectrum visible light. At a power density of 75 mW cm^-2, a 50% incidence of gross necrosis of the tail skin (ED₅₀) occurred at a light dose of 130 J cm^-2. For haematoporphyrin derivative at the same administered dose, the ED₅₀ was 33 J cm^-2. Increasing the interval between TPPS and light from 1 to 34 days did not spare the tail skin, in terms of an increase in ED₅₀. The slopes of curves of incidence of tail necrosis vs light dose were steep: 1/slope ranged between 20 and 50 J cm^-2.     

PULSED RADIATION STUDIES OF PHOTODYNAMIC SENSITISERS: THE NATURE OF DHE

Abstract Laser flash photolysis has previously been used to study the nature of DHE via measurements of photophysical parameters which are dependent on the molecular weight of the system being studied. These results to date allow only a lower limit to be established for DHE which imply that in some environments such as detergents more than two porphyrin units are linked. We have now determined the triplet extinction coefficient of DHE by the pulse radiolysis technique via an energy transfer method which allows the triplet extinction of DHE to be estimated independent of the molecular weight. The combined techniques allow the actual molecular weight of DHE to be established at about 4200. Laser flash techniques have also now been used to determine, for a number of potential photodynamic sensitisers, the quantum yield of triplet state formation (θ_T) and, using the direct luminescence of singlet oxygen at 1270 nm, the quantum yield of singlet oxygen formation (θ_δ). For many of the porphyrins studied θ_δ is less than θ_T. For DHE itself there is a substantial increase in θ_δ in detergent compared to buffer. The θ_δ yields for a number of related systems including 'simple'systems such as haematoporphyrin, for linked porphyrin-chlorin systems, (including DHE in which the end porphyrin is reduced to a chlorin–DHEC), and for phthalocyanines are compared. For the DHEC the θ_δ is close to that of DHE itself which may imply that such chlorins could be of use in photodynamic therapy (PDT).     

ULTRASTRUCTURAL STUDIES ON THE MECHANISM OF THE PHOTODYNAMIC THERAPY OF TUMORS

Abstract Balb/c mice bearing a transplanted MS-2 fibrosarcoma were injected with 2.5 mg kg ¹ of either tetra(4-sulfonatophenyl/porphine (TPPS) in phosphate-buffered saline or 0.5 mg kg⁻¹ of Zn²⁺-phthalocyanine (Zn-Pc) incorporated into unilamellar liposomes of dipalmitoyl-phosphatidylcholine. Chromatographic studies showed that TPPS is mainly transported in the serum by globulins and albumin, while Zn-Pc is specifically bound by lipoproteins. Exposure of the injected mice to red light (300 J cm⁻²) caused extensive tumor necrosis. The ultrastructural analysis of tumor specimens taken from mice at 15 h after PDT showed that TPPS photoinduces a preferential necrosis of the neoplastic cells, while Zn-Pc causes severe photodamage to both the vascular system and the neoplastic cells. The different modes of tumor photosensitization by TPPS and Zn-Pc are discussed on the basis of the transport mechanism of the two dyes.     

HEMATOPORPHYRIN DERIVATIVE UPTAKE BY ATHEROMA IN ATHEROSCLEROTIC RABBITS: THE SPECTRA OF FLUORESCENCE FROM HEMATOPORPHYRIN DERIVATIVE DEMONSTRATED BY AN EXCIMER DYE LASER

Abstract A new diagnostic and therapeutic endoscopic system consisting of an excimer pulse dye laser is presented. This report demonstrates the accumulation of hematoporphyrin derivative (HpD) in atheroma as shown by the fluorescence of HpD using this equipment. Atheroma was induced in the aorta of WHHL (Watanabe heritable hyperlipidemic) rabbits, 5 mg kg⁻¹ HpD was injected intravenously and the rabbits were sacrificed 24 h later. The aorta was dissected and the localization of HpD was examined. Characteristic peaks of the fluorescence of HpD at 630, 665 and 690 nm wavelength were detected in the atheromatous lesion. However, in the fatty plaque, the emission peak at 630 nm was lower and the 665 nm peak faded away. No fluorescence with peaks was detected in the normal area. The ratio of fluorescence intensity in atheroma, border zones and normal areas was 10.4 : 5.0 : 1.0. On normal rabbits made atherosclerotic by diet and balloon damage, an ultra thin endoscopic catheter was inserted from the descending aorta of atherosclerotic rabbits under anesthesia. Essentially the same data was obtained by these studies in vivo as was obtained in the in vitro studies. The above data suggests the possibility of future applications of this equipment for diagnosis of atheroma.     

Ca2+ INFLUX INDUCED BY PHOTODYNAMIC ACTION IN HUMAN CEREBRAL GLIOMA (U-87 MG) CELLS: POSSIBLE INVOLVEMENT OF A CALCIUM CHANNEL

Abstract The plasma membrane has been implicated as a critical target of photodynamic action on cells. We have observed that the photosensitization of human cerebral glioma (U-87 MG) cells by hematoporphyrin derivative (HpD) causes a large increase in intracellular calcium [Ca²⁺]. This increase in [Ca²⁺]_i was solely due to the influx of extracellular Ca²⁺ through the plasma membrane and showed a dependence on HpD concentration, light dose and concentration of calcium in the extracellular medium. The magnitude of the Ca²⁺ influx decreased with increasing postirradiation time, which suggests that the cell membrane partially recovers from the photodynamic injury. The photoinduced Ca²⁺ influx was inhibited by the Ca²⁺ channel blocker diltiazem and the reducing agent dithioerythritol. These findings are discussed in terms of possible activation of a Ca²⁺ channel as a result of photosensitization.     

PHOTOSENSITIZATION OF MURINE TUMOR, VASCULATURE and SKIN BY 5-AMINOLEVULINIC ACID-INDUCED PORPHYRIN

Abstract— The effects of topical and systemic administration of 5-aminolevulinic acid (ALA) were examined in several murine tumor systems with regard to porphyrin accumulation kinetics in tumor, skin and blood, vascular and tumor cell photosensitization and tumor response after light exposure. Marked, transient increases in porphyrin levels were observed in tumor and skin after systemic and topical ALA. Rapid, transient, dose-dependent porphyrin increases were also observed in blood; these were pronounced after systemic ALA injection and mild after topical application. They were highest within 1 h after ALA injection, thereafter declining rapidly. This matched the clearing kinetics of injected exogenous protoporphyrin IX (PpIX). Initially, vascular photosensitivity changed inversely to blood porphyrin levels, increasing gradually up to 5 h post-ALA, as porphyrin was clearing from the bloodstream. This pattern was again matched by injected, exogenous PpIX. After therapeutic tumor treatment vascular disruption of the tumor bed, while observed, was incomplete, especially at the tumor base. Minimal direct tumor cell kill was found at low photodynamic therapy (PDT) doses (250 mg/kg ALA, 135 J/cm² light). Significant, but limited (<1 log) direct photodynamic tumor cell kill was obtained when the PDT dose was raised to 500 mg/kg systemic ALA, followed 3 h later by 270 J/cm², a dose that was however toxic to the animals. The further reduction of clonogenic tumor cells over 24 h following treatment was moderate and probably limited by the incomplete disruption of the vasculature. Tumor responses were highest when light treatment was carried out at the time of highest tumor porphyrin content rather than at the time of highest vascular photosensitivity. Tumor destruction did not reach the tumor base, regardless of treatment conditions.     

PHOTOINACTIVATION OF CHINESE HAMSTER CELLS BY HEMATOPORPHYRIN DERIVATIVE AND RED LIGHT

Abstract— The use of hematoporphyrin derivative (HpD) has previously been demonstrated to be beneficial in clinical cancer therapy. This paper describes cell culture studies used to examine HpD phototherapy in Chinese hamster ovary cells (line CHO). Survival curves have been obtained for both direct HpD toxicity and HpD induced photoinactivation. Examination of HpD induced photoinactivation as a function of stage in the cell growth cycle has also been performed, as has the quantitative measurement of HpD uptake in cells (using ³H-HpD) as a function of cellular incubation time, serum concentration in the incubation medium, and cell cycle position. In the absence of light, no toxicity was observed for HpD incubation levels of up to 400 μg/m/ when incubations times were 3 h or less. Exposure of cells to light alone (> 590 nm, 4.0 mW/cm²) for 9 min was also found to be completely nontoxic. Survival curves obtained for exponentially growing cells labeled with various concentrations of HpD and subsequently illuminated with red light exhibited a threshold or shoulder region at short exposure times followed by exponential killing at longer exposure times. The cell cycle response curves for HpD induced photoinactivation of synchronized CHO cells was nearly flat, indicating no variation in sensitivity for cells treated at time periods from 6 to 15 h after mitosis. Additon of serum to the incubation medium resulted in improved plating efficiency and reproducible survival curves but decreased cellular uptake of HpD.     

STUDIES ON THE PHOTOPROTECTIVE MECHANISM OF BUTYLATED HYDROXYTOLUENE

Abstract It has been suggested that butylated hydroxytoluene (BHT) ^‡ , a phenolic antioxidant that conveys photoprotective properties to skin, may act by altering epidermal parameters that influence the level of ultraviolet light (UV) reaching critical target sites. The potential diminution of effective UV dose could account for the observed inhibition of UV-induced ornithine decarboxylase (ODC). Thus studies were conducted to examine the possibility that BHT was capable of promoting epidermal proliferation, just as others have shown with lung epithelial cells. UV-induction of ODC, in unrestrained Skh-Hr-I mice irradiated with Westinghouse FS–20 lamps, was shown to be linearly responsive from 0.3 to 0.6, J/cm²/dose. The degree of inhibition afforded by dietary BHT was approximately constant over this dose range. In addition, intraperitoneal administration of BHT (400 mg/kg) inhibited UV-induced ODC as greatly as when provided dietarily. Greatest inhibition occurred when BHT was administered at least 46 h prior to UV exposure, with the inhibitory event(s) requiring over 20 h to become effective. However, BHT had no effect, during this time interval, upon the incorporation of [³H]thymidine or leucine into DNA and protein, respectively. Nor were BHT effects reflected in the epidermal labeling index. These data suggest that BHT does not induce epidermal proliferation. An alternate explanation, in which the antioxidant properties of BHT play a major role, is proposed.     

PHOTOCHEMICAL PROPERTIES OF PORPHYRIN c: AN AGENT FOR USE IN TUMOR PHOTOTHERAPY

Abstract— The absorption and fluorescence properties of porphyrin c (P c ), the porphyrin chromophore present in cytochrome c , have been determined in several solvents and micellar environments. In aqueous buffer solutions at pH 7.5 Pc may exist in both a fluorescent monomeric form with quantum yield of fluorescence, (Φ_f,) ∼ 0.03, and fluorescence lifetime, (τ_f) ∼ 8 ns, and as a non-fluorescent aggregate. The proportion of monomeric form is higher in organic solvents and micelles but is reduced with increasing porphyrin concentrations in aqueous solutions. Porphyrin c readily complexes with Zn²⁺ to produce a fluorescent chelate (Zn-P c ) with Φ_f, ∼ 0.02 and τ_f, ∼ 2 ns at pH 7.5. The yields of singlet excited oxygen formation from Pc and the Zn-P c complex are higher than observed for hematoporphyrin derivative (HpD). Both P c and Zn-P c are effective agents in tumor phototherapy and do not induce the prolonged cutaneous photosensitivity observed with the use of HpD.     

IMPROVING THE EFFICIENCY OF HEMATOPORPHYRIN PHOTOTHERAPY OF OCULAR TUMORS

Malignant melanoma of the choroid is the most common primary intraocular tumor in adult humans. Controversy exists over which is the most effective therapy. One therapeutic modality that has not been thoroughly investigated is hematoporphyrin derivative phototherapy (HPdPRT), a technique used successfully in the clinic on many non-ocular tumors.
The effect of HPd PRT on an ocular, amelanotic melanoma was evaluated using Greene melanoma implanted on pigmented rabbit iris. The parameters used clinically on non-ocular tumors (intravenous 2.5 mg HPd kg^-1 body weight and irradiation at 633 nm 48 h later) were totally ineffective in killing Greene melanoma implanted on the iris. The dose of dye (2.5–5 mg kg^-1), wavelength of light (500-700 nm), and illumination intensity were varied to determine the most efficient parameters for treating this tumor. The most important parameter was dye dose; increasing it to 5 mg kg^-1 resulted in some control of tumor growth. Administering 100% O₂ prior to and during irradiation also improved HPd PRT cytotoxicity. The use of pulsed light (pulses of 1 or 2 min) further enhanced killing and reduced the length of irradiation needed. These studies suggested that HPd PRT might be used efficiently on ocular melanomas.     

PHOTODYNAMIC THERAPY OF B16 PIGMENTED MELANOMA WITH LIPOSOME-DELIVERED Si(IV)-NAPHTHALOCYANINE

The possibility of extending photodynamic therapy to the treatment of highly pigmented neoplastic lesions was tested by using Si(IV)-naphthalocyanine (SiNc) as a tumor-localizing agent. Si(IV)-naphthalocyanine displays intense absorbance at 776 nm (ɛ= 5 × 10⁵ M⁻¹ cm⁻¹), where melanin absorption becomes weaker. As an experimental model we selected B16 pigmented melanoma subcutaneously transplanted to C57BL mice. Upon injection of 0.5 or 1 mg kg⁻¹ of liposome-incorporated SiNc, maximal accumulation of the photosensitizer in the tumor was observed at 24 h with recoveries of 0.35 and 0.57 μg g⁻¹, respectively. However, the tumor targeting by SiNc shows essentially no selectivity, since the photosensitizer concentrations in the skin (peritumoral tissue) were very similar to those found in the tumor at all postinjection times examined by us. Irradiation of SiNc-loaded melanoma with 776 nm light from a diode laser at 24 h postinjection induces tumor necrosis and delay of tumor growth. The effect appears to be of purely photochemical nature at dose rates up to 260 mW cm⁻²; at higher dose rates, thermal effects are likely to become important.     

SUPEROXIDE DISMUTASE AS AN AMPLIFIER OF THE CHEMICAL REACTIVITY OF PORPHYRIN RADICAL-CATIONS

Abstract— Porphyrin radical-cations have been produced using laser flash photolysis via oxidation of the porphyrin triplets by metronidazole. This radical-cation reacts with OJ as shown by its increased half-life in the presence of native superoxide dismutase. Comparable results are obtained when porphyrin radical-cations are formed by Br₂^-O₂^-oxidation of porphyrins produced in pulse radiolysis of oxygen-saturated aqueous solutions containing 20 mM Br^-O^-. These results provide an explanation for the enhancement by superoxide dismutase of the photosensitizing capacity of porphyrins in the presence of electrophilic nitroimidazoles (Bazin and Santus, 1986). They may also apply to porphyrin radical-cations formed by monophotonic or biphotonic photoionization processes.     

THE EFFECT OF CHEMICAL STRUCTURE ON THE PHOTOSENSITIZING EFFICIENCIES OF PORPHYRINS

Abstract— A kinetic investigation was performed on the photooxidation of methionine sensitized by various porphyrins at different oxygen concentrations. The rate of photooxidation was found to be strongly dependent on the nature of the sensitizer. In the case of hematoporphyrin, chelation of Mg²⁺ and Zn²⁺ and especially of Cu²⁺ and Fe²⁺ caused a significant decrease of the photosensitizing efficiency. Fluorescence and/or flash photolysis studies showed that such a decrease is ascribed to an enhancement of the non-radiative decay of the first excited singlet state as well as to a reduction of the triplet lifetime. The sensitizing efficiency is also dependent on the nature of the porphyrin side chains. A reaction mechanism involving ¹O₂ as the oxidizing agent is proposed.     

HIGH DOSE PHOTOIRRADIATION THERAPY OF HUMAN TUMORS USING THE GOLD METAL VAPOR LASER

Abstract The gold metal vapor laser (GMVL; Quentron, Adelaide, South Australia) produces up to 6.0 W of power and this has allowed the study of high dose and high dose rate photoirradiation therapy. We have undertaken 35 treatments in 31 patients with tumors in the rectum (4), esophagus (8), brain (17) and malignant ascites (2). All patients were carefully monitored for complications and, where possible, assessed for response. Dose rates of 1.2-2.2 W were used and doses were estimated to be between 72 and 245 J cm⁻². The major complications noted were in the patients with esophageal cancer and included fever 1-10 days following therapy in 5/8. There was one early death at 10 days and 3/8 patients developed mediastinitis. Of the 4 anorectal tumors treated (8 treatments) there was one bleed 10 days after treatment and no other local complications. One of 2 patients with malignant ascites developed a bowel obstruction immediately after treatment. Of the 17 patients with glioma there was 1 early death at day 10 and no local complications. We conclude that phototherapy of the gastrointestinal tract with the GMVL is tolerable using high dose rates and doses but significant complications occur. Therapy of intra-cranial tumors with similar doses and dose rates is not associated with local complications. The basis for this may be the different anatomy, the content of HpD in different sites, and use of steroids in cerebral tumors, or the better control of hyperthermia in the brain.     

Monte Carlo Model of Stricture Formation in Photodynamic Therapy of Normal Pig Esophagus

Photodynamic therapy (PDT) is FDA-approved for use in patients with Barrett's esophagus using porfimer sodium (2 mg per kg) and a recommended light dose of 130 J cm⁻¹ for high grade dysplasia. Despite uniform drug and light doses, the clinical outcome of PDT is variable. A significant number of PDT cases result in esophageal strictures, a side effect related to excessive energy absorption. The purpose of this project was to model esophageal stricture formation with a Monte Carlo simulation. An original multilayer Monte Carlo computer simulation was developed for esophageal PDT. Optical absorption and scattering coefficients were derived for mucosal and muscle layers of normal porcine esophagus. Porfimer sodium was added to each layer by increasing the absorption coefficient by the appropriate amount. A threshold-absorbed light dose was assumed to be required for stricture formation and ablation. The simulation predicted irreversible damage to the mucosa with a 160 J cm⁻¹ light dose and damage to the muscle layer with an additional 160 J cm⁻¹ light dose for a tissue porfimer sodium content of 3.5 mg kg⁻¹. The simulation accurately modeled photodynamic stricture formation in normal pig in vivo esophageal tissue. This preliminary work suggests that the absorbed light threshold for stricture formation may be between 2 and 4 J per gram of tissue.