THE EFFECTS OF ASPIRIN ON MICROVASCULATURE AFTER PHOTODYNAMIC THERAPY

Abstract— The effects of aspirin (acetylsalicylic acid: ASA) on vessel behavior and tumor response were measured during and after photodynamic therapy (PDT). Changes to vessel constriction, macromolecular leakage, tumor interstitial pressure, and tumor response were examined. Animals were randomly placed into treatment groups and injected with 0–25 mg/kg Photofrin® and given 0 or 135 J/cm² light treatment. The light treatment was standardized to 75 mW/cm² at 630 nm over a 30 min treatment interval (135 J/cm²). The treatment groups were further subdivided to receive Photofrin® alone or Photofrin® plus 100 mg/kg ASA. A cremaster muscle model in Sprague-Dawley rats was used to directly observe microvascular response and changes in vessel permeability to macromolecules. A tumor interstitial pressure model was designed to measure pressure changes in a chondrosarcoma tumor over time. This model indirectly measures macromolecular leakage, among other factors, in the tumor tissue. Groups of 10–20 rats were implanted subcutaneously with chondrosarcoma and were subjected to PDT to assess tumor response to the various treatments. Statistically significant differences in vessel leakage and changes in interstitial pressure were observed between animals given ASA plus PDT as compared to animals given PDT alone. The administration of ASA significantly inhibited venule leakage of albumin and reduced increases in interstitial pressure after treatment. The use of ASA had no effect on vessel constriction or tumor response after PDT. These findings suggest that the increases in vessel permeability observed during and after PDT, using Photofrin®, do not significantly contribute to tumor response.     

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.     

PHOTODYNAMIC THERAPY: EFFECT ON THE ENDOTHELIAL CELL OF THE RAT AORTA

Previous studies in our laboratory have demonstrated that photodynamic therapy (PDT) of experimental bladder tumors leads to rapid destruction of the endothelial lining within the tumor microvasculature. Endothelial cell death during PDT may be a consequence of direct cell injury resulting from retention of photosensitizer within the endothelial cell or, alternatively, result from intravascular activation of circulating photosensitizer with subsequent indirect endothelial damage. In the experiments described here, we investigated the possibility that photosensitizer retained within the endothelial cell was sufficient to cause endothelial cell injury in the absence of circulating drug. The experimental model was rat aorta photosensitized in vivo via the intravenous injection of tin(II) etiopurpurin dichloride (SnET2), and subsequent in situ or in vitro (in explant culture) light (670 nm) treatment from an argon pumped dye laser. Damage to the lining of the aorta was assessed morphometrically by determining the areal density of silver stained endothelial cells. Results indicate that purpurin SnET2-PDT directly damages the endothelial lining.     

IMPROVED SURVIVAL FROM INTRACAVITARY PHOTODYNAMIC THERAPY OF RAT GLIOMA

The effectiveness of intratumoral photoradiation in photodynamic therapy (PDT) using a polyporphyrin photosensitizer was studied in the RT-2 rat glioma model. One week after intracerebral implantation of RT-2 cells, experimental rats received a single i.p. injection of 2 mg/kg of Photofrin. After administration of the photosensitizer (48 h), the tumors were partially resected and the exposed cavity was irradiated with 15 J of laser light at a wavelength of 630 nm. Further treatment with a large craniectomy significantly enhanced rat survival. Control rats which received no photosensitizer but were treated with surgery, alone or in combination with laser irradiation, succumbed from early tumor recurrence. Photodynamic therapy without decompressive surgery resulted in hemorrhagic infarction of residual tumor and adjacent brain with focal cerebral edema which resulted in cerebral herniation and early death. Our results indicate that photodynamic therapy is effective in treating residual brain tumor but at the expense of brain tissue surrounding the tumor. Unless relieved, intracranial pressure from photodynamic therapy-associated cerebral edema in this animal model resulted in shortened survival.     

THE EVOLUTION OF PHOTODYNAMIC THERAPY TECHNIQUES IN THE TREATMENT OF INTRAOCULAR TUMORS

Abstract The techniques of photodynamic therapy (PDT) and the indications for its use in the treatment of intraocular tumors have evolved during the years in which it has been assessed in patients at our institution. It is now clear that transcorneal PDT delivered at a subthermal dose-rate to the surface of a pigmented lesion such as choroidal melanoma has little effect. In the absence of pigment, however, as in the case of retinoblastoma or amelanotic melanoma of the iris or choroid, the tumor kill attributed to PDT alone is significant. Data from animal tumor models in our institution and from patient studies elsewhere suggest that the addition of heat with the light delivery will predictably improve the outcome of the treatment of pigmented lesions. Ocular PDT delivered in conjunction with heat will be useful clinically as an adjunct to scleral plaque therapy by reducing the height of a lesion and concurrently the dose of radiation necessary at the base of the tumor for sterilization. Since the clinical tumoricidal effect of PDT is now known to be due at least in part to vascular damage, trans-scleral application of light to the base of melanomas and occlusion of its blood supply holds significant promise of efficacy with continued improvement of the light delivery system. Finally, a transpupillary approach to occlusion of the choroidal vascular supply to a melanoma by surrounding the tumor with photodynamic lesions may provide the best approach for ocular PDT as a primary therapy.     

NORMAL BRAIN TISSUE RESPONSE TO PHOTODYNAMIC THERAPY USING ALUMINUM PHTHALOCYANINE TETRASULFONATE IN THE RAT

Abstract— The effects of photodynamic therapy (PDT) on normal brain tissue and depth of brain necrosis were evaluated in rats receiving 2.5 mg/kg aluminum phthalocyanine tetrasulfonate. Twenty-four hours later brains were irradiated with 675 nm light at a power density of 50 mW/cm² and energy doses ranging from 1.6 to 121.5 J/cm². Brains were removed 24 h after PDT and evaluated microscopically. When present, brain lesions consisted of well-demarcated areas of coagulation necrosis. When plotting the depth of necrosis against the natural log of energy dose, the data fit a piecewise linear model, with a changepoint at 54.6 J/cm² and an x intercept of 7.85 J/cm². The slopes before and after the changepoint were 2.04 and 0.21 mm/In J cm^-2, respectively. The x intercept suggests a minimum light dose below which necrosis of normal brain will not occur, whereas the changepoint indicates the energy density corresponding to an approximate maximum depth of necrosis.     

CLINICAL LASER PHOTODYNAMIC THERAPY IN THE TREATMENT OF BLADDER CARCINOMA

Abstract The treatment of bladder carcinoma using dihematoporphyrin ether (DHE) and laser photodynamic therapy (PDT) is described herein. Patients selected for this study have cytology- and biopsy-proven transitional cell carcinoma, no histologic evidence of muscle invasion, and negative excretory urograms. Sixteen patients have been treated, with follow-up from 6 to 36 months. Eleven have had a complete response, and 3 a partial response in that they required re-treatment for recurrence. Two of these patients have not recurred at this time. One of the patients who recurred had tumor extension into the prostatic urethra and has been successfully re-treated (disease-free at 6 months). There was one treatment failure and 1 patient lost to follow-up. Photosensitivity for up to 4 weeks is a known side-effect, but unexpected morbidity included a transient but significant increase in urinary frequency, urgency, and occasionally hematuria which spontaneously resolved within 3-4 weeks. Careful placement of the fiberoptic tip in the centre of the bladder, bladder distension during treatment with saline rather than water, the instillation of the minimum volume required to “smooth out” the mucosa for complete bladder photoradiation, and delivered energy of 25 J cm’or less may have prevented the more severe complications (i.e. bladder shrinkage) reported by Dougherty and Nseyo (personal communication). We also feel that there is some early evidence that a heightened immune response (similar to intravesical BCG) may potentially play some role in explaining the efficacy of PDT in long disease-free intervals, although this is just a histologic observation at present. It appears the PDT offers another practical treatment modality for non-invasive transitional cell carcinoma in patients refractory to standard surgical and chemotherapeutic regimens, and has been addressed by numerous other investigators such as Benson (1985) and Hisazumi (1983). We are presently recommending to our patients in these categories to undergo a course of PDT prior to relinquishing to cystectomy.     

TRANSPORT OF LIGHT IN TISSUE IN PHOTODYNAMIC THERAPY

Abstract The dose rate in photodynamic therapy is proportional to the energy fluence rate and the concentration of the photosensitizer. Calculations of the energy fluence rate have been performed in slab, cylindrical and spherical geometries with the discrete ordinates transport method and diffusion theory. The attentuation of the energy fluence rate is least in slab geometry and greatest in spherical geometry. Violet (405 nm) light is attenuated much more rapidly than red (630 nm) light. Small tissue dimensions or narrow beam irradiation further decrease the energy fluence rate with radius and depth. Anisotropic scattering increases the energy fluence rate at large depths, but decreases it near the source. Measurements of the absolute energy fluence rate vs depth in a mouse tumor model exhibit an order of magnitude attenuation through the skin and a 3 mm thick tumor. Calculations of the energy fluence rate of the DHE fluorescence have been carried out to guide measurement of the concentration. Violet light excitation is much more efficient than red light excitation.     

CRITICAL IMPORTANCE OF THE TRIPLET LIFETIME OF PHOTOSENSITIZER IN PHOTODYNAMIC THERAPY OF TUMOR

The relation between the lifetimes of the triplet states of various porphyrins and their photosensitizing effects on the photodynamic therapy (PDT) of tumor has been examined. Diethylene-triamine pentaacetic acid ester of 4-[1-(2-hydroxy-ethyloxy)ethyl]-2-vinyl deuteroporphyrin-IX gallium (III) complex (Ga-DP), zinc (II) complex (Zn-DP), and manganese (III) complex (Mn-DP) and Photofrin II (PII) are used as the photosensitizer. The triplet lifetimes have been measured for the samples adsorbed on filter paper (FP) and found to be 57 ms (Ga-DP), 26 ms (Zn-DP), less than or equal to 10 microseconds (Mn-DP) and 9 ms (PII). The phosphorescence of Ga-DP in tumor-bearing golden hamsters are measured both in tumor tissue and in liver. They show bi-exponential decay with the lifetimes of about 5 and 20 ms. From the values, the generation rate, kct[3O2], of singlet molecular oxygen in living animal tissue may be estimated to be an order of 10(2) s-1. The PDT effects have been quantitatively investigated for in vitro experiments; upon irradiation the growth inhibitions of mouse p388 leukemia cells are obtained as a function of concentration of Ga-DP, Zn-DP, Mn-DP and PII. The experimental results indicate that the PDT effects depend essentially on the triplet lifetimes of the photosensitizers.     

EFFICACY OF PHOTODYNAMIC THERAPY ON ORIGINAL AND RECURRENT RAT MAMMARY TUMORS

Abstract— Photodynamic therapy has demonstrated efficacy toward primary, metastatic and recurrent human tumors. Here, we investigated the ability of photodynamic therapy, using Photofrin, to inhibit growth of R3230AC mammary adenocarcinomas when tumors were treated as original implants and again as lesions recurring at the initial treatment site. The results demonstrate that both initial implants and lesions recurring after the first photodynamic treatment respond similarly to the same photodynamic therapy protocol, with mean tumor volume doubling times of ˜ 11 days in both cases. Cells cultured from original tumor implants or tumors that recurred after photodynamic treatment accumulate equivalent amounts of [¹⁴C]polyhematoporphyrin. Single cell suspensions prepared from either original or recurrent tumors from animals administered 5 mg/kg Photofrin and exposed to light in vitro displayed comparable phototoxicity. Additionally, examination of tumors by light microscopy revealed no morphological differences between the original tumor implants and the recurrent lesions. Taken together, these data indicate that lesions which recurred at the site of the initial photodynamic treatment were not resistant to a second identical course of photodynamic therapy.     

THE PREPARATION OF RADIOLABELED PORPHYRINS and THEIR USE IN STUDIES OF PHOTODYNAMIC THERAPY

Abstract— We tested water-soluble sulfonated phthalocyanine and three metal chelate derivatives for their tumoricidal effect on the EMT-6 mammary tumor in mice exposed to red light. The metal-free sulfophthalocyanine had little effect, whereas the aluminum complex and the lower sulfonated fraction of the gallium complex exhibited tumoricidal activity similar to hematoporphyrin-based photosensitizer (Photofrin II). The higher sulfonated fractions of the gallium complex were less active as compared to the lower sulfonated fraction. The cerium complex was the most active sensitizer in terms of dye and light doses required to induce tumor necrosis and cure but also showed the highest phototoxicity towards healthy skin. These results suggest that sulfonated phthalocyanines will offer a new alternative in photodynamic therapy of light-accessible neoplasms.     

PHOTOBLEACHING OF PORPHYRINS USED IN PHOTODYNAMIC THERAPY AND IMPLICATIONS FOR THERAPY

Abstract— The development of an extraction procedure to quantitate dihematoporphyrin ether (DHE) concentration in tissues correlated to fluorescence measurements from instrumentation developed for in vivo fluorimetry was examined. In vivo fluorometric results from mouse mammary carcinoma (SMT-F) were calibrated against results of the chemical extraction assay quantitated spectrophotometrically. Fluorescence and drug extractable levels increase in a linear fashion with injected dose. Loss of porphyrin fluorescence (photobleaching) and intra-tumoral porphyrin level has been demonstrated both in vitro (NHIK cells) and in vivo (SMT-F tumor) during illumination with light following exposure to Hpd or DHE. This process is essentially independent of porphyrin tumor level in vivo and could lead to tumor protection at very low porphyrin levels. On the other hand, this photobleaching process which occurs concurrent with cellular inactivation and tissue damage due to the photodynamic process can be exploited to protect normal tissue during photodynamic therapy (PDT) and thus greatly enhance the therapeutic ratio. This has been demonstrated in patients undergoing PDT.     

TUMOR DESTRUCTION IN PHOTODYNAMIC THERAPY

Abstract The effects of photodynamic therapy (PDT) on the tumor microvasculature in the first few hours after treatment was studied at the light microscope (LM) and electron microscope (EM) levels in DBA/2Ha mice bearing SMT-F tumors. Animals received intraperitoneal injections of 10 mg kg⁻ of Photofrin II and 24 h later tumors were treated with 100 J cm⁻² of light (630 nm). Animals were sacrificed and their tumors removed at time 0, 30 min, 1, 2, 4, 8, 16 and 24 h after treatment. The results indicate that the effects of PDT are initially direct destruction of the microfibrils in the subendothelial zone of the tumor capillaries with subsequent tumor cell death secondary to hemorrhage and vascular collapse.     

ADRIAMYCIN RESISTANCE IN CHINESE HAMSTER FIBROBLASTS FOLLOWING OXIDATIVE STRESS INDUCED BY PHOTODYNAMIC THERAPY

Photodynamic therapy (PDT) generates reactive oxygen species that are responsible for the initial cytotoxic events produced by this treatment. An extended (16 h) porphyrin incubation prior to light irradiation increased expression of the 75, 78 and 94 kDa glucose-regulated stress proteins (GRP), as well as the cognate form of the 70 kDa heat shock protein. However, these stress proteins were not induced following isoeffective PDT doses using a short (1 h) porphyrin incubation protocol. In the current study, Chinese hamster fibroblasts were used to examine sensitivity to adjunctive PDT and adriamycin as previous reports indicate a correlation between stress protein synthesis and a decrease in adriamycin cytotoxicity. Treatments that either induced GRP ( i.e . PDT with an extended porphyrin incubation or exposure to the calcium ionophore A23187) or did not induce GRP ( i.e . PDT with a short porphyrin incubation or UV irradiation) were followed at increasing time intervals with a 1 h adriamycin incubation. A time-dependent decrease in adriamycin cytotoxicity was observed when cells were first exposed to either of the PDT protocols or to A23187. Alterations in intracellular drug levels did not account for the change in adriamycin sensitivity. Likewise, intracellular glutathione concentrations and antioxidant enzyme activities were not significantly altered following PDT or A23187. Parameters associated with altered adriamycin sensitivity included a decrease in the percentage of S phase cells following PDT and A23187 as well as a depletion of intracellular ATP after PDT using the extended porphyrin incubation. These results demonstrate that PDT can be added to the growing list of diverse stresses producing transient resistance to adriamycin and that stress protein induction is not universally associated with all oxidative treatments inducing this resistance.     

PHOTODYNAMIC ALTERATIONS OF THE CELL ENVELOPE OF PROTEUS MIRABILIS AND THEIR REPAIR

Abstract— Photodynamic treatment of Proteus mirabilis gives rise to changes in the cell envelope as can be detected by the behaviour of cells during lysis: lysis of cells proceeds more readily than that of untreated cells, measured by the liberation of labelled DNA. After photodynamic treatment the cell envelope is restored to the control levels. This repair process takes place within 5 min in broth as well as in buffer solutions.     

PHOTODYNAMIC THERAPY DOSIMETRY IN POSTMORTEM AND in vivo RAT TUMORS AND AN OPTICAL PHANTOM

Dosimetry in photodynamic therapy as currently practiced is empirical in that it does not account for optical properties of the target lesion. However, since light attenuation in tissue is unpredictable, measurements of optical properties are needed to ensure optimal light dose delivery. Further improvements in the uniformity of light dose distribution in tumors can be afforded by implanting multiple light sources. A technique is described in which the use of multiple cylindrical sources was combined with measurements of light energy fluence rate in the tumor. Six sources were placed within translucent plastic needles, which were inserted into tumors in a parallel array. Tumor attenuation characteristics were measured by placing a miniature light detector in one needle, while illuminating a cylindrical source in another, nearby, needle. This process was repeated for different needle pairs. In one postmortem and two in vivo tumors the absorption coefficient, transport scattering coefficient and penetration depth ranged from 0.56–0.81 cm ¹, 9.4–15.2 cm ¹ and 1.7–2.3 mm. respectively. Apparent penetration depths for in vivo tumors changed with time, during experiments. Predictions of dosimetry were generally consistent with direct measurements of light in tumors. Somewhat better agreement was observed in an optical phantom.     

SYNTHESIS OF POSITIVELY CHARGED PHTHALOCYANINES and THEIR ACTIVITY IN THE PHOTODYNAMIC THERAPY OF CANCER CELLS

Positively charged zinc containing or metal free phthalocyanines 6a-c and 7a-c were prepared via a three step procedure starting from 4-nitrophthalonitrile. The phthalocyanines contain alkyl chains of different length in order to influence the hydrophilic vs lipophilic character of the compounds. The partition between a hydrophilic (water) and lipophilic (octanol-1) phase was determined, and the photoredox activities were investigated. Initial results on the photodynamic activity of these compounds were compared with those of Dougherty's Photofrin II on different malignant and non-malignant cell lines (XP 29MAmal, CX1, HeLa, S180 and NO17). Positively charged phthalocyanines in vitro showed a higher photodynamic activity than Photofrin II.     

EFFECTS OF LIGHT BEAM SIZE ON FLUENCE DISTRIBUTION AND DEPTH OF NECROSIS IN SUPERFICIALLY APPLIED PHOTODYNAMIC THERAPY OF NORMAL RAT BRAIN

The light fluence distributions of 632.8 nm light incident on the exposed surface of normal rat brain in vivo have been measured using an interstitial, stereotactically-mounted optical fiber detector with isotropic response. The dependence of the relative fluence rate on depth and the spatial distribution of fluence were compared for incident beam diameters of 3 and 5 mm. The fluence rate at depth of 1-6 mm along the optical axis within the brain tissue was approximately 70% greater for a 5 mm diameter beam than for a 3 mm beam, at the same incident fluence rate, although the plots of the relative fluence rate vs depth were parallel over the depth range 1-6 mm. The depths of necrosis resulting from photodynamic treatment of brain tissue using the photosensitizer Photofrin and irradiation by 632 nm light with 3 and 5 mm incident beams were also measured. The observed difference in necrosis depths was consistent with the measured difference in fluence. The importance of beam size in photodynamic treatment with small diameter incident light fields is discussed.     

OCTA-ALKYL ZINC PHTHALOCYANINES: POTENTIAL PHOTOSENSITIZERS FOR USE IN THE PHOTODYNAMIC THERAPY OF CANCER

The synthesis, characterization and electronic spectra of a series of nine 1,4,8,11,15,18,22,25-octa-alkyl zinc phthalocyanines (ZnPc), potential photosensitizers for the photodynamic therapy of cancer, are described. The substituents on the phthalocyanine (Pc) macrocycle “red-shift” the absorbance maximum, in cyclohexane, of all nine members of this series to a value of 703 ± 2 nm, with a corrected fluorescence emission maximum for the octadecyl derivative of 715 nm. The solubilities and degree of aggregation of six examples in cyclohexane have been measured. The highest homologue, the octadecyl derivative, remains essentially unaggregated to a concentration of 1.5 ± 10^?4 mol dm^?3. The photostability of this Pc has been examined and the compound shown to be sensitive to photooxidation processes, which lead to its decomposition to 3,6-fcw-decylphthalimide. Known singlet oxygen quenchers inhibit the photodecomposition. In a comparative study, the octadecyl ZnPc underwent a more rapid photodecomposition than the corresponding metal-free derivative.