The effects of photodynamic therapy on human neutrophil migration using bacteriochlorin a

Bacteriochlorin a photodynamic therapy (BCA-PDT) caused inhibition of interleukin (IL)-8-activated neutrophil migration, at concentrations that did not induce membrane damage. Random migration and migration induced by other chemoattractants were also inhibited, indicating that the effect of BCA-PDT was not at the level of chemoattractant-receptor interaction but down stream. The BCA-PDT completely blocked superoxide production of phorbol ester-stimulated neutrophils indicating that superoxide production by neutrophils present in the tumor before and during BCA-PDT is not the cause of inactivation of tumor cells. Both type I and type II quenchers prevented inhibition by BCA-PDT but only in electroporated cells. Confocal laser scanning microscopy showed that the fluorescence of BCA was located inside the cell. These results show that the effects of BCA-PDT are intracellular and of a mixed type I/type II character and that the neutrophils present in the tumor during illumination probably do not contribute to tumor eradication by releasing reactive oxygen species.     

Historical development of photodynamic therapy

Photodynamic therapy is based on the accumulation of photosensitizing drugs in tumours and subsequent activation by visible light, leading to the release of singlet oxygen in photochemical reactions. Besides the treatment of precancerous lesions and malignant tumours in superficial sites, new experimental indications, such as psoriasis, are being investigated. The development of new photosensitizing agents for topical application and appropriate light sources has led to increasing interest in this promising treatment modality among dermatologists. This historical review deals with the scientific investigations of photodynamic therapy and diagnosis that started with the experiments of Oscar Raab at the end of the nineteenth century.     

Advances in ophthalmological photodynamic therapy

Photodynamic therapy is a new experimental therapeutic technique which is attracting increasing attention. Its biopharmacological basis of action is the specific interaction of a photosensitizing compound with the cellular elements of pathological lesions. The photosensitizer is thought to enter specifically into the pathologic cells, where it accumulates. The lesion is then irradiated with a sensitizing laser-beam of specific wave-length to activate the photosensitizer, which then becomes a generator of free oxygen radicals. These radicals destroy the sensitizer-harboring pathological cells. The advantage of specifically destroying pathological lesions without affecting surrounding normal tissue is obvious. Recently, many experimental studies have been conducted to test the usefulness of photodynamic therapy for ocular disorders, mainly advanced age-related macular degeneration and uveal melanoma. Results so far are encouraging.     

Induction of tumor immunity by photodynamic therapy

The mechanism of tumor destruction by photodynamic therapy (PDT) incorporates a variety of events leading to inactivation of tumor cells. The unique feature of PDT is the mobilization of the host to participate in the eradication of treated cancer. A critical element is the induced inflammation at the treated site associated with massive invasion of activated myeloid cells. In addition to further destruction of cancer cells, conditions are created for the presentation of tumor antigens with subsequent activation of lymphoid cells, leading to tumor-specific immunity. This inflammation-primed immune development process results in generation of tumor-specific immune memory cells that appear to be elicited against both strongly and poorly immunogenic PDT-treated cancers. Once generated by PDT, it is conceivable that these immune cells (especially if further expanded and activated by adjuvant immunotherapy) can be engaged in additional eradication of disseminated and/or metastatic lesions of the same cancer. A number of immunotherapy regimens have already been proven effective in enhancing the curative effect of PDT with various animal tumor models. Inflamed cancerous tissue at the PDT-treated site appears to exert powerful attracting signals for immune cells activated by different immunotherapy regimens.     

Reduction of tumour oxygenation during and after photodynamic therapy in vivo: effects of fluence rate

It has been proposed that the generation of O2 during photodynamic therapy (PDT) may lead to photochemical depletion of ambient tumour oxygen, thus causing acute hypoxia and limiting treatment effectiveness. We have studied the effects of fluence rate on pO2, in the murine RIF tumour during and after PDT using 5 mg kg(-1) Photofrin and fluence rates of 30, 75 or 150 mW cm(-2). Median pO2 before PDT ranged from 2.9 to 5.2 mmHg in three treatment groups. Within the first minute of illumination, median tumour pO2 decreased with all fluence rates to values between 0.7 and 1.1 mmHg. These effects were rapidly and completely reversible if illumination was interrupted. During prolonged illumination (20-50 J cm(-2)) pO2 recovered at the 30 mW cm(-2) fluence rate to a median value of 7.4 mmHg, but remained low at the 150 mW cm(-2) fluence rate (median pO2 1.7 mmHg). Fluence rate effects were not found after PDT, and at both 30 and 150 mW cm(-2) median tumour pO2 fell from control levels to 1.0-1.8 mmHg within 1-3 h after treatment conclusion. PDT with 100 J cm(-2) at 30 mW cm(-2) caused significantly (P = 0.0004) longer median tumour regrowth times than PDT at 150 mW cm(-2), indicating that lower fluence rate can improve PDT response. Vascular perfusion studies uncovered significant fluence rate-dependent differences in the responses of the normal and tumour vasculature. These data establish a direct relationship between tumour pO2, the fluence rate applied during PDT and treatment outcome. The findings are of immediate clinical relevance.     

Hydroxyphthalocyanines as potential photodynamic agents for cancer therapy

A series of benzyl-substituted phthalonitriles, substituted at the 3-, 4-, and 4,5-positions, underwent varied condensations with phthalonitrile to give a series of protected (monohydroxy- and polyhydroxyphthalocyaninato)zinc(II) derivatives which were readily cleaved to give several hydroxyphthalocyanines (ZnPc) (phthalocyanine phenol analogues). Their efficacy as sensitizers for the photodynamic therapy (PDT) of cancer was evaluated on the EMT-6 mammary tumor cell line. In vitro, the 2-hydroxy ZnPc (32) was the most active, followed by the 2,3- and 2,9-dihydroxy ZnPc (39 and 45), with the 2,9,16-trihydroxy ZnPc (33) exhibiting the least activity. In vivo, the monohydroxy derivative 32 and the 2,3-dihydroxy derivative 39 were both efficient in inducing tumor necrosis at 1 micromol kg-1, but complete tumor regression was poor, even at 2 micromol/kg. In contrast, the 2,9-dihydroxy isomer 45, at 2 micromol kg-1, induced tumor necrosis in all animals treated, with 75% complete regression. These results underline the importance of the position of the substituents on the Pc macrocycle to optimize tumor response and confirm the PDT potential of the unsymmetrical Pcs bearing functional groups on adjacent benzene rings.     

An inexpensive light source for oncologic photodynamic therapy

The advantages obtained in vaginal surgery and caesarean section using spinal anesthesia led us to test this anesthesia to verify feasibility, problems and advantages in abdominal surgery. Spinal anesthesia was performed in 60 patients between 21 and 87 years of age. Thirty-seven total abdominal hysterectomies with or without adnexectomy, 5 laparotomic miomectomies, 3 adnexectomies, 5 colposacropexies, 2 hysterectomies with lymphadenectomy, 7 Burch colposuspension with or without hysterectomy and 1 laparoscopy for sterilization were performed. No significant problems during surgery and the postoperative period were observed. Resumption of the different physiologic functions were more rapid, hospital stay shorter and compliance greater than with general anesthesia.     

Two years'' experience with photodynamic therapy of bladder carcinoma

Emergent cardiac catheterization was performed on a 70-year-old female patient who was admitted for further evaluation of acute myocardial infarction. Coronary angiography didn't reveal any significant stenotic lesion, but levogram showed extensively abnormal contractility around the center of the apex region. On the second hospital day, 99mTc-PYP/201TlCl dual SPECT gave findings similar to those found in acute myocardial infarction, but myocardium--released enzyme stayed within the normal range. Two weeks after, 201TlCl myocardial scintigraphy showed disappearance of the perfusion defect, and normal contractility was observed on the levogram of the chronic phase. Since this case was clinically denied to be myocardial infarction, it was considered a typical case of stunned myocardium which showed prolonged left ventricular abnormal contractility with transient myocardial ischemia. This is a case suggestive for estimations of myocardial reversibility in patients with myocardial perfusion and metabolic disorder in dual SPECT.     

Cooperative effects of photodynamic treatment of cells in microcolonies

Microcolonies of 2-8 Madison-Darby canine kidney cells (MDCK II) and Chinese hamster lung fibroblasts (V79) cells were incubated with the photosensitizer Photofrin and exposed to light, and the resulting number of dead cells per colony was determined. The distribution of this number was found to be incompatible with the assumption that cells are inactivated independently. The experimental distributions were significantly different from the binomial distribution expected from this assumption, but in accordance with a model in which an inactivated cell can inactivate adjacent cells with a certain probability. These findings are contrary to the common view that damage caused by radiation is limited to the cell in which the primary damage takes place. Our findings clearly indicate some kind of cooperativity between cells treated with Photofrin and light.     

Light distribution by linear diffusing sources for photodynamic therapy

The distribution of the light emitted by linear light diffusers commonly employed in photodynamic therapy (PDT) has been investigated. A device is presented which measures the angular distribution of the exiting light at each point of the diffuser. With these data the fluence rate in air or in a cavity at some distance from the diffuser can be predicted. The results show that the light is scattered from the diffuser predominantly in the forward direction. Experiments and calculations show that the fluence rate in air and in a cavity of scattering tissue at some distance from the diffuser has a maximum near the tip of the diffuser, instead of near the middle. However, the fluence rate resulting from an interstitial diffuser in a purely scattering tissue phantom shows a maximum in the bisecting plane of the diffuser as would be predicted when the diffuser emitted light isotropically. The scattering nature of the tissue is expected to cancel the anisotropy of the diffuser.     

Potentiation of the 5-aminolevulinic acid-based photodynamic therapy with cyclophosphamide

We have investigated the efficacy of the Photodynamic Therapy (PDT) from 5-aminolevulinic acid (ALA) in combination with an antineoplastic agent using an in vitro-in vivo model developed in our laboratory. The alkylant cyclophosphamide (CY) was chosen because there is evidence of the porphyrinogenic properties of this drug. Male BALB/c mice bearing a transplantable mammary adenoarcinoma were given two doses of 35 mg de CY/kg wt. i.p. and 9 mg/kg wt intratumorally. At 16, 22 and 40 hrs after the last injection of CY the animals were sacrificed and explants of 2 mg of tumor were incubated 2 hrs in a medium containing 0.6 mM ALA; and then irradiated with a He-Ne laser. Innocula of 1 mm3 of irradiated and non-irradiated tissue were then injected subcutaneously under the right and left flanks of a normal mouse, respectively. The efficacy of the treatment was determined following the growth of the tumor from day 10 after tumor implantation. Under the present conditions a 30% increased efficacy was observed in the case of the explants treated with CY 40 hrs after the last i.p. injection. Porphyrins in the liver and tumor and other tissues of the injected mice were also determined; except for a slight increase in tumor and liver, 40 and 22 hrs after CY i.p. injection respectively, no other changes were observed in any tissue, as compared with not CY treated mice. These results indicate that future treatment, combining the tumor localizing properties of endogenously formed porphyrins from ALA and antineoplasic drugs such as cyclophosphamide, should be encouraged.     

Improvements in the design of linear diffusers for photodynamic therapy

The angular radiance distribution of several linear diffusers used for photodynamic therapy (PDT) was measured. The forward scattering found previously was not observed for these designs. The improved isotropy leads to a better agreement between intended treatment site and actual maximum of the fluence rate profile when the linear diffuser is used in a hollow, cylindrical organ.     

Feasibility of photodynamic therapy using endogenous photosensitization for colon cancer

A novel approach to photodynamic therapy (PDT) involves endogenous photosensitization by the oral administration of delta-aminolevulinic acid (ALA), a naturally occurring substance that is the precursor of protoporphyrin IX (PpIX). A 60-year-old man with adenocarcinoma of the sigmoid colon received ALA, 60 mg/kg by mouth. Six hours later, when the plasma level of PpIX had peaked, the tumor was exposed locally to red light at 633 nm to activate PpIX. Endoscopy and biopsy findings subsequent to this treatment showed unequivocal visible changes and necrosis. Six months later, the patient again underwent successful treatment without adverse effects. This report suggests a role for PDT using endogenous photosensitization in certain circumstances involving adenocarcinoma of the large intestine.     

Meso-tetraphenylporphyrin dimer derivative as a potential photosensitizer in photodynamic therapy

Studies on the synthesis, preliminary in vivo biological activity, singlet oxygen and fluorescence yields of a dimeric porphyrin (D1) are described. The pharmacokinetic behavior and photodynamic properties of the dimer D1 were examined in Balb/c mice bearing an MS-2 fibrosarcoma. Compound D1 shows a high selectivity for tumor localization (tumor/peritumoral tissue ratios of dye concentration ranging between ca 100 and 140 at 24 h after drug administration of 5.0-1.0 mg kg-1 into DL-alpha-dipalmitoylphosphatidylcholine liposomes). The phototherapeutic efficiency of dimer D1 was evaluated by following the growth curves of fibrosarcoma irradiated with red light (600-700 nm) with a total dose of 400 J cm-2, at 24 h after intravenous injection. Photodynamic therapy-treated tumors showed a significant delay in growth as compared to untreated control mice. The results obtained suggest that the porphyrin dimer D1 may be a promising candidate for further use in PDT experiments.     

Regeneration processes in rabbit endometrium: a photodynamic therapy model

The origin and process of regeneration in rabbit endometrium was evaluated following photodynamic epithelial destruction using topically applied aminolevulinic acid (ALA). Selective destruction of endometrial epithelium was performed using photodynamic therapy (PDT). ALA was diluted to 200 mg/ml dextran 70 shortly prior to administration. A volume of 1.2 ml was injected into the left uterus. Intrauterine illumination (wavelength 630 nm, light dose 40-80 J/cm2) was performed 3 h after drug administration. Tissue morphology was evaluated by light and scanning electron microscopy 1, 3, 7 and 28 days post-treatment (three animals at each time-point). Regeneration of the endometrium following epithelial ablation by PDT was fully activated after 24 h and was completed after 72 h. Endometrial surface generation occurred by proliferation, originating primarily in deeper regions of the glands. Findings from our morphological follow-up study support the origin of endometrial regeneration being mainly from undifferentiated stem cells and residual glandular epithelium.