Monitoring in situ dosimetry and protoporphyrin IX fluorescence photobleaching in the normal rat esophagus during 5-aminolevulinic acid photodynamic therapy

Experimental therapies for Barrett's esophagus, such as 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT), aim to ablate the premalignant Barrett's epithelium. However, the reproducibility of the effects should be improved to optimize treatment. Accurate irradiation with light of a proper wavelength (633 nm), fluence and fluence rate has shown to be critical for successful ALA-PDT. Here, we have used in situ light dosimetry to adjust the fluence rate measured within the esophagus for individual animals and monitored protoporphyrin IX (PpIX) fluorescence photobleaching simultaneously. Rats were administered 200 mg kg-1 ALA (n = 14) or served as control (n = 7). Animals were irradiated with an in situ measured fluence rate of 75 mW cm-2 and a fluence of 54 J cm-2. However, this more accurate method of light dosimetry did not decrease the variation in tissue response. Large differences were also observed in the dynamics of PpIX fluorescence photobleaching in animals that received the same measured illumination parameters. We found that higher PpIX fluorescence photobleaching rates corresponded with more epithelial damage, whereas lower rates corresponded with no response. A two-phased decay in PpIX fluorescence could be identified in the response group, with a rapid initial phase followed by a slower rate of photobleaching. Non-responders did not show the rapid initial decay and had a significantly lower rate of photobleaching during the second phase of the decay (P = 0.012).     

Intraperitoneal photodynamic therapy in the Fischer 344 rat using 5-aminolevulinic acid and violet laser light: a toxicity study

OBJECTIVE: Our study was designed to investigate 5-aminolevulinic acid (ALA) as a candidate for intraperitoneal photodynamic therapy (IP-PDT). The toxicity of IP-PDT and the effects of IP-PDT on abdominal and pelvic organs, particularly the small intestine, were investigated after ALA administration and illumination with violet laser light. STUDY DESIGN AND RESULTS: The toxicity of IP-PDT was evaluated in Fischer 344 rats in two ways. In the first part of the study local PDT effects on the intestine were analyzed histologically. Violet laser light (lambda: 406-415 nm) was applied as a 2 cm diameter spot on the intestine 3 h after intraperitoneal (i.p.) administration of 50 mg/kg ALA. (A) Histological tissue samples were taken 0 min, 6 h and 1, 2 and 3 days after treatment (optical dose 3.2 J/cm(2)). Immediately after local PDT (3.2 J/cm(2), 50 mg/kg ALA) showed no effect on the intestine. However, 6 h post PDT there was complete destruction of the mesothelial lining and the outer (longitudinal) smooth muscle. Ganglion cells of the myenteric (Auerbach) plexus were also destroyed. The inner circular smooth muscle, the muscularis mucosa and the lamina propria were unharmed. Marked lymphectasia was present at this time. (B) To determine the threshold light dose of tissue destruction caused by PDT, different optical doses (1.6, 3.2, 6.4 J/cm(2)) were administered and histologic analysis of tissue samples were obtained 1 day post treatment. Destruction of the entire external musculature, submucosal structures and muscularis mucosa of the intestine at the illumination site could be observed above 1.6 J/cm(2) (50 mg/kg ALA). In the second part of the study whole peritoneal cavity PDT (WPC-PDT) was performed by illumination of the whole peritoneal cavity with 1.6 J/cm(2) violet light 3 h after ALA administration using different drug doses (200, 100 and 50 mg/kg). WPC-PDT showed lethal toxicity with a drug dose above 50 mg/kg ALA at 1.6 J/cm(2). The probable cause of death in the first 3 days after IP-PDT was rhabdomyolysis, whereas when death occurred at longer time intervals, megaintestine associated with significant damage could be observed; however, without perforation of the intestinal wall. CONCLUSION: In rats WPC-PDT with 50 mg/kg ALA, 1.6 J/cm2 at lambda=415 nm was the maximum tolerable light dose. This dose is likely to be above the threshold of destruction of ovarian cancer micrometastasis.     

Protoporphyrin IX fluorescence photobleaching and the response of rat Barrett's esophagus following 5-aminolevulinic acid photodynamic therapy

Barrett's esophagus (BE) can experimentally be treated with 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT), in which ALA, the precursor of the endogenous photosensitizer protoporphyrin IX (PpIX) and subsequent irradiation with laser light are applied to destroy the (pre)malignant tissue. Accurate dosimetry is critical for successful ALA-PDT. Here, in vivo dosimetry and kinetics of PpIX fluorescence photobleaching were studied in a rat model of BE. The fluence and fluence rate were standardized in vivo and PpIX fluorescence was measured simultaneously at the esophageal wall during ALA-PDT and plotted against the delivered fluence rather than time. Rats with BE were administered 200 mg kg(-1) ALA (n = 17) or served as control (n = 4). Animals were irradiated with 633 nm laser light at a measured fluence rate of 75 mW cm(-2) and a fluence of 54 J cm(-2). Large differences were observed in the kinetics of PpIX fluorescence photobleaching in different animals. High PpIX fluorescence photobleaching rates corresponded with tissue ablation, whereas low rates corresponded with no damage to the epithelium. Attempts to influence tissue oxygenation by varying balloon pressure and ventilation were shown not to be directly responsible for the differences in effect. In conclusion, in vivo dosimetry is feasible in heterogeneous conditions such as BE, and PpIX fluorescence photobleaching is useful to predict the tissue response to ALA-PDT.     

New 5-aminolevulinic acid esters--efficient protoporphyrin precursors for photodetection and photodynamic therapy

Photodetection (PD) and photodynamic therapy (PDT) with 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) accumulation are approaches to detect and treat dysplasia and early cancer in the gastrointestinal tract and in the urinary bladder. Because ALA-induced PPIX production is limited, we synthesized ALA ester hydrochlorides 3-22 and tested them in two different in vitro models (gastrointestinal tract: HT29-CCD18; urinary bladder: J82-UROTSA). PPIX accumulation after incubation with 0.12 mmol/L for 3 h and PPIX accumulation as a function of different incubation times were measured using flow cytometry. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays were performed to check cellular dark toxicity. Phototoxicity after irradiation was tested. ALA nonafluorohexylester hydrochloride 11, ALA thiohexylester hydrochloride 13 and ALA dibenzyldiester dihydrochloride 19 induced appreciably increased PPIX levels and showed improved phototoxicity compared with the references ALA hydrochloride 1, ALA hexylester hydrochloride 3 and ALA benzylester hydrochloride 4. Thus, the new compounds 11, 13 and 19 are promising compounds for PD and PDT.     

Susceptibility to 5-aminolevulinic acid based photodynamic therapy in WHO I meningioma cells corresponds to ferrochelatase activity

5-Aminolevulinic acid (5-ALA) is a natural precursor of protoporphyrin IX (PpIX), which can be used as a photosensitizer in photodynamic therapy (PDT). Accumulation of PpIX in benign meningioma cells has been observed previously, its exploitation for PDT, however, was discouraged by inconsistent results. To evaluate PDT for benign meningiomas, we investigated PpIX synthesis in two human meningioma cell lines (HBL-52 and BEN-MEN-1), their respective extracellular loss of PpIX and corresponding ferrochelatase (FECH) activity as well as their susceptibility to PDT. We demonstrated PpIX production after 5-ALA administration and minor loss to the extracellular space in both cell lines. However, significantly more (up to five times) PpIX was accumulated in BEN-MEN-1 as compared with HBL-52 cells. FECH activity was 2.7-fold higher in HBL-52 compared with BEN-MEN-1 cells and accordingly higher FECH levels could be shown in HBL-52 cells by Western blot analysis. BEN-MEN-1 cells were much more sensitive to PDT and cells could be almost completely killed by irradiation doses of 2 J cm(-2) , whereas HBL-52 showed only an insufficient response at this irradiation dose. We conclude that differences in intracellular PpIX concentrations between HBL-52 and BEN-MEN-1 benign meningioma cells were mainly due to differences in FECH activity and that these differences correspond to their susceptibility to 5-ALA-induced PDT.     

Oxygen monitoring during 5-aminolaevulinic acid induced photodynamic therapy in normal rat colon. Comparison of continuous and fractionated light regimes

Currently, the clinical use of 5-aminolaevulinic acid (ALA) induced protoporphyrin IX (PPIX) for photodynamic therapy (PDT) is limited by the maximum tolerated oral ALA dose (60 mg/kg). Attempts have been made to enhance this treatment modality without increasing the administered dose of ALA. One way to do this is through light dose fractionation, where the irradiation is interrupted at a particular point for a short period of time. This can produce up to three times more necrosis than with the same light dose delivered without a break. An oxygen microelectrode was employed to study the effect of continuous and fractionated light regimes on the level of oxygen in the colon of normal Wistar rats during ALA PDT. A rapid decline in pO2 occurred close to the irradiation fibre as soon as the light dose commenced. With the fractionated regime, a partial recovery in pO2 was observed during the dark interval which was reversed soon after the second light fraction commenced. We have shown that the level of tissue oxygen at the treatment site is affected differently when the light dose is fractionated, than when continuous illumination is employed. This factor may at least partially explain the difference in outcome of these two treatment regimes. Further, oxygen measurements might prove to be a useful way of monitoring PDT treatments if they can predict whether tissue is likely to be viable following treatment.     

Effects of photodynamic therapy with topical application of 5-aminolevulinic acid on normal skin of hairless guinea pigs.

Photodynamic therapy (PDT) is a relatively new approach to the treatment of neoplasms which involves the use of photoactivatable compounds to selectively destroy tumors. 5-Aminolevulinic acid (ALA) is an endogenous substance which is converted to protoporphyrin IX (PpIX) in the synthetic pathway to heme. PpIX is a very effective photosensitizer. The goal of this study was to evaluate the effect of PDT using topical ALA on normal guinea pig (g.p.) skin and g.p. skin in which the stratum corneum was removed by being tape-stripped (TS). Evaluation consisted of gross examination, PpIX fluorescence detection, reflectance spectroscopy, and histology. There was no effect from the application of light or ALA alone. Normal non-TS g.p. skin treated with ALA and light was unaffected unless high light and ALA doses were used. Skin from which the stratum corneum was removed was highly sensitive to treatment with ALA and light: 24 h after treatment, the epidermis showed full thickness necrosis, followed by complete repair within 7 d. Time-dependent fluorescence excitation and emission spectra were determined to characterize the chromophore and to demonstrate a build-up of the porphyrin in the skin. These data support the view that PDT with topical ALA is a promising approach for the treatment of epidermal cutaneous disorders.     

5-aminolevulinic acid-based photodynamic therapy in leukemia cell HL60

A study to explore the optimal experimental parameters and the photosensitization of 5-aminolevulinic acid (ALA)-based photodynamic therapy (PDT) in promyelocytic leukemia cell HL60 has been conducted, in which HL60 cells and their control groups, peripheral blood mononuclear cell (PBMC), first are incubated with different concentrations of ALA in dark for different periods of time and then followed by irradiating with different wavebands for different fluences. Fluorescence microscope and spectrofluorometer have been used to detect the fluorescence of protoporphyrin IX (PpIX) endogenously produced by ALA. The response of the cells to ALA-PDT was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2-5-diphenyl-2H-tetrazolium bromide (MTT) assay (interval between irradiation and the MTT assay is 24 h) and by flow cytometry (the length of time between irradiation and the flow assay is 30 min). MTT results will reflect the relative number of metabolically active mitochondria in the population. Propidium iodide uptake in flow cytometry will test for membrane damage. The results of parameter experiments were obtained: 1 x 10(5)/mL HL60 cell was first incubated with 1 mmol/L ALA in dark for 4 h and the maximum fluorescence of PpIX level appeared; then irradiated with 410 nm (4 mW/cm2) for 14.4 J/cm2 and maximum photodamage to membrane and mitochondrial function of HL60 cell resulted. With the normal granulocytes, such response was not detected. Therefore a hypothetical idea can be brought forward that ALA-based PDT can be used for inactivation of leukemia cell HL60 and these optimal parameters may be useful for clinical application.     

Photodynamic therapy of human glioma spheroids using 5-aminolevulinic acid

The response of human glioma spheroids to 5-aminolevulinic acid (ALA)-mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (< or = 50 J cm-2), lower fluence rates are more effective. At a fluence of 50 J cm-2, near-total spheroid kill is observed at fluence rates of as low as 10 mW cm-2. The fluence rate effect is not as pronounced at higher fluences (> 50 J cm-2), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.     

The vascular response to photodynamic therapy with ATX-S10Na(II) in the normal rat colon

The mechanism of tissue damage from photodynamic therapy (PDT) may be cellular, vascular or both, depending on the photosensitising agent and the treatment conditions. Well established photosensitisers like porfimer sodium have an optimum drug light interval of two days and may cause skin photosensitivity lasting several weeks. ATX-S10Na(II) is a new photosensitiser that remains largely in the vasculature after systemic administration and clears from the body within a few hours. The present study looks at the factors controlling the extent of PDT necrosis using ATX-S10Na(II) and correlates these with changes in the circulation after PDT. Normal Wistar rats were sensitised with ATX-S10Na(II), 2 mg/kg. At laparotomy, a laser fibre was positioned just touching the colonic mucosa and 50 J light at 670 nm delivered varying the drug light interval (0.5-24 h) and light delivery regime (100 mW continuous, 20 mW continuous or 100 mW in five fractions). Some animals were killed at three days to document the area of necrosis, others received fluorescein shortly prior to death (from a few minutes to three days after PDT) to outline the zone of PDT induced vascular shutdown. Maximum necrosis was seen with the shortest drug light interval (0.5 h), with no effect by 6 h. Fractionating the light or lowering the power did not increase the necrosis. The area of fluorescein exclusion increased over the first 2 h after PDT (in contrast to the re-perfusion seen with other photosensitisers) and correlated with the area of necrosis. PDT with ATX-S10Na(II) is most effective with a drug light interval of less than one hour. It induces irreversible vascular shutdown that extends after completion of light delivery and which is largely independent of the light delivery regime.     

Two-photon photodynamic therapy of C6 cells by means of 5-aminolevulinic acid induced protoporphyrin IX

Photodynamic therapy (PDT) has received increased attention as a treatment modality for malignant tumors as well as non-oncologic diseases such as age-related macular degeneration (AMD). An alternative to excite the photosensitizer by the common one-photon absorption is the method of two-photon excitation (TPE). This two-photon photodynamic therapy has the potential of improving the therapeutic outcome due to a highly localized photodynamic effect. The present study investigated the two-photon excited PDT performing in vitro experiments where C6 rat glioma cells were irradiated with a pulsed and focused fs Ti:sapphire laser emitting light at 800 nm. The irradiance distribution of the laser beam was carefully analyzed before the experiment and the applied irradiance was known for each position within the irradiated cell layer. Cells were divided into four groups and one group was incubated with 5-ALA and irradiated 4-5h later. The survival of this group was tested after irradiation by means of ethidium bromide and acridine orange staining and compared to a control group, which was irradiated under the same conditions, but not incubated with 5-ALA before. Both groups showed necrotic areas depending on the applied irradiance, the value of which at the margin of the necrotic area could be deduced from its size. 5-ALA incubated cells became necrotic after irradiation with a mean irradiance above 6.1 x 10(10) W/cm(2), while non-incubated cells remained viable. Cells of both groups became necrotic when treated with an irradiance above 10.9 x 10(10) W/cm(2). The observed affected area of the cell layers was between 0.13 mm(2) and 1.10 mm(2). Since the irradiation of non-incubated cells below the mean power density of 10.9 x 10(10) W/cm(2) induced no necrosis, apparently no thermal damage was induced in the cells and necrosis of the 5-ALA incubated cells can be ascribed to the photodynamic effect induced by two-photon excitation. The successful photodynamic treatment of a large area of a monolayer cell culture induced by two-photon excitation offers new perspectives for photodynamic treatment modalities.     

Enhanced tumour selectivity of photodynamic therapy in the rat colon using a radioprotective agent.

Radioprotective agents have been found to protect normal tissues during photodynamic therapy (PDT). We have investigated a phosphorylated thiol protectant WR-77913 (W7) with the photosensitizer aluminium sulphonated phthalocyanine (AISPc). We compared the effects of PDT on normal and tumour tissue in the rat colon, with and without this protectant. In normal colon no necrosis was seen in sites treated after administration of the W7. Necrosis of mean diameter 4.2 mm was seen in those given the protectant after light exposure. At tumour sites the area of necrosis was similar after light exposure before and after the administration of the protective agent. These results suggest a possible role for W7 in enhancement of selectivity of PDT action. Several mechanisms of protection against porphyrin phototoxicity by these drugs have been proposed, including acceleration of photobleaching. We used fluorescence to detect AISPc in strips of rat colon before and after laser treatment, with and without W7. However, a primary role for the photobleaching of AISPc as the mechanism for the protection shown is not supported by these observations.     

Angiogenesis induced by photodynamic therapy in normal rat brains

Angiogenesis promotes tumor growth and invasiveness in brain. Because brain injury often induces expression of angiogenic-promoting molecules, we hypothesize that oxidative insult induced by photodynamic therapy (PDT) could lead to an endogenous angiogenic response, possibly diminishing the efficacy of PDT treatment of tumors. Therefore, we sought to establish whether PDT induced an angiogenic response within the nontumored brain. PDT using Photofrin as a sensitizer at an optical dose of 140 J/cm2 was performed on normal rat brain (n = 30). Animals were sacrificed at 24 h, and 1, 2, 3 and 6 weeks after PDT treatment. Fluorescein isothiocyanatedextran perfusion was performed, and brains were fixed for immunohistological study. Immunostaining revealed that vascular endothelial growth factor (VEGF) expression increased within the PDT-treated hemisphere 1 week after treatment and remained elevated for 6 weeks. Three-dimensional morphologic analysis of vasculature within PDT-treated and contralateral brain demonstrated PDT-induced angiogenesis, as indicated by a significant increase in vessel connectivity (P < 0.001) concomitant with decreased (P < 0.05) mean segment length compared with vessels within the contralateral hemisphere. Volumetric measurement of angiogenic regions indicate that neovascular expansion continued for 4 weeks after PDT. These data demonstrate that PDT induces VEGF expression and neovascularization within normal brain. Because angiogenesis promotes growth and invasiveness of tumor, antagonizing this endogenous angiogenic response to PDT may present a practical means to enhance the efficacy of PDT.     

The influence of temperature on photodynamic cell killing in vitro with 5-aminolevulinic acid

Cell survival was investigated after exposing cells in vitro to different temperatures before or after photodynamic therapy with 5-aminolevulinic acid. The photodynamic process was found to be temperature dependent. Cells exposed for 1h to 41 degrees C before light exposure or to 7 degrees C after light exposure showed decreased survival. Furthermore, the photobleaching rate of protoporphyrin IX in the cells was found to increase with increasing temperature during the light exposure. Thus, the photodynamic effect with 5-aminolevulinic acid may be enhanced by heating the tumour area before, and by cooling it immediately after the treatment.     

Treatment of HPV infection-associated cervical condylomata acuminata with 5-aminolevulinic acid-mediated photodynamic therapy

The aim of this study was to investigate the efficacy of 5-aminolaevulinic acid (ALA)-mediated photodynamic therapy (PDT) in treatment of human papillomavirus (HPV)-associated cervical condylomata. A total of 56 patients with cervical and external condylomata lesions were recruited for this open-label study. HPV genotyping of exfoliated cells collected from the cervix and external lesions was performed. Cervical lesions were treated with PDT by applying ALA gel (10%) to the surface of the cervix for 4 h followed by irradiating with a 635 nm laser at 100 J cm(-2). PDT was repeated at 2-week intervals if lesion and HPV infection remained. Patients were followed up for 6-24 months. Genotyping analysis revealed four HPV subtypes (HPV6, 11, 16 and 18). The overall complete remission rate of 1-4 sessions of treatments was 98.2% and the corresponding HPV clearance rate was 83.9%. Ten cases showed complete removal of cervical lesions and HPV infection after a single treatment. Recurrence rate was 3.6%. Adverse effects were minimal and no structural complications were reported. In conclusion, topical ALA PDT is safe and effective for eradicating cervical HPV infection and eliminating condylomata lesion. Its definitive role in treating cervical condylomata deserves further investigation.     

Topical application of 5-aminolevulinic acid hexyl ester and 5-aminolevulinic acid to normal nude mouse skin: differences in protoporphyrin IX fluorescence kinetics and the role of the stratum corneum

An important limitation of topical 5-aminolevulinic acid (ALA)-based photodetection and photodynamic therapy is that the amount of the fluorescing and photosensitizing product protoporphyrin IX (PpIX) formed is limited. The reason for this is probably the limited diffusion of ALA through the stratum corneum. A solution to this problem might be found in the use of ALA derivatives, as these compounds are more lipophilic and therefore might have better penetration properties than ALA itself. Previous studies have shown that ALA hexyl ester (ALAHE) is more successful than ALA for photodetection of early (pre)malignant lesions in the bladder. However, ALA pentyl ester slightly increased the in vivo PpIX fluorescence in early (pre)malignant lesions in hairless mouse skin compared to ALA. The increased PpIX fluorescence is located in the stratum corneum and not in the dysplastic epidermal layer. In the present study, ALA- and ALAHE-induced PpIX fluorescence kinetics are compared in the normal nude mouse skin, of which the permeability properties differ from the bladder. Application times and ALA(HE) concentrations were varied, the effect of a penetration enhancer and the effect of tape stripping the skin before or after application were investigated. Only during application for 24 h, did ALAHE induce slightly more PpIX fluorescence than ALA. After application times ranging from 1 to 60 min, ALA-induced PpIX fluorescence was higher than ALAHE-induced PpIX fluorescence. ALA also induced higher PpIX production than ALAHE after 10 min of application with concentrations ranging from 0.5 to 40%. The results of experiments with the penetration enhancer and tape stripping indicated that the stratum corneum acts a barrier against ALA and ALAHE. Use of penetration enhancer or tape stripping enhanced the PpIX production more in the case of ALAHE application than in the case of ALA application. This, together with the results from the different application times and concentrations indicates that ALAHE diffuses more slowly across the stratum corneum than ALA.     

The influence of photodynamic therapy on the ultrastructure of the normal rat bladder.

Reduced bladder capacity is a major side effect for patients receiving photodynamic therapy (PDT) for bladder cancer. A rat bladder model has been developed to address both the vascular and tissue effects of the photodynamic treatment of the urinary bladder. Bladders were exteriorized and positioned in a plexiglass tissue bath. Effects on microvasculature were assessed during PDT of the bladder by recording luminal diameter changes in arterioles and venules. Animals receiving Photofrin II (10 mg kg-1) 30 min prior to PDT scored a statistically significant reduction in the diameter of the red blood cell column in the vessels, whereas administration of Photofrin II 48 h prior to PDT was ineffective. Morphological changes included significant endothelial and vascular myocyte damage in the 30 min PDT group alone. Among the other tissue components, the mucosal lining was minimally affected and the response of the muscularis was highly variable. Smooth muscle cell changes ranged from mild contraction to frank necrosis with many of the affected cells located near the altered vascular beds. These data suggest that the clinical symptoms of reduced bladder capacity can be accounted for by vascular damage and myocyte sensitivity. Further refinements in the Photofrin II and light doses used in therapy may reduce bladder complications and allow for better management of bladder cancer.     

Using enzymatic reactions to enhance the photodynamic therapy effect of porphyrin dityrosine phosphates

This paper reports the synthesis and photodynamic therapy (PDT) effect of a porphyrin derivative containing tyrosine phosphate, which promises a new, useful approach to develop PDT agents.     

Comparison of distribution and photodynamic effects of di- and tetra-sulphonated aluminium phthalocyanines in normal rat colon

We have previously reported photodynamic therapy of normal rat colon using aluminium sulphonated phthalocyanine (AISPc). In that study, the AISPc used was a mixture of phthalocyanines of different degrees of sulphonation. Phthalocyanines of defined degrees of sulphonation have recently become available and we compared the distribution of the di- and tetra-sulphonates (AIS2Pc and AIS4Pc) in rat colon and colon wall structures employing both chemical extraction and fluorescence photometry using a charge coupled device imaging system. Also, the photodynamic effects produced by these components in rat colon were compared at various times after photosensitization. After intravenous photosensitizer administration using equimolar doses, the concentration of AIS2Pc in colon fell off more rapidly with time than AIS4Pc. Differences were noted in the microscopic distribution of these compounds, with the di-sulphonate exhibiting peak fluorescence in colon wall structures by 1 h after photosensitization, while mucosal fluorescence with the tetra-sulphonate peaked at 5 h. Fluorescence was also lost from the colon wall much more slowly with the tetra-sulphonate, which tended to be retained in the submucosa. Maximum photosensitizing capability was seen at 1 h with AIS2Pc and no lesions could be produced with photodynamic therapy at 1 week, with up to 5.65 mumol/kg. With AIS4Pc (5.65 mumol/kg), while no lesions could be produced with light treatment at 1 h, photodynamic therapy at 1 week produced lesions only slightly smaller than those produced with treatment at 48 h (the time of maximum effect), and significant photosensitization was present at 2 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)