Photodynamic therapy with porphyrin and phthalocyanine sensitisation: quantitative studies in normal rat liver

Selective sensitisation of malignant tumours to monochromatic light (photodynamic therapy, PDT) is a promising approach to cancer treatment, but current sensitisers are unsatisfactory and the parameters controlling effects produced in normal and neoplastic tissue are poorly understood. To quantify the effects in a relatively homogeneous organ, we carried out experiments in the livers of normal rats following systemic sensitisation with haematoporphyrin derivative (HpD) and a new sensitiser, a sulphonated aluminium phthalocyanine (AlSPc) using light from an Argon pumped tunable dye laser. Damage from PDT (dominant at 100 mW laser power) could be distinguished from that due to local hyperthermia (dominant at 400 mW). For both sensitisers, the extent of PDT necrosis increased with the applied light energy and was abolished by occluding the hepatic blood flow during therapy. With HpD, the extent of PDT necrosis was maximum with only a few hours between sensitisation and therapy, and was not detectable when this interval was increased to a week. With AlSPc, the extent of necrosis in liver changed little with sensitisation times from 1 h to 1000 h (6 weeks), and declined slowly thereafter, matching the amount of AlSPc measurable by alkali extraction, although prolonged photosensitisation was not seen with AlSPc in muscle. Less cutaneous photosensitivity was seen with AlSPc than with HpD. AlSPc is easier to produce and handle than HpD, has a more appropriate strong absorption peak (at 675 nm) and from these results, warrants further study as a photosensitiser for PDT.     

Photodynamic therapy in the normal rat colon with phthalocyanine sensitisation

Photodynamic therapy (PDT) involves the interaction of light with an administered photosensitising agent to produce cellular destruction. It has promising potential for the local and endoscopic treatment of gastrointestinal cancer. There is however little data on the response of normal intestine to PDT. We have investigated the use of a new photosensitiser chloro aluminum sulphonated phthalocyanine (AlSPc) for colonic PDT. The peak concentration of AlSPc in the colon measured by alkali extraction occurred 1 h after i.v. injection. The cellular uptake demonstrated by laser fluorescence microscopy was greater in the mucosa than in the muscle. AlSPc was activated in the tissues by light from an argon ion pumped dye laser at 675 nm. The laser power was set at 100 mW and the fibre placed touching the mucosa. In control animals no macroscopic damage was seen. Temperature measurement using a microthermocouple array showed no temperature rise during light exposure. The energy (fluence), dose of sensitiser and time from sensitisation to phototherapy were altered and the area of necrosis measured. The geometry of the colon made theoretical analysis of the correlation between laser energy and size of lesion difficult. However, following direct measurement of the relative light intensity (fluence rate) in the colon we were able to confirm that there was a threshold fluence for colonic necrosis. The area of photodynamic damage seen 72 h after phototherapy fell with the fall in tissue concentration of AlSPc from 1 h to 1 month after i.v. injection. However, maximum tissue necrosis occurred when treatment was performed immediately after i.v. injection. In this situation, intense vascular spasm was seen and any light transmitted through the colon which fell on the small bowel mesentery caused a lethal ischaemic necrosis. The initial histological changes after PDT were vascular, followed by full thickness necrosis at 72 h. Healing by regeneration was complete by 2-3 weeks. Despite full thickness necrosis there was no reduction in the colonic bursting pressure at any time. Colon treated by hyperthermia had a reduced bursting pressure. Specific collagen stains showed that PDT did not alter the submucosal collagen architecture whereas hyperthermia did.     

Liposome-delivered Si(IV)-naphthalocyanine as a photodynamic sensitiser for experimental tumours: pharmacokinetic and phototherapeutic studies

The pharmacokinetic behaviour and phototherapeutic effectiveness of bis(di-isobutyloctadecylsil-oxy)-2,3-naphthalocyanatosilicon (iso-BOSiNc) incorporated into dipalmitoyl-phosphatidylcholine (DPPC) liposomes have been studied in Balb/c mice bearing an MS-2 fibrosarcoma. We found that iso-BOSiNc i.v.-injected at a dose of 0.5 mg kg-1 b.w. is preferentially transported by serum lipoproteins; in particular, the photosensitiser is associated with LDL (57.8% of total recovery in the serum) and HDL (35.7%) while minor amounts are associated to VLDL (2.63%) and other serum proteins (3.89%), Iso-BOSiNc concentrations greater than 1 microgram g-1 of tissue are recovered from the tumour at 12-48 h after administration while the ratio of iso-BOSiNc concentration in tumour and peritumoral tissue is greater than 10. Upon increasing the injected dose, the additional iso-BOSiNc is almost exclusively bound by HDL, which leads to large uptake of the photosensitiser by liver and spleen. The efficiency of iso-BOSiNc as a photodynamic agent was measured upon irradiation with a different dose-rate for a total light dose of 450 J cm-2. The extent of tumour necrotic area increases as a function of the time after the end of PDT treatment and reaches a maximum level after about 24 h. Moreover, the necrotic area is linearly dependent on the irradiation dose-rate up to 100 mW cm-2. In all there is substantial evidence that iso-BOSiNc delivered in a liposomal dispersion is a highly effective photosensitizer for PDT of tumours.     

Oral versus intravenous administration of 5-aminolaevulinic acid for photodynamic therapy.

Endogenously synthesised protoporphyrin IX (PpIX) following the administration of 5-amino-laevulinic acid (ALA) is an effective photosensitiser for photodynamic therapy (PDT). Following intravenous administration, PpIX accumulates predominantly in mucosa of hollow viscera and on light exposure, mucosal ablation results with relative sparing of the submucosa and muscularis layers. Oral administration is effective with ALA in contrast to conventional exogenous photosensitisers such as haematoporphyrin derivative and phthalocyanines. Oral administration of ALA is also simpler, safer, cheaper and more acceptable to patients. We studied the porphyrin sensitisation kinetics profile in the stomach, colon and bladder in normal rats following enterally and parenterally administered ALA using microscopic fluorescence photometric studies of frozen tissue sections. Mucosal cells in all three organs exhibit higher fluorescence levels as compared with underlying smooth muscle following both intravenous and oral administration. Peak concentration were seen 4 h after sensitisation at the highest doses used (200 mg kg-1 i.v., 400 mg kg-1 oral), and slightly earlier with lower doses. The temporal kinetics of both routes of administration were similar although a higher oral dose was required to achieve the same tissue concentration of PpIX. The highest level of fluorescence was achieved in the gastric mucosa and in decreasing levels, colonic and bladder mucosa. A similar degree of mucosal selectivity was achieved in each organ with each route of administration but an oral dose in excess of 40 mg kg-1 was required to achieve measurable PpIX sensitisation. In a pilot clinical study, two patients with inoperable rectal adenocarcinomas were given 30 mg kg-1 and one patient with sigmoid colon carcinoma was given 60 mg kg-1 ALA orally. Serial biopsies of normal and tumour areas were taken over the subsequent 24 h. Fluorescence microscopy of these specimens showed maximum accumulation of PpIX 4 to 6 h after administration of 30 mg kg-1 ALA. There was greater PpIX accumulation in tumour than adjacent normal mucosa in two patients. Preferential PpIX accumulation in tumour was greater in the patient receiving 60 mg kg-1 ALA.     

Photodynamic therapy with WST09 (Tookad): quantitative studies in normal colon and transplanted tumours

Photodynamic therapy (PDT) is attracting increasing interest for the safe destruction of localised tumours in a range of organs. However, most photosensitising drugs require a delay of hours to days between drug administration and light activation with skin photosensitivity that may last for weeks. WST09 (Tookad) is a new faster acting photosensitiser that clears within a few hours. In normal rat colon, after sensitisation with an intravenous bolus of WST09, light was delivered to a single point on the mucosa and the extent of PDT necrosis measured 3 days later. The lesion diameter was greatest with the highest dose of drug and light and the shortest drug light interval (DLI), falling rapidly with a DLI more than 5 min. In tumours transplanted subcutaneously or into the colon, the extent of necrosis only started falling with a DLI greater than 15 min, suggesting a possible window for tumour selectivity. Histological changes 3 days after PDT were essentially the same as those seen with longer acting photosensitisers. The lesion dimensions were comparable to the largest ones seen with other photosensitisers under similar experimental conditions. We conclude that WST09 is a powerful photosensitiser that produces PDT effects similar to those seen with longer acting drugs, but with the major advantages of a short DLI and rapid clearance.     

Photodynamic therapy: An effective,but non-selective treatment for superficial cancers of the oral cavity

It has often been claimed that photodynamic therapy (PDT) produces selective destruction of small cancers without affecting the adjacent normal tissue. The objective of our work was to treat small cancers of the oral cavity with PDT and subsequently excise the treated areas for histological studies of tumour and adjacent normal tissue exposed to the same light dose. Eleven patients with histologically proven T1NO oral squamous-cell carcinomas were treated with PDT, using Photofrin as a sensitiser. The tumours plus a surrounding cuff of normal tissue were exposed to 50 J/cm² non-thermal laser light at 630 nm delivered by surface illumination and the treated areas subsequently excised. Histological staining and image analysis were used to determine the nature and extent of injury. No macroscopic distinction was evident between tumour and normal tissue exposed to light. Histologically, replacement of superficial epithelium, tumour and connective tissue with a fibrinous necrotic slough was seen. There was also loss of endothelium from small vessels, with haemorrhage and thrombosis. Preservation of subepithelial collagen and elastin was demonstrated with EVG staining. No evidence of selective tumour necrosis was found. Although depth of injury was variable, full thickness mucosal necrosis occurred in all cases.Int. J. Cancer 71: 937-942, 1997. © 1997 Wiley-Liss Inc.     

The significance of the nature of the photosensitizer for photodynamic therapy: quantitative and biological studies in the colon

Photodynamic therapy (PDT) depends on the interaction of light with an administered photosensitiser to produce a local cytotoxic effect. The most widely used photosensitiser is haematoporphyrin derivative (HpD), but newer photosensitisers such as aluminium sulphonated phthalocyanine (A1SPc) are promising. HpD and A1SPc have been compared as photosensitisers for colonic PDT in the rat. Quantitative analysis showed that following injection of a standard photosensitiser dose, A1SPc produced more damage than HpD with increasing energy (fluence). Alteration of the injected dose of photosensitiser did not produce a clear difference. There was a loss of reciprocity for photosensitiser/light combinations at low injected dose (0.5 mg kg-1), both HpD and A1SPc producing no damage. Similarly at high photosensitiser dosage (25 mg kg-1) there was no quantitative difference between A1SPc and HpD. Photosensitiser photodegradation at low photosensitiser doses, and light attenuation by high tissue concentrations of A1SPc account for these findings. PDT with either agent produced the same histological damage and full thickness necrosis produced no mechanical weakening of the colon measured by the bursting pressure. The submucosal collagen was preserved and healing was by regeneration.     

Distribution and photodynamic effects of meso-tetrahydroxyphenylchlorin (mTHPC) in the pancreas and adjacent tissues in the Syrian golden hamster.

Photodynamic therapy (PDT) has the potential to destroy small tumours with safe healing of adjacent normal tissue. This study looks at the effects of PDT on the normal pancreas and adjacent tissues in hamsters using the photosensitiser meso-tetrahydroxyphenylchlorin (mTHPC). Pharmacokinetic studies used fluorescence microscopy on sections of pancreas, stomach and duodenum 1 h to 6 days after mTHPC. Highest levels of sensitiser were seen in the gastric and duodenal mucosa and in the acinar pancreas after 2-4 days. For PDT, light at 652 nm was delivered by placing a 0.2 mm diameter bare-ended fibre against the tissue. An energy of 50 J was used 2 or 4 days after 0.1 or 0.3 mg kg-1 mTHPC and animals killed 1 to 7 days later. Maximum necrosis was seen 3 days after PDT with lesions up to 4 mm in pancreas, 4.5 mm in duodenum and 2.5 mm in stomach. By fractionating the light dose, the lesion size could be increased by 30%. The main complication was free or sealed duodenal perforation (avoided by shielding the duodenum). Partial, reversible bile duct obstruction was seen occasionally. There was no macroscopic damage to the bile ducts or major blood vessels. Apart from the duodenum, all lesions healed safely. In this animal model, only the duodenum was at risk of serious, irreversible damage. Treatment is likely to be safer in the much thicker human duodenum. mTHPC is a powerful photosensitiser and suitable for further study for tumours in the region of the pancreas although care is required near the duodenum.     

The sensitivity of normal brain and intracranially implanted VX2 tumour to interstitial photodynamic therapy.

The applicability and limitations of a photodynamic threshold model, used to describe quantitatively the in vivo response of tissues to photodynamic therapy, are currently being investigated in a variety of normal and malignant tumour tissues. The model states that tissue necrosis occurs when the number of photons absorbed by the photosensitiser per unit tissue volume exceeds a threshold. New Zealand White rabbits were sensitised with porphyrin-based photosensitisers. Normal brain or intracranially implanted VX2 tumours were illuminated via an optical fibre placed into the tissue at craniotomy. The light fluence distribution in the tissue was measured by multiple interstitial optical fibre detectors. The tissue concentration of the photosensitiser was determined post mortem by absorption spectroscopy. The derived photodynamic threshold values for normal brain are significantly lower than for VX2 tumour for all photosensitisers examined. Neuronal damage is evident beyond the zone of frank necrosis. For Photofrin the threshold decreases with time delay between photosensitiser administration and light treatment. No significant difference in threshold is found between Photofrin and haematoporphyrin derivative. The threshold in normal brain (grey matter) is lowest for sensitisation by 5 delta-aminolaevulinic acid. The results confirm the very high sensitivity of normal brain to porphyrin photodynamic therapy and show the importance of in situ light fluence monitoring during photodynamic irradiation.     

Foscan uptake and tissue distribution in relation to photodynamic efficacy

Clinical photodynamic therapy (PDT) schedules are based on the assumption that optimum drug-light intervals are times at which there is a maximum differential between photosensitiser retention in the tumour and surrounding normal tissue. However, vascular-mediated effects contribute to tumour destruction by PDT; therefore, plasma sensitiser levels and endothelial cell drug exposure could also be important determinants of PDT response. The purpose of this study was to investigate the influence of tumour, tissue and plasma concentrations of the photosensitiser Foscan (meta-tetrahydroxyphenylchlorin, mTHPC) on PDT response. Groups of BalbC nude mice, bearing human mesothelioma xenografts (H-MESO1) were injected (i.v.) with a single dose of (14)C-labelled mTHPC, or with two doses, separated by 72 h. Drug levels in plasma, tumour and normal tissues were measured at 5 min to 120 h after drug administration. The PDT tumour and skin responses were evaluated by illuminating separate groups mice at intervals of 5 min to 120 h after injection of Foscan (nonlabelled). Drug levels in both tumour and skin increased during the first 24 h after a single injection, and remained almost constant for at least 120 h. The second injection produced a further, rapid increase in mTHPC levels in tumours and skin, with steady state being maintained from 20 min to 120 h. By contrast, PDT response of both tumours and skin were maximal for illumination at 1-3 h after drug, with very little response when illumination was given 48-120 h after drug. There was no significant correlation between tumour or skin drug level and PDT response. There was, however, a significant correlation between plasma drug levels and tumour or skin response, excluding an initial distribution time of 20 min. These studies demonstrate a pronounced disassociation between tumour drug levels and optimum drug-light intervals for PDT response with Foscan. We suggest that the PDT effect, in both tumours and normal tissues, is largely mediated via vascular damage and that the selectivity of PDT is not based on differential tumour drug uptake.     

Photodynamic therapy using 5-aminolaevulinic acid for experimental pancreatic cancer--prolonged animal survival.

Experimental studies have been carried out using 5-aminolaevulinic acid (ALA) to induce transient porphyrin photosensitisation for photodynamic therapy (PDT) in a pancreatic cancer model in Syrian golden hamsters. ALA was given either intravenously or orally (in bolus or fractionated doses) with the laser light delivered by means of a bare fibre touching the tissue surface or external irradiation using a light-integrating cylindrical applicator. Animals were killed 1-24 h after ALA administration for pharmacokinetic studies and 3-7 days after light exposure to study PDT-induced necrosis. A separate survival study was also performed after a fractionated oral dose of ALA and external irradiation. Protoporphyrin IX sensitisation in the tumour tissue as measured by quantitative fluorescence microscopy was highest after intravenous administration of 200 mg kg-1 ALA and then in decreasing order after oral fractionated and oral bolus doses (both 400 mg kg-1). Laser light application at 630 nm to give 12-50 J from the bare fibre or 50 J cm-2 using surface illumination with the cylindrical applicator resulted in tumour necrosis up to 8 mm in depth. In larger tumours a rim of viable tumour was observed on the side opposite to illumination. In a randomised study, survival of treated animals was significantly longer than in the untreated control group (log-rank test, P < 0.02), although all animals died of recurrent tumour. This technique shows promise in the treatment of small volumes of tumour in the pancreas.     

In vivo fluorescence and photodynamic activity of zinc phthalocyanine administered in liposomes.

Zinc(II) phthalocyanine, a hydrophobic photosensitiser, was incorporated in unilamellar liposomes and studied in vivo for fluorescence kinetics and photodynamic activity. An observation chamber mounted in a dorsal skinfold of female WAG/Rij rats was used as a model system. In the chamber, an isogeneic mammary carcinoma was transplanted in the subcutaneous tissue. Phthalocyanine fluorescence was excited at 610 nm with a power density of 0.25 mW cm-2 and was detected above 665 nm through a high-pass filter using a two-stage image intensifier coupled to a charge-coupled device (CCD) camera. Following i.v. administration of 0.14 mg kg-1 of the drug, the fluorescence pharmacokinetics of the dye in vasculature, normal tissue and tumour tissue was determined as a function of time. Tumour fluorescence increased slowly to a maximum about 3 h post injection (p.i.), and remained well above the normal tissue fluorescence till 24 h p.i. Fluorescence in the circulation was always stronger than in the tissues. A treatment light dose at a wavelength of 675 nm was delivered 24 h p.i. One group of six animals received a total light dose of 150 J cm-2 (100 mW cm-2). A second group of six animals received a total light dose of 450 J cm-2 at the same dose rate. Vascular damage resulting from treatment was observed only at the final stages of the irradiation, despite the relatively high levels of fluorescence in the circulation. Immediate post-treatment (re)transplantation of the content of the chamber into the flank always resulted in tumour regrowth, confirming the presence of viable tumour cells following photodynamic therapy (PDT). When the chamber was left intact, the light dose of 450 J cm-2 yielded complete tissue necrosis. The role of the dye-carrier complex in shielding the vascular surrounding from photoproducts was studied in a third group of animals. The presence of peroxides was demonstrated in the serum of these animals after PDT with zinc phthalocyanine in liposomes (ZnPc-lip) using a total light dose of 450 J cm-2. This ex vivo observation supports the previously reported observations in vitro that the carrier complex is able to quench the photoproducts resulting from photoactivation of the photosensitiser which is present in the circulation.     

Fluorescence and photodynamic effects of bacteriochlorin a observed in vivo in 'sandwich' observation chambers.

Bacteriochlorin a (BCA), a derivative of bacteriochlorphyll a, is an effective photosensitiser in vitro and in vivo. BCA has a major absorption peak at 760 nm where tissue penetration is optimal. This property, together with rapid tissue clearance promises minor skin photosensitivity. The tissue localising and photodynamic properties of BCA were studied using isogeneic RMA mammary tumours, transplanted into subcutaneous tissue in transparent ''sandwich'' observation chambers on the back of WAG/Rij rats. The fluorescence kinetics following an i.v. administration of 20 mg kg-1 BCA was assessed in blood vessels, tumour and normal tissue. Subsequently, the development of vascular- and tissue damage after a therapeutic light dose (760 nm, 600 J cm-2) was observed. Fifteen minutes post injection (p.i.), the fluorescence of BCA in the tumour reached a plateau value of 2.5 times the fluorescence in the normal tissue. From 1 h post injection the tumour fluorescence diminished gradually; after 24 h, the tumour fluorescence signal did not exceed that of the normal tissue. Following photodynamic therapy (PDT), 24 h p.i., complete vascular stasis was observed 2 h post treatment in the tumour only, with subsequent recovery. The presence of viable tumour cells following PDT was assessed by histology and re-transplantation of treated tumour tissue from the chamber into the flank immediately or 7 days after treatment. In both cases tumour regrowth was observed. BCA-PDT (20 mg kg-1, 760 nm, 100 J cm-2) 1 h after BCA administration, an interval which gives the optimal differential between tumour and normal tissue, was sufficient to prevent tumour regrowth. However, this only occurred when re-transplantation was performed 7 days after PDT. During PDT, 1 h p.i., vascular damage in tumour and normal tissue was considerable. Complete vascular shut-down was observed in the tumour 2 h after therapy and in the surrounding tissues at 24 h. Circulation damage was associated with vascular spasm and occlusion probably due to thrombi formation. Oedema was notable, especially following PDT with 600 J cm-2 at 24 h p.i.     

Cell uptake, distribution and response to aluminium chloro sulphonated phthalocyanine, a potential anti-tumour photosensitizer

The uptake, retention and effects of aluminium chloro sulphonated phthalocyanine (AlSPc) were measured in two cell lines, UV-2237 a murine fibrosarcoma and the non-tumorigenic NIH/3T3 fibroblast line. The behaviour of cells treated with AlSPc was compared with that of those treated with haematoporphyrin derivative (HpD), a photosensitizer often used in photodynamic therapy (PDT) of cancer. AlSPc absorbs light strongly in the red region, is taken up by cells in a dose dependent fashion and is retained in vitro over a period of days (5 days after exposure greater than 40% remains cell-associated versus less than 25% of HpD). Additionally AlSPc was less cytotoxic to cells, maintained in darkness or exposed to room light, compared to HpD (100% viability versus 0% viability 3 days after 60 min exposure to room light). However red light (approximately 600-700 nm) caused greater toxicity in AlSPc-treated cells (100%) than in similarly exposed HpD-treated cells (less than 60%). No significant differences were detected between the responses of the fibrosarcoma and the fibroblast cell lines. These characteristics of AlSPc suggest that it may prove to be a useful photosensitizer for PDT of cancer and this possibility is discussed.     

meso-Tetra(hydroxyphenyl)porphyrins, a new class of potent tumour photosensitisers with favourable selectivity

We compared para-, meta- and ortho-isomers of meso-tetra(hydroxyphenyl)porphyrin (p-, m- and o-THPP) and the potassium salt of the para compound (K-p-THPP) with haematoporphyrin derivative (HpD) and Photofrin II in their ability to sensitise tumours, skin and brain to light. HpD and Photofrin II induced modest tumour photosensitisation at the cost of substantial skin and brain sensitisation. At doses low enough to keep sensitisation of these normal tissues within acceptable limits, tumour sensitisation was sufficient to give necrosis only approximately 2 mm deep after exposure to 10 J cm-2 light. In contrast, doses of p-THPP, K-p-THPP and m-THPP that produced skin and brain sensitivity within acceptable limits sensitised tumours enough to give 4-9 mm necrosis after 10 J cm-2 light. m-THPP was, on a molar basis, about 25-30 times as potent as HpD and Photofrin II in sensitising tumours. o-THPP was also a potent tumour photosensitiser, but induced a prohibitive degree of skin photosensitivity even at low doses. It is unlikely that these differences in relative selectivity are due to differences in such photophysical parameters as optimum activating wavelength (which would affect tissue penetration by light), or light absorption, and physicochemical factors that determine tissue localisation may be involved. The high tumour sensitising potency and favourable tissue selectivity of m-THPP, p-THPP and K-p-THPP make them promising candidates for clinical tumour phototherapy.     

Evaluation of porphyrin C analogues for photodynamic therapy of cerebral glioma.

A series of monomeric porphyrins (2-8) based on porphyrin C (1) have been tested as sensitisers for photodynamic therapy (PDT) of cerebral glioma using the in vitro/in vivo C6 intracerebral animal tumour model. The in vivo screening, consisting of cytotoxicity, phototoxicity (red light) and subcellular localisation studies, revealed two sensitisers (porphyrin 7, molecular weight 863 Da and porphyrin 8, molecular weight 889 Da), which had greater photoactivity than porphyrin C and similar photoactivity to haematoporphyrin derivative (HpD) although at a 5-fold higher dose than HpD. Both sensitisers showed intracellular localisation to discrete organelle sites and exhibited considerably less ''dark'' cytotoxicity than HpD. The kinetics of uptake of porphyrins 7 and 8 was studied in the mouse C6 glioma model as well as in biopsy samples from normal brain, liver, spleen and blood. Maximal drug uptake levels in tumour occurred 9 and 6 h after intraperitoneal injection for 7 and 8 respectively, at which time the tumour to normal brain ratios were 15:1 and 13:1 respectively. The effect of PDT using porphyrin 7 activated by the gold metal vapour laser tuned to 627.8 nm was studied in Wistar rats bearing intracerebral C6 glioma. At a drug dose of 10 mg porphyrin 7 kg-1 body weight and laser doses of up to 400 J cm-2 light, selective tumour kill with sparing of normal brain was achieved, with a maximal depth of tumour kill of 1.77+/-0.40. mm. Irradiation following a higher drug dose of 75 mg porphyrin 7 kg-1 body weight resulted in a greater depth of tumour kill, but also significantly increased the likelihood and extent of necrosis in normal brain.     

In vivo fluorescence kinetics and photodynamic therapy using 5-aminolaevulinic acid-induced porphyrin: increased damage after multiple irradiations.

The kinetics of fluorescence in tumour (TT) and subcutaneous tissue (ST) and the vascular effects of photodynamic therapy (PDT) were studied using protoporphyrin IX (PpIX), endogenously generated after i.v. administration of 100 and 200 mg kg-1 5-aminolaevulinic acid (ALA). The experimental model was a rat skinfold observation chamber containing a thin layer of ST in which a small syngeneic mammary tumour grows in a sheet-like fashion. Maximum TT and ST fluorescence following 200 mg kg-1 ALA was twice the value after 100 mg kg-1 ALA, but the initial increase with time was the same for the two doses in both TT and ST. The fluorescence increase in ST was slower and the maximum fluorescence was less and appeared later than in TT. Photodynamic therapy was applied using green argon laser light (514.5 nm, 100 J cm-2). Three groups received a single light treatment at different intervals after administration of 100 or 200 mg kg-1 ALA. In these groups no correlation was found between the fluorescence intensities and the vascular damage following PDT. A fourth group was treated twice and before the second light treatment some fluorescence had reappeared after photobleaching due to the first treatment. Only with the double light treatment was lasting TT necrosis achieved, and for the first time with any photosensitiser in this model this was accomplished without complete ST necrosis.     

A study of the effects of photodynamic therapy on the normal tissues of the rabbit jaw.

Photodynamic therapy (PDT) is an anti-cancer treatment which involves the systemic administration of a photosensitising drug which is preferentially absorbed by tumour tissue. Relatively little drug should be absorbed by the surrounding normal tissues. Tumour destruction is achieved when the tumour is illuminated with light of a wavelength which activates the photosensitising drug thereby inducing a cytotoxic reaction. However studies in many tissues have shown that the hoped for tumour selectivity is rarely achieved. Using the rabbit mandible and gingiva as our models we have studied the effects of various doses of PDT on the tissues of the oral cavity, namely mucosa, bone, muscle and salivary gland. The photosensitiser used was di-sulphonated aluminium phthalocyanine. Results show that whereas bone is extremely resistant to PDT the other tissues are vulnerable to it. In the case of muscle and salivary gland this susceptibility is very much dose related. In salivary tissue necrotising sialometaplasia was observed in areas of the gland adjacent to those that had undergone necrosis. All tissues were noted to heal or regenerate well following PDT injury.     

Distribution and photodynamic effect of disulphonated aluminium phthalocyanine in the pancreas and adjacent tissues in the Syrian golden hamster.

Necrosis of small volumes of tumour tissue with photodynamic therapy (PDT) can be achieved relatively easily. For this to be clinically relevant, it is essential to know what the same treatment parameters do to adjacent normal tissues into which the tumour has spread. For pancreatic cancers, local spread to vital structures is common. We have studied chemical extraction, microscopic fluorescence kinetics and photodynamic effects of disulphonated aluminum phthalocyanine (AlS2Pc) in normal pancreas and adjacent tissues in hamsters. Chemical extraction exhibited a peak duodenal concentration of AlS2Pc 48 h after sensitisation, with levels much higher than in stomach and pancreas. With microscopic fluorescence photometry highest levels were seen in duodenal submucosa and bile duct walls 48 h after photosensitisation. Pancreatic ducts, duodenal mucosa and gastric mucosa and submucosa exhibited intermediate fluorescence with relatively weak fluorescence in pancreatic acinar tissue and the muscle layer of the stomach. As expected, on the basis of fluorescence intensity and chemical extraction studies, the duodenal and bile duct wall were the most vulnerable tissues to photodynamic therapy. When the dose of 5 mumol kg-1 of sensitiser was used, duodenal perforations, gastric ulcers and transudation of bile from the bile duct occurred. However, the lesions in the stomach and bile duct healed without perforation or obstruction, so only the duodenum was at risk of serious, irreversible damage. Using a lower dose of photosensitiser markedly reduced damage.     

Photodynamic therapy of a mouse glioma: intracranial tumours are resistant while subcutaneous tumours are sensitive

Subcutaneous and intracranial VMDk tumours were treated with photodynamic therapy (PDT) using a new sensitiser, m-THPP. Subcutaneous tumours were highly sensitive to PDT but intracranial tumours were much more resistant, requiring a 30-fold increase in sensitiser dose to produce equivalent levels of necrosis. Resistance of intracerebral tumours was not due to failure of the sensitiser to enter tumours. Necrosis of intracranial tumours was increased when mice breathed 100% oxygen during PDT while subcutaneous tumour necrosis was unaffected.