Photodynamic therapy on the normal rabbit larynx with phthalocyanine and 5-aminolaevulinic acid induced protoporphyrin IX photosensitisation.

Photodynamic therapy (PDT) is a promising technique for the treatment of small tumours in organs where it is essential to minimise damage to immediately adjacent normal tissue as PDT damage to many tissues heals by regeneration rather than scarring. As preservation of function is one of the main aims of treating laryngeal tumours, this project studied the effects of PDT on the normal rabbit larynx with two photosensitisers, endogenous protoporphyrin IX (PPIX) induced by the administration of 5-aminolaevulinic acid (ALA) and disulphonated aluminium phthalocyanine (AIS2Pc). The main aims of the study were to examine the distribution of protoporphyrin IX and AIS2Pc by fluorescence microscopy in the different regions of the larnyx and to assess the nature and subsequent healing of PDT damage. Peak levels of PPIX were found 0.5-4 h after administration of ALA (depending on dose) with highest levels in the epithelium of the mucosa. With 100 mg kg-1, PDT necrosis was limited to the mucosa, whereas with 200 mg kg-1 necrosis extended to the muscle. With 1 mg kg-1 AIS2Pc, 1 h after administration, the drug was mainly in the submucosa and muscle, whereas after 24 h, it was predominantly in the mucosa. PDT at 1 h caused deep necrosis whereas at 24 h it was limited to the mucosa. All mucosal necrosis healed by regeneration whereas deeper effects left some fibrosis. No damage to cartilage was seen in any of the animals studied. The results of this study have shown that both photosensitisers are suitable for treating mucosal lesions of the larynx, but that for both it is important to optimise the drug dose and time interval between drug and light to avoid unacceptable changes in normal areas

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

Enhancement of photodynamic therapy with 5-aminolaevulinic acid-induced porphyrin photosensitisation in normal rat colon by threshold and light fractionation studies.

5-Aminolaevulinic acid (ALA)-induced prophyrin photosensitisation is an attractive option for photodynamic therapy (PDT) since skin photosensitivity is limited to 1-2 days. However, early clinical results on colon tumours using the maximum tolerated oral dose of 60 mg kg-1 showed only superficial necrosis, presumably owing to insufficient intratumoral porphyrin levels, although inadequate light dosimetry may also be a factor. We undertook experiments using ALA, 25-400 mg kg-1 intravenously, to establish the threshold doses required for a PDT effect. Laser light at 630 nm (100 mW, 10-200 J) was delivered to a single site in the colon of photosensitised normal Wistar rats at laparotomy. The animals were killed 3 days later and the area of PDT-induced necrosis measured. No lesion was seen with 25 mg kg-1. The lesion size increased with larger ALA doses and with the light dose but little benefit was seen from increasing the ALA dose above 200 mg kg-1 or the light dose above 100 J. Thus there is a fairly narrow window for optimum doses of drug and light. Further experiments showed that the PDT effect can be markedly enhanced by fractionating the light dose. A series of animals was sensitized with 200 mg kg-1 ALA and then treated with 25 J. With continuous irradiation, the lesion area was 13 mm2, but with a single interruption of 150 s the area rose to 94 mm2 with the same total energy. Results were basically similar for different intervals between fractions (10-900 s) and different numbers of fractions (2-25). This suggests that a single short interruption in the light irradiation may dramatically reduce the net light dose required to achieve extensive necrosis

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 of the normal rat stomach: a comparative study between di-sulphonated aluminium phthalocyanine and 5-aminolaevulinic acid.

Dysplasia in the upper gastrointestinal tract carries a risk of invasive malignant change. Surgical excision of the affected organ is the only treatment available. Photodynamic therapy has been shown to be promising in the treatment of early and superficial tumours and may be useful for the ablation of dysplastic mucosa. Because of the diffuse nature of the disease, such treatment would necessarily involve destruction of large areas of mucosa and it is desirable to confine its effect to the mucosa in order that safe healing can take place. By means of photometric fluorescence microscopy, we have studied the pattern of photosensitisation in the normal rat stomach using di-sulphonated aluminium phthalocyanine (AlS2Pc) and 5-aminolaevulinic acid (ALA) as photosensitisizers. AlS2Pc resulted in a panmural photosensitisation of the gastric wall with the highest level encountered in the submucosa. The mucosa and muscularis propria were sensitised to equal extent. Following light exposure, a full thickness damage resulted. ALA is a natural porphyrin precursor and exogenous administration gave rise to accumulation of protoporphyrin IX (PPIX) in the cells. The resultant pattern of photosensitisation was predominantly mucosal and its photodynamic effect was essentially confined to the mucosa. ALA produced a selective photosensitisation of the gastric mucosa for its photodynamic ablation with sparing the underlying tissue layers

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

Interstitial laser hyperthermia: a new approach for treating liver metastases.

The palliative management of hepatic metastases remains unsatisfactory. There is a need for a simple non invasive technique which can stop or retard the rate of tumour growth. In principle, Interstitial Laser hyperthermia may fulfil such a role. In experimental studies, this technique produced precise in situ necrosis within solid organs which healed safely. In a pilot feasibility study, we treated ten patients with a total of 18 hepatic metastases on 31 occasions using a percutaneous approach to achieve an overall objective response rate of 44%. The treatment proved simple to perform, was well tolerated and produced radiological evidence of necrosis in small metastases (diameter less than or equal to 3 cm). However, further research is required before the technique can be regarded as established. Its future role in most cases will be to control the growth of discrete hepatic metastases unsuitable for resection. In instances where the extent of necrosis can be matched accurately to tumour volume, the potential for cure exists

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

Photodynamic therapy with phthalocyanine sensitisation: quantitative studies in a transplantable rat fibrosarcoma.

Photodynamic therapy (PDT) is a promising approach to the local destruction of malignant tumours, but little work has been done to determine which factors control the extent of tissue necrosis produced. Using a new photosensitiser, a sulphonated aluminium phthalocyanine (AlSPc) and light from an argon ion pumped dye laser at 675 nm, we quantified the effects of interstitial PDT in a transplantable fibrosarcoma in rats. At 100mW laser power, thermal effects were comparable to those of PDT, so subsequent studies were carried out at 50 mW, where thermal effects were minimal. The depth of PDT necrosis increased with the logarithm of the applied energy. Tissue concentration of AlSPc was measured by alkali extraction and at all times after sensitisation, correlated well with the necrosis produced with a given light dose. Peak tumour concentration of AlSPc occurred 24-48 h after sensitisation compared with a peak at 3 h in muscle. The peak ratio tumour:muscle was 2:1 at 24 h. Apart from a different time interval to reach the peak sensitiser concentration, the extent of tumour damage varied with the light and sensitiser parameters in a similar way to that found in normal liver, although the optical penetration depth was greater in the tumour (2.5 mm vs. 1.8 mm). At doses of AlSPc below 1 mg kg-1 the diameter of necrosis increased with the logarithm of the dose of sensitiser, and doubling the dose from 0.25 to 0.5 mg kg-1 increased the depth of necrosis by 50%. However, at higher doses, the changes were smaller and increasing the dose from 2.5 to 5 mg kg-1 only increased the necrosis by 10% for the same light dose. In all dose ranges, a given percentage increase in the tissue concentration of AlSPc gave a much smaller percentage increase in the extent of necrosis for the same light dose, suggesting that selectivity of necrosis between tumour and normal tissue is likely to be much less than the selectivity of retention of the photosensitiser. From these results, the extent of PDT necrosis in this fibrosarcoma is as predictable as it is in normal liver if the light dose, tissue concentration of AlSPc and optical penetration depth of the tissue are known. Further studies are now required on different tumour models to establish how tumours respond compared with adjacent normal tissue when the tumour is growing in its organ of origin rather than the non-physiological situation using a transplantable tumour as in this study