S. G. Bown

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.

Fluorescence distribution and photodynamic effect of ALA-induced PP IX in the DMH rat colonic tumour model.

Aminolaevulinic acid (ALA) is the first committed step in haem synthesis. In the presence of excess ALA the natural regulatory feedback system is disrupted allowing accumulation of protoporphyrin IX (PP IX) the last intermediate product before haem, and an effective sensitiser. This method of endogenous photosensitisation of cells has been exploited for photodynamic therapy (PDT). We have studied the fluorescence distribution and biological effect of induced PP IX in normal and tumour tissue in the rat colon. Fluorescence in normal colonic tissue was at a peak of 4 h with a rapid fall off by 6 h. The fluorescence had returned to background levels by 24 h. All normal tissue layers followed the same fluorescence profile but the mucosa showed fluorescent levels six times higher than the submucosa, with muscle barely above background values. At 6 h the ratio of fluorescence levels between normal mucosa and viable tumour was approximately 1:6. At this time laser treatment showed necrosis of normal mucosa and tumour with sparing of normal muscle. There was good correlation between the fluorescence distribution and the biological effect of ALA-induced photosensitisation on exposure to red light. ALA may be superior to conventional sensitisers for tumours that produce haem as the PP IX is synthesised in malignant cells while the other sensitisers mainly localise to the vascular stroma of tumours. There is also a greater concentration difference between the PP IX levels in tumours and in normal mucosa and normal muscle than with the other photosensitisers raising the possibility of more selective necrosis in tumours.

NATURE OF THE BLEEDING VESSEL IN RECURRENTLY BLEEDING GASTRIC-ULCERS

An unselected consecutive series of 826 patients admitted for acute upper gastrointestinal bleeding underwent urgent endoscopy. Peptic ulcers were found in 402 (49%). Of the 329 ulcer craters that could be fully examined, visible vessels were identified in 156 (47%), other stigmata of recent hemorrhage in 66, and no stigmata of recent hemorrhage in 107. One hundred twenty-nine patients with stigmata of recent hemorrhage (93 of whom had visible vessels) randomly allocated to no endoscopic treatment were observed for evidence of further bleeding. Fifty-four of the 93 patients (58%) with visible vessels rebled, compared with 2 of 36 (6%) with other stigmata of recent hemorrhage. No patient without stigmata of recent hemorrhage rebled. Twenty-seven patients in whom a visible vessel in a gastric ulcer was identified at endoscopy underwent urgent partial gastrectomy because of recurrent bleeding. The vessel identified at endoscopy was found in 26 of 27 resection specimens (96%). The arterial vessel wall protruded above the surface of the ulcer crater in 10 specimens, and clot in continuity with a breach in the vessel wall protruded in a further 10 specimens. Postoperative angiography, when technically possible, showed that the breached artery ran across the base of the ulcer in all of these specimens. Pathological changes were common in the bleeding artery and included arteritis in 24 of 29 (83%) eroded arteries found in these specimens, with aneurysmal dilatation in 14 of 27 (52%) bleeding points that could be fully examined. The ulcer had penetrated to serosa in 13 specimens (45%). The bleeding artery had a mean external diameter of 0.7 mm with a range of 0.1-1.8 mm. This study provides new information about the nature of the bleeding vessel in gastric ulcers, and some of this information is relevant in planning studies of endoscopic therapy for bleeding peptic ulcers. It validates the endoscopic identification of a visible vessel, and confirms that such identification has a high predictive value for the development of recurrent hemorrhage.

Biological Effects of Intrahepatic Neodymium:Yttrium-Aluminum-Garnet Laser Photocoagulation in Rats

The effects of low-power neodymium:yttrium-aluminum-garnet laser light delivered intrahepatically in normal rats have been studied. Power settings of 0.5-2.0 W and exposures of 50-2400 s produced well-defined, highly reproducible necrotic lesions of up to 16 mm in diameter. The diameter of the necrotic lesions depended upon both the power setting and total energy delivered. Histologic examination showed coagulative necrosis that healed by granulation with a small residual fibrous nodule by 60 days. Microthermocouples embedded within the liver indicated temperatures of up to 100 degrees C close to the fiber tip for the higher power settings, and temperatures at the periphery of the necrotic zones were consistent with tissue damage by a purely thermal effect. Radiologic examination of photocoagulated lobes of liver in which arterial tree had been filled with a radiopaque polymer demonstrated loss of all small and some large vessels in the treated area. The technique could be used to induce necrosis of intrahepatic and other tumors.

CONTROLLED TRIAL OF Nd-YAG LASER PHOTOCOAGULATION IN BLEEDING PEPTIC ULCERS

The efficacy of Nd-YAG laser photocoagulation in the endoscopic control of haemorrhage from peptic ulcers was shown in a controlled trial. 527 patients admitted consecutively with acute upper gastrointestinal haemorrhage underwent urgent endoscopy. Peptic ulcers were seen in 260. All 138 ulcer patients with stigmata of recent haemorrhage (SRH) accessible to laser therapy were included in the trial (26 inaccessible, 96 no SRH). Patients were stratified into three groups--those with a visible vessel, those with other SRH, and those with clot that could not be washed off before therapy. Laser and control groups were well matched for other factors known to influence prognosis. Overall, 7/70 laser-treated and 27/68 control ulcers rebled (p less than 0.001). Rebleeding occurred from 6/39 treated and 23/43 control ulcers with a visible vessel (p less than 0.001); 0/17 treated and 1/13 ulcers with other SRH (NS); and 1/13 treated and 2/11 control ulcers with overlying clots (NS). 7/70 treated but 24/68 controls required emergency surgery (p less than 0.005). 1 treated patient but 8 control patients died after an episode of rebleeding (p less than 0.05).

CONTROLLED TRIAL OF ARGON LASER PHOTOCOAGULATION IN BLEEDING PEPTIC ULCERS

The efficacy of argon laser photocoagulation in the endoscopic control of haemorrhage from peptic ulcers was tested in a controlled trial at two centres in London. Of 330 patients consecutively admitted with acute upper gastrointestinal tract haemorrhage, 76 were seen at endoscopy to have a peptic ulcer accessible to laser therapy and stigmata of recent haemorrhage. These patients were included in the trial. Identification of the exact source of haemorrhage within the ulcer crater was achieved by use of standard endoscopes with careful washing. Patients were stratified into three groups: ulcers with a visible vessel (52 total, 11 actively spurting); those without a vessel (17); and those with an overlying clot persisting after washing (7). Of the 52 patients with visible vessel, 8 of 24 treated with the laser and 17 of 28 control patients had a further haemorrhage. 7 control patients died after an episode of rebleeding, but no treated patients. Treated and control patients were well matched for other factors known to influence the case fatality rate. This is the first controlled trial to demonstrate a significant reduction in mortality by the use of a non operative treatment in patients with bleeding peptic ulcers.

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.

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.

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.

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.