pheophorbide-a and Pancreatic-Neoplasms

The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model.. Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis.. PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session.. PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.

Topics: Animals; Apoptosis; Cell Division; Chlorophyll; Electrophoresis; Humans; Male; Mice; Mice, Nude; Pancreatic Neoplasms; Photochemotherapy; Porphyrins; Radiation-Sensitizing Agents; Tumor Cells, Cultured

Laser-induced fluorescence (LIF) of pheophorbide-a (Ph-a) was used for imaging of a rat pancreatic tumor. Using a dimensionless function (the ratio of Ph-a fluorescence by bluish autofluorescence), the fluorescence contrasts between excised tumors and their paired pancreas were investigated up to 48 h after a 9 mg kg-1 Ph-a intravenous administration. Among five tested excitation wavelengths, 355 and 610 nm excitations gave the best distinctive contrasts, both 48 h after dye injection. The LIF imaging of six intrapancreatic tumors and six healthy pancreas was carried out in vivo using two laser excitations: 355 nm (Nd:YAG + tripling) for bluish autofluorescence and 610 nm (rhodamine 6G dye) for reddish autofluorescence and dye emission. Images were recorded through bandpass filters at 470 and 640 nm (autofluorescence) and at 680 nm (dye + autofluorescence) with an intensified charged-coupled device camera. Autofluorescence as Ph-a fluorescence images did not allow accurate LIF diagnosis of pancreatic carcinoma. An image processing, including for each pixel a computed division of Ph-a fluorescence (after subtraction of reddish autofluorescence) by bluish autofluorescence intensity generated poorly contrasted tumor images in five of six and false tumor localization in one of three of the tumor-bearing pancreas. A fitting of the digital 640 nm autofluorescence up to the mean 680 nm fluorescence intensity in pancreas prior to subtraction allowed a safe diagnosis to be made with well-contrasted tumor images. To assess automation ability of the processing, a same fitting coefficient (mean of individual values) was applied. In this way, false-negative (one of six) and false-positive (two of six) images were present in tumor-bearing animals as false-positive in one-half of the controls. A successful standardized procedure was then applied with a normalization of 640 and 680 nm pancreas intensities to a same set threshold prior processing. In opposition to thin-layered hollow organs, such as bronchial tube or digestive tract, LIF imaging of carcinoma inserted in a compact organ is exhausting. The use of a dye excitable in the red wavelength range (610 nm for Ph-a) may partly solve this problem, rendering LIF imaging more accurate and potentially automated.

Topics: Animals; Chlorophyll; Fluorescence; Lasers; Pancreas; Pancreatic Neoplasms; Radiation-Sensitizing Agents; Rats; Spectrometry, Fluorescence

Laser-induced fluorescence of pheophorbide a (Ph-a) was used for in vitro photodynamic imaging (PDI) of a rat pancreatic acinar tumor. A 400 nm excitation induced a 470 nm autofluorescence and a 678 nm dye fluorescence in tumors and their surrounding pancreas 24 h after a 9 mg kg-1 body weight Ph-a intravenous administration. With lower intensities in these blood-rich tumors than in pancreas, Ph-a fluorescence signals are unable to provide tumor images. A dimensionless function (the ratio of Ph-a fluorescence by autofluorescence, called Rt for the tumor and Rp for the pancreas) was used for fluorescence contrast calculation (C = Rt/Rp) between six tumors and their paired pancreas. Among five available laser excitation wave-lengths, only the 355 nm excitation gave a distinctive contrast (C = 1.5). The PDI of six intrapancreatic tumors and their intraperitoneal metastasis and of two control normal pancreas was thus performed ex vivo using a 355 nm excitation source delivered by a tripled Nd:YAG laser and a charged-coupled device camera. Fluorescence images were recorded at 680 nm (dye), 640 nm (background) and 470 nm (autofluorescence) through three corresponding 10 nm width bandpass filters. Computed division for each pixel of Ph-a fluorescence values by autofluorescence generated false color image. In this way, contrasted tumor images were obtained. But in five out of six animals false-positive images were present due to an autofluorescence decrease in some normal pancreatic areas. A 470 nm autofluorescence imaging on the same tumors gave in all cases false-positive image and false-negative in half of the cases. These observations suggest that autofluorescence alone is unable to achieve accurate PDI of pancreatic carcinoma and that using Ph-a as a PDI dye needs strong improvements.

Topics: Animals; Chlorophyll; Evaluation Studies as Topic; Fluorescence; Image Processing, Computer-Assisted; Lasers; Pancreatic Neoplasms; Photosensitizing Agents; Rats; Rats, Inbred Lew

Selective histological necrosis of experimental pancreatic carcinoma by photodynamic therapy (PDT) has been successful with haematoporphyrin derivatives and phthalocyanine as photosensitizers. This report describes the feasibility of PDT with pheophorbide A as the photosensitizer to treat azaserine-induced pancreatic rat carcinoma and analyses survival of the animals. An organ distribution study 24 h after pheophorbide A administration (9 mg/kg intravenously) gave a selectivity ratio of 13.5:1 between tumour and surrounding tissue. Light of 660 nm and 100 J/cm2 induced selective necrosis of the tumour. Six of nine rats were cured in 120 days whereas all 36 control animals died within 35 days (P < 0.01). The pancrease and hepatic pedicle were relatively unaffected by PDT, but the duodenum was injured.

Topics: Adenocarcinoma; Animals; Chlorophyll; Duodenum; Necrosis; Pancreas; Pancreatic Neoplasms; Photochemotherapy; Radiation-Sensitizing Agents; Rats; Rats, Inbred Lew; Survival Analysis; Time Factors

The in vivo administration and distribution of a potent new photosensitizer, pheophorbide A (PH-A), was investigated in rats. The spectral characteristics were determined. This hydrophobic compound was solubilized by an ethanol/phosphate-buffered saline (PBS) mixture (v/v) and sonicated immediately before i.v. administration. Tissue distribution and the affinity of PH-A for an acinar pancreatic tumor were determined in Lewis rats for up to 48 h after a single i.v. administration of 3 mg kg-1 body wt. Methanol-extracted PH-A was quantitatively determined by fluorescence spectrophotometry at 665.6 nm. The PH-A uptake pattern showed that the reticulo-endothelial system is the major target of PH-A, followed by the gut and then the lung and pancreas. PH-A concentrations in skin were very low. The presence of an enterohepatic cycle was suggested by the PH-A biliary output, intestinal uptake and blood concentrations. Tumor PH-A retention was longer than pancreatic retention. The ratio of tumoral to peri-tumoral pancreas PH-A was 6.7:1, 24 h after i.v. administration. With its similar tissue pattern, better absorption spectrum and lower skin toxicity, PH-A could be a more potent photosensitizer than hematoporphyrin derivatives.

Topics: Animals; Chlorophyll; Molecular Structure; Pancreas; Pancreatic Neoplasms; Photochemotherapy; Photosensitizing Agents; Porphyrins; Rats; Rats, Inbred Lew; Spectrometry, Fluorescence; Tissue Distribution