[Spectral properties of hematoporphyrin derivative after interacted with human serum albumin].

The spectral properties of photosensitizer HpD, human serum albumin (HSA) and their complex have been studied. The results show that HpD can form HpD-HSA complex with HSA in physiological condition. Compared with pure HpD, the maximum absorption and the fluorescence peaks for HpD-HSA complex had 8-10 nm red-shift. When HpD-HSA complex was excited by the light of corresponding to excitation peaks of HSA (228 and 279 nm) and HpD (394 nm), it was found that the absorption of HSA and HpD both contributed to the emission of HpD-HSA complex at 622 nm. The complex of HpD-HSA was individually excited by wavelengths corresponding to HpD absorption peaks of 402, 502, 537 and 570 nm, and the excitation efficiency of HpD-HSA at pumping wavelength of 537 and 570 nm was higher than that of HpD. The results demonstrate that the red-shift caused by the interaction of HpD and the special proteins in blood should be considered in selecting the excitation and emission wavelength in photodynamic diagnosis and therapy. The results also indicate that the excitation efficiency of porphyrin-protein complex in vivo is higher than that of HpD in vitro when a longer wavelength light corresponding to the week absorption peaks of porphyrin-protein complex is used in photodynamic therapy.

Determination of endogenous porphyrins and the maximal HpD tumor/normal skin ratio in SKH-1 hairless mice by light induced fluorescence spectroscopy.

The treatment of skin tumors is an application of photochemotherapy (PCT) which involves an initial administration of a photosensitizer (PS) followed by irradiation with a light beam that causes the PS to produce cytotoxic oxygen species within the tumors. As the PS is also present in normal skin, it is necessary to know how it is distributed between the two tissues. In this study, we have used SKH-1 hairless mice bearing papillomas or carcinomas chemically induced. The biodistribution of hematoporphyrin derivative (HpD) and the tissue autofluorescence measurements were studied by light induced fluorescence spectroscopy. The tumor and normal autofluorescence spectra measured on control mice with papillomas or carcinomas had a very similar shape. However, the principal endogenous porphyrin peak at about 630 nm showed a fluorescence signal amplitude 2 (for papilloma) and 1.5 (for carcinoma)-fold higher than the one found for the normal skin. Moreover, the fluorescence intensity of carcinoma spectrum is 1.4-fold lower than the one of papilloma spectrum at 630 nm. The tissue autofluorescence can be used to distinguish tumor from normal skin and benign from malignant tumor. This difference in fluorescence intensity at 630 nm was directly related to the concentration of endogenous porphyrins in the tumor. Fluorescence intensity ratios between tumor and normal skin were measured 4, 8, 24, 48, 72 and 96 hours after intraperitoneal injection of HpD (5 mg/kg body weight). The best tumor/normal skin ratio was 6.2 for HpD and the time required to reach this ratio was 48 h. HpD showed a moderate selectivity since the ratio was higher than 1 during the four first days. Photodynamic therapy with the same dose of HpD used in this biodistribution study must also be carried out to verify that the maximal tumor/skin ratio corresponds to the maximal efficiency of HpD.

A solubilization technique for photosensitizer quantification in ex vivo tissue samples.

The determination of the photosensitizer concentration in ex vivo tissue samples is commonly used for pharmacokinetic and dosimetric studies of photodynamic therapy, both clinically and pre-clinically. In this report, a new method is presented based on tissue solubilization and subsequent fluorometry. This method has the advantages of good sensitivity, accuracy and reproducibility, as well as low cost and ease of handling of the tissue samples. The method was tested for six different photosensitizers in a variety of tissues. The accuracy and concentration detection limits are compared with those of other published extraction methods.

Chromatographic analysis of photodynamically significant porphyrin dimers and trimers.

Two porphyrin dimers, dihematoporphyrin dimer (DHD) and divinyl dimer (DVD), and two porphyrin trimers, dihematoporphyrin trimer (DHT) and divinyl trimer (DVT), have been analyzed utilizing isocratic ion-pair reversed-phase high-performance liquid chromatography. Results indicate that the vinyl porphyrins can be distinguished by three peaks appearing near 15, 38, and 42 min. The hematoporphyrin complexes are identified by the appearance of a peak located at 35 min. The DVT and DVD complexes present unique chromatographic markers at 28 and 15 min, respectively. Based on the location of these chromatographic markers, it was found that the Photofrin drug must contain the DVD and the DHT complexes, but does not contain the DVT complex. The purity of the DVT complex is compromised by the presence of DHD and DHT impurities.

Study of porphyrin fluorescence in tissue samples of tumour-bearing mice.

A time-gated fluorescence-imaging technique was applied to study the distribution of sensitizer in porphyrin-treated tumour-bearing mice. The animals were administered with either haematoporphyrin derivative (HpD) or Photofrin and sacrificed 4 or 12 h later. Fluorescence images were acquired from tumour, skin, muscle, fat, brain, lymph node, bowel and bone of both treated and untreated mice. The results obtained with HpD and Photofrin are similar. In images acquired 30 ns after excitation a bright exogenous fluorescence allows clear detection of the tumour. Nevertheless, the images show that porphyrins localize with different concentrations in all the examined tissues except the brain. Moreover, an appreciable long-living endogenous emission was observed in the bone.

Studies of porphyrin-containing specimens using an optical spectrometer connected to a confocal scanning laser microscope.

A spectrometer has been developed for use with a confocal scanning laser microscope. With this unit, spectral information from a single point or a user-defined region within the microscope specimen can be recorded. A glass prism is used to disperse the spectral components of the recorded light over a linear CCD photodiode array with 256 elements. A regulated cooling unit keeps the detector at 277 K, thereby allowing integration times of up to 60 s. The spectral resolving power, lambda/delta lambda, ranges from 350 at lambda = 400 nm to 100 at lambda = 700 nm. Since the entrance aperture of the spectrometer has the same size as the detector pinhole used during normal confocal scanning, the three-dimensional spatial resolution is equivalent to that of normal confocal scanning. Light from the specimen is deflected to the spectrometer by a solenoid controlled mirror, allowing fast and easy switching between normal confocal scanning and spectrometer readings. With this equipment, studies of rodent liver specimens containing porphyrins have been made. The subcellular localization is of interest for the mechanisms of photodynamic therapy (PDT) of malignant tumours. Spectroscopic detection is necessary to distinguish the porphyrin signal from other fluorescent components in the specimen. Two different substances were administered to the tissue, Photofrin, a haematoporphyrin derivative (HPD) and delta-amino levulinic acid (ALA), a precursor to protoporphyrin IX and haem in the haem cycle. Both are substances under clinical trials for PDT of malignant tumours. Following administration of these compounds to the tissue, the potent photosensitizer and fluorescent compound Photofrin, or protoporphyrin IX, respectively, is accumulated.(ABSTRACT TRUNCATED AT 250 WORDS)

[The distribution of hematoporphyrin derivatives in the animal body]. / Raspredelenie proizvodnykh gematoporfirina v organizme zhivotnykh.

The pharmacokinetic distribution of haematoporphyrin derivative (HPD) and synthetic oligomeric haematoporphyrin (OG) which showed various lipophilicity was studied in animal tumor and normal tissues. The accumulation of dyes in tissue and their tumor-seeking capacity depended on the degree of polymerization, lipophilicity, and ratio of dye components. The difference in the absolute concentrations of HPD and OG (6.9 and 16.1 micrograms per g tissue, respectively) in tumor after dye injection in an adequate concentration (10 mg/kg b. w.) indicates that synthetic OG is beneficial for tumor diagnosis and photodynamic therapy.

Effect of photodynamic therapy on the endothelium-dependent relaxation of isolated rat aortas.

The early vascular effects of photodynamic therapy (PDT) include transient vasoconstriction and platelet aggregation. Since endothelium-derived relaxing factor (EDRF) is a potent vasodilator and inhibitor of platelet aggregation, we questioned whether PDT impairs the production of EDRF. To study this possible effect of PDT, endothelium-dependent relaxations of thoracic aortas obtained from male Wistar rats were determined. The aortic rings were connected to a isometric force transducer, exposed to various doses of Photofrin porfimer sodium (Photofrin) (0.1-1.0 microgram/ml), and illuminated with red light (wavelength > 610 nm, 14.6 +/- 1.5 mW/cm2) for different time periods (5-25 min). Endothelium-dependent relaxation was induced by acetylcholine in precontracted aortic rings. This EDRF-mediated relaxation was decreased after PDT in a light dose- and drug dose-dependent manner. Light microscopic examination did not show loss of endothelial cells. Similar results were obtained with rat aortas exposed to Photofrin in vivo and illuminated in vitro. Direct smooth muscle relaxation induced with sodium nitroprusside was not impaired, showing that PDT did not reduce the ability of smooth muscles to relax. No effect on the contractile responses was found either. We conclude that PDT impairs the production or release of EDRF by the endothelium. This could play an important role in the initial events occurring in vivo during and after PDT.

Assessing Photofrin uptake in atherosclerosis with a fluorescent probe: comparison with photography and tissue measurements.

The purpose of this study was to assess Photofrin porfimer sodium (P*) concentration in atherosclerotic plaque (ASP) using a fluorescence detector (Fluoroprobe) compared with fluorescent photography and chemical extraction of P*. ASP was created in the aortoiliac segments of Yucatan miniswine by a combination of balloon endothelial injury and 2% cholesterol and 15% lard diet for 7 weeks. At that time, swine were given P* I.V. in one of the following single dosages: Group I, 2.5; Group II, 1.0; or Group III, 0.5 mg/kg. Swine were sacrificed 24 hours later and aortoiliac and control carotid artery segments removed. Fluorescence was determined from these segments using photographic techniques, the Fluoroprobe, and a spectrofluorometer after chemical extraction. ASP were identified in all swine using photography and the Fluoroprobe. The intensity of fluorescence measured with the Fluoroprobe for Groups I to III was 1,098 +/- 524, 471 +/- 337, and 295 +/- 173 units, respectively (P < 0.01). The tissue concentration of P* in ASP from each group was 130.4 +/- 82.7, 10.0 +/- 1.2, and 9.1 +/- 0.6 ng/g, respectively (P < 0.01). There was a linear correlation between the fluorescence intensity measured with the Fluoroprobe and the extracted tissue concentration (r = 0.88, P < 0.0001). This study showed that a fluorescent detector such as the Fluoroprobe accurately detects the uptake of P* into atherosclerotic plaque.