Tolyporphin: a natural product from cyanobacteria with potent photosensitizing activity against tumor cells in vitro and in vivo.

Tolyporphin (TP), a porphyrin extracted from cyanobacteria, was found to be a very potent photosensitizer of EMT-6 tumor cells grown both in vitro as suspensions or monolayers and in vivo in tumors implanted on the backs of C.B17/Icr severe combined immunodeficient mice. Thus, during photodynamic treatment (PDT) of EMT-6 tumor cells in vitro, the photokilling effectiveness of TP measured as the product of the reciprocal of D50 (the light dose necessary to kill 50% of cells) and the concentration of TP is approximately 5000 times higher than that of Photofrin II (PII), the only PDT photosensitizer thus far approved for clinical trials. TP almost exclusively localizes in the perinuclear region and specifically in the endoplasmic reticulum (ER), as shown by microspectrofluorometry on single living EMT-6 cells costained with the ER and/or Golgi fluorescent vital probes, 3,3'-dihexyloxacarbocyanine iodide and N-[4,4-difluoro-(5,7-dimethyl-BODIPY)-1-pentanoyl]-D-erythro-sphin gosine (Molecular Probes, Eugene, OR). As a result, the singlet oxygen-mediated photodynamic activity of TP induces an effective inactivation of the acyl CoA:cholesterol-O-acyltransferase, a sensitive marker of ER membrane integrity and alterations of the nuclear membrane. In vivo, with the EMT-6 mouse tumor model, an exceptional effectiveness is also observed as compared to that of PII and other second generation photosensitizers of the pheophorbide class, which are themselves much more potent than PII. The outstanding PDT activity of TP observed in vivo may be due to its unique biodistribution properties, in particular much less extraction by the liver, resulting in a higher delivery to other tissues, including tumor.

Prediction of tumour hypoxia and radioresistance with nuclear medicine markers.

Second-generation nuclear medicine markers of tumour hypoxia have been synthesised and screened for hypoxic marking activity in cell cultures and in mouse tumours (EMT-6). Markers of the iodinated azomycin nucleoside class with greater water solubility and faster plasma clearance rates relative to iodoazomycin arabinoside (IAZA) were of particular interest. The test systems used to characterise hypoxic marking activity of compounds included (1) covalent linkage of radiolabelled markers to cells in suspension culture equilibrated with specific O2 concentrations; (2) biodistribution of radiolabelled markers in EMT-6 tumour-bearing mice; and (3) biodistribution in R3327-AT tumour-bearing rats by nuclear medicine procedures. Of the iodinated azomycin nucleosides produced to date, beta-D-iodoazomycin galactoside (beta-D-IAZG) and beta-D-iodoazomycin xylopyranoside (beta-D-IAZXP) exhibited high metabolism-dependent hypoxic cell uptake, rapid clearance kinetics from the blood and excellent tumour marking activity in vivo. Tumour-blood (T/B) ratio (a measure of tumour hypoxic fraction) was dependent upon EMT-6 tumour size and implantation site. The radioresistance of individual tumours was measured by in vivo/in vitro assay and correlated well with the T/B ratio of hypoxic marker. These studies have identified beta-D-IAZG and beta-D-IAZXP as effective hypoxic markers for planar and single photon emission computerised tomography (SPECT) imaging studies of tumour oxygenation.

Uptake by cells and photosensitizing effectiveness of novel pheophorbide derivatives in vitro.

Pheophorbide a prepared from the algae Spirulina was derivatized at the C(7)-carboxylic group by linking amino alkyls of various lengths and terminal functional groups. The compounds were purified by thin-layer chromatography (TLC) and by high-pressure liquid chromatography (HPLC). Solubilization of compounds by serum lipoproteins, the kinetics of compound uptake into mammalian cells, and photosensitizing effectiveness when activated by 673 nm laser light have been studied. Optimal photosensitizer uptake into cells and the greatest photosensitizing activity were observed with compounds having side-chain lengths of 4-6 carbon atoms which terminated in -OH and -CH3 groups. The most effective compounds were 3 orders of magnitude more potent than Photofrin in the degree of photoinactivation of cultured EMT-6 tumor cells. HDL and LDL significantly promoted the efflux of these photosensitizing drugs from cells, suggesting that their long-term retention in normal tissues in vivo would be minimal and produce little phototoxicity.

Polarographic needle electrode measurements of oxygen in rat prostate carcinomas: accuracy and reproducibility.

PURPOSE: The oxygenation status of tumors may be important for predicting tumor response to therapy. Previous studies with the anaplastic (R3327-AT) and well-differentiated (R3327-H) Dunning rat prostate tumors using indirect assays of tumor oxygenation indicated the relative hypoxic and radioresistant nature of the anaplastic tumor. We now report direct measurements of oxygen in these tumors made with the pO2 histograph to determine: (a) whether a significant difference in oxygenation status could be detected between them: (b) whether sequential measurements on the same tumor gave similar values; and (c) whether tumor oxygenation correlated with tumor volume. METHODS AND MATERIALS: R3327-AT and R3327-H tumors were grown in Fischer X Copenhagen rat to volumes of 1.0-7.0 cm3. Electrode measurements (100-200) were made in tumors in anesthetized animals along two parallel tracks. Repeat measurements were made at 1-5 days along different parallel tracks. Oxygen partial pressures of muscle tissue were measured and served as a normal tissue control. Statistical analyses were applied to determine whether tumor oxygen levels were different between the two tumor histologies, whether sequential measurements in the same tumor were reproducible, and whether tumor oxygenation correlated with tumor volume. RESULTS: The average median pO2 of the well-differentiated (n = 15) and the anaplastic (n = 15) tumors was 6.0 mmHg (SE +/- 1.3) and 2.2 mmHg (SE +/- 0.3), respectively. The average median pO2 of normal rat muscle (n = 15) was 23.6 mmHg (SE +/- 2.0). These values represent highly significant differences in oxygen concentration between the two tumors and rat muscle. The differences in average mean pO2 values were also highly significant. Repeat measurements in the same tumors on different days gave average median values of 4.7 and 2.2 mmHg in the R3327-H (n = 15) and R3327-AT (n = 15) tumors, respectively. For these repeat measurements, median pO2 values decreased in 15 and increased in 15 tumors, and were not significantly different from the first measurements. The average differences observed in median pO2 were 37% (SE +/- 7) and 58% (SE +/- 10) for the R3327-H and R3327-AT tumors, respectively. No significant correlation was observed between pO2 levels and the tumor volumes investigated in this study. CONCLUSIONS: The median pO2 values of the anaplastic Dunning tumors were significantly lower than those of the well-differentiated tumors (p < 0.001). Oxygen levels in both tumors were significantly lower than those measured in normal rat muscle (p < 0.00005). Repeat measurements of median pO2 in the same tumors were not significantly different for either tumor model (p > 0.5). The changes observed in pO2 distributions within individual tumors from day to day may indicate true dynamics of its oxygenation status and/or the limits of electrode measurements, by sampling along only two insertion sites. The electrode measurements of pO2 in these tumor models are reproducible and confirm previously detected oxygenation differences between the anaplastic and well-differentiated tumors.

Photodosimetry of interstitial light delivery to solid tumors.

Both anaplastic and well-differentiated Dunning prostate adenocarcinomas were illuminated in anesthetized Fischer X Copenhagen rats by single-fiber and multiple-fiber illuminators. Each illuminator consisted of a 2-cm laterally diffusing optical fiber placed within a plastic brachytherapy needle which was implanted into a tumor. Light attenuation coefficients for various wavelengths were obtained from measures of the radial falloff of intensity with distance from single fibers. These coefficients served as input to a 2-dimensional (2-D) photodosimetry computer code which calculated relative light intensities in planes perpendicular to single-fiber and various multiple-fiber configurations. These calculations assumed uniform optical property of tissue throughout each tumor, uniform and equal illuminance from diffusing fibers, and precise needle implantation. Relative light intensities along specific tumor tracks were measured and compared with those predicted by the 2-D photodosimetry code. Agreement within +/- 14% was observed for all configurations studied. Variations in relative intensity in tumor planes perpendicular to a standard seven-fiber illuminator were determined as a function of the distance between the implanted needles. Light wavelengths of 700 nm and greater produced relatively uniform light fields (approximately +/- 20%) in the R3327-AT tumor with needle spacings of at least 1 cm. The addition of two fibers at the periphery of this illuminator (a nine-fiber illuminator) improved the uniformity of light delivery to the encompassed tumor volumes. The importance of precision photodosimetry for interstitial applications of photodynamic therapy is discussed.

Optical properties of experimental prostate tumors in vivo.

The optical properties of tumor tissue provide important information for optimizing treatment plans in photodynamic therapy, especially when interstitial application by multiple fibers is planned. Near infrared light, required to activate novel photosensitizers, should facilitate improved light penetrance of tumor tissue compared with 630 nm light used for activating Photofrin II. We have measured light energy fluence rates for 630 and 789 nm light along radial tracks from a single laterally diffusing optical fiber centrally implanted into Dunning R3327-AT and R3327-H rat prostate tumors in anesthetized rats. A total of 20 R3327-AT and 10 R3327-H tumors were used in this study with volumes from 2.6 to 13.3 cm3. Light track data were analyzed by an empirical model that described light attenuation. At 630 nm, light attenuation coefficients (LAC) were approximately 1.9 x higher than those at 789 nm for both tumors with the well-differentiated, well-perfused tumor (R3327-H) attenuating to a greater extent than did the rapidly growing anaplastic tumor (R3327-AT). The intertumor variation of LAC was greater than the spatial variations observed within individual tumors. LAC were a function of tumor volume for only 630 nm light in the R3327-AT tumors.