Malcolm S. McPhee

UPTAKE KINETICS AND INTRACELLULAR LOCALIZATION OF HYPOCRELLIN PHOTOSENSITIZERS FOR PHOTODYNAMIC THERAPY: A CONFOCAL MICROSCOPY STUDY

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The intracellular localization and uptake kinetics of hypocrellin B and several selected hypocrellin congeners were determined semiquantitatively by fluorescence confocal microscopy in monolayer cultures of EMT6/Ed murine tumor cells. Each compound had unique uptake kinetics. Although no compound tested to date has demonstrated nuclear labeling, most could be detected in lysosomes, Golgi, endoplasmic reticulum and, to a minor extent, in cellular membranes. No two compounds gave identical labeling distributions. The differences are assumed to originate in physicochemical properties characteristic of each compound, which may ultimately impact upon the primary modality of phototoxicity.

Analysis of tissue optical coefficients using an approximate equation valid for comparable absorption and scattering

New photosensitizers activated by longer wavelengths than 630 nm light used with Photofrin II are under evaluation by various groups for the treatment of malignancies. Any increase in tumour volume destroyed by these agents as compared to Photofrin II will be partly determined by tissue penetrance at the longer wavelengths. Attenuation coefficients were measured for various tissues at 630 nm and the more penetrative near infrared wavelength of 789 nm. A new model of light propagation in tissue is shown to be accurate for arbitrary ratios of absorption and scattering, by comparison with a rigorous solution to the transport equation. Absorption and transport scattering coefficients of tissues at 630 and 789 nm were obtained by fitting this model to optical attenuation measurements. In vitro tissues included bovine heart, kidney and tongue, pig liver and fat, and chicken muscle; in vivo tissues included Dunning R3327-AT and R3327-H tumours. The penetration depth was found to be 1.35-2.25 times greater at 789 than 630 nm, depending on tissue type. The greatest differences in penetration between the two wavelengths were in the highly pigmented tissues. These substantial increases in penetration in the infrared may be important in future applications of photodynamic therapy.

Hypocrellins as photosensitizers for photodynamic therapy : a screening evaluation and pharmacokinetic study

Abstract Hypocrellin compounds were selected as potential photosensitizers for photodynamic therapy (PDT) owing to their high quantum yields of singlet oxygen (1O2), and facility for site-directed chemical modification to enhance phototoxicity, pharmacokinetics, solubility, and light absorption in the red spectral region, among other properties. Parent hypocrellins A and B share an absorption peak at 658 nm. These molecules may therefore be considered useful progenitors of derivatives which absorb more strongly in the red, considering that the ideal sensitizer should absorb in the 650–800 nm range, beyond the absorption range of hemoglobin and melanin, and where light penetration in tissues is maximized through reduced scattering. A series of pure, monomeric hypocrellin derivatives was tested for properties of dark cytotoxicity and photosensitizing potential by clonogenic assay in monolayer cultures of EMT6/Ed murine tumor cells. Their respective toxicities are reported on a molar basis. The in vitro screening assay has, to date, resulted in the selection of four hypocrellin derivatives for further development as photosensitizers for PDT. Cellular uptake for photosensitizing doses of selected compounds was determined by fluorimetry. Dose escalation studies in rodents indicate that potentially photosensitizing doses promote no demonstrable systemic toxicity.

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 intertitial 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 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 T1.9 × 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.

Optical dosimetry for interstitial photodynamic therapy

An approach to photodynamic treatment of tumors is the interstitial implantation of fiber optic light sources. Dosimetry is critical in identifying regions of low light intensity in the tumor which may prevent tumor cure. We describe a numerical technique for calculating light distributions within tumors, from multiple fiber optic sources. The method was tested using four translucent plastic needles, which were placed in a 0.94 X 0.94 cm grid pattern within excised Dunning R3327-AT rat prostate tumors. A cylindrical diffusing fiber tip, illuminated by 630 nm dye laser light was placed within one needle and a miniature light detector was placed within another. The average penetration depth in the tumor region between the two needles was calculated from the optical power measured by the detector, using a modified diffusion theory. Repeating the procedure for each pair of needles revealed significant variations in penetration depth within individual tumors. Average values of penetration depth, absorption coefficient, scattering coefficient, and mean scattering cosine were 0.282 cm, 0.469 cm-1, 250 cm-1 and 0.964, respectively. Calculated light distributions from four cylindrical sources in tumors gave reasonable agreement with direct light measurements using fiber optic probes.

PHOTODYNAMIC THERAPY‐INDUCED HYPOXIA IN RAT TUMORS and NORMAL TISSUES

Abstract The administration of misonidazole (MISO) to Fischer x Copenhagen rats whose R3327‐H prostate tumors were treated with photodynamic therapy (PDT) produced enhanced tumor growth delays and cures. This potentiation of PDT by MISO was previously observed with R3327‐AT tumors and was postulated to result from drug cytotoxicity of naturally‐occurring and PDT‐induced hypoxic cells. Radioactively‐labelled MISO has been developed as a marker for tissue p02 at the cellular level and [3H]MISO was administered to R3327‐AT and R3327‐H tumor‐bearing rats before and after standard PDT treatments. The amount of 3H in tissues 24 h after drug administration was a measure of‘bound MISO’which reflects average tissue oxygenation. [3H]MISO retained in R3327‐AT tumors was ˜4x and in liver tissue ˜2x that retained in muscle, heart, brain and R3327‐H tumors (1x). Tumors treated with Photofrin II and lased with 1000 J showed a 6‐fold increase in retained [3H]MISO in R3327‐H tumors and a 2‐fold increase in retained [3H]MISO in R3327‐AT tumors. The absolute levels of retained 3H in both tumors after PDT were similar. These data provide direct evidence that PDT induces rapid hypoxia in both tumors. When the gastrocnemius muscle of the rat leg was similarly treated, the amount of [3H]MISO retained was ˜4x greater than that in untreated muscle. This result suggests that PDT‐induced hypoxia is not selective to just tumor tissue. These data suggest that the hypoxia‐inducing property of PDT might be exploited in combination with hypoxic cell cytotoxins to produce improved tumor responses and cures.

In vivo light transmission spectra in EMT6/Ed murine tumors and Dunning R3327 rat prostate tumors during photodynamic therapy

Variations in the optical coefficients in tissue and the photosensitizer during photodynamic therapy (PDT) will require adjustment of the light dose during the course of therapy. We have studied the dynamics using light transmission spectra for two different tumor models when tetrasulfonated aluminum phthalocyanine (AlPcS4) was used as photosensitizer.

Photodynamic therapy dosimetry in postmortem and in vivo rat tumors and an optical phantom

Dosimetry in photodynamic therapy as currently practiced is empirical in that it does not account for optical properties of the target lesion. However, since light attenuation in tissue is unpredictable, measurements of optical properties are needed to ensure optimal light dose delivery. Further improvements in the uniformity of light dose distribution in tumors can be afforded by implanting multiple light sources. A technique is described in which the use of multiple cylindrical sources was combined with measurements of light energy fluence rate in the tumor. Six sources were placed within translucent plastic needles, which were inserted into tumors in a parallel array. Tumor attenuation characteristics were measured by placing a miniature light detector in one needle, while illuminating a cylindrical source in another, nearby, needle. This process was repeated for different needle pairs. In one postmortem and two in vivo tumors the absorption coefficient, transport scattering coefficient and penetration depth ranged from 0.56–0.81 cm 1, 9.4–15.2 cm 1 and 1.7–2.3 mm. respectively. Apparent penetration depths for in vivo tumors changed with time, during experiments. Predictions of dosimetry were generally consistent with direct measurements of light in tumors. Somewhat better agreement was observed in an optical phantom.

Intracellular uptake kinetics of hypocrellin photosensitizers for photodynamic therapy

Hypocrellins are naturally occurring compounds with photosensitizing properties in biological systems. We have prepared synthetic derivatives of hypocrellin B, which have promise as photosensitizers in the clinical application of photodynamic therapy. The inherent fluorescence of four selected compounds has provided a means of determining their uptake and distribution in monolayer cultures of EMT6/Ed murine tumor cells, via fluorescence confocal microscopy.

Anisotropy of radiance in tissue phantoms and Dunning R3327 rat tumors: Radiance measurements with flat cleaved fiber probes

The goal of this study is to determine if flat cleaved fiber probes are appropriate for interstitial measurements of radiance in tissue. Flat cleaved probes have the advantage of high responsivity, and they are easy to insert into tissue. Owing to the non‐isotropic response of flat cleaved probes, a calibration function is required, taking the anisotropy in the radiance in tissue into account.