Analysis of effective molecular diffusion rates for verteporfin in subcutaneous versus orthotopic Dunning prostate tumors

Photosensitizer biodistribution change inside tissue is one of the dominant factors in photodynamic therapy efficacy. In this study, the pharmacokinetics of a benzoporphyrin derivative (BPD), delivered in verteporfin for injection formulation, have been quantified in the rat Dunning prostate tumor MAT-LyLu model, using both subcutaneous and orthotopic sites. Blood plasma sampling indicated that BPD had a bi-exponential metabolic lifetime in vivo, with the two lifetimes being 9.6 min and 8.3 h. The spatial distributions in the tumor were quantified as a function of distance from the perfused blood vessels, using fluorescence histologic images of the tumor. A fluorescent vascular marker was used to obtain locations and shapes of perfused capillaries at a wavelength of emission different from that of BPD and to allow colocalized images to be acquired of vessel and BPD locations. Using the BPD fluorescence images obtained 15 min after intravenous administration, a forward finite-element solution to the diffusion equation was used to predict the drug distribution by matching the fluorescence intensity images observed microscopically. An inverse solver was used to minimize the root mean square error between the image of simulated diffusion and the experimental image, resulting in estimation of the diffusion coefficient of BPD in the tumor models. Effective diffusion coefficients were 0.88 and 1.59 microm2/s for the subcutaneous and orthotopically grown tumors, respectively, indicating that orthotopic tumors have significantly higher vascular extravasation rates as compared with subcutaneous tumors. This analysis supports the hypothesis that leakage rates of the photosensitizer vary considerably. Thus, although varying the time between injection and optical irradiation may be used to vary the targeting between vascular and less vascular areas, the precise time of treatment will depend on the nature of the permeability of the vasculature in the tissue being treated.     

Absorption and scattering imaging of tissue with steady-state second-differential spectral-analysis tomography

A novel approach to reconstructing both the absorption and the scattering properties of a turbid medium simultaneously from steady-state broadband spectral measurements is presented that utilizes second-differential fitting to the water spectrum to estimate the optical path length in tissue. Theoretical and experimental evidence is provided to demonstrate the robust accuracy of the spectroscopy approach and reconstructed absorption images. The steady-state broadband CCD system has the potential to provide accurate chromophore imaging without the technological complexity of time- or frequency-domain systems.     

Rotating sector study of the gas phase photochlorination of 2,2-dichloro-1,1,1-trifluoroethane

The rate constant for the combination of 2,2-dichloro-1,1,1-trifluoroethyl radicals has been measured by applying the rotating sector technique to the gas phase photochlorination of 2,2-dichloro-1,1,1-trifluoroethane at 315°K. The observed value is 6.89 × 10¹² cc/mole.sec. This value is in excellent agreement with measurements by Wampler and Kuntz which yielded a temperature-independent value of 6.6 × 10¹² cc/mole.sec. The measurement by Wampler and Kuntz was determined from the photochemical system (CF₃CCl₃ + C-C₆H₁₂ + hν). The Arrhenius parameters for the reaction CF₃CCl₂· + Cl₂ → CF₃CCl₃ + Cl were found to be given by the expression log k₃ = 12.10 ? 5830/2.3RT (units in mole, cc, and sec). This is a relatively high activation energy for a chlorination reaction and makes the reaction ever slower than the chlorination of chloroform.     

Protoporphyrin IX level correlates with number of mitochondria, but increase in production correlates with tumor cell size

Protoporphyrin IX (PpIX) is produced in cells via the heme synthesis pathway, from the substrate aminolevulinic acid (ALA), and can be used for tumor detection, monitoring or photodynamic therapy. PpIX production varies considerably between tumor cell types, and determining the cell types and methods to optimize production is a central issue in properly utilizing this drug. A panel of eight cancer cell types was examined for PpIX production capacity, including breast, prostate, and brain cancer tumors, and the production varied up to 10-fold among cell types. A positive correlation was seen between mitochondrial content and naturally occurring PpIX prior to ALA administration, but mitochondrial content did not correlate to the yield of PpIX resulting from the addition of ALA. Interestingly, total cell size was positively correlated to the yield of PpIX from ALA administration. Addition of an iron chelator, 1,2-dimethyl-3-hydroxy-4-pyridone (L1) in combination with ALA allows the final step in the heme synthesis pathway, conversion of PpIX to heme, to be delayed, thereby further increasing the yield of PpIX. Those cell types that had the lowest ALA to PpIX production without L1 showed the largest percentage increase in production with L1. The study indicates that use of L1 in tumors with a lower innate production of PpIX with ALA alone may be the most productive approach to this combined delivery.     

Assessment of photosensitizer dosimetry and tissue damage assay for photodynamic therapy in advanced-stage tumors

Photodynamic therapy (PDT) efficacy is a complex function of tissue sensitivity, photosensitizer (PS) uptake, tissue oxygen concentration, delivered light dose and some other parameters. To better understand the mechanisms and optimization of PDT treatment, we assessed two techniques for quantifying tissue PS concentration and two methods for quantifying pathological tumor damage. The two methods used to determine tissue PS concentration kinetic were in vivo fluorescence probe and ex vivo chemical extraction. Both methods show that the highest tumor to normal tissue PS uptake ratio appears 4 h after PS administration. Two different histopathologic techniques were used to quantify tumor and normal tissue damage. A planimetry assessment of regional tumor necrosis demonstrated a linear relationship with increasing light dose. However, in large murine tumors this finding was complicated by the presence of significant spontaneous necrosis. A second method (densitometry) assessed cell death by nuclear size and density. With some exceptions the densitometry method generally supported the planimetry results. Although the densitometry method is potentially more accurate, it has greater potential subjectivity. Finally, our research suggests that the tools or methods we are studying for quantifying PS levels and tissue damage are necessary for the understanding of PDT effect and therapeutic ratio in experimental in vivo tumor research.     

Photobleaching-based dosimetry predicts deposited dose in ALA-PpIX PDT of rodent esophagus

An improved method to estimate dose to esophageal tissue was investigated in the setting of photodynamic therapy with aminolevulinic acid-induced protoporphyrin IX (PpIX) treatment. A model of treatment-induced edema in the esophagus mucosa proved to be a well controlled and useful way to test the dosimetry model, and the light from the treatment laser together with the PpIX fluorescence intensity could be quantified reliably in real time. Dosimetry calculations based upon the detected fluorescence and bleaching kinetics were used to calculate the "effective" dose to the tissue, and a correlation was shown to exist between this metric and the edema induced in the esophagus. The difference between animals with no detectable treatment effect and those with significant edema was predictable based upon the dose calculation. The underlying assumption in the interpretation of the data is that rapid photobleaching of PpIX occurs when there is ample oxygen supply, and this bleaching is not present when oxygen is limited. This leads to the prediction that integration of the light and drug dose, in intervals where appreciable photobleaching occurs, should provide a prediction of the relative dose of singlet oxygen produced. This detection system and rodent model can be used for prospective dosimetry studies that focus on optimization of esophageal PDT.     

Endoscopic, rapid near-infrared optical tomography

This is believed to be the first demonstration of near-infrared (NIR) optical tomography employed at the endoscope scale and at a rapid sampling speed that allows translation to in vivo use. A spread-spectral-encoding technique based on a broadband light source and linear-to-circular fiber bundling was used to provide endoscopic probing of many source-detector fibers for tomography as well as parallel sampling of all source-detector pairs for rapid imaging. Endoscopic NIR tomography at an 8 Hz frame rate was achieved in phantoms and tissue specimens with a 12 mm probe housing eight sources and eight detectors. This novel approach provides the key feasibility studies to allow this blood-based contrast imaging technology to be attempted in detection of cancer in internal organs via endoscopic interrogation.     

In vivo NADH fluorescence monitoring as an assay for cellular damage in photodynamic therapy.

In this study the endogenous fluorescence signal attributed to reduced nicotinamide adenine dinucleotide (NADH) has been measured in response to photodynamic therapy (PDT)-induced damage. Measurements on cells in vitro have shown that NADH fluorescence decreased relative to that of controls after treatment with a toxic dose of PDT, as measured within 30 min after treatment. Similarly, assays of cell viability indicated that mitochondrial function was reduced immediately after treatment in proportion to the dose delivered, and the proportion of this dose response did not degrade further over 24 h. Measurements in vivo were used to monitor the fluorescence emission spectrum and the excited state lifetime of NADH in PDT-treated tissue. The NADH signal was defined as the ratio of the integrated fluorescence intensity of the 450 +/- 25 nm emission band relative to the fluorescence intensity integrated over the entire 400-600 nm range of collection. Measurements in murine muscle tissue indicated a 22% reduction in the fluorescence signal immediately after treatment with verteporfin-based PDT, using a dose of 2 mg/kg injected 15 min before a 48 J/cm2 light dose at 690 nm. Control animals without photosensitizer injection had no significant change in the fluorescence signal from laser irradiation at the same doses. This signal was monotonically correlated to the deposited dose used here and could provide a direct dosimetric measure of PDT-induced cellular death in the tissue being treated.