Michele M. Kim

Explicit dosimetry for 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a-mediated photodynamic therapy: macroscopic singlet oxygen modeling

Abstract. Type II photodynamic therapy (PDT) is based on the photochemical reactions mediated through an interaction between a photosensitizer, ground-state oxygen ([O32]), and light excitation at an appropriate wavelength, which results in production of reactive singlet oxygen ([O12]rx). We use an empirical macroscopic model based on four photochemical parameters for the calculation of [O12]rx threshold concentration ([O12]rx,sh) causing tissue necrosis in tumors after PDT. For this reason, 2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a (HPPH)-mediated PDT was performed interstitially on mice with radiation-induced fibrosarcoma (RIF) tumors. A linear light source at 665 nm with total energy released per unit length of 12 to 100  J/cm and source power per unit length (LS) of 12 to 150  mW/cm was used to induce different radii of necrosis. Then the amount of [O12]rx calculated by the macroscopic model incorporating explicit PDT dosimetry of light fluence distribution, tissue optical properties, and HPPH concentration was correlated to the necrotic radius to obtain the model parameters and [O12]rx,sh. We provide evidence that [O12]rx is a better dosimetric quantity for predicting the treatment outcome than PDT dose, which is proportional to the time integral of the products of the photosensitizer concentration and light fluence rate.

Erratum: Macroscopic singlet oxygen modeling for dosimetry of Photofrin-mediated photodynamic therapy: an in-vivo study

Abstract. Although photodynamic therapy (PDT) is an established modality for cancer treatment, current dosimetric quantities, such as light fluence and PDT dose, do not account for the differences in PDT oxygen consumption for different fluence rates (φ). A macroscopic model was adopted to evaluate using calculated reacted singlet oxygen concentration ([O21]rx) to predict Photofrin-PDT outcome in mice bearing radiation-induced fibrosarcoma tumors, as singlet oxygen is the primary cytotoxic species responsible for cell death in type II PDT. Using a combination of fluences (50, 135, 200, and 250  J/cm2) and φ (50, 75, and 150  mW/cm2), tumor regrowth rate, k, was determined for each condition. A tumor cure index, CI=1−k/kcontrol, was calculated based on the k between PDT-treated groups and that of the control, kcontrol. The measured Photofrin concentration and light dose for each mouse were used to calculate PDT dose and [O21]rx, while mean optical properties (μa=0.9  cm−1, μs′=8.4  cm−1) were used to calculate φ for all mice. CI was correlated to the fluence, PDT dose, and [O21]rx with R2=0.35, 0.79, and 0.93, respectively. These results suggest that [O21]rx serves as a better dosimetric quantity for predicting PDT outcome.

In-vivo singlet oxygen threshold doses for PDT

Abstract Objective: Dosimetry of singlet oxygen (1O2) is of particular interest because it is the major cytotoxic agent causing biological effects of type-II photosensitizers during photodynamic therapy (PDT). An in-vivo model was developed to determine the singlet oxygen threshold dose, [1O2]rx,sh, for PDT. Material and methods: An in-vivo radiation-induced fibrosarcoma (RIF) tumor mouse model was used to correlate the radius of necrosis to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include five photosensitizer-specific photochemical parameters along with [1O2]rx,sh. Photosensitizer-specific model parameters were determined for benzoporphyrin derivative monoacid ring A (BPD) and compared with two other type-II photosensitizers, Photofrin® and m-tetrahydroxyphenylchlorin (mTHPC) from the literature. In order to discuss the possible influence of vascular vs. apoptotic cell killing mechanisms on the singlet oxygen threshold dose, the [1O2]rx,sh values for BPD with 3 h and 15 min drug-light intervals, with the latter being known to have a dominantly vascular effect, were compared. Results: The mean values (standard deviation) of the in-vivo [1O2]rx,sh are approximately 0.56 (0.26) and 0.72 (0.21) mm (or 3.6×107 and 4.6×107 singlet oxygen per cell to reduce the cell survival to 1/e) for Photofrin® and BPD3 h, respectively, assuming that the fraction of generated singlet oxygen that interacts with the cell is 1. The [1O2]rx,sh value for BPD15 min (0.12) was substantially lower than that for a DLI of 3 h. While the values for the photochemical parameters (ξ, σ, g, β) used for BPD were preliminary and may need further refinement, there is reasonable confidence for the values of [1O2]rx,sh. For mTHPC-PDT, the [1O2]rx,sh value derived from in-vivo mouse study was reported to be 0.4 mm. In comparison, the singlet oxygen required per cell was reported to be 9×108 per cell per 1/e fractional kill in an in-vitro mTHPC-PDT study on a rat prostate cancer cell line (MLL cells) and was reported to be 7.9 mm for a multicell in-vitro EMT6/Ro spheroid model for mTHPC-PDT. Conclusions: The experimental results of [1O2]rx,sh in an in-vivo RIF tumor model for Photofrin®, BPD, and mTHPC are about 20 times smaller than those observed in vitro. These results are consistent with the knowledge that factors other than singlet oxygen-mediated tumor cell killing can contribute to PDT damage in-vivo.

Comparison of singlet oxygen threshold dose for PDT

Macroscopic modeling of singlet oxygen (1O2) is of particular interest because it is the major cytotoxic agent causing biological effects for type II photosensitizers during PDT. We have developed a macroscopic model to calculate reacted singlet oxygen concentration ([1O2]rx for PDT. An in-vivo RIF tumor mouse model is used to correlate the necrosis depth to the calculation based on explicit PDT dosimetry of light fluence distribution, tissue optical properties, and photosensitizer concentrations. Inputs to the model include 4 photosensitizer specific photochemical parameters along with the apparent singlet oxygen threshold concentration. Photosensitizer specific model parameters are determined for several type II photosensitizers (Photofrin, BPD, and HPPH). The singlet oxygen threshold concentration is approximately 0.41 - 0.56 mM for all three photosensitizers studied, assuming that the fraction of singlet oxygen generated that interacts with the cell is (f = 1). In comparison, value derived from other in-vivo mice studies is 0.4 mM for mTHPC. However, the singlet oxygen threshold doses were reported to be 7.9 and 12.1 mM for a multicell in-vitro EMT6/Ro spheroid model for mTHPC and Photofrin PDT, respectively. The sensitivity of threshold singlet oxygen dose for our experiment is examined. The possible influence of vascular vs. apoptotic cell killing mechanism on the singlet oxygen threshold dose is discussed using the BPD with different drug-light intervals 3 hrs vs. 15 min. The observed discrepancies between different experiments warrant further investigation to explain the cause of the difference.

Explicit macroscopic singlet oxygen modeling for benzoporphyrin derivative monoacid ring A (BPD)-mediated photodynamic therapy.

Photodynamic therapy (PDT) is an effective non-ionizing treatment modality that is currently being used for various malignant and non-malignant diseases. In type II PDT with photosensitizers such as benzoporphyrin monoacid ring A (BPD), cell death is based on the creation of singlet oxygen (1O2). With a previously proposed empirical five-parameter macroscopic model, the threshold dose of singlet oxygen ([1O2]rx,sh]) to cause tissue necrosis in tumors treated with PDT was determined along with a range of the magnitude of the relevant photochemical parameters: the photochemical oxygen consumption rate per light fluence rate and photosensitizer concentration (ξ), the probability ratio of 1O2 to react with ground state photosensitizer compared to a cellular target (σ), the ratio of the monomolecular decay rate of the triplet state photosensitizer (β), the low photosensitizer concentration correction factor (δ), and the macroscopic maximum oxygen supply rate (g). Mice bearing radiation-induced fibrosarcoma (RIF) tumors were treated interstitially with a linear light source at 690nm with total energy released per unit length of 22.5-135J/cm and source power per unit length of 12-150mW/cm to induce different radii of necrosis. A fitting algorithm was developed to determine the photochemical parameters by minimizing the error function involving the range between the calculated reacted singlet oxygen ([1O2]rx) at necrosis radius and the [1O2]rx,sh. [1O2]rx was calculated based on explicit dosimetry of the light fluence distribution, the tissue optical properties, and the BPD concentration. The initial ground state oxygen concentration ([3O2]0) was set to be 40μM in this study. The photochemical parameters were found to be ξ=(55±40)×10-3cm2mW-1s-1, σ=(1.8±3)×10-5μM-1, and g=1.7±0.7μMs-1. We have taken the literature values for δ=33μM, and β=11.9μM. [1O2]rx has shown promise to be a more effective dosimetry quantity for predicting necrosis than either light dose or PDT dose, where the latter is simplistically a temporal integral of the products of the photosensitizer concentration and light fluence rate.

Comparison of PDT parameters for RIF and H460 tumor models during HPPH-mediated PDT

Singlet oxygen (1O2) is the major cytotoxic species producing PDT effects, but it is difficult to monitor in vivo due to its short life time in real biological environments. Mathematical models are then useful to calculate 1O2 concentrations for PDT dosimetry. Our previously introduced macroscopic model has four PDT parameters: ξ, σ, β and g describing initial oxygen consumption rate, ratio of photobleaching to reaction between 1O2 and cellular targets, ratio of triplet state (T) phosphorescence to reaction between T and oxygen (3O2), and oxygen supply rate to tissue, respectively. In addition, the model calculates a fifth parameter, threshold 1O2 dose ([1O2]rx,sd). These PDT parameters have been investigated for HPPH using radiation-induced fibrosarcoma (RIF) tumors in an in-vivo C3H mouse model. In recent studies, we additionally investigated these parameters in human non-small cell lung carcinoma (H460) tumor xenografts, also using HPPH-mediated PDT. In-vivo studies are performed with nude female mice with H460 tumors grown intradermally on their right shoulders. HPPH (0.25 mg/kg) is injected i.v. at 24 hours prior to light delivery. Initial in vivo HPPH concentration is quantified via interstitial HPPH fluorescence measurements after correction for tissue optical properties. Light is delivered by a linear source at various light doses (12-50 J/cm) with powers ranging from 12 to 150 mW per cm length. The necrosis radius is quantified using ScanScope after tumor sectioning and hematoxylin and eosin (H and E) staining. The macroscopic optimization model is used to fit the results and generate four PDT parameters. Initial results of the parameters for H460 tumors will be reported and compared with those for the RIF tumor.

Parameter determination for singlet oxygen modeling of BPD-mediated PDT

Photodynamic therapy (PDT) offers a cancer treatment modality capable of providing minimally invasive localized tumor necrosis. To accurately predict PDT treatment outcome based on pre-treatment patient specific parameters, an explicit dosimetry model is used to calculate apparent reacted 1O2 concentration ([1O2]rx) at varied radial distances from the activating light source inserted into tumor tissue and apparent singlet oxygen threshold concentration for necrosis ([1O2]rx, sd) for type-II PDT photosensitizers. Inputs into the model include a number of photosensitizer independent parameters as well as photosensitizer specific photochemical parameters ξ σ, and β. To determine the specific photochemical parameters of benzoporphyrin derivative monoacid A (BPD), mice were treated with BPDPDT with varied light source strengths and treatment times. All photosensitizer independent inputs were assessed pre-treatment and average necrotic radius in treated tissue was determined post-treatment. Using the explicit dosimetry model, BPD specific ξ σ, and β photochemical parameters were determined which estimated necrotic radii similar to those observed in initial BPD-PDT treated mice using an optimization algorithm that minimizes the difference between the model and that of the measurements. Photochemical parameters for BPD are compared with those of other known photosensitizers, such as Photofrin. The determination of these BPD specific photochemical parameters provides necessary data for predictive treatment outcome in clinical BPD-PDT using the explicit dosimetry model.

A compact fiber-optic probe-based singlet oxygen luminescence detection system

This paper presents a novel compact fiberoptic based singlet oxygen near‐infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single‐photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal‐to‐noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic‐coupled superconducting nanowire single‐photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo. figureWiley-VCH Verlag & Co.KGaA

In vivo outcome study of BPD-mediated PDT using a macroscopic singlet oxygen model

Macroscopic modeling of the apparent reacted singlet oxygen concentration ([1O2]rx) for use with photodynamic therapy (PDT) has been developed and studied for benzoporphryin derivative monoacid ring A (BPD), a common photosensitizer. The four photophysical parameters (ξ, σ, β, δ) and threshold singlet oxygen dose ([1O2]rx, sh) have been investigated and determined using the RIF model of murine fibrosarcomas and interstitial treatment delivery. These parameters are examined and verified further by monitoring tumor growth post-PDT. BPD was administered at 1 mg/kg, and mice were treated 3 hours later with fluence rates ranging between 75 – 150 mW/cm2 and total fluences of 100 – 350 J/cm2. Treatment was delivered superficially using a collimated beam. Changes in tumor volume were tracked following treatment. The tumor growth rate was fitted for each treatment condition group and compared using dose metrics including total light dose, PDT dose, and reacted singlet oxygen. Initial data showing the correlation between outcomes and various dose metrics indicate that reacted singlet oxygen serves as a good dosimetric quantity for predicting PDT outcome.

PDT Dose Dosimeter for Pleural Photodynamic Therapy

PDT dose is the product of the photosensitizer concentration and the light fluence in target tissue. Although existing systems are capable of measuring the light fluence in vivo, the concurrent measurement of photosensitizer in the treated tissue so far has been lacking. We have developed and tested a new method to simultaneously acquire light dosimetry and photosensitizer fluorescence data via the same isotropic detector, employing treatment light as the excitation source. A dichroic beamsplitter is used to split light from the isotropic detector into two fibers, one for light dosimetry, the other, after the 665 nm treatment light is removed by a band-stop filter, to a spectrometer for fluorescence detection. The light fluence varies significantly during treatment because of the source movement. The fluorescence signal is normalized by the light fluence measured at treatment wavelength. We have shown that the absolute photosensitizer concentration can be obtained by an optical properties correction factor and linear spectral fitting. Tissue optical properties are determined using an absorption spectroscopy probe immediately before PDT at the same sites. This novel method allows accurate real-time determination of delivered PDT dose using existing isotropic detectors, and may lead to a considerable improvement of PDT treatment quality compared to the currently employed systems. Preliminary data in patient studies is presented.