Translucent poly(vinyl alcohol) cryogel dosimeters for simultaneous dose buildup and monitoring during chest wall radiation therapy.

Chest wall radiation therapy treatment delivery was monitored using a 5 mm thick radiochromic poly(vinyl alcohol) cryogel that also provided buildup material. The cryogels were used to detect positioning errors and measure the impact of shifts for a chest wall treatment that was delivered to a RANDO phantom. The phantom was shifted by ± 2, ± 3, and ± 5 mm from the planned position in the anterior/posterior (A/P) direction; these shifts represent setup errors and the uncertainty associated with lung filling during breath-hold. The two-dimensional absolute dose distributions measured in the cryogel at the planned position were compared with the distributions at all shifts from this position using gamma analysis (3%/3 mm, 10% threshold). For shifts of ± 2, ± 3, and ± 5 mm the passing rates ranged from 94.3% to 95.6%, 74.0% to 78.8%, and 17.5% to 22.5%, respectively. These results are consistent with the same gamma analysis performed on dose planes calculated in the middle of the cryogel and on the phantom surface using our treatment plan-ning system, which ranged from 94.3% to 95.0%, 76.8% to 77.9%, and 23.5% to 24.3%, respectively. The Pinnacle dose planes were then scaled empirically and compared to the cryogel measurements. Using the same gamma metric, the pass rates ranged from 97.0% to 98.4%. The results of this study suggest that cryogels may be used as both a buildup material and to evaluate errors in chest wall treat-ment positioning during deep-inspiration breath-hold delivery. The cryogels are sensitive to A/P chest wall shifts of less than 3 mm, which potentially allows for the detection of clinically relevant errors.

Validation and application of a model of oxygen consumption and diffusion during photodynamic therapy in vitro.

The photophysical parameters for the photosensitizer Pd(II) meso-Tetra(4-carboxyphenyl) porphine (PdT790) acquired in a previous study were incorporated into the PDT oxygen diffusion models for cell suspensions and cell monolayers. The time-dependent phosphorescence signals generated by the diffusion models are shown to match signals previously measured by M.A.W. and M.S.P. when reasonable physical and photophysical parameters are used. Simulations were performed to investigate the effects of metabolic and photodynamic oxygen consumption rates on the PDT dose in each of the treatment geometries. It was found that in cell suspensions of <1 million cells per mL, PDT should not be inhibited by hypoxia if the photodynamic consumption rate is <1 mm s(-1). For cell monolayers the optimal photodynamic oxygen consumption rate was found to depend on the metabolic rate of oxygen consumption. If cells remained well oxygenated in the absence of PDT, then maximum PDT dose was delivered with the lowest practical photodynamic oxygen consumption rate. Simulations of PDT treatments for multicell tumor spheroids showed that large anoxic cores develop within the spheroids and, as a consequence, less PDT dose is delivered in comparison with similar treatments in cell suspensions and cell monolayers.

Measurement of intracellular oxygen concentration during photodynamic therapy in vitro.

A technique is introduced that monitors the depletion of intracellular ground state oxygen concentration ([(3)O(2)]) during photodynamic therapy of Mat-LyLu cell monolayers and cell suspensions. The photosensitizer Pd(II) meso-tetra(4-carboxyphenyl)porphine (PdT790) is used to manipulate and indicate intracellular [(3)O(2)] in both of the in vitro models. The Stern-Volmer relationship for PdT790 phosphorescence was characterized in suspensions by flowing nitrogen over the suspension while short pulses of 405 nm light were used to excite the sensitizer. The bleaching of sensitizer and the oxygen consumption rate were also measured during continuous exposure of the cell suspension to the 405 nm laser. Photodynamic therapy (PDT) was conducted in both cell suspensions and in cell monolayers under different treatment conditions while the phosphorescence signal was acquired. The intracellular [(3)O(2)] during PDT was calculated by using the measured Stern-Volmer relationship and correcting for sensitizer photobleaching. In addition, the amount of oxygen that was consumed during the treatments was calculated. It was found that even at large oxygen consumption rates, cells remain well oxygenated during PDT of cell suspensions. For monolayer treatments, it was found that intracellular [(3)O(2)] is rapidly depleted over the course of PDT.

Effect of 1O2 quencher depletion on the efficiency of photodynamic therapy.

Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4) fluorescence and photodynamic oxygen consumption were monitored during AlPcS4-photodynamic therapy (PDT) of Mat LyLu cells in suspension. These measurements were used to calculate the PDT efficiency, which is defined as the oxygen consumption rate divided by the sensitizer concentration. As a function of the intracellular oxygen concentration consumed by PDT, the normalized PDT efficiency fell off more quickly at lower photosensitizer concentrations. The changes in PDT efficiency were compared to models of PDT in which the photosensitizer (PS) and singlet oxygen quencher (A) were either free to diffuse or were fixed. The model in which PS and A are free to diffuse did not agree with the experimental data because this model predicts that the reduction in PDT efficiency is independent of [PS]. A Monte Carlo model was written to simulate PDT when both PS and A are stationary. This model was found to describe the experimental data when the initial intracellular [A] = 90 mM and when the initial and final (i.e. after all A has been depleted) singlet oxygen lifetimes were 0.4 and 1.2 µs respectively.

Monitoring oxygen concentration during photodynamic therapy using prompt photosensitizer fluorescence.

A novel technique is described that uses either time-resolved or steady state prompt photosensitizer fluorescence to measure local oxygen concentration. Solution experiments conducted with Al(III) phthalocyanine chloride tetrasulfonic acid confirmed that the steady state fluorescence signal is dependent on the oxygen concentration and fluence rate. A relationship between prompt sensitizer fluorescence and sensitizer triplet quenching efficiency is derived which does not require knowledge of the Stern-Volmer constant. Similar relationships are also derived for sensitizer delayed fluorescence and phosphorescence. An explicit photodynamic therapy (PDT) dose metric that incorporates light dosimetry, sensitizer dosimetry, and triplet quenching efficiency is introduced. All components of this metric can be determined by optical measurements.

Calculation of singlet oxygen dose using explicit and implicit dose metrics during benzoporphyrin derivative monoacid ring A (BPD-MA)-PDT in vitro and correlation with MLL cell survival.

Photodynamic therapy (PDT) oxygen consumption, clonogenic cell survival, fluorescence photobleaching and photoproduct formation were investigated during benzoporphyrin derivative monoacid (BPD-MA)-PDT of MAT-LyLu cells in vitro. Cells were incubated with BPD-MA concentrations of 0.1, 0.5 or 2.5 µg mL(-1) for 2 h and then treated with 405 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment, and photobleaching and photoproduct generation were quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony-forming assay. The amount of oxygen consumed by PDT per photon absorbed decreased with BPD-MA intracellular concentration. Survival was correlated with the total amount of oxygen consumed by PDT per unit volume, which is assumed to be equivalent to the amount of singlet oxygen that reacted. A photobleaching-based singlet oxygen dose metric was also found to predict survival independent of intracellular BPD-MA concentration. The BPD-MA photoproduct was bleached during the treatment. Two singlet oxygen dose metrics based on photoproduct kinetics could not be correlated with cell survival over the full range of intracellular BPD-MA concentrations used.

Rapid access towards follow-up NOP receptor agonists using a knowledge based approach.

A knowledge based approach has been adopted to identify novel NOP receptor agonists with simplified hydrophobes. Substitution of the benzimidazol-2-one piperidine motif with a range of hydrophobic groups and pharmacophore guided bio-isosteric replacement of the benzimidazol-2-one moiety was explored. Compound 51 was found to be a high affinity, potent NOP receptor agonist with reduced affinity for the hERG channel.

Simple photodynamic therapy dose models fail to predict the survival of MLL cells after HPPH-PDT in vitro.

Fluorescence photobleaching, photodynamic therapy (PDT) oxygen consumption and clonogenic cell survival were investigated during 2-(1-hexyloxethyl)-2-devinyl pyropheophoribde-a (HPPH) PDT of MAT-LyLu cells in vitro. Cells were incubated with HPPH concentrations of 0.24, 1.2, 3.6 or 12 microm for 4 h and then treated with 650 nm light under oxygenated and hypoxic conditions. Fluorescence spectra were acquired during treatment and photobleaching was quantified using singular value decomposition of the spectra. Cell survival was measured at set times during the treatment using a colony forming assay. Intracellular fluorescence lifetime measurements were also performed at each incubation concentration. The photobleaching kinetics did not follow first- or second-order kinetics and the fluorescence lifetime was similar for all intracellular concentrations. As the intracellular concentration of drug was increased, the amount of singlet oxygen and the absorbed quanta per cell required to achieve the same cell kill increased. Singlet oxygen dose was calculated using one- and two-compartment models of HPPH intracellular distribution. It was found that a two-compartment model, in which a PDT-sensitive binding site saturates at low concentrations, accounts for the observed photobleaching, oxygen consumption and cell survival.

Synthesis and evaluation of N-3 substituted phenoxypropyl piperidine benzimidazol-2-one analogues as NOP receptor agonists with analgesic and sedative properties.

A series of 3-phenoxypropyl piperidine benzimidazol-2-one analogues have been discovered as novel NOP receptor agonists. Structure-activity relationships have been explored via N-3 substitution of the benzimidazol-2-one with a range of functionality. The N-methyl acetamide derivative (+)-7f was found to be a high-affinity, potent NOP agonist with greater than 100-fold selectivity over the MOP receptor. Furthermore (+)-7f was shown to be both antinociceptive and sedative when administered iv to rodents.

Synthesis and SAR studies of 3-phenoxypropyl piperidine analogues as ORL1 (NOP) receptor agonists.

A series of 3-phenoxypropyl piperidine analogues have been discovered as novel ORL1 receptor agonists. Structure-activity relationships have been explored around the 3-phenoxypropyl region with several potent and selective analogues identified.