THE VALIDATION OF A NEW VASCULAR DAMAGE ASSAY FOR PHOTODYNAMIC THERAPY AGENTS

The therapeutic effect of photodynamic therapy (PDT: photodynamic sens itizer + light) is partly due to vascular damage. This report describe s a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blo od perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer use s chopped monochromatic light from an argon laser as a source for exci ting fluorescein fluorescence. The fluorescent Light emitted from the tissue is collected by a six element fiberoptic array, filtered and de livered to a photodiode detector coupled to a phase-locked amplifier f or conversion to a voltage signal for recording. This arrangement perm its a rather simple, inexpensive construction and allows for the simul taneous use of the argon laser by other investigators. The routine ass ay for characterizing a specific photosensitizer at a standard dose co nsists of the sequential allocation of eight mice to a set of differen t light doses designed to span the dose-response range of fluorescein fluorescence exclusion (measured 8-10 min after fluorescein injection) . The assay validation experiment used an anionic photosensitizer, 2-[ 1-hexyloxyethyl]-2-devinyl pyropheophorbide-a at a dose of 0.4 mu mol/ kg. The parameter estimates (n = 34 mice) from fitting the standard Hi ll dose-response model to the data were: median fluorescence exclusion light dose FE(50) = 275 +/- 8.3 J/cm(2) and Hill sigmoidicity paramet er m = -3.66 +/- 0.28. Subsets of the full data set randomly selected to simulate a standard eight mice experiment yielded similar parameter estimates, The new assay provides reliable estimates of PDT vascular damage with a frugal sequential experimental design.