title: The validation of a new vascular damage assay for photodynamic therapy agents.
abstract: The therapeutic effect of photodynamic therapy (PDT: photodynamic sensitizer + light) is partly due to vascular damage. This report describes a new vascular photodamage assay for PDT agents and a validation of the assay. The method described here quantitates changes in tissue blood perfusion based on the relative amount of injected fluorescein dye in treated and untreated tissues. A specially designed fluorometer uses chopped monochromatic light from an argon laser as a source for exciting fluorescein fluorescence. The fluorescent light emitted from the tissue is collected by a six element fiberoptic array, filtered and delivered to a photodiode detector coupled to a phase-locked amplifier for conversion to a voltage signal for recording. This arrangement permits a rather simple, inexpensive construction and allows for the simultaneous use of the argon laser by other investigators. The routine assay for characterizing a specific photosensitizer at a standard dose consists of the sequential allocation of eight mice to a set of different 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 mumol/kg. The parameter estimates (n = 34 mice) from fitting the standard Hill dose-response model to the data were: median fluorescence exclusion light dose FE50 = 275 +/- 8.3 J/cm2 and Hill sigmoidicity parameter 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.
authors.affiliation: Department of Radiation Biology, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Bellnier
authors.forename: W R
authors.initials: WR
authors.lastname: Potter
authors.forename: L A
authors.initials: LA
authors.lastname: Vaughan
authors.forename: T M
authors.initials: TM
authors.lastname: Sitnik
authors.forename: J C
authors.initials: JC
authors.lastname: Parsons
authors.forename: W R
authors.initials: WR
authors.lastname: Greco
authors.forename: J
authors.initials: J
authors.lastname: Whitaker
authors.forename: P
authors.initials: P
authors.lastname: Johnson
authors.forename: B W
authors.initials: BW
authors.lastname: Henderson
mesh_terms: D000818:Animals; D001808:Blood Vessels / Q000187:drug effects* / Q000528:radiation effects; D002734:Chlorophyll / Q000031:analogs & derivatives / Q000494:pharmacology; D019793:Fluorescein; D005452:Fluoresceins; D051379:Mice; D008809:Mice, Inbred C3H; D010778:Photochemotherapy; D017319:Photosensitizing Agents / Q000494:pharmacology; D015203:Reproducibility of Results; D012867:Skin / Q000098:blood supply / Q000187:drug effects / Q000528:radiation effects; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000706:statistics & numerical data
chemical_list: D005452:Fluoresceins; D017319:Photosensitizing Agents; D002734:Chlorophyll; C083998:2-(1-hexyloxyethyl)-2-devinyl pyropheophorbide-a; D019793:Fluorescein