We have investigated the in vivo fluorescence of human skin with UV excitation and the effect of UV irradiation on the UV fluorescence. A particular chromophore was found to be very sensitive to suberythemogenic UV radiation. This chromophore has the spectral characteristics of tryptophan residues in proteins and is characterized by a fluorescence excitation maximum at 295 nm. The fluorescence of this chromophore in human epidermis has an excitation maximum that is coincident with the maximum of the action spectrum of most UV-induced photobiologic responses to human skin. The fluorescence of the chromophore was found to increase with UV exposure. The action spectrum was determined by following the increase of the emission at 345 nm with excitation at 295 nm as a function of skin exposure to a number of wavelengths in the UV region of the spectrum. The results show that irradiation in the UVB (290-320 nm) is more effective in producing the change in the fluorescence of tryptophan. Irradiation in the UVA (320-380 nm) was found to be capable of producing the increase but to a smaller extent. Whereas tryptophan fluorescence is found in both the epidermis and the dermis, it is only the epidermal component that increases with UV exposure. The change in 295 nm fluorescence with UV exposure was determined to be oxygen dependent. The fluorescence of tryptophan moieties measured in situ was found to increase when epidermal proliferation increases. This was verified by inducing epidermal repair after mechanical insult (tape stripping). The results suggest two possible scenarios for the UV-induced increase of the fluorescence: a prompt photooxidation of tryptophan moieties or a fast proliferation response to the insult created by UV irradiation.

The goal of this investigation was to establish methodology to determine and prevent phototoxic responses of normal skin to photodynamic therapy (PDT). The drug used was a second-generation photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA). The dependence of skin phototoxicity on drug dose (0.5-2.0 mg/kg), fluence (1.2-390 J/cm2), and wavelength (690 nm and 458 nm) was studied in the New Zealand albino rabbit in the first 5 h after injection. Skin responses were recorded for 2 wk after irradiation. Noninvasive measurements of drug fluorescence were made on unexposed skin sites during the first 5 h after drug injection. Immediate responses to PDT included erythema induced by 458 nm light and blanching induced by 690 nm light. Delayed reactions included edema on the day of exposure, purpura at 24 h, eschar by day 2 or 3, and scar by the end of follow-up. The threshold fluence for immediate responses correlated strongly with the threshold fluence for delayed reactions. The induction of threshold purpura on day 1 was a reliable index for skin phototoxicity that led to necrosis. The minimum purpura dose on day 1 after irradiation increased exponentially with the interval between drug injection and irradiation, independent of irradiation wavelength, for all drug doses. The action spectrum for threshold purpura mimics closely the absorption spectrum of BPD-MA. The in vivo drug fluorescence correlated with skin phototoxicity, thus allowing predictive dosimetry. This model system defines the safety limits for skin phototoxicity of PDT with BPD-MA.

In an ideal world, photodynamic therapy (PDT) of abnormal tissue would reliably spare the surrounding normal tissue. Normal tissue responses set the limits for light and drug dosimetry. The threshold fluence for necrosis (TFN) was measured in normal skin following intravenous infusion with a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) Verteporin as a function of drug dose (0.25-2.0 mg/kg), wavelength of irradiation (458 and 690 nm) and time interval (0-5 h) between drug administration and irradiation. The BPD-MA levels were measured in plasma and skin tissue to elucidate the relationship between TFN, drug kinetics and biodistribution. The PDT response of normal skin was highly reproducible. The TFN for 458 and 690 nm wavelengths was nearly identical and the estimated quantum efficiency for skin response was equal at these two wavelengths. Skin phototoxicity, quantified in terms of 1/TFN, closely correlated with the plasma pharmacokinetics rather than the tissue pharmacokinetics and was quadratically dependent on the plasma drug concentration regardless of the administered drug dose or time interval between drug and light exposure. This study strongly suggests that noninvasive measurements of the circulating drug level at the time of light treatment will be important for setting optimal light dosimetry for PDT with liposomal BPD-MA, a vascular photosensitizer.