Dominic J. Robinson

Fluorescence Photobleaching of ALA‐induced Protoporphyrin IX during Photodynamic Therapy of Normal Hairless Mouse Skin: The Effect of Light Dose and Irradiance and the Resulting Biological Effect

The photobleaching of 5‐aminolaevulinic acid (ALA)‐induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT‐induced dam‐age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo‐bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo‐bleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50–150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT‐induced dam‐age. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photo‐bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Improved response of plaque psoriasis after multiple treatments with topical 5-aminolaevulinic acid photodynamic therapy.

We investigated the clinical response of 10 patients with plaque psoriasis to multiple treatments with photodynamic therapy, using topical application of 5-aminolaevulinic acid followed by exposure to broad-band visible radiation. Treatment was performed up to 3 times per week, with a maximum of 12 treatments, using a light dose of 8 Jcm(-2) delivered at a dose-rate of 15 mW cm(-2). Eight patients showed a clinical response. Out of 19 treated sites, 4 cleared, 10 responded but did not clear and 5 showed no improvement. Of the 4 sites that cleared only 1 did so fully, after 7 treatments, 45 days after the start of therapy. Of the 10 sites that responded partially, the greatest reduction in scale, erythema and induration index occurred after a minimum of 3 and a maximum of 8 treatments. The intensity of 5-aminolaevulinic acid-induced protoporphyrin IX fluorescence, recorded prior to the first treatment, varied between sites on the same patient as well as between patients. There was also a variation in fluorescence intensity recorded from the same site immediately prior to subsequent treatments, although the pretreatment levels generally decreased as the study progressed and then increased as psoriasis relapsed. Biopsies confirmed that fluorescence was localized throughout the epidermis and stratum corneum, but the level was not consistent between sections taken within the same biopsy. We also observed fluorescence at sites distant from the ones that received 5-aminolaevulinic acid, which was not present prior to the start of the treatment programme, but found no evidence of elevated levels of plasma porphyrins. The level of discomfort associated with this therapy increased with increasing values of the calculated photodynamic dose, defined as the product of the initial photosensitizer concentration and the percentage reduction in fluorescence following irradiation. Therefore, although clinical efficacy improved with multiple treatments, unpredictable response and patient discomfort make ALA-PDT unsuitable for the treatment of psoriasis.

Large patches of Bowen's disease treated by topical aminolaevulinic acid photodynamic therapy

Large patches of Bowens disease (intraepidermal carcinoma in situ) can be difficult to treat by conventional methods. Photodynamic therapy (PDT) uses the combination of a photosensitizer, which preferentially accumulates in malignant cells, and photoactivation by visible light to kill the malignant cells, 5‐aminolaevulinic acid (ALA) PDT uses excess exogenous ALA, which produces, via the haem synthesis pathway, a build up of the photosensitizer protoporphyrin IX. We describe the use of topical ALA PDT to treat three patients with three especially large patches of Bowens disease. Following two treatments all three lesions achieved a complete clinical and histological response with a good cosmetic result. ALA PDT is a simple, effective and well tolerated treatment for large patches of Bowens disease.

In vivo pharmacokinetics of protoporphyrin IX accumulation following intracutaneous injection of 5-aminolevulinic acid

Photodynamic therapy with 5-aminolevulinic acid (ALA) derived protoporphyrin IX (PpIX) as photosensitizer is a promising treatment for basal cell carcinomas. Until now ALA has been administered topically as an oil-in-water cream in most investigations. The disadvantage of this administration route is insufficiënt penetration in deeper, nodular tumours. Therefore we investigated intracutaneous injection of ALA as an alternative administration route. ALA was administered in 6-fold in the normal skin of three 6-week-old female Dutch pigs by intracutaneous injection of an aqueous solution of ALA (pH 5.0) in volumes of 0.1-0.5 ml and concentrations of 0.5-2% and by topical administration of a 20% ALA cream. During 8 h fluorescence of ALA derived PpIX was measured under 405 nm excitation. For the injection the measured fluorescence was shown to be dose dependent. All injected doses of 3 mg ALA or more lead to a faster initial increase rate of PpIX synthesis and significantly greater fluorescence than that measured after topical administration of ALA. Irradiation (60 Jcm(-2) for 10 min) of the spots was performed at 3.5 h after ALA administration. After 48 and 96 h visual damage scores were evaluated and biopsies were taken for histopathological examination. After injection of 2 mg ALA or more the PDT damage after illumination was shown to be significantly greater than after topical application of 20% ALA. An injected dose of 10 mg ALA (0.5 ml of a 2% solution) resulted in significantly more tissue damage after illumination than all other injected doses.

In vivo monitoring of photosensitizer fluorescence during photodynamic therapy

A method is presented of monitoring the low level fluorescence emitted by the photosensitizing agent protoporphyrin IX during superficial photodynamic therapy of skin carcinomas, using 630 nm illumination. A fiber optic probe samples the light field which is filtered and recorded by an optical spectrum analyzer. The technique is minimally invasive and can proceed concurrently with light dosimetry measurements. This paper presents in vitro data that define the sensitivity and selectivity of the technique, along with preliminary in vivo measurements. These indicate that it is the rate of phototransformation of the photosensitizer, rather than the total light dose, that determines the optimum treatment duration. Clinically effective treatment therefore depends upon achieving a threshold concentration of drug throughout the volume of the lesion. In this way the effect of phototransformation does not inactivate the drug before complete tumor necrosis occurs.

Rate-equation analysis of Protoporphyrin IX photo-oxidation

The process of photo-degradation, during photodynamic therapy, has important implications for both the diagnostic and therapeutic potential of specific photosensitizers. Monitoring photo-degradation may provide a useful indicator of the local concentration of singlet oxygen, and therefore a direct measure of photodynamic effectiveness. The spectroscopic properties of protoporphyrin IX (PpIX), the photosensitizer generated by the precursor 5-aminolevulinic acid, have been well documented. By recording the fluorescence emission spectrum of PpIX during illumination (at 630 nm) the photo-degradation of the sensitizer can be recorded. The rate of PpIX photo-degradation is dependent on the concentrations of both the sensitizer and molecular oxygen, but the decay cannot be described by a simple function that is valid under a variety of experimental conditions. By numerically solving differential equations describing the instantaneous concentrations of species in the photo-oxidation pathway of PpIX, we have been able to model the dynamics of sensitizer fluorescence under varying conditions of sensitizer concentration, oxygen concentration and illumination irradiance. Results are consistent with those measured in aqueous solution.

Light Fractionated ALA-PDT: From Pre-Clinical Models to Clinical Practice

Photodynamic therapy of superficial basal cell carcinoma using topical 5-aminolevulinic acid and a light fluence of 75-100 J cm−2 yields unsatisfactory long term clinical response rates. In a range of pre-clinical models illumination with two light fractions separated by 2 hours apart was considerably more effective than single illumination. Response is further enhanced if the fluence of the first light fraction is reduced while the cumulative fluence is maintained. We have demonstrated that these encouraging pre-clinical results are also evident for the clinical ALA-PDT of the treatment of superficial basal cell carcinoma. In a large scale randomised study including 505 primary sBCC we have shown that therapy using two light fractions of 20 and 80 Jcm−2 performed 4 and 6 hours after the application of a single dose of 20% ALA results in a significant increase in complete response (P 0.002, log-rank test). Twelve months after therapy, complete response rate following a two-fold illumination is 97% whereas the complete response to a single illumination is 89%. Numerous studies are underway investigating the mechanism underlying the increase in tissue response. Increased efficacy is not simply associated with an increasing PpIX content of the tissues during the treatment scheme and there is no direct relationship between the total amount of PpIX utilised and efficacy. We have shown that fractionated illumination does not enhance the efficacy of PDT using methyl-ester derivatives of ALA despite almost identical PpIX fluorescence kinetics during therapy. Our most recent data suggest that the in-vivo distribution of MAL and ALA and the exact site of PDT induced damage, is an important parameter in the mechanism underlying fractionated illumination for ALA-PDT. There is significant potential for the future use of light fractionation in other organs.

Establishment of treatment parameters for ALA-PDT of plaque psoriasis

We report an investigation into the use of photodynamic therapy (PDT), following topically applied 5-aminolaevulinic acid (ALA), as a treatment for plaque psoriasis. Treatment was performed 4 hours post-ALA, using white light doses of 2 - 16 J cm-2 delivered at 10 - 40 mW cm-2. The fluorescence emission of protoporphyrin IX was used as an indicator of the relative concentration of photosensitizer within each plaque before, during, and after therapy. Results show that the rate of sensitizer photo- oxidation is proportional to both pre-treatment fluorescence intensity and surface irradiance, consistent with a rate- equation analysis. A correlation of fluorescence measurements with clinical response of plaques indicates that the effectiveness of PDT is dominated by the level of PpIX at the onset of treatment, and is much less dependent upon light dose. Using these findings we have established a PDT treatment protocol that involves the delivery of 8 J cm-2 of white light, at a rate of 15 mW cm-2. The possibility of ALA-PDT being established as the therapy of choice is discussed.

Correction of fluorescence for depth-specific optical and vascular properties using reflectance and differential path-length spectroscopy during PDT

Introduction: The rate of PpIX fluorescence photobleaching is routinely used as a dose metric for ALA-PDT. Diffuse reflection spectroscopy is often used to account for variations in tissue optical properties at the photosensitizer excitation and emission bands. It can be used to quantify changes in vascular parameters, such as blood volume fraction and saturation, and can aid understanding of tissue response to PDT. The volume and(/or) depth over which these signals are acquired are critical. The aim of this study is to use quantitative reflectance spectroscopy (DPS) to correct fluorescence for changes in tissue optical properties and monitor PDT. Materials & Methods: ALA was topically applied to hairless mice skin and the incubated spot was treated with PDT according to fractionated illumination schemes. DPS measurements of vascular parameters and optical properties were performed directly before and after illumination. Both the differential signal, delivery-and-collection-fiber signal and the collection fiber signal, which all probe different measurement volumes, are analyzed. Results & Conclusions: Analysis of DPS measurements shows that at the depth where most fluorescence originates, there is almost no blood present. During PDT vascular parameters at this depth stay constant. In more oxygenated layers of the tissue, the optical properties do change during PDT, suggesting that only a small part of PpIX fluorescence originates from the interesting depths where vascular response occurs. Correcting fluorescence emission spectra for optical changes at specific depths and not for the total of changes in a larger volume, as is usually done now, can improve PpIX photobleaching based treatment monitoring.

Flourescence analysis of ALA-induced Protoporphyrin IX in psoriatic plaque

The success reported for the treatment of superficial skin carcinomas by photodynamic therapy (PDT), following topical application of 5-aminolaevulinic acid (ALA), has therapeutic implications for the treatment of other skin disorders. This presentation describes the accumulation of the photosensitizing agent protoporphyrin IX (PpIX) in areas of psoriatic plaque, by monitoring the fluorescence emission induced by low-intensity laser excitation at 488 nm. We present the results from 15 patients, with a total of 42 plaques. These results show that PpIX fluorescence increases in intensity within the 6 hour period following application of ALA, which implies there is a potential for PDT. The emission is localized to the area of ALA application and the effect of occlusion appears insignificant. Also, the rate of increase, and maximum intensity of fluorescence emission, is not directly related to the applied quantity of ALA. The variability of the fluorescence intensity is as great between plaques at different sites on the same patient as between different patients. We also present measurements of the depletion in intensity of fluorescence emission during PDT treatment, using white light, at an irradiance of 25 mW cm-2, that is a consequence of the molecular photo-oxidation of PpIX. The use of fluorescence measurements in predicting the therapeutic effect of treating plaque psoriasis by ALA-PDT is discussed.