Hugo J. van Staveren

Photodynamic therapy for esophageal lesions: selectivity depends on wavelength, power, and light dose

BACKGROUND Photodynamic therapy with 5-aminolevulinic acid-induced photosensitization could selectively eliminate esophageal epithelial lesions. This study aimed at optimizing laser parameters for 5-aminolevulinic acid photodynamic therapy of the normal rat esophagus. METHODS Sixty rats received 200 mg/kg 5-aminolevulinic acid orally and were illuminated 3 hours later with either 633 or 532 nm light (n = 30 for each group) through an endoesophageal balloon catheter. Rats received either 8.3 or 25 J/cm diffuser, applied with a 33, 100, or 300 mW/cm diffuser. During illumination, tissue fluorescence measurements and light dosimetry were done. Rats were sacrificed at 48 hours after photodynamic therapy. RESULTS During illumination, protoporphyrin IX fluorescence declined faster when a higher power output was used. Fluence rate at the esophageal surface was highest for 633-nm light. At 532 nm, light caused less damage to the epithelium and muscle than 633-nm light. Illumination with 33 mW resulted in selective epithelial ablation, whereas illumination with 300 mW caused muscle damage with minor epithelial damage. CONCLUSIONS The assumed selective epithelial damage of 5-aminolevulinic acid photodynamic therapy in the esophagus largely depends on the combination of wavelength, power, and light dose applied. Most selective epithelial damage was found when low-power 633-nm light was used.

Fluorescence Imaging and Spectroscopy of Ethyl Nile Blue A in Animal Models of (Pre)malignancies

Discrimination between normal and premalignant tissues by fluorescence imaging and/or spectroscopy may be enhanced by a tumor‐localizing fluorescent drug. Ethyl Nile Blue A (EtNBA), a dye with no phototoxic activity, was investigated for this purpose. The pharmacokinetics and tissue‐localizing properties were investigated in a rat palate model with chemically induced premalignant mucosal lesions (0.5 mg/kg EtNBA intravenous [i.v.]), a hairless mouse model with UVB‐induced premalignant skin lesions (1 mg/kg EtNBA intraperitoneal) and in a rat skin‐fold observation chamber model on the back of a rat with a transplanted solid tumor (2.5 mg/kg EtNBA i.v.). Fluorescence images and spectra were recorded in vivo (600 nm excitation, 665–900 nm detection) and in frozen tissue sections at several time points after EtNBA administration. In the rat palate the EtNBA fluorescence was maximum almost immediately after injection, whereas in the mouse skin and the observation chamber the fluorescence maximum was reached between 2 and 3 h after injection. EtNBA cleared from tissues after 8–24 h. EtNBA localizes in the transplantable solid tumor, but is not targeted specifically to the dysplastic location in the rat palate and mouse skin. However, in the rat palate the EtNBA fluorescence increased significantly with increasing dysplasia, apparently due to the increasing thickness of the upper keratinized layer of the epithelium where the dye was found to localize. Localization in this layer occurred both in the rat palate and in hairless mouse skin.

Fractionated illumination in oesophageal ALA-PDT: effect on ferrochelatase activity.

BACKGROUND AND OBJECTIVE Administration of 5-aminolevulinic acid (ALA) induces accumulation of the photosensitive compound protoporphyrin IX (PpIX) in certain tissues. PplX can be used as photosensitizer in photodynamic therapy (PDT). More selective or higher PpIX accumulation in the area to be treated could optimize the results of ALA-PDT. Porphobilinogen deaminase (PBGD) is rate-limiting in PpIX formation whereas ferrochelatase converts PpIX into haem by chelation of ferrous iron into PpIX. This results in a moment of close interaction (ferrochelatase binding to PpIX) during which ferrochelatase could selectively be destroyed resulting in an increased PpIX concentration. The aim of the present study was to investigate whether illumination before PDT can selectively destroy ferrochelatase. and whether this results in higher PpIX accumulation and thereby increases the PDT effect. Furthermore, the effect of a second ALA dose was tested. STUDY DESIGN/MATERIALS AND METHODS Oesophageal tissue of 60 rats were allocated to 2 groups of 30 animals each. In one group, enzyme and PpIX measurements were performed after ALA administration (200 mg/kg orally, n=20), or a second dose of 200 mg/kg ALA at 4 h (n=10), half of each group with and without illumination at 1 h with 12.5 J/cm diffuser length. In the second group, PDT was performed. Ten animals were illuminated at 3 h after ALA administration with 20 (n=5) or 32.5 J/cm (n=5), 10 animals were illuminated at 1 h (12.5 J/cm) and received intra-oesophageal PDT treatment (20 J/cm) at 3 h (n=5) or 4 h (n=5) after ALA. Additionally, 10 animals received a second dose of 200 mg/kg ALA at 4 h and were illuminated (20 J/cm) at 7 h after the first dose of ALA with (n=5) or without (n=5) illumination at 4 h (12.5 J/cm). RESULTS Illumination with 12.5 J/cm at 1 h after ALA administration caused inhibition of the activity of ferrochelatase at 3 and 4 h after ALA (P=0.02 and P<0.001, respectively), but not at 7 h (P=0.3). In animals sacrificed at 4 h the ratio PBGD:ferrochelatase was higher in animals illuminated at 1 h compared to non-illuminated animals (P<0.001). PpIX concentration was highest (42.7 +/- 3.2 pmol/mg protein) at 3 h after ALA administration and did not increase by illumination at 1 h. Administration of a second dose of ALA did not result in higher PpIX accumulation. After PDT, no difference in epithelial or muscular damage was found between the various groups. CONCLUSION Illumination at 1 h after ALA administration can cause selective destruction of ferrochelatase, resulting in a higher ratio of PBGD:ferrochelatase. This does not result in accumulation of more porphyrins, even when a second dose of ALA is given. Therefore, under the conditions used in this study fractionated illumination does not enhance ALA-PDT-induced epithelial ablation of the rat oesophagus.

Whole bladder wall photodynamic therapy using 5-ALA: an experimental study in pigs

The agent 5-aminolevulinic acid (5-ALA) can be an alternative drug in whole bladder wall (WBW) photodynamic therapy (PDT), as its good tumor selectivity and the short time skin photosensitivity after systemic administration are advantageous for clinical use. To determine the maximum drug and light doses for reversible normal tissue damage, a pre-clinical study was performed using an in vivo normal piglet bladder model. First, the kinetics of PpIX production in 2 pigs was determined in vitro after oral administration of 75 and 150 mg/kg ALA respectively. The concentration of PpIX in plasma, and erythrocytes was determined by reversed phase high-performance liquid chromatography (HPLC) and the maximum was reached at approximately equals 5 hours after the administration of ALA. This provided a guideline for the optimum interval between ALA administration and light application. Next, various ALA doses were either administered orally or instilled in the bladder and different light doses were applied. Bladder biopsies were taken at regular intervals and normal tissue damage was investigated histologically. Reversible tissue damage was obtained using 60 mg/kg of 5-ALA in combination with a light dose of 100 J cm-2 (non-scattered plus scattered 630 nm wavelength light) in the case of oral administration. In the case of intravesical instillation, a drug dose of 2.5 gram and a light dose of 100 J cm-2 are still too high to obtain reversible tissue damage.

Depth-sensitive fluorescence detection of dyes in tissue phantoms

It is well known that tumors can be detected by fluorescence imaging after administration of suitable photosensitizers. The tumors thickness, however, an important diagnostic parameter, cannot be assessed by optical methods up to now. We show that depth resolution can be achieved in the case of a fluorescent dye distributed homogeneously in a superficial region of a light scattering medium. Diffusion theory shows that the ratio of the fluorescence measured at different illumination angles is over a wide range sensitive to the thickness of the dye containing layer only, regardless of the dye concentration. For the experimental proof, a new temporally and mechanically stable tissue phantom system was developed. The experimental results on tissue phantoms confirm the predictions of diffusion theory.

Integrating sphere effect in whole bladder wall photodynamic therapy at violet, green, and red wavelengths

Fluence rates were measured in vivo at a piglet bladder wall during whole bladder wall (WBW) light irradiation at 458, 488, 514, 532, and 630 nm wavelengths. Bladder optical properties, the absorption-, scattering-, and anisotropy coefficient, were determined in vitro at these wavelengths using a double integrating sphere set-up. Monte Carlo (MC) computer simulations for WBW photodynamic therapy (PDT) were performed in a spherical geometry representing the bladder. The in vivo measured fluence multiplication factor ((beta) ) decreases from approximately equals 5 at 630 nm to approximately equals 1.5 at 458 nm. The simulated (beta) values, using the in vitro optical properties and non-absorbing (saline) bladder contents, are consistently larger with a minimum at 514/532 nm and a maximum at 458 and 630 nm. Simulations with slightly light absorbing bladder contents show that the inevitable urine in the cavity can at least partly be responsible for the lower in vivo values. Whereas the MC simulations use an in vitro absorption coefficient, the in vivo observed phenomenon might be attributed to additional light absorption by hemoglobin in the bladder tissue. Thus, WBW-PDT with red light is technically more advantageous than with green or blue light as this gives the strongest integrating sphere effect.

Effect of wavelength and light dose in photodynamic therapy of the normal rat esophagus with ALA-induced photosensitization

Red light (approximately 630 nm) is almost universally used in photodynamic therapy (PDT) as it is the most penetrating of the porphyrin excitation wavelengths. For the treatment of superficial (pre)malignant oesophageal lesions however green light might be more efficient than red in destroying this thin lesions. This study aimed to compare several powers and doses of red and green light for photodynamic therapy with 5- aminolevulinic acid (ALA)-induced photosensibilization of the normal rat oesophagus. Sixty rats received 200 mg/kg ALA orally. Ten untreated rats served as controls. In groups of five rats, at three hours after ALA administration the oesophagi were illuminated with red (633 nm) or green (532 nm) light, using a 1 cm cylindric diffuser, which was placed in the center of an inflated balloon catheter. Several treatment schemes were applied: 33 mW/cm diffuser during 250 and 750 seconds (providing 8.3 and 25 J/cm diffuser), 100 mW/cm diffuser during 83 and 250 seconds (providing 8.3 and 25 J/cm diffuser) and 300 mW/cm diffuser during 83 and 250 seconds (providing 25 and 75 J/cm diffuser). During illumination, fluorescence in the oesophagus was measured. The rats were sacrificed at 48 hours after illumination. During illumination, fluorescence declined in a rate that was dependent on the light-power that was used. A higher power resulted in a faster decline of fluorescence intensity. Both the red-light control group the and green-light control group did not show any damage histologically. In the red light group selective epithelial damage was seen most pronounced in the groups treated with 33 mW/cm diffuser. Illumination during 750 seconds however, also resulted in considerable muscle damage. In the green light group in all subgroups less damage was seen. Only illumination with 300 mW/cm diffuser during 250 seconds resulted in good PDT effect in 2 out of 5 rats. In the rat oesophagus, the efficacy of ALA-PDT treatment depends both on wavelength and illumination scheme. In this study best results were obtained using red light, in a power setting of 33 mW/cm diffuser. The optimal light-dose probably lies between 8.3 and 25 J/cm diffuser.

ALA-PDT of the normal rat esophagus: efficiency and safety largely depends on the timing of illumination

Photodynamic therapy (PDT) is an experimental treatment modality for (pre)malignant oesophageal lesions. 5- Aminolevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)- mediated photo-sensitization could be very useful as ALA- induced porphrin accumulation selectively occurs in the oesophageal epithelium. The present study aimed to optimize the time between illumination and the administration of ALA. 200 mg/kg ALA was given orally to 24 rats (allocated to 6 groups of 4 animals each). Four animals served as controls and received phosphate buffered saline orally. The animals were illuminated at various time-points (either 1, 2, 3, 4, 6, or 12 hours) after ALA administration. Illumination was performed with a cylindrical diffuser placed in a balloon catheter. The device was originally made for percutaneous transluminal coronary angioplasty and consisted of a semi-flexible catheter and an inflatable cylindric optically clear balloon. The diffuser was placed centrally in the catheter. The same illumination parameters (633 nm, 25 J radiant energy/cm diffuser, power output 100 mW/cm diffuser) were used for each animal. During illumination, fluorescence measurements and light dosimetry were performed. The animals were sacrificed at 48 hours after PDT for histological assessment. Highest PpIX fluorescence was found at 2, 3, and 4 hours after ALA administration. Dosimetric measurements showed a 2 - 3 times higher in vivo fluence rate compared to the estimated fluence rate. Histology at 48 hours after PDT showed diffuse epithelial damage at the laser site only in rats illuminated at 2 hours after ALA administration. Illumination at 3, 4, and 6 hours after ALA administration resulted in diffuse epithelial damage in only one of four rats. In none of the rats illuminated at 1 and 12 hours after administration of ALA epithelial damage was found. These results show that illumination at 2 hours after oral ALA administration provides an efficient and safe scheme for ALA-PDT in the normal rat oesophagus. Illumination at other time points results in incomplete epithelial damage.

Online-monitoring of light dosimetry and PpIX fluorescence during PDT with oral ALA in the normal rat esophagus

5-Aminolevulinic acid (ALA)-induced, protoporphyrin IX (PpIX)-mediated photodynamic therapy (PDT) is a treatment modality for (pre)malignant esophageal lesions (Barretts esophagus).

Light dosage in whole bladder wall photodynamic therapy at 532 and 630 nm

The optical absorption, scattering and anisotropy coefficient of piglet and diseased human bladder tissue were determined in vitro in the wavelength range of 500 - 650 nm. Monte Carlo simulations for whole bladder wall (WBW) photodynamic therapy (PDT) have been performed using the optical parameters determined in vitro. The calculated light dose rate values are in agreement with those measured in clinical WBW-PDT, previously performed at 630 nm. The light dose rate at the bladder wall can differ by a factor of 4, due to variations in optical properties of the tissue. This demonstrates the necessity of in situ light dosimetry during clinical WBW-PDT. WBW-PDT with red light (630 nm) will be technically more advantageous than with green light (532 nm), because of a higher integrating sphere effect.