Tetra(m-hydroxyphenyl)chlorin clinical photodynamic therapy of early bronchial and oesophageal cancers

There have been few studies to date of clinical photodynamic therapy (PDT) with tetra(m-hydroxyphenyl)chlorin (mTHPC). This paper describes the results of the authors' experience with this second-generation photosensitizer, used in the treatment of 40 early cancers of the oesophagus and the bronchi. Surface illumination of the tumour was performed, in most cases, 4 days after intravenous injection of 0.15 mg kg^–1 of mTHPC, using 652 nm or 514 nm continuous wave laser light. Endoscopic follow-up with biopsies and brushings was possible for 35 tumours: 27 (77%) showed no recurrence after disease-free follow-ups that ranged from 3 to 38 months. Major complications, all of which were after red light illumination, included one bronchial stenosis, one oesophagotracheal fistula and two probable occult perforations of the oesophagus. Photodynamic therapy with green light renders such perforations of the oesophageal wall essentially impossible at the applied conditions, and appears not to reduce the efficacy of the treatment. Skin photosensitization, which was never observed later than the first week after injection, occurred in 12 patients. Hence, PDT with mTHPC is a safe and effective treatment for early carcinomas of the oesophagus and the tracheobronchial tree.     

Applicator for light delivery and in situ light dosimetry during endobronchial photodynamic therapy: First measurements in humans

This paper presents a design of an applicator for light delivery and light dosimetry during endobronchial photodynamic therapy (EB-PDT). The design incorporates a linear diffuser that is fixed in the centre of the lumen by a steel spring basket that does not block air flow. An isotropic light detector is included in this design, to measure the light fluence actually delivered to the bronchial mucosa surface. The applicator is designed for use with common bronchoscopy equipment, and can be used with bronchoscopes with a large biopsy channel (≈3 mm). The first clinical measurements were performed and caused no additional discomfort to the (nonphotosensitized) patients. The data showed considerable inter-patient variability of the light fluence rate measured as a result of fixed output power of the diffuser. This fact and the expected strong dependence of the fluence rate on the lumen diameter stress the importance of in situ fluence rate measurement for a proper evaluation of the relationship between light fluence and the biological response of EB-PDT.     

The Calibration of fibre optic probes used for light dosimetry measurements during photodynamic therapy

We describe the calibration of fibre optic probes used to perform in vivo light dosimetry studies during the treatment of skin lesions by photodynamic therapy. Results from six individual detectors show that the linearity of the calibration and the calculated radiant energy fluence rate within a liquid phantom are independent of the type and sensitivity of the probe. The method of calibration is also shown to yield the optical interaction coefficients of the phantom.     

Light delivery and light dosimetry for photodynamic therapy

Photodynamic therapy (PDT) has attracted attention because it was considered to be a selective form of cancer treatment causing minimal damage to normal tissues. This is not exactly true, because the ratio between the photosensitizer concentrations in tumour and surrounding normal tissues is not always much more than one. Nevertheless, tumour destruction by PDT with relatively little damage to normal tissue is possible in many cases. This requires sophisticated light delivery and/or light dosimetry techniques. In this respect the limited penetration of light into biological tissues can sometimes be useful. In this paper a qualitative and sometimes quantitative discussion is given of the physical phenomena determining the energy fluence in a biological tissue. Most important is light scattering, the contribution of which depends on the geometrical conditions. Finite beam surface irradiation, irradiation of hollow organs (bladder) and interstitial irradiation are discussed separately. The emphasis is on light dose and light dose distribution. It is emphasized that PDT dosimetry in general is complicated by photosensitizer distribution (which is usually not known), by photobleaching of the sensitizer, by possible effects of hyperthermia, and by changes in optical properties during and as a result of PDT.     

Photofrin-mediated photodynamic therapy of rat palatal mucosa: Normal tissue effects and light dosimetry

Photodynamic therapy (PDT) is a treatment modality with potential application for premalignant lesions and squamous cell carcinoma of the oral mucosa. PDT in principle has dual selectivity. This may result from a preferential retention of the photosensitizer in target tissue. In addition, the photodynamic activity will be limited to the irradiated area because PDT will not affect tissues in the absence of excitation light. The specificity of PDT is limited by the fact that normal tissues also retain the photosensitizer to some degree, which makes these tissues susceptible to PDT damage. To optimize PDT for oral malignancies, a study was undertaken on normal tissue to investigate the responses in rat palatal mucosa and surrounding anatomical structures. Eighty male Wistar rats were used in the study. Photofrin was administered i.v. at four doses (0, 2.5, 5 or 10 mg kg^–1 body weight). Irradiation for PDT was performed 24 h later. An argon pumped dye laser system was used to produce light of two different treatment wavelengths (514.5 and 625 nm), and various energy density levels (0, 25, 50, 100 or 200 J cm^–2). Early effects of PDT were studied at 2 days and late effects at 2 months after treatment. Twenty-four hours after i.v. administration of Photofrin, it was found that PDT affects normal tissues of the oral cavity both macroscopically and microscopically. Combinations of photosensitizer doses 5 mg kg^–1 and light doses100 J cm^–2 caused severe and permanent damage to the palatal mucosa and adjacent normal structures such as palatal bone and dentition.Light scattering and internal reflection usually raise the fluence rate in tissue above the irradiance of the incident beam. In an additional study using six male Wistar rats, the energy fluence rate at two treatment wavelengths (514.5 and 625 nm) was measured ex vivo in the palatal mucosa and adjacent anatomical structures. As expected, the energy fluence rates were wavelength, tissue and depth dependent. At the air-mucosa boundary, light of 625 nm was found to have a three-times higher fluence rate than the primary incident beam. Under similar conditions, the fluence rate of 514.5 nm was found to be less, but still twice as high as the primary incident beam. At deeper levels of the rat maxilla, fluence rates were still elevated compared with the incident beam. For 625 nm light, this phenomenon was observed up to the level of the nasal cavity. These increased fluence rates could largely explain the pattern of damage to normal mucosa and surrounding anatomical structures.     

Quantitative light dosimetry in vitro and in vivo

Knowledge of the radiant energy fluence rate in tissues during laser irradiation is important for the understanding and improvement of the results of preclinical as well as clinical treatments. Quantitative data are extremely rare, however. In this paper, quantitative measurements of energy fluence rates are reported, in vitro as well as in vivo, with emphasis on light dosimetry for photodynamic therapy. Examples are given of fluence rate distributions that may occur during surface irradiation, intracavity irradiation of hollow organs (bladder) and interstitial irradiation. For the same incident irradiance, the energy fluence rate in tissue may vary considerably, depending on type of tissue, wavelength and geometry. During experimental and clinical interstitial PDT-treatments a considerable decrease in light penetration into tumours was observed, apparently indicating changes in optical properties as a result of treatment. This demonstrates the importance of in vivo light dosimetry.     

Coagulation of blood at the tip of optical fibres used for light delivery in photodynamic therapy

Optimal delivery of light to the tumour is of considerable importance in photodynamic therapy. The most effective way of delivering laser light to the tumour tissue is through an implanted optical fibre. In order to investigate the possible effects taking place at the tips of fibres implanted in tissue, fibres were used to deliver light to human blood and the transmission of light by the blood was measured at different power levels. The maximum power level which could be delivered without charring or coagulation at the fibre tip was measured for five different fibres. Three plane cut fibres and two with 1.5 cm long diffusing tips were studied. Charring and coagulation, which resulted in practically no light being delivered more than 0.5 mm from the fibre tip, were observed at relatively low-output powers (70-130 mW) for all the plane-cut fibres. This is less than the level required to deliver a clinically useful dose in a reasonable time. In contrast, neither charring nor coagulation was observed at the diffusing tips for output powers up to 1.1 W and consequently these should be the fibres of choice for interstitial photodynamic therapy. Observed changes in light transmission through the blood with increasing output power indicate that, for accurate light dosimetry, a means of monitoring delivered light in vivo during photodynamic therapy is essential.     

The optical properties of skin tumours measured during superficial photodynamic therapy

Accurate measurement of light distribution in tissue can improve the knowledge of in vivo tissue optical properties, which is essential for precise dosimetry calculations during photodynamic therapy (PDT). In our application of PDT, superficial skin lesions are treated by topical administration of 5-aminolaevulinic acid followed by surface illumination using 630 nm laser light. Several small detector probes inserted under the skin surface prior to illumination record the light intensity at different depths throughout the treatment. Results from 11 patients are presented. These results indicate light distributions described by the diffusion theory for light transport in tissue. The accumulated data do not imply one specific set of optical parameters for the skin, but indicate that although the depth of light penetration is constant for all patients, the variation in skin colour is significant when performing dosimetry calculations. The average value of effective attenuation coefficient was found to be 0.359 mm⁻¹ (±9.5%) and the coefficientk that is used to quantify the build-up of subsurface fluence rate varies between 0.12 and 8.23.     

A comparison of novel light sources for photodynamic therapy

A diode laser, light-emitting diode (LED) array bandwidth 25 nm, full width half maximum (FWHM) and filtered arc lamp (bandwidth 40 nm, FWHM), all with peak emission at about 650 nm, suitable for the photosensitizer tetra(meta-hydroxyphenyl)chlorin (mTHPC), were compared with a copper vapour laser pumped dye laser, using depth of necrosis in normal rat liver as a measure of photodynamic effect. A three-way comparison between a DL10K dye laser, the LED array and the filtered arc lamp resulted in mean depths of necrosis of 4.64, 4.29 and 4.04 mm, respectively, at 20 J cm^-2, the values for the laser and arc lamp being significantly different at the 5% level. A further comparison of a narrower linewidth DL20K dye laser with the LED array, using a light dose of 20 J cm^-2, showed a significant difference between the mean depths of necrosis of 4.97 and 4.05 mm, respectively (p=0.01). A final study, comparing the DL20K dye laser with the diode laser and a light dose of 10 J cm^-2, demonstrated no significant difference in depths of necrosis (3.23 and 3.25 mm, respectively). The results obtained in the three studies are attributed to the relative bandwidths of light emission for the various sources. A simple mathematical model is presented explaining the results in terms of the relative activation of the photosensitizer and the consequent threshold fluence required for the induction of necrosis. It is concluded that, in order to achieve the same depth of effect as a laser when using the broad band sources, the incident fluence would have to be approximately doubled. However, when the low cost and ease of use of the non-laser sources are taken into consideration, these devices are likely to find widespread applications in clinical photodynamic therapy.     

Wavelength-dependent effect of tetra(m-hydroxyphenyl)chlorin for photodynamic therapy in an ‘early’ squamous cell carcinoma model

The purpose of the present study was to correlate the wavelength of the irradiation source with the phototoxic activity of tetra(m-hydroxyphenyl)chlorin (mTHPC) in healthy and neoplastic mucosae. The hamster tumour model for early squamous cell carcinoma was used in these experiments. In vitro and in vivo studies have shown that mTHPC absorbs significantly at 652 nm (1, 2). This wavelength is used currently in clinical mTHPC photodynamic therapy (PDT) trials. In order to study the wavelength dependence of the phototoxic effect on normal and tumour tissues, irradiation tests were performed 4 days after injection of 0.5mg kg^-1 mTHPC. An argon-ion pumped dye laser was used as the light source. The light dose of 12 J cm^-2 was delivered at a light dose rate of 150 mW cm^-2. The wavelength was varied between 642.5 and 665 nm at 2.5-nm increments. The PDT damage was evaluated in serial Haematoxylin and Eosin stained sections using a tissue-damage scale. Light between 647.5 and 652.5 nm induced the highest damage to both the healthy and tumour mucosae. At wavelengths equal to or below 645 nm, and equal to or above 655 nm, tissue damage decreased. Wavelengths below 642 nm and above 660 nm did not induce any visible tissue damage. These results suggest that the in vivo optimal wavelength range for PDT with mTHPC is between 647 and 652 nm. This information is essential for selecting an appropriate light source.     

光动力疗法治疗鲜红斑痣的疗效

目的探讨光动力疗法（photodynamic therapy,PDT）治疗鲜红斑痣的临床疗效和安全性。方法采用PDT治疗鲜红斑痣125例。根据患者体重,静脉推注相应的光敏剂PSD-007后,皮损局部行铜蒸气激光照射,至红斑肿胀渗出为治疗结点;每次治疗前采集红斑图像以评价治疗效果。2个月后复诊,采用5级分类法评价前次治疗的效果,并进行患者满意度调查,根据红斑减退变平等情况决定治疗结束或继续下一个疗程治疗。结果PDT治疗鲜红斑痣临床效果明显,治疗后即可出现明显的畸形血管肿胀封闭,经过4个疗程的治疗,治愈率达49．60％,显效率达96．00％,其中经过不到2个疗程的治疗,治愈率即达到13．60％。在整个治疗过程中,患者满意率为95．20％。结论PDT治疗鲜红斑痣疗效显著,美容效果好,不良反应小,值得临床推广应用。     

Optical dosimetry in photodynamic therapy

The diffusion approximation for the radiant flux distribution in a tissue layer has been applied to optical dosimetry in photodynamic therapy of malignant tumors. The model assumes that tumor eradication requires a minimum absorbed energy by the localized photosensitizer, taken as 0.19 J/cm3 for hematoporphyrin derivative (HPD) at 630 nm. The analysis leads to the required incident irradiance for front surface illumination as a function of the tumor depth, the optical penetration depth of the tumor, and the concentration of localized sensitizer. The effect of HPD photobleaching on the required light dose and drug dose levels is considered. The results are given in tabular form for typical clinical applications.     

Sensitization and photodynamic therapy of normal pancreas,duodenum and bile ducts in the hamster using 5-aminolaevulinic acid

Photodynamic therapy (PDT) using 5-aminolaevulinic-acid-(ALA)-induced protoporphyrin IX (PPIX) increases survival in hamsters with pancreatic cancer. However, experiments with other photosensitizers on this model show a high risk of duodenal perforation. In this paper, the pharmacokinetics and PDT effects of ALA on normal tissues in the pancreatobiliary region are presented. Using quantitative fluorescence microscopy, maximum PPIX fluorescence was seen in the bile ducts, less in the duodenal mucosa and least in the muscularis propria and pancreas. For PDT, light was delivered either using a bare fibre touching the tissue (single-point illumination), or irradiating a 1.5 cm diameter circular area. Single-point PDT (50 J) produced only localized reversible damage without perforation. Surface irradiation of the whole periampullary region (50 J cm^–2) caused extensive damage, sometimes with perforation. Before PDT can be used safely to treat tumours of the pancreas and bile duct, further studies are necessary to understand its effect on larger areas of normal tissue.     

Laser light application and light monitoring for photodynamic therapy in hollow organs

Photodynamic therapy (PDT) of tumours in hollow organs requires light application devices which enable homogeneous illumination of the tissue surface in hollow organs. This paper presents two laser light application systems generating a homogeneous light distribution and one monitoring unit which detects variations in the applied laser light intensity during treatment. A cylindrical light diffuser has been developed for PDT of cylindrical organs (e.g. bronchus, oesophagus). This system guarantees a defined homogeneity of the laser light intensity. An exact positioning to a defined treatment area is possible. Adjacent tissue is prevented from laser light irradiation. A special lens system has been developed for the irradiation of flat surfaces (e.g. skin, carina, organ wall of the large bowel and the stomach). The illumination area is defined by the distance to the tissue and the large aperture. The size of the system allows for it to pass through the biopsy channel of conventional endoscopes. A monitoring device recently developed helps to detects of the fibre, changes in the medium around the fibre tip, and variations of the laser output power during laser treatment. All devices serve for evaluating an accurate light dosimetry in clinical PDT.     

Standardization of intralipid for light scattering in clinical photodynamic therapy

Intralipid is a light scattering medium used in photodynamic therapy (PDT) in both phantom studies and to obtain optimum light distributions from clinical light delivery systems. Light delivery to a target is one variable in PDT over which we have direct control, and should be as accurate as possible. Variations in the scattering ability of Intralipid from batch to batch will lead to an unpredictable light distribution and undesirable PDT effects. Furthermore, 10% and 20% stock solutions, when diluted to similar final concentrations, have different scattering abilities which will lead to inaccuracies in studies measuring scattering coefficients and imprecision in light distributions from light delivery systems. Batch to batch variations of similar stock solutions should lead to minimal differences in outputs from light delivery systems. A simple and quick assay system is described to define the concentration of Intralipid based on its light scattering ability, which enables testing of solutions prior to use. In the clinical setting where accurate light distribution is required, we suggest that only one stock solution (e.g. 20%) is used and that the final dilution scattering ability is checked using this assay system.     

Photophysics and photochemistry of photodynamic therapy: fundamental aspects

Photodynamic therapy (PDT) is a treatment modality for cancer and various other diseases. The clinical protocol covers the illumination of target cells (or tissue), which have been loaded with a photoactive drug (photosensitizer). In this review we describe the photophysical and primary photochemical processes that occur during PDT. Interaction of light with tissue results in attenuation of the incident light energy due to reflectance, absorption, scattering, and refraction. Refraction and reflection are reduced by perpendicular light application, whereas absorption can be minimized by the choice of a photosensitizer that absorbs in the far red region of the electromagnetic spectrum. Interaction of light and the photosensitizer can result in degradation, modification or relocalization of the drug, which differently affect the effectiveness of PDT. Photodynamic therapy itself, however, employs the light-induced chemical reactions of the activated photosensitizer (triplet state), resulting in the production of various reactive oxygen species, amongst them singlet oxygen as the primary photochemical product. Based on these considerations, the properties of an ideal photosensitizer for PDT are discussed. According to the clinical experience with PDT, it is proposed that the innovative concept of PDT is most successfully implemented into the mainstream of anticancer therapies by following an application-, i.e. tumor-centered approach with a focus on the actual clinical requirements of the respective tumor type.     

In vivo to post-mortem change in tissue penetration of red light

For precise light dosimetry in photodynamic therapy (PDT), the light penetration characteristics of the tissue concerned need to be assessed. Several attenuation coefficients have so far been measured using 633 nm wavelength red light and human tissue obtained from autopsy or surgery. However, the validity of such ex vivo derived values remains uncertain, and hence is evaluated further in this study. Using a rat thigh muscle model, the tissue transmittance of 633 nm red light was compared between in vivo and ex vivo. Transmittance (in vivo) following preliminary injection of 40 mg kg^–1 haematoporphyrin derivative (HPD) was also measured in muscle and liver. The effective attenuation coefficient (_eff) ex vivo in muscle was 0.98±0.06 mm^–1 and in vivo 0.97 ± 0.05 mm^–1. Values agreed within experimental error indicating that in this tissue, changes from in vivo to post-mortem produced no alteration in optical penetration at 633 nm. Preliminary injection of HPD did not influence the penetration depth in muscle although significant changes were observed in an organ of high porphyrin avidity, the liver (0.68±0.08 mm vs 0.40±0.08 mm with HPD). Until techniques for in vivo measurements of light attenuation coefficient become available, the use of ex vivo derived values with 633 nm wavelength light would seem appropriate.     

The study of endogenous porphyrins in human skin and their potential for photodynamic therapy by laser induced fluorescence spectroscopy

Human skin shows a strong autofluorescence in the red spectral region when excited by the 407 nm radiation of a krypton ion laser. The spectrum consists of three main peaks around 600, 620 and 640 nm, which are typical for metalloporphyrins such as Zn-protoporphyrin, coproporphyrin and free protoporphyrin IX, and perhaps represent a mixture of these compounds. The fluorescence is located in sebaceous follicles which contain large amounts of the porphyrin-producing skin bacterium Propionibacterium acnes. Irradiation, especially with violet light, reduces both the integral fluorescence intensity and the number of living bacteria. The process of photobleaching is oxygen-dependent. In addition, irradiation results in the formation of fluorescent photoproducts with spectral bands similar to photo-protoporphyrin. It seems to be possible to use the endogenous porphyrins for a photodynamic therapy of acne vulgaris and to monitor the therapeutic effect by the simultaneous measurement of spectral changes.