Characterization of a murine model for the rapid assessment of acute photodynamic response in tumour and muscle

A murine implanted colorectal tumour model is described in which a measurement of tumour depth of necrosis is combined with a simultaneous measurement of muscle damage. The response of this model to a range of photodynamic therapy parameters was characterized using the chlorin photosensitizer mTHPC (temoporfin, Foscan®R). Both tumour depth of necrosis (measured directly and on histology) and muscle swelling correlate with the dose of photosensitizer and light, and are shown to be repeatable and consistent with published values obtained under similar conditions using established models.     

Tissue photosensitizer detection by low-power remittance fluorimetry

A simple adaptation of a commercial spectrofluorimeter which allows the semiquantitative determination of photodynamic therapy photosensitizer fluorescence in accessible tissues is described. Light from a xenon lamp is directed via a monochromator onto the tissue surface by a bifurcated random fibre bundle. Tissue fluorescence is directed to the emission monochromator and photomultiplier of the fluorimeter by the second limb of the fibre bundle. Although relatively simple, this device can be used to carry out a wide range of useful measurements in clinical and experimental photodynamic therapy. The sensitivity and reproducibility of the measurements were determined using mouse tumour and muscle tissue fluorescence measured in vivo compared with photosensitizer content measured by high performance liquid chromatography. As an illustration of the potential applications of such systems, the time courses of fluorescence in the skin of patients treated with the photosensitizers Photofrin® and metatetra(hydroxyphenyl)chlorin (mTHPC) (temoporfin) and the photobleaching of 5-aminolaevulinic acid-derived protoporphyrin IX during treatment, are described.     

Near infra-red diode laser for photodynamic tumour therapy using bacteriochlorina

Clonogenic cell survivals were performed in order to assess the feasibility of tumour cell kill with an experimental diode laser emitting 250 mW of light at λ = 779 nm using the photosensitizer bacteriochlorina(BCA). The AlGaAs diode laser is based on organometallic vapour epitaxial crystal growth technology. The electrical to optical conversion efficiency amounts to 21% and the beam divergence is 47° by 7.0° full width at half maximum. BCA was proved to be an effective non-toxic photosensitizer in vitro and in vivo. It has a major absorption peak at 760 nm where tissue penetration of light is optimal. Clonogenic T24 human bladder carcinoma cell survivals were photosensitizer concentration and light dose dependent. A 0.1% survival rate was obtained with an illumination intensity of 50 mWcm⁻² for 90 s (4.5 Jcm⁻²) and a BCA concentration of 6 μgml⁻¹. Illumination without BCA at energy levels exceeding the PDT levels with a factor 10, or BCA alone without illumination had no effect on the cells in the clonogenic cell survivals. The combination of BCA with a near infra-red diode laser is most promising for photodynamic tumour therapy as a result of the reliability, compactness and relatively low price of the illumination device, the high transmittance of near infra-red light in tissue and the tumour killing potential of BCA.     

Selective necrosis in hamster pancreatic tumours using photodynamic therapy with phthalocyanine photosensitization.

Photodynamic therapy (PDT) is often thought to be able to effect selective tumour necrosis. This therapeutic selectivity, based on transient differences in tumour: normal tissue photosensitizer concentration ratios, is rarely useful clinically in extracranial tumours, although PDT itself may be of value by virtue of the nature of the damage produced and healing of normal tissue by regeneration. This report describes the effects of PDT on normal pancreas and chemically induced pancreatic cancers in the hamster, where a different mechanism of selective necrosis may be seen. Photosensitizer distribution in normal and neoplastic pancreas was studied by chemical extraction and fluorescence microscopy. Correlation of distribution studies with necrosis produced by PDT shows that the photodynamic dose (product of tissue concentration of sensitizer and light dose) threshold for damage is seven times as high for normal pancreas as for pancreatic cancer. Tumour necrosis extended to the point where tumour was invading normal areas without damaging the normal tissue. In rat colonic cancer, photodynamic dose thresholds in tumour and normal tissue are similar and so such marked selectivity of necrosis is not possible. The reason for this selectivity in the pancreas is not clear, but recent evidence has suggested a difference in response to PDT between normal and neoplastic pancreatic cell lines and the presence of a singlet oxygen scavenger in normal pancreas is postulated. Furthermore, the present fluorescence microscopy studies suggest that tumour stroma contains the highest level of photosensitizer and thus receives the highest photodynamic dose during PDT. These results suggest a possible role for PDT in treating small pancreatic tumours or as an adjuvant to other techniques, such as surgery, that reduce the main bulk of tumours localized to the pancreas.     

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.     

Chromatographic analysis and tissue distribution of radiocopper-labelled haematoporphyrin derivatives

The photosensitizers haematoporphyrin derivative (HPD) and dihaematoporphyrin ether/ester (DHE) have been labelled with the radionuclide copper-64 to allow non-invasive measurement of the concentration of photosensitizer in tumour and normal tissues. The effects of labelling have been assessed by gel and high-performance liquid chromatography. Direct comparison of the tissue uptake and clearance of⁶⁴Cu-labelled HPD/DHE with those of [¹⁴C]HPD/DHE has been made in mice bearing intramuscular radiation-induced fibrosarcoma (RIF) tumours, by gamma and beta counting of tumour, skin, liver, muscle, lung, kidney, brain, and blood samples at 1–72 h after intraperitoneal injection of test and control drug. The uptake and clearance curves of⁶⁴Cu-labelled HPD or DHE in tumour, liver and blood agree to within 20% with those for the¹⁴C-labelled control. Non-invasive quantification of⁶⁴Cu-labelled DHE by gamma counting in vivo in rabbits bearing VX2 tumours in the ear shows good agreement with the photosensitizer concentrations in tumour and liver measured post-mortem in tissue samples.     

A comparison of photosensitizer administration routes for interstitial photodynamic therapy of the liver

Accurate localization of laser light within a tumour lessens the need for selective tumour retention of the photosensitizer. The aim of this study was to investigate different routes of photosensitizer administration for interstitial photodynamic therapy (IPDT) of the liver. Sprague-Dawley rats were photosensitized with HPD 5 mg kg⁻¹ intravascularly at 48 h or by regional administration 60 min prior to light delivery or by interstitial injection (0.04 mg, 0.15 ml) directly into the hepatic parenchyma at 10 and 60 min prior to light delivery. Thirty-two joules of light from a helium-neon (HeNe) laser were delivered interstitially into the median lobe of the liver via a 200-μm optical fibre. Four days after light delivery the liver was harvested, sectioned and stained with haematoxylin and eosin (HE). The maximum cross-sectional area of photodamage was estimated for each photosensitizer administration route in six livers. Both conventional PDT and interstitial routes of administration of the photosensitizer showed comparable areas (±s.e.m.) of bioactivity (8.32±2.03 mm² and 9.5±1.44 mm²) that were greater than those for control livers treated with light only (1.89±0.39 mm²,p<0.01). The maximum area of biological effect was noticed in livers regionally photosensitized by the portal vein or hepatic artery 60 min prior to light delivery (intraportal vein 13.32±1.52 mm² and intrahepatic artery 14.21±4.19 mm²,p<0.01). These results suggest that for IPDT, regional administration of a photosensitizer may achieve the greatest biological effect. This route may be the most appropriate route for interstitial PDT using a selective light delivery system within the liver.     

Photodynamic therapy of tumours and other diseases using porphyrins

Photodynamic therapy (PDT) with porphyrins and red light (620–630 nm) is finding increasing clinical application for both the eradication of relatively small tumours and the palliation of inoperable or obstructive tumours. PDT also shows some promise for the sterilization of the tumour bed after surgical removal of neoplastic masses. Several porphyrins have been found to be accumulated and retained by tumour tissues; however, a chemically prepared derivative of haematoporphyrin, termed HpD, and a purified form of HpD, termed DHE (dihaematoporphyrin ether or ester), are most frequently used in clinical practice owing to their optimal tumour-localizing properties and low systemic toxicity in the dark. The efficiency of HpD/DHE photoactivation by red light is very low, since their extinction coefficient at wavelengths above 600 nm is below 10³ m ⁻¹ cm⁻¹. Therefore, a large number of investigations are being performed in order to improve the efficacy of PDT. One approach involves the use of porphyrin analogs (e.g., chlorins, phthalocyanines) which retain a high affinity for tumours and possess intense absorption bands in the red spectral region. Moreover, the selectivity of tumour targeting can be enhanced by transport of the photosensitizing drug with some types of lipoproteins or monoclonal antibodies. These developments are of interest also in view of the proposed extension of PDT to the treatment of other diseases, including viral and microbial infections, atheroma and psoriasis.     

Fluorescence detection of small gastrointestinal tumours: principles, technique, first clinical experience

Photodynamic therapy (PDT) is a form of cancer treatment based on the selective accumulation of a photosensitizer in neoplastic tissue. The fluorescent properties of a photosensitizer permit diagnostic localization of primary tumours and/or metastasis. Occult lesions are hard to detect and can easily be missed during routine laparoscopy. Fluorescence observation offers additional optical information and the ability to detect these occult tumours. Clinically, we used 5-aminolevulinic acid for peritoneal staining and tumour demarcation via tumour-specific fluorescence induced by protoporphyrin IX. For laparoscopic observations, a "D-Light" system was used; the conventional white light source was equipped with an optical blocking filter that transmits at the excitation wavelength (380-450 nm) and blocks all other parts of the spectrum. With the aid of a suitable observation filter, the relevant fluorescence was detectable. With the help of this fluorescence we increased the capacity to detect occult tumours, that were missed with white-light observation (9/26). In the gastrointestinal tract, we used a krypton laser at 405 nm for PP IX fluorescence induction. Although there were high sensitivity rates for neoplasms (81% peritoneal carcinomas, 60% gastric cancer), no exact histopathological statement could be achieved at because of false-positive fluorescence, mainly caused by inflammation (6/32). Current clinical goals and the future perspectives of photodynamic diagnostic are discussed.     

A review of photoradiation therapy in the management of central nervous system tumours

Photoradiation therapy depends on the selective retention of a photosensitizer within the tumour followed by activation of the sensitizer by irradiating the tumour with light of the appropriate wavelength. The present methods of treatment of cerebral glioma are inadequate and the possible benefit of utilizing photoradiation therapy to obtain improved local control of the tumour has been studied in the laboratory and in clinical trials. The biological basis for photoradiation therapy and the laboratory studies and clinical trials involving the use of photoradiation therapy to treat cerebral tumours are discussed.     

Experimental photodynamic therapy with tetrapropyl-porphycene: Ultrastructural studies on the mechanism of tumour photodamage

Red light irradiation of a transplanted MS-2 fibrosarcoma in mice at 24 h after injection of liposome-zbound tetra-n-propyl-porphycene (TPP, 2mg kg^–1 b.w.) caused an efficient tumour necrosis. Electron microscopy analysis of tumour specimens taken at different times after the phototherapeutic treatment showed the development of direct damage of malignant cells between 3 and 6 h; the earliest detectable alterations occurred at the level of mitochondria. The endocellular damage gradually progressed with extensive vacuolization of the cytoplasm and, at later stages, formation of pyknotic nuclei. On the other hand, the vascular system of the tumour appeared to be well preserved up to about 9 h, when several endothelial alterations were detected. The damage of the tumour tissue was essentially complete 24 h after the phototreatment. The pattern of tumour modification is consistent with a preferential transport and tumour release of the liposome-bound TPP by low-density lipoproteins.     

Fluorescence detection and photodynamic treatment of photosensitized tumours in special consideration of urology

Most methods of modern laser tumour therapy are physically based on the conversion of light to heat. Recently tumours have also been treated using ionizing processes for tissue ablation. Photodynamic laser therapy (PDT), however, involves light-induced non-thermal biochemical processes and the use of a photosensitizer.Several drugs are known to be stored selectively in tumours after systemic application. This transient marking can be used for diagnostic and therapeutic procedures. The marker most commonly used is dihaematoporphyrin ether/ester (DHE) intravenously injected at doses of 0.2–3.0 mg/kg bodyweight for diagnosis and therapy, respectively. The corresponding clearance intervals after injection of DHE range from 3–48 h to 25–75 h.Detection of photosensitized tumours might offer great advantages. The highly sensitive two-wavelength laser excitation method with computerized fluorescence imaging recently has been transferred to the hospital for clinical tests.Photoinduced production of singlet oxygen is claimed to be the initial process which leads to later tumour destruction and therapy. PDT has been applied to 20 patients suffering from superficial tumours (TIS GII–III) recurred after application of other treatments. The results after PDT were evaluated by three-monthly check-ups (endoscopy, cytology, bladder mapping, renal ultrasonography) as well as by computed tomography (CT) examination at 8–13 month intervals. In six patients treated by PDT no tumour recurrence has been found over the whole observation period of up to 5 years. Four patients have remained free of tumour (12 and 14 months) after repeated transurethral resection (TUR) and Nd-YAG laser therapy following PDT. Due to an initial application of insufficient irradiation four patients required a second PDT. In one patient a circumscribed dysplasia appeared at the left ostium 26 months following PDT and was treated successfully by means of thermal Nd-YAG laser irradiation following TUR. In six patients slight mucosal atypia persisted for a period of at least 2.5 years. One cystectomy had to be performed because of bladder shrinkage. The dissected bladder, however, was free of tumour.These preliminary results suggest that PDT is justified in patients who are in a worst-case situation with cystectomy recommended in case of recurrent superficial TIS bladder carcinoma and indicate the future potential of photodynamic therapy of tumours.Homogeneous irradiation of the area to be treated and a reliable light dosimetry are prerequisites for an effective tumour therapy. Standard instruments for a routine application do not exist, but are under development.     

Thermal damage and haematoporphyrin-derivative-sensitized photochemical damage in laser irradiation of rabbit retina

In photodynamic therapy of solid tumours, 630 nm light is used to activate the photosensitizer haematoporphyrin derivative (HpD) in vivo, resulting in photochemical toxicity. This form of treatment has been used in a wide range of tumour types and sites, including ocular tumours such as malignant melanoma and retinoblastoma. In addition to HpD-sensitized photochemical effects, direct thermal damage may occur if the light exposure is sufficiently high. In this study, the thresholds for immediate and delayed thermal damage to the normal rabbit retina have been measured for a range of incident optical power and energy densities. HpD-sensitized photochemical damage has been demonstrated at light levels below these thermal thresholds. Thus, with appropriate selection of light power levels and exposure times, photodynamic response can be achieved without accompanying thermal damage.     

Laser-induced in vivo fluorescense of bacteriochlorina: Preliminary results

Female WAG/RIJ rats with isologous RMA-mammary or rhabdomyosarcoma tumours transplanted subcutaneously on the flank and thigh were given 10 mg kg⁻¹ bacteriochlorina intravenously (IV). In vivo fluorescence properties of this new photosensitizer were studied in a first attempt by using intensified fluorescence imaging. Analysis of the digitized images yielded tumour/muscle fluorescence ratios. Thirty minutes after IV injection a ratio of about 2.4 was reached, which was maintained for at least 48 h.     

Photodynamic treatment of neoplastic lesions of the gastrointestinal tract

Photodynamic therapy (PDT) is a form of cancer treatment based on the selective accumulation of a photosensitizer (by exogenous or endogenous means) in neoplastic tissue. Subsequent activation of the photosensitizer by a specific wavelength of light results in tumor cell death. Activation of a photosensitizer to the appropriate energy state results in the production of singlet oxygen, a powerful oxidizing agent. PDT can kill cells by three mechanisms: direct cell death by photooxidation, apoptosis, or as a consequence of vascular shutdown. The toxicity of PDT is site specific and dependent on the organ being irradiated and the selectivity of the photosensitizer for target tissue over normal tissue. However, there are also reactions related to the sensitizer per se that are independent of those related to the treatment site. Such reactions include cutaneous photosensitization, nausea, vomiting, hypotension, and altered liver 'function' tests. Excitation of photosensitizer by an incident photon produces reemission of a fluorescent photon, which can be used to detect a tumor that is not ordinarily evident. The major limiting factor in using PDT is the depth of tumor kill. The majority of clinical experience involving PDT of the gastrointestinal tract involves patients who are considered to be poor operative risks, and reported follow-ups after treatment are not only limited but also variable.     

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.     

The study of the characteristic of photocytotoxicity under high peak power pulsed irradiation with ATX-S10Na(II) in vitro

We studied hydrophilic photosensitizer ATX-S10Na(II) mediated photocytotoxicity against macrophage-like cell under pulsed irradiation. We found that photocytotoxicity suppression under high intensity irradiation was directly induced by a decrease in the Type-II photoreaction. We showed that this decrease was not attributable to absorption saturation with the high intensity irradiation. We found the cell lethality change from 70% to 13% with the pulse peak power density ranging from 0.29 MW/cm² to 1.36 MW/cm², at the light dose of 20 J/cm² and the pulse repetition rate at 40 Hz. To investigate the Type-II reaction, we measured the photobleaching, oxygen consumption and singlet oxygen luminescence of the photosensitizer solution. The transient absorption from the photosensitizer during the irradiation was measured with the pump-and-probe technique. We believe that the photocytotoxicity suppression induced by the high intensity irradiation might be useful for the treatment of depth-controlled photodynamic therapy without the wall damage of a hollow organ.     

Photodynamic therapy of malignant brain tumours

Experience with intraoperative PDT in 50 patients with malignant supratentorial tumours is reported; in 33 cases the tumour was recurrent. In 45 patients the tumour was a cerebral glioma and in five cases a solitary cerebral metastasis. There were 29 males and 17 females with an age range of 17–73 (mean 48) years. All patients received either haematoporphyrin derivative (HPD) or dihaematoporphyrin ether (DHE) 18–24 h preoperatively. A photoilluminating device, of the authors' design, was coupled to an argon dye pump laser in order to deliver light at 630 nm to a tumour cavity created by radical tumour resection and/or tumour cyst drainage. The total light energy delivered ranged from 440 to 3888 J and the light energy density ranged from 8 to 175 J/cm². In eight patients a line fibre(s) was used to administer interstitial light as a supplement to the cavitary photoillumination. The additional light dose ranged from 60 to 945 J/cm.There were two postoperative deaths as the consequence of haematoma accumulation in the tumour resection cavity. In three patients neurological function was worse postoperatively and did not recover. Postoperative cerebral oedema was pronounced in some cases and required second craniotomy in two patients (the histology from both showed haemorrhagic necrosis of residual tumour). Four patients developed wound infections; two of these required surgical treatment. Four patients, two of whom were hemiparetic, developed deep vein thrombosis and required anticoagulant therapy. There were no adverse systemic reactions to the administration of either photosensitizer and only three skin photosensitivity reactions.Follow up ranged from 1 to 30 months. In the group of 45 patients with gliomas the death rate per observation year was 0.92 for the interval between PDT and death. For the interval between first diagnosis and death the rate was 0.41 deaths per observation year. The median survival was 8.6 months with a 1 and 2 year actuarial survival rate of 32% and 18%, repectively.In 12 patients a complete or near complete CT scan response was identified post PDT. These patients tended to have a tumour geometry (e.g. cystic) that allowed complete or near complete light distribution to the tumour. The median survival for this group was 17.1 months with a 1 and 2 year actuarial survival of 62% and 38%, respectively. In the 33 cases without a complete response the median survival was 6.5 months with a 1 and 2 year actuarial survival of 22% and 11%, respectively.Photodynamic therapy of malignant brain tumours can be carried out with acceptable risk. Good responses appear to be related to adequate light delivery to the tumour.     

Possible advantages of aluminum-chloro-tetrasulfonated phthalocyanine over hematoporphyrin derivative as a photosensitizer in photodynamic therapy

The potency of aluminum-chloro-tetrasulfonated phthalocyanine (AlS4Pc) as a photosensitizer in photodynamic therapy was evaluated in in vitro and in vivo studies. Compared with hematoporphyrin derivative (HpD), the following advantages of AlS4Pc were revealed: (1) AlS4Pc was less toxic than HpD in vitro without hight irradiation. (2) AlS4Pc showed more photodynamic-dependent cytotoxicity and anti-tumor effect in the red area of the spectrum (>660 nm) at which tissue penetration is high. (3) AlS4Pc appeared to be removed more rapidly from normal tissues such as muscle and skin. (4) AlS4Pc showed less photodynamic-dependent cytotoxicity in vitro and milder cutaneous phototoxicity in vivo with UVA irradiation. On the basis of these observations, AlS4Pc shows considerable promise as a photosensitizer for PDT.     

Human pancreatic carcinoma cells are sensitive to photodynamic therapy in vitro and in vivo.

BACKGROUND: The aim of this study was to assess the efficiency of photodynamic therapy (PDT) on human pancreatic cancer cells in vitro and in an animal model. METHODS: Human pancreatic tumour cell lines were submitted to PDT with pheophorbide a (Ph a), a chlorophyll derivative, in culture and after grafting into athymic mice. Ph a was tested in culture (10-10-10-5 mol/l) with a 5-J/cm2 energy treatment and on tumour-bearing Nude mice (30 mg/kg intraperitoneally) with a 100-J/cm2 PDT session. The effect of PDT was assessed in vitro using proliferative, apoptotic and clonogenic tests and in vivo on tumour growth and on the induction of tumour necrosis. RESULTS: PDT inhibited tumour cell growth in culture by affecting DNA integrity. This tumour cell photodamage started at low concentration (10-7 mol/l) as corroborated by clonogenic and tumour growth tests. A strong necrosis was achieved in vivo with a single PDT session. CONCLUSION: PDT destroyed human pancreatic carcinoma after low photosensitizer supply and weak energy application. It exerted this tumoricidal effect via apoptosis induction with a gentle protocol, and apoptosis and/or necrosis with a stronger protocol.