WAVELENGTH-DEPENDENT EFFECTS OF BENZOPORPHYRIN DERIVATIVE MONOACID RING A in vivo AND in vitro

Abstract Benzoporphyrin derivative monoacid ring A (BPD-MA) is a chlorin-like photosensitizer currently in clinical trials for cancer and psoriasis. It has maximal absorption peaks at both 630 and 690 nm and can be activated at both these wavelengths. In vitro phototoxicity tests using the P8 15 murine mastocytoma cell lines conducted over wavelengths of light between 678 and 700 nm emitted by an argon-ion pumped dye laser showed that equivalent cell kill could be achieved between 682 and 690 nm. Tests on in vivo phototoxicity of normal skin of DBN2 mice injected with 2 mg/kg of BPD-MA and exposed to light at 125 J/cm², between 620 and 700 nm, demonstrated peaks of normal skin damage occurring at 630–640 nm and 680–690 nm. In tests carried out with light between 620 and 700 nm, at 10 nm increments, it was seen that light delivered at 680–690 nm caused slightly more damage to normal skin than light delivered at 630–640 nm. When lower doses of light between 675 and 705 nm were tested using smaller increments, it was determined that equivalent skin damage occurred over a range of 68–95 nm. Antitumor efficacy in tumor-bearing DBN2 mice was tested between 683 and 695 nm. It was found that equivalent antitumor efficacy, determined by assessing tumor-free status at 20 days posttreatment, occurred at wavelengths between 685 and 693 nm. When tumor-bearing animals injected with BPD-MA at 2 mdkg and exposed to light 3 h later were treated with either 630 or 690 nm light at various doses, it was observed that 690 nm light was more effective at tumor ablation than was 630 nm light, demonstrating that while similar damage to normal skin may be effected by equivalent doses of light at either wavelength, tumor ablation was greater at 690 nm. Further, our data suggest that alternative light sources with bandwidths greater than those of the argon-ion pumped dye laser (±0.3 nm) may have equivalent efficacy with this photosensitizer.     

Pharmacokinetics of Tetra (m-hydroxyphenyl)chlorin in Human Plasma and Individualized Light Dosimetry in Photodynamic Therapy

The pharmacokinetics of the photosensitizer 5,10,15,20-tetra( m -hydroxyphenyl) chlorin(mTHPC) was investigated in the plasma of 20 patients by absorption and fluorescence spectroscopy. The temporal behavior was characterized by a rapid decrease in concentration during the first minutes after intravenous injection of 0.15 mg/kg mTHPC. A minimum concentration in the plasma was reached after about 45 min. The drug concentration then increased again, attaining a maximum after about 10 h, after which it decreased again with a halflife of about 30 h. Irradiation tests in the oral cavity at different time intervals after the injection revealed that the tissue re-action was only partially correlated with the mTHPC plasma level. The tissue response was stronger at later drug-light intervals (1–4 days) than during the first hours after injection even though the mTHPC plasma concentration was higher at the shorter times. Relative mTHPC concentrations were also measured in the mucosae of the oral cavity, the esophagus and the bronchi of 27 patients by light-induced fluorescence spectroscopy using an optical fiber-based spectrometer. These measurements were performed prior to photodynamic therapy (PDT), 4 days after injection of the photosensitizer. Highly significant linear correlations were found between the relative mTHPC concentrations in the mucosae of these three organs. Likewise, the plasma levels of mTHPC measured just before PDT were significantly correlated with the mTHPC concentrations in the three types of mucosae mentioned above. These results indicate that mTHPC plasma levels measured just before PDT can be used for PDT light dosimetry.     

Photodynamic Synovectomy Using Benzoporphyrin Derivative in an Antigen-induced Arthritis Model for Rheumatoid Arthritis

Experimental photodynamic therapy (PDT) has recently been adapted for the treatment of inflammatory and rheumatoid arthritis. The biodistribution of benzoporphyrin derivative monoacid ring A (BPD-MA) and the effect of percutaneous light activation via intra-articular bare cleaved optical fibers was investigated using a rabbit-antigen-induced arthritis model. Qualitative evaluation of intra-articular photosensitizer clearance was performed with laser-induced fluorescence from 0 to 6 h following intravenous injection. The compound was rapidly taken up within the joint and then cleared steadily over the 6 h interval. Biodistribution was determined by fluorescence microscopy and spectrofluoroscopic extraction techniques 3 h following intravenous injection of 2 mg/kg BPD-MA. The biodistribution study demonstrated elevated levels of BPD-MA in synovium (0.35 μg/g) and muscle (0.35μg/g). Fluorescence microscopy demonstrated presence of the compound within pathologic synovium but absence of the photosensitizer within meniscus, ligament, bone and articular cartilage. Tissue effects were evaluated histologically at 2 and 4 weeks posttreatment. BPD-MA-mediated PDT caused synovial necrosis in the region of light activation in 50% of treatment knees at 2 weeks and 43% at 4 weeks. No damage to nonpathologic tissues was observed. These studies indicate that selective destruction of synovium can be achieved by the light-activated photosensitizing agent BPD-MA without damage to articular cartilage or periarticular soft tissues. PDT needs to be further evaluated to optimize treatment parameters to provide for a new minimally invasive synovectomy technique.     

Benzophenothiazine and Benzoporphyrin Derivative Combination Phototherapy Effectively Eradicates Large Murine Sarcomas

Abstract— The tumoricidal effects of photochemotherapy with two photosensitizers, 5-ethylamino-9-diethylaminobenzo[ a ] phenothiazinium chloride (EtNBS) and benzoporphyrin derivative monoacid ring A (BPD-MA), were evaluated separately and in combination against the EMT-6 fibrosarcoma implanted subcutaneously in BALB/c mice. Animals carrying tumors 8-10 mm in diameter were divided into eight different groups (∼20/group) and subjected to various photoirradiation and drug conditions. The tumor response to photodynamic therapy (PDT) was measured as the mean tumor wet weight 2 weeks post-PDT. The combination treatment with 5.25 mg/kg EtNBS and 2.5 mg/kg BPD-MA followed by photoirradiation with 100 J/cm² at 652 nm and then by 100 J/cm² at 690 nm resulted in a 95% reduction in the average tumor weights compared to controls (no light, no drugs) with 76% of the mice being tumor free 2 weeks post-PDT. Because treatment with EtNBS or BPD-MA at twice the light dose and drug concentration resulted in either no significant reduction in tumor weights or increased the lethality of treatment, respectively, the data suggest that the enhanced PDT effect observed with the combination of drugs is synergistic rather than additive. Histology of tumors 24 h post-PDT with the combination of drugs showed nearly complete destruction of the tumor mass with little or no damage to the vasculature and no extravasation of red blood cells. There was no damage to the normal skin adjacent to the tumor. Fluorescence microscopy of EMT-6 cells incubated in vitro with the two photosensitizers revealed that they were localized to different intracellular compartments. The fluorescence pattern from frozen tumor tissue slices following the in vivo administration of the photosensitizers indicated a greater intracellular localization for EtNBS vs BPD-MA.     

Damage Threshold of Normal Rat Brain in Photodynamic Therapy

Normal brain tissue response to photodynamic therapy (PDT) must be quantified in order to implement PDT as a treatment of brain neoplasm. We therefore calculated the threshold for PDT-induced tissue necrosis in normal brain using Photofrin (porfimer sodium, Quadralogic Technologies Inc., Vancouver, BC) as the photosensitizer. The absolute light fluence-rate distribution for superficial irradiation and effective attenuation depth were measured in vivo using an invasive optical probe. Photosensitizer uptake in cerebral cortex was measured with chemical extraction and fluorometric analysis. Photodynamic therapy-induced lesion depths at various drug dose levels were measured as a biological end point. The PDT threshold for normal brain necrosis was calculated as in the magnitude of 10¹⁶ photons/cm³. Thus normal rat brain is extremely vulnerable to PDT damage. This suggests that extra precautions must be exercised when PDT is used in brain.     

Light Sources and Dosimetry Techniques for Photodynamic Therapy

Effective treatment delivery in photodynamic therapy (PDT) requires coordination of the light source, the photosensitizer, and the delivery device appropriate to the target tissue. Lasers, light-emitting diodes (LEDs), and lamps are the main types of light sources utilized for PDT applications. The choice of light source depends on the target location, photosensitizer used, and light dose to be delivered. Geometry of minimally accessible areas also plays a role in deciding light applicator type. Typically, optical fiber-based devices are used to deliver the treatment light close to the target. The optical properties of tissue also affect the distribution of the treatment light. Treatment light undergoes scattering and absorption in tissue. Most tissue will scatter light, but highly pigmented areas will absorb light, especially at short wavelengths. This review will summarize the basic physics of light sources, and describe methods for determining the dose delivered to the patient.     

Techniques for delivery and monitoring of TOOKAD (WST09)-mediated photodynamic therapy of the prostate: clinical experience and practicalities

Photodynamic therapy of solid organs requires sufficient PDT dose throughout the target tissue while minimizing the dose to proximal normal structures. This requires treatment planning for position and power of the multiple delivery channels, complemented by on-line monitoring during treatment of light delivery, drug concentration and oxygen levels. We describe our experience in implementing this approach in Phase I/II clinical trials of the Pd-bacteriophephorbide photosensitizer TOOKAD (WST09)-mediated PDT of recurrent prostate cancer following radiation failure. We present several techniques for delivery and monitoring of photodynamic therapy, including beam splitters for light delivery to multiple delivery fibers, multi-channel light dosimetry devices for monitoring the fluence rate in the prostate and surrounding organs, methods of measuring the tissue optical properties in situ, and optical spectroscopy for monitoring drug pharmacokinetics of TOOKAD in whole blood samples and in situ in the prostate. Since TOOKAD is a vascular-targeted agent, the design and implementation of the techniques are different than for cellular-targeted agents. Further development of these delivery and monitoring techniques will permit full on-line monitoring of the treatment that will enable real-time, patient-specific and optimized delivery of PDT.     

Treatment of Malignant Melanoma by High-Peak-Power 1064 nm Irradiation Followed by Photodynamic Therapy

Irradiation of B16 pigmented melanoma subcutaneously transplanted in C57 mice with a single 650 mj pulse (10 ns) of 1064 nm light from a Q-switched Nd: YAG laser caused instantaneous bleaching of the pigmented tissue. Visual and histological examination of the resulting gray-colored tumor revealed the breakdown of melanosomes with no detectable alteration of the normal and tumor-overlying skin. Histological examination of the irradiated tumor showed some degree of vascular damage; the depth of the photodamage was not affected by the successive delivery of three consecutive light pulses. The bleached tumor grew at a modestly slower rate but the high-peak-power (HPP) laser treatment did not affect the tumor concentration of a photodynamic sensitizer Si(IV)-naphthalocyanine (isoBO-SiNc) intravenously injected 24 h before Nd : YAG irradiation. Treatment of the B16 pigmented melanoma by photodynamic therapy (PDT: 1 mg/kg isoBO-SiNc, 300 mW/cm², 520 J/cm²) from a 774 nm diode laser immediately after the 1064 nm irradiation resulted in a 16 day delay of tumor regrowth, which was markedly longer than the delay (ca 6 days) obtained after PDT under identical conditions without the preirradia-tion. Thus, pretreatment of pigmented tumors with HPP 1064 nm light appears to enhance their susceptibility to conventional PDT. The tumor response was further enhanced by repeating the combined HPP/PDT treatment at an interval of 10 days (regrowth delay: 27 days), as well as by applying hyperthermia immediately after HPP/PDT (regrowth delay: ca 34 days).     

In Vivo Fluence Rate Effect in Photodynamic Therapy of Early Cancers with Tetra(m-hydroxyphenyl)chlorin

Abstract— Several parameters affect clinical trials in photodynamic therapy and influence the therapeutic outcome. Beside drug dose, light dose, drug-light interval and other variables, the fluence rate is a parameter that can influence the therapeutic results. In this study we have evaluated the fluence rate effect with a second-generation photosensitizer, tetra( m -hydroxyphenyl)chlorin (mTHPC) using a 7,12-dimethylbenz(a)anthracene induced early squamous cell carcinoma of the Syrian hamster cheek pouch as a tumor model. Following injection of 0.5 mg/kg of mTHPC, irradiation tests were performed at two drug-light intervals, 4 and 8 days. Wavelength and light dose were adapted from those applied routinely in clinical trials. Irradiations at 652 nm were carried out with fluences ranging from 8 to 20 J/cm² delivered at fluence rates of 15 and 150 mW/cm². Similar tests were also performed at 514 nm with a fluence of 80 J/cm² delivered at fluence rates ranging from 25 to 125 mW/cm². At both wavelengths and drug-light intervals for a given fluence, the higher fluence rates resulted in less tissue damage in tumor and healthy mucosae. However, the lower fluence rates yielded slightly less therapeutic selectivity. This study confirms that the fluence rate is of major importance in clinical PDT.     

Photodynamic therapy for malignant mesothelioma: preclinical studies for optimization of treatment protocols

Effective photodynamic therapy (PDT) depends on the optimization of factors such as drug dose, drug-light interval, fluence rate and total light dose (or fluence). In addition sufficient oxygen has to be present for the photochemical reaction to occur. Oxygen deficits may arise during PDT if the photochemical reaction consumes oxygen more rapidly than it can be replenished, and this could limit the efficacy of PDT. In this study we investigated the influence of the drug-light interval, illumination-fluence rate and total fluence on PDT efficacy for the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC). The effect of increasing the oxygenation status of tumors during PDT was also investigated. PDT response was assessed from tumor-growth delay and from cures for human malignant mesothelioma xenografts grown in nude mice. Tumor-bearing mice were injected intravenously with 0.15 or 0.3 mg.kg-1 mTHPC, and after intervals of 24-120 h, the subcutaneous tumors were illuminated with laser light (652 nm) at fluence rates of 20, 100 or 200 mW.cm-2. Tumor response was strongly dependent on the drug-light interval. Illumination at 24 h after photosensitization was always significantly more effective than illumination at 72 or 120 h. For a drug-light interval of 24 h the tumor response increased with total fluence, but for longer drug-light intervals even high total fluences failed to produce a significant delay in tumor regrowth. No fluence-rate dependence of PDT response was demonstrated in these studies. Nicotinamide injection and carbogen breathing significantly increased tumor oxygenation and increased the tumor response for PDT schedules with illumination at 24 h after photosensitizer injection.     

Fluorescence imaging and phototoxicity effects of new formulation of chlorin e6-polyvinylpyrrolidone

Evaluations of the efficiency of a new formulation of chlorin consisting of a complex of trisodium salt chlorin e6 (Ce6) and polyvinylpyrrolidone (PVP) in photodynamic therapy (PDT) and fluorescence diagnosis was performed on poorly differentiated human bladder carcinoma murine model with the following specific aims: (i) to qualitatively evaluate the fluorescence accumulation in human bladder tumor, (ii) to determine fluorescence distribution of Ce6-PVP using the tissue extraction technique and fluorescence imaging technique, (iii) to compare the fluorescence distribution of Ce6, Ce6-PVP and Photofrin in skin of nude mice, and (iv) to investigate phototoxicity caused by different parameters (drug-light interval, drug dose, irradiation fluence rate and total light fluence) in PDT. The fluorescence of the Ce6-PVP formulation was determined either by fluorescence imaging measurements or by chemical extraction from the tissues displaying similar trends of distribution. Our results demonstrated that the Ce6-PVP formulation possesses less in vivo phototoxic effect compared to Ce6 alone. The phototoxicity revealed a strong dependence on the drug and light dosimetry as well as on the drug-light interval. In PDT, the Ce6-PVP compound was most toxic at the 1h drug-light interval at 200J/cm(2), while Ce6 alone was most toxic at a light dose of more that 50J/cm(2) at the 1 and 3h drug-light interval. We also confirmed that Ce6-PVP has a faster clearance compared to Ce6 alone or Photofrin. This eliminates the need for long-term photosensitivity precautions. In conclusion, the Ce6-PVP formulation seems to be a promising photosensitizer for fluorescence imaging as well as for photodynamic treatment.     

Reactive Oxygen Species Explicit Dosimetry for Photofrin-mediated Pleural Photodynamic Therapy

Explicit dosimetry of treatment light fluence and implicit dosimetry of photosensitizer photobleaching are commonly used methods to guide dose delivery during clinical PDT. Tissue oxygen, however, is not routinely monitored intraoperatively even though it is one of the three major components of treatment. Quantitative information about in vivo tissue oxygenation during PDT is desirable, because it enables reactive oxygen species explicit dosimetry (ROSED) for prediction of treatment outcome based on PDT-induced changes in tumor oxygen level. Here, we demonstrate ROSED in a clinical setting, Photofrin-mediated pleural photodynamic therapy, by utilizing tumor blood flow information measured by diffuse correlation spectroscopy (DCS). A DCS contact probe was sutured to the pleural cavity wall after surgical resection of pleural mesothelioma tumor to monitor tissue blood flow (blood flow index) during intraoperative PDT treatment. Isotropic detectors were used to measure treatment light fluence and photosensitizer concentration. Blood-flow-derived tumor oxygen concentration, estimated by applying a preclinically determined conversion factor of 1.5 × 10⁹ μMs cm⁻² to the blood flow index, was used in the ROSED model to calculate the total reacted reactive oxygen species [ROS]rx. Seven patients and 12 different pleural sites were assessed and large inter- and intrapatient heterogeneities in [ROS]rx were observed although an identical light dose of 60 J cm⁻² was prescribed to all patients.     

Photodynamic therapy in lung and gastrointestinal cancers

Twelve central bronchial carcinoma patients and two gastrointestinal (GI) tract (oesophageal and colonic) early-stage cancer patients were treated with photodynamic therapy (PDT). Haematoporphyrin (HP/5, Jacopo Monico, Italy) at a dose of 5 mg kg-1 body weight was used as photosensitizer. Laser light at 628.2-630 nm generated by two different laser systems (gold vapour laser (I.P. Optics, Sofia, Bulgaria) in lung cancer cases and an argon dye laser system (Spectra Physics, Mountain View, U.S.A.) in GI tract cancers) was used. Lung cancers were irradiated 48 h after drug administration and GI tract cancers were irradiated 72 h after infusion of the photosensitizer. Both tumour sites were treated with a total energy dose in the range 350-600 J cm-2. Efficiency of PDT in lung cancer was evaluated by X-rays and endoscopic and functional respiratory tests for bronchial de-obstruction. Complete remission after PDT of GI tract cancers was considered to be tumour eradication (histologically and cytologically proved) and a tumour-free interval of at least 12 months.     

Assessment of cutaneous photosensitivity of TOOKAD (WST09) in preclinical animal models and in patients

TOOKAD (WST09) is a new, long-wavelength palladium bacteriopheophorbide photosensitizer that targets tissue vasculature. The cutaneous phototoxicity of TOOKAD was assessed in normal rat and pig animal models and in patients in a Phase-I trial of TOOKAD-mediated photodynamic therapy (PDT) for recurrent prostate cancer. Controlled skin exposures were administered using solar-simulated light at various times after drug administration. Two different spectral ranges were used. In the first, the UV portion of the spectrum was removed (UV(-)) because UV irradiation in nondrugged control animals produced an erythema response at incident energy densities (J/cm(2)) lower than those required to induce a PDT response. In the second, the full solar spectrum (UV(+)) was used, and the potentiation by the photosensitizer of the UV-mediated minimum erythema dose was assessed. Results showed that the PDT skin response was negligible at clinical drug doses of 2 mg/kg for any period after administration at light doses of 128 J/cm(2) in the animal models. In patients, there was no observed UV(-) skin response at doses of up to 2 mg/kg, drug-light intervals of 1-3 h or greater and light exposures up to 128 J/cm(2). At higher drug doses in the rat and pig models, the duration of skin phototoxicity was found to be approximately 3 h and less than 1 h, respectively. Using the full spectrum of solar-simulated light, the presence of TOOKAD did not measurably enhance the UV(+)-induced erythema in the rats, pigs or patients.     

Monitoring photoproduct formation and photobleaching by fluorescence spectroscopy has the potential to improve PDT dosimetry with a verteporfin-like photosensitizer

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug-light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm2 at 686 nm and a fluence rate of 70 mW/cm2. Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.     

pH-Dependent Chalcogenopyrylium Dyes as Potential Sensitizers for Photodynamic Therapy: Selective Retention in Tumors by Exploiting pH Differences between Tumor and Normal Tissue

Ideal photosensitizers have long-wavelength absorption and strong tumor selectivity with rapid clearance from normal tissues. The telluroselenopyrylium dye 1 that absorbs light at 795 nm (epsilon = 285,000 M-1 cm-1) has a novel property that enhances the tumor specificity and normal tissue clearance. After intralesional injection to both tumors and surrounding skin, it disappeared from the normal skin of BALB/c mice faster than it did from subcutaneously implanted Colon 26 tumors, which resulted in therapeutic selectivity. In vivo reflectance spectroscopy showed that the half-life in tumor was about 50 min while in skin it was around 12 min. This phenomenon appears to be related to the pH differences in normal skin versus tumor, because the rates of drug hydrolysis in solution were shown to be sensitive to changes in pH. Inhibition of tumor regrowth following intratumoral photosensitizer administration depended on both light dose and drug dose, as well as the time interval between dye injection and irradiation; selectivity depended on the time interval. Although treatment parameters were not optimized efficacy was superior to systemic Photofrin under our standard conditions. We discuss how new, more optimal, photosensitizers can be designed that use rates of hydrolysis to exploit the differences in pH between normal tissue and tumor.     

Preclinical studies in normal canine prostate of a novel palladium-bacteriopheophorbide (WST09) photosensitizer for photodynamic therapy of prostate cancers

Photodynamic therapy (PDT) uses light to activate a photosensitizer to achieve localized tumor control. In this study, PDT mediated by a second-generation photosensitizer, palladium-bacteriopheophorbide WST09 (Tookad) was investigated as an alternative therapy for prostate cancer. Normal canine prostate was used as the animal model. PDT was performed by irradiating the surgically exposed prostate superficially or interstitially at 763 nm to different total fluences (100 or 200 J/cm2; 50, 100 or 200 J/cm) at 5 or 15 min after intravenous administration of the drug (2 mg/kg). Areas on the bladder and colon were also irradiated. The local light fluence rate and temperature were monitored by interstitial probes in the prostate. All animals recovered well, without urethral complications. During the 1 week to 3 month post-treatment period, the prostates were harvested for histopathological examination. The PDT-induced lesions showed uniform hemorrhagic necrosis and atrophy, were well delineated from the adjacent normal tissue and increased linearly in diameter with the logarithm of the delivered light fluence. A maximum PDT-induced lesion size of over 3 cm diameter could be achieved with a single interstitial treatment. There was no damage to the bladder or rectum caused by scattered light from the prostate. The bladder and rectum were also directly irradiated with PDT. At 80 J/cm2, a full-depth necrosis was observed but resulted in no perforation. At 40 J/cm2, PDT produced minimal damage to the bladder or rectum. On the basis of optical dosimetry, we have estimated that 20 J/cm2 is the fluence required to produce prostatic necrosis. Thus, the normal structure adjacent to the prostate can be safely preserved with careful dosimetry. At therapeutic PDT levels, there was no structural or functional urethral damage even when the urethra was within the treated region. Hence, Tookad-PDT appears to be a promising candidate for prostate ablation in patients with recurrent, or possibly even primary, prostate cancer.     

In vivo photodynamic characteristics of the near-infrared photosensitizer 5,10,15,20-tetrakis(M-hydroxyphenyl) bacteriochlorin

This paper describes the photodynamic characteristics of the new near-infrared photosensitizer 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (mTHPBC or SQN400) in normal rat and mouse tissues. A rat liver model of photodynamic tissue necrosis was used to determine the in vivo action spectrum and the dose-response relationships of tissue destruction with drug and light doses. The effect of varying the light irradiance and the time interval between drug administration and light irradiation on the biological response was also measured in the rat liver model. Photobleaching of mTHPBC was measured and compared with that of its chlorine analog (mTHPC) in normal mouse skin and an implanted mouse colorectal tumor. The optimum wavelength for biological activation of mTHPBC in rat liver was 739 nm. mTHPBC was found to have a marked drug-dose threshold of around 0.6 mg kg-1 when liver tissue was irradiated 48 h after drug administration. Below this administered drug dose, irradiation, even at very high light doses, did not cause liver necrosis. At administered doses above the photodynamic threshold the effect of mTHPBC-PDT was directly proportional to the product of the drug and light doses. No difference in the extent of liver necrosis produced by mTHPBC was found on varying the light irradiance from 10 to 100 mW cm-2. The extent of liver necrosis was greatest when tissue was irradiated shortly after mTHPBC administration and necrosis was absent when irradiation was performed 72 h or later after drug administration, suggesting that the drug was rapidly cleared from the liver. In vivo photobleaching experiments in mice showed that the rate of bleaching of mTHPBC was approximately 20 times greater than that of mTHPC. It is argued that this greater rate of bleaching accounts for the higher photodynamic threshold and this could be exploited to enhance selective destruction of tissues which accumulate the photosensitizer.     

In vivo Spectroscopic Evaluation of the Intraperitoneal Cavity in Canines

As part of a preclinical trial for the treatment of peritoneal carcinomatosis (PC) with photodynamic therapy (PDT), we have assessed changes in optical properties, tissue oxygenation and drug concentration as a result of benzoporphyrin derivative (BPD)-mediated PDT using diffuse reflectance and fluorescence measurements. PDT can effectively treat superficial disease spread, but treatment efficacy is influenced by physical properties of the treated tissue which can change over the treatment time. In this study, healthy canines were given BPD and irradiated with 690 nm light during a partial bowel resection, and spectroscopic and fluorescence measurements were made using an in-house built spectroscopic probe. Hemoglobin concentration, oxygenation and optical properties were determined to be highly heterogeneous between canines and at different anatomical locations within the same subject, so further development of PDT dosimetry systems will need to address this patient and location-specific dose optimization. Compared to other photosensitizers, we found no apparent BPD photobleaching after PDT.     

Photodynamic parameters in the chick chorioallantoic membrane (CAM) bioassay for topically applied photosensitizers

The relative efficacy of Photofrin-based photodynamic therapy (PDT) has been compared with that of the second-generation photosensitizers 5-aminolevulinic acid (ALA), sulfonated chloro-aluminum phthalocyanine (AlPcSn), benzoporphyrin derivative monoacid ring A (BPD-MA), and lutetium texaphyrin (Lutex). PDT-induced vascular damage in the chick chorioallantoic membrane (CAM) is measured following topical application of the photosensitizers. In order to make meaningful comparisons, care is taken to keep treatment variables the same. These include light dose (5 and 10 J/cm2), power density (33 and 100 mW/cm2), and drug uptake time (30 and 90 min). The drug dose ranges from 0.1 microgram/cm2 for BPD to 5000 micrograms/cm2 for ALA. Results are also analyzed statistically according to CAM vessel type (arterioles versus venules), vessel diameter, and vessel development (embryonic age). For each photosensitizer, the order of importance for the various PDT parameters is found to be unique. The differences between the sensitizers are most likely due to variation in biophysical and biochemical characteristics, biodistribution, and uptake kinetics.