A direct sensitized fluorimetric determination of 5,10,15,20-tetra(m-hydroxyphenyl)chlorin [m-THPC (Foscan)] in human plasma using a cyclodextrin inclusion complex.

The 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) (Foscan) is a photosensitizer used in the photodynamic therapy (PDT) of cancers which is currently under clinical trial. The formation of a m-THPC inclusion complex with dimethyl-beta-cyclodextrin (Me-beta-CD) in solution was demonstrated on the basis of circular dichroism experiments. A 1:2 complex stoichiometry was found and an inclusion constant beta 2 = 2.8(+/- 0.4) x 10(10) M-2 was determined. The formation of such a complex was shown to enhance the m-THPC fluorescence intensity. It could be exploited to improve the sensitivity of the direct m-THPC detection in human plasma. Optimization of the operating conditions shows that the best results were obtained by the addition of 100 microL of a concentrated Me-beta-CD solution (3.2 x 10(-2) M) to 1 mL plasma samples. Compared to the standard conditions, a 90% increase in sensitivity was obtained. The proposed analytical method which showed a linear response function [0-300 ng mL-1 (440 pM)] and a low limit of detection [1.5 ng mL-1 (2 pM) (S/N = 3)] appears, especially due to the absence of metabolism, a simple and specific method suitable for pharmacokinetics studies in patients.     

Synthesis,and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.     

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.     

The high photoactivity of m-THPC in photodynamic therapy. Unusually strong retention of m-THPC by RIF-1 cells in culture

5,10,15,20-Tetra(m-hydroxyphenyl)chlorin (m-THPC, Foscan) is an extremely powerful photosensitizer showing up to 200 times the photodynamic activity of Photofrin in patients, in terms of drug/light dose. The influence of treatment conditions on the photodynamic efficacy of m-THPC has been compared to polyhematoporphyrin (PHP), a Photofrin equivalent, and a cationic pyridinium zinc (II) phthalocyanine (PPC), using the RIF-1 cell line. As predicted, the presence of serum during sensitizer incubation reduced the photodynamic efficacy of all three sensitizers. However, the presence of serum during the illumination period only had an inhibitory effect with PHP and PPC but not m-THPC. Quantification of the intracellular levels of sensitizer revealed that this was due to the efflux of PPC and PHP but not m-THPC into the medium, suggesting that m-THPC is tightly sequestered on entering the cell. This may partially explain the high efficacy of m-THPC in clinical photodynamic therapy and also highlights the importance not only of incubation conditions but also illumination conditions when in vitro comparisons are performed.     

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.     

A comparative study of the cellular uptake and photodynamic efficacy of three novel zinc phthalocyanines of differing charge

Three novel substituted zinc (II) phthalocyanines (one anionic, one cationic and one neutral) were compared to two clinically used photosensitizers, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin (m-THPC) and polyhematoporphyrin (PHP), as potential agents for photodynamic therapy (PDT). Using the RIF-1 cell line, photodynamic efficacy was shown to be related to cellular uptake. The cationic phthalocyanine (PPC, pyridinium zinc [II] phthalocyanine) had improved activity over the other two phthalocyanines and slightly improved activity over PHP and m-THPC. The initial subcellular localization of each photosensitizer was dependent upon the hydrophobicity and plasma protein binding. The phthalocyanines had a punctate distribution indicative of lysosomes, whereas m-THPC and PHP had a more diffuse cytoplasmic localization. A relocalization of phthalocyanine fluorescence was observed in some cases following low-level light exposure, and this was charge dependent. The anionic phthalocyanine (TGly, tetraglycine zinc [II] phthalocyanine) relocalized to the nuclear area, the localization of the hydrophobic phthalocyanine (TDOPc, tetradioctylamine zinc [II] phthalocyanine) was unchanged, whereas the distribution of the cationic phthalocyanine (PPC) became more cytoplasmic. This suggests that relocalization following low-level irradiation is a critical factor governing efficacy, and a diffuse cytoplasmic distribution may be a determinant of good photodynamic activity.     

m-THPC-Mediated Photodynamic Therapy (PDT) Does Not Induce Resistance to Chemotherapy, Radiotherapy or PDT on Human Breast Cancer Cells In Vitro

Photodynamic therapy (PDT) uses laser light to activate a photosensitizer that has been absorbed preferentially by cancer cells after systemic administration. A photo-toxic reaction ensues resulting in cell death and tissue necrosis. Some cells, however, may survive PDT. This study was performed to determine if surviving human breast cancer cells (MCF-7) can become resistant to PDT, chemotherapy or radiotherapy. The MCF-7 cells were cultured under standard conditions prior to being exposed to the photosensitizer, 5,10,15,20-meta-tet-ra(hydroxyphenyl)chlorin (zn-THPC), for 24 h and then irradiated with laser light (652 nm). Surviving cells were allowed to regrow by allowing a 2 week interval between each additional PDT. After the third and final treatment, colony formation assays were used to evaluate the sensitivity of cultured cells to ionizing radiation and PDT and the ATP cell viability assay tested in vitro chemosen-sitivity. Flow cytometry was used to analyze the cell cycle. No alterations in the cell cycle were observed after three cycles of PDT with m-THPC. Similar responses to chemotherapy and ionizing radiation were seen in control and treatment groups. The m-THPC-sensitized PDT did not induce resistance to subsequent cycles of PDT, chemo- or radiotherapy. Photodynamic therapy with m-THPC may represent a novel adjunctive treatment of breast cancer that may be combined with surgery, chemotherapy or ionizing radiation.     

新型光敏剂32-(4-甲氧基苯基)-152-天冬氨酸-二氢卟吩e6的药代动力学及组织分布特征

二氢卟吩类衍生物3²-(4-甲氧基苯基)-15²-天冬氨酸-二氢卟吩e6(DYSP-C34)是从程海湖螺旋藻中提取并合成的新型光敏剂。研究DYSP-C34在生物体内的药代动力学及组织分布过程对光动力疗法(PDT)的有效性和安全性至关重要。该文运用高效液相色谱-紫外(HPLC-UV)检测技术,建立了大鼠血浆中DYSP-C34的检测方法。采用沉淀蛋白-液液萃取法处理血浆和组织样品,采用Unitary C₁₈色谱柱(250 mm×4.6 mm, 5 μm)分离,流动相为甲醇-5 mmol/L四丁基磷酸氢铵缓冲盐溶液(70∶30, v/v),流速为1.0 mL/min,进样量为20 μL,检测波长为400 nm,柱温为40 ℃。实验结果表明,大鼠血浆药物质量浓度在1~200 μg/mL范围内线性良好,判定系数(r²)为0.9941。在低、中、高(8、40、120 μg/mL)3个添加水平下的提取回收率分别为74.39%、69.71%和65.89%,日内和日间相对标准偏差(RSD)均在5%以内。运用此方法测定静脉注射DYSP-C34(16 mg/kg)后大鼠血浆中以及荷瘤小鼠组织中的药物浓度,采用DAS 2.0计算出药物半衰期t_1/2z为6.98 h,药-时曲线下面积AUC_(0-∞)为1025.01 h·mg/L,平均驻留时间MRT_(0-∞)为9.19 h。DYSP-C34在荷瘤小鼠体内的分布结果显示,DYSP-C34可以在肿瘤组织中蓄积,并具有一定的滞留作用。综上,该文建立了大鼠血浆中DYSP-C34的HPLC-UV测定方法,并进行了方法学验证,此方法简便、快速,结果准确。阐明了DYSP-C34在静脉给药方式下大鼠体内药代动力学和荷瘤小鼠组织中的分布特征,对临床合理用药和药效学研究具有重要意义。     

Skin Necrosis due to Photodynamic Action of Benzoporphyrin Depends on Circulating Rather than Tissue Drug Levels: Implications for Control of Photodynamic Therapy

In an ideal world, photodynamic therapy (PDT) of abnormal tissue would reliably spare the surrounding normal tissue. Normal tissue responses set the limits for light and drug dosimetry. The threshold fluence for necrosis (TFN) was measured in normal skin following intravenous infusion with a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA) Verteporfin as a function of drug dose (0.25-2.0 mg/kg), wavelength of irradiation (458 and 690 nm) and time interval (0–5h) between drug administration and irradiation. The BPD-MA levels were measured in plasma and skin tissue to elucidate the relationship between TFN, drug kinetics and biodistribution. The PDT response of normal skin was highly reproducible. The TFN for 458 and 690 nm wavelengths was nearly identical and the estimated quantum efficiency for skin response was equal at these two wavelengths. Skin phototoxicity, quantified in terms of 1/ TFN, closely correlated with the plasma pharmacokinetics rather than the tissue pharmacokinetics and was quadratically dependent on the plasma drug concentration regardless of the administered drug dose or time interval between drug and light exposure. This study strongly suggests that noninvasive measurements of the circulating drug level at the time of light treatment will be important for setting optimal light dosimetry for PDT with liposomal BPD-MA, a vascular photosensitizer.     

Elevated serum cytokine levels in mesothelioma patients who have undergone pleurectomy or extrapleural pneumonectomy and adjuvant intraoperative photodynamic therapy

Patients treated on a Phase-I clinical trial of photodynamic therapy (PDT) developed a systemic capillary leak syndrome that constituted the dose-limiting toxicity. We examined serum samples from patients treated at the maximally tolerated dose level for evidence of a systemic, cytokine-mediated inflammatory response. Patients underwent pleurectomy or extrapleural pneumonectomy (EPP) followed by intraoperative PDT of the thorax using Foscan at a dose of 0.1 mg/kg 6 days before surgery and 652 nm red light at a dose of 10 J/cm2. Levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, IL-8, IL-10 and IL-12 were assayed before Foscan administration; after anesthetic induction, surgical resection and light delivery; in postoperative recovery and the day after the surgery. Of the analyzed patients, eight underwent a pleurectomy and one an EPP followed by PDT. IFN-gamma, TNF-alpha and IL-12 showed no elevation, but IL-1beta, IL-6, IL-8 and IL-10 levels were elevated after surgery and PDT. IL-1beta showed a statistically significant variation from baseline after surgery and IL-6, after PDT. The results suggest a systemically mediated inflammatory response resulting from thoracic surgery followed by PDT. Further investigation of specific mechanisms is warranted.     

PEG-m-THPC-mediated photodynamic effects on normal rat tissues

Photodynamic therapy (PDT) of malignancies uses light to activate a photosensitizer preferentially accumulated in cancer cells. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21-23-[H]-porphyrin (PEG-m-THPC), was evaluated in non-tumor-bearing rats. The aim of this study was to assess the photodynamic threshold for damage and its sequelae in normal rat tissue. Thirty-five Fischer rats were sensitized with 3, 9 or 30 mg/kg body weight PEG-m-THPC. Colon, vagina and perineum were irradiated with laser light of 652 nm wavelength and an optical dose of 50, 150 or 450 J/cm fiber length. Temperature in the pelvis was measured during PDT. Three days following PDT the effect on skin, vagina, colon, striated muscle, connective tissue, nerves and blood vessels was assessed by histology. The healing of the above-mentioned tissues was assessed on two rats 3 and 8 weeks after PDT using 9 mg/kg PEG-m-THPC activated with 450 J/cm laser light. No dark toxicity was observed. PDT using 30 mg/kg PEG-m-THPC induced severe necrosis irrespective of the optical dose. Body weight of 9 or 3 mg/kg activated with less than 450 J/cm induced moderate or no damage. No substantial increase in body temperature was seen during PDT. Tissues with severe PDT-induced damage seem to have a good tendency to regenerate. We conclude that within the dose required for tumor treatment PEG-m-THPC is a safe photosensitizer with promising properties. PDT of the colon mucosa below 9 mg/kg PEG-m-THPC and 150 J/cm seems to be safe. All other tissues can be exposed to 9 mg/kg PEG-m-THPC activated with less than 450 J/cm laser light with little side effects.     

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.     

Photodynamic activity of substituted zinc trisulfophthalocyanines: role of plasma membrane damage

We recently reported that variations in cellular phototoxicity among a series of alkynyl-substituted zinc trisulfophthalocyanines (ZnPcS3Cn) correlates with their hydrophobicity, with the most amphiphilic derivatives showing the highest cell uptake and phototoxicity. In this study we address the role of the plasma membrane in the photodynamic response as it relates to the overall hydrophobicity of the photosensitizer. The membrane tracker dye 1-[4(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), which is incorporated into plasma membranes by endocytosis, was used to establish plasma membrane uptake by EMT-6 cells of the ZnPcS3C, by colocalization, and TMA-DPH membrane uptake rates after photodynamic therapy were used to quantify membrane damage. TMA-DPH colocalization patterns show plasma membrane uptake of the photosensitizers after short 1 h incubation periods. TMA-DPH plasma membrane uptake rates after illumination of the photosensitizer-treated cells show a parabolic relationship with photosensitizer hydrophobicity that correlates well with the phototoxicity of the ZnPcS3C,. After a 1 h incubation period, overall phototoxicity correlates closely with the postillumination rate of TMA-DPH incorporation into the cell membrane, suggesting a major role of plasma membrane damage in the overall PDT effect. In contrast, after a 24 h incubation, phototoxicity shows a stronger but imperfect correlation with total cellular photosensitizer uptake rather than TMA-DPH membrane uptake, suggesting a partial shift in the cellular damage responsible for photosensitization from the plasma membrane to intracellular targets. We conclude that plasma membrane localization of the amphiphilic ZnPcS3C6-C9 is a major factor in their overall photodynamic activity.     

Fluorescence imaging of Foscan and Foslip in the plasma membrane and in whole cells

A fluorescence microscope equipped with a condenser for total internal reflection (TIR) illumination was combined with a pulsed laser diode and a time-gated image intensifying camera for fluorescence lifetime measurements of single cells. In particular, fluorescence patterns, decay kinetics, and lifetime images of the lipophilic photosensitizers Foscan and Foslip were studied in whole cells as well as in close vicinity to their plasma membranes. Fluorescence lifetimes of both photosensitizers in cultivated HeLa cells decreased from about 8 ns at an incubation time of 3 h to about 5 ns at an incubation time of 24 h. This seems to result from an increase in aggregation (or self-quenching) of the photosensitizers during incubation. Selective measurements within or in close proximity to the plasma membrane indicate that Foscan and Foslip are taken up by the cells in a similar way, but may be located in different cellular sites after an incubation time of 24 h. A combination of TIR and fluorescence lifetime imaging microscopy (FLIM), described for the first time, appears to be promising for understanding some key mechanisms of photodynamic therapy (PDT).     

Time-Resolved Studies of the Excited-State Dynamics of meso-Tetra(hydroxylphenyl)chlorin in Solution

Meso-tetra(hydroxyphenyl)chlorin (m-THPC) is a new photosensitizer developed for potential use in photodynamic therapy (PDT) for cancer treatment. In PDT, the accepted mechanism of tumor destruction involves the formation of excited singlet oxygen via intermolecular energy transfer from the excited triplet-state dye to the ground triplet-state oxygen. Femtosecond transient absorption measurements are reported here for the excited singlet state dynamics of m-THPC in solution. The observed early time kinetics were best fit using a triple exponential function with time constants of 350 fs, 80 ps and > or = 3.3 ns. The fastest decay (350 fs) was attributed to either internal conversion from S2 to S1 or vibrational relaxation in S2. Multichannel time-resolved absorption and emission spectroscopies were also used to characterize the excited singlet and triplet states of the dye on nanosecond to microsecond time scales at varying concentrations of oxygen. The nanosecond time-resolved absorption data were fit with a double exponential with time constants of 14 ns and 250 ns in ambient air, corresponding to lifetimes of the S1 and T1 states, respectively. The decay of the T1 state varied linearly with oxygen concentration, from which the intrinsic decay rate constant, ki, of 1.5 x 10(6) s-1 and the biomolecular collisional quenching constant, kc, of 1.7 x 10(9) M-1 s-1 were determined. The lifetime of the S1 state of 10 ns was confirmed by fluorescence measurements. It was found to be independent of oxygen concentration and longer than lifetimes of other photosensitizers.     

MALDI-TOF mass spectrometric analysis for the characterization of the 5,10,15,20-tetrakis- (m-hydroxyphenyl)bacteriochlorin (m-THPBC) photoproducts in biological environment

Photoproducts formation upon irradiation (739 nm) of 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (m-THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption spectroscopy and MALDI-TOF mass spectrometry. The experiments were performed with a freshly prepared PBS-HSA solution of m-THPBC and with a PBS-HSA m-THPBC solution incubated for 6 h at 37 degrees C. The incubation of m-THPBC solution leads to the dye monomerisation, whereas in the freshly prepared solution, m-THPBC is under an aggregated form. Regardless of the incubation condition, photobleaching experiments carried out by absorption spectroscopy demonstrate the degradation of the photosensitizer and its phototransformation in m-THPC. Moreover, m-THPC was the sole photoproduct detected using absorption spectroscopy. Together with a degradation of m-THPBC and formation of m-THPC, MALDI-TOF mass spectrometry evidenced several other photoinduced modifications. Photoproducts such as dihydroxy m-THPBC and dihydroxy m-THPC were detected in both conditions; however, the formation of hydroxylated photoproducts was significantly greater in incubated solution. In addition, small molecules arising from the degradation of the photosensitizer and identified as dipyrin derivatives and dipyrrolic synthon were observed.     

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.     

Effect of photosensitizer dose on fluence rate responses to photodynamic therapy

Photodynamic therapy (PDT) regimens that conserve tumor oxygenation are typically more efficacious, but require longer treatment times. This makes them clinically unfavorable. In this report, the inverse pairing of fluence rate and photosensitizer dose is investigated as a means of controlling oxygen depletion and benefiting therapeutic response to PDT under conditions of constant treatment time. Studies were performed for Photofrin-PDT of radiation-induced fibrosarcoma tumors over fluence rate and drug dose ranges of 25-225 mW cm(-2) and 2.5-10 mg kg(-1), respectively, for 30 min of treatment. Tumor response was similar among all inverse regimens tested, and, in general, tumor hemoglobin oxygen saturation (SO2) was well conserved during PDT, although the highest fluence rate regimen (225 mWx2.5 mg) did lead to a modest but significant reduction in SO2. Regardless, significant direct tumor cell kill (>1 log) was detected during 225 mWx2.5 mg PDT, and minimal normal tissue toxicity was found. PDT effect on tumor oxygenation was highly associated with tumor response at 225 mWx2.5 mg, as well as in all other regimens tested. These data suggest that high fluence rate PDT can be carried out under oxygen-conserving, efficacious conditions at low photosensitizer dose. Clinical confirmation and application of these results will be possible through use of minimally invasive oxygen and photosensitizer monitoring technologies, which are currently under development.     

Identification and partial characterization of an unusual distribution of the photosensitizer meta-tetrahydroxyphenyl chlorin (temoporfin) in human plasma

Temoporfin (m-THPC) is an extremely powerful photosensitizing drug, more than 100-fold more photocytotoxic than Photofrin and many other drugs. The reasons for this are not yet known but are likely to be associated with the mechanism of uptake of the drug and its intratumoral and intracellular localization. Uptake itself is likely to be dependent upon the plasma binding of the drug following administration. In the current work, we have shown that the addition of m-THPC to human plasma in vitro at clinically relevant doses of sensitizer and administration solvent (diluant) gives rise to a protein-binding pattern quite different to that of Photofrin and other hydrophobic drugs as judged by density-gradient ultracentrifugation. Analysis of the binding immediately after addition to human plasma has shown that lipoprotein binding accounts for only a minor proportion of the sensitizer, which is mainly associated with a high-density protein fraction that is not coincident with serum albumin. The m-THPC protein complex does not fluoresce significantly even on dilution. This binding pattern is highly dependent on administration conditions and storage. Over a period of 6-8 h at 37 degrees C the m-THPC that is associated with this unidentified fraction redistributes to the plasma lipoproteins. Plasma collected from rats after intravenous administration of m-THPC also contains this low fluorescent complex, showing that this phenomenon is not limited to human plasma and also occurs in vivo. It is postulated that the m-THPC bound to the unknown protein fraction is highly aggregated and that it is likely to be taken up into tissues in this form. This unusual uptake may possibly be associated with the very high activity of m-THPC and also to the recent finding of a second peak in the plasma pharmacokinetics of the drug.     

Highly Selective Targeting of Ovarian Cancer with the Photosensitizer PEG-m-THPC in a Rat Model

Photodynamic therapy (PDT) uses light to activate a photosensitizer that has been absorbed or retained preferentially by cancer cells after systemic administration. The first pegylated photosensitizer, tetrakis-(m-methoxypolyethylene glycol) derivative of 7,8-dihydro-5,10,15,20-tetrakis(3-hydroxyphenyl)-21,23-[H]-porphyrin (PEG-m-THPC), was evaluated to target selectively unresectable pelvic ovarian cancer bulks. Our goals were two-fold: (1) to establish an ovarian cancer model suitable for the development of debulking techniques and (2) to characterize the pharmacokinetics and tumor selectivity of PEG-m-THPC by fluorescence microscopy. NuTu-19 ovarian cancer cells were injected into the caudal part of the right psoas muscle of Fisher rats. Five weeks later, 30 mg/kg body weight of PEG-m-THPC was injected intravenously. Necropsy was performed between 4 and 10 days following drug application, and fluorescence of the tumor and various abdominal organs was measured. All rats developed bulky pelvic tumors with an average diameter of 2.6 cm (+/- 0.6 SD). Tumor masses were encompassing and infiltrating pelvic organs in a similar manner to ovarian cancers in humans. Fluorescence of cancer tissue was maximal 8-10 days following drug application. At 8 days, the tumor-to-tissue ratio was 40:1 (+/- 12 SE) for most abdominal organs. We conclude that this tumor model may be used for the study of new pelvic debulking techniques, and that the tumor selectivity of PEG-m-THPC is exceptionally high 8 days after drug application. Based on these data, we are currently developing a PDT-based minimally invasive debulking technique for advanced ovarian cancer.