光动力疗法在消化道恶性肿瘤中的研究进展

光动力疗法(Photodynamic therapy,PDT)是近年来新型的微创治疗技术,光敏剂注入机体一段时间后,会特异性地聚集在肿瘤组织内,此时以特定波长的光照射肿瘤部位,光敏剂发生光动力学反应,杀死肿瘤细胞。随着内镜引导技术的不断进步及新型光敏剂的不断涌现,目前PDT在治疗消化道恶性肿瘤方面取得了明显的进步,且疗效显著。本文针对PDT的原理进行介绍,并聚焦于PDT在消化道恶性肿瘤的临床应用,为临床治疗及科研提供参考。     

The tyrosine kinase inhibitor imatinib mesylate enhances the efficacy of photodynamic therapy by inhibiting ABCG2.

PURPOSE: The ATP-binding cassette protein ABCG2 (breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) and, thus, may confer resistance to this treatment modality. Tyrosine kinase inhibitors (TKI) can block the function of ABCG2. Therefore, we tested the effects of the TKI imatinib mesylate (Gleevec) on photosensitizer accumulation and in vitro and in vivo PDT efficacy. EXPERIMENTAL DESIGN: Energy-dependent photosensitizer efflux and imatinib mesylate's effects on intracellular accumulation of clinically used second- and first-generation photosensitizers were studied by flow cytometry in murine and human cells with and without ABCG2 expression. Effects of ABCG2 inhibition on PDT were examined in vitro using cell viability assays and in vivo measuring photosensitizer accumulation and time to regrowth in a RIF-1 tumor model. RESULTS: Energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX (PpIX) synthesized from 5-aminolevulenic acid, and the benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) was shown in ABCG2+ cell lines, but the first-generation multimeric photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were minimally transported. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2+ cells, but not in ABCG2- cells, and enhanced PDT efficacy both in vitro and in vivo. CONCLUSIONS: Second-generation clinical photosensitizers are transported out of cells by ABCG2, and this effect can be abrogated by coadministration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other ABCG2 transport inhibitors may enhance efficacy and selectivity of clinical PDT.     

Photodynamic therapy: a means to enhanced drug delivery to tumors

Using the photosensitizer 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a, we have determined that photodynamic therapy (PDT) can be used to facilitate the delivery of macromolecular agents. PDT regimens that use low fluences and fluence rates were the most successful. This effect was demonstrated for fluorescent microspheres with diameters ranging from 0.1 to 2 microm. Such treatment given immediately before administration of Doxil, a liposomally encapsulated formulation of doxorubicin with an average diameter of 0.1 microm, significantly enhanced its accumulation in transplanted murine Colo 26 tumors. The combination of PDT and Doxil led to a highly significant potentiation in tumor control without concomitant enhancement of systemic or local toxicity. Interestingly, concentration-effect modeling suggested that the enhanced cure rate was greater than what was predicted based on the increase in intratumor Doxil concentration. In summary, we have developed a novel PDT treatment that enhances the delivery and efficacy of macromolecule-based cancer therapies such as Doxil.     

Antitumor effect of photodynamic therapy with chlorin-based photosensitizer DH-II-24 in colorectal carcinoma

While photodynamic therapy (PDT) has been recognized as a promising therapeutic modality for the treatment of various cancers and diseases, developments of effective photosensitizers are highly desired to improve the prospect for the use of PDT. In this study, we evaluated DH-II-24, a new photosensitizer, for antitumor PDT in vitro and in vivo . Loaded into human colorectal carcinoma cells (HCT116), DH-II-24 was primarily accumulated in mitochondria, lysosomes, and endoplasmic reticula. Administration of DH-II-24 followed by light exposure induced necrotic cell death in a dose-dependent manner, whereas DH-II-24 in the absence of light induced minimal cell death. In order to investigate the distribution and phamacokinetics of the photosensitizer in vivo , DH-II-24 was intravenously injected to female BALB/c nude mice. Fluorescence imaging in vivo showed that DH-II-24 was rapidly distributed across the entire body and then mostly eliminated at 24 h. Next, effectiveness of DH-II-24-mediated PDT was examined on colorectal carcinoma xenografts established subcutaneously in BALB/c nude mice. DH-II-24 (1 mg/kg, i.v. administration) followed by light exposure significantly suppressed growth of xenograft tumors, compared to light exposure or DH-II-24 alone. Histological examination revealed necrotic damage in PDT-treated tumors, concomitantly with severe damage of tumor vasculature. These results suggest that DH-II-24 is a potential photosensitizer of photodynamic therapy for cancer. ( Cancer Sci 2009; 100: 2431–2436)     

Pretreatment photosensitizer dosimetry reduces variation in tumor response

Purpose: To compensate for photosensitizer uptake variation in photodynamic therapy (PDT), via control of delivered light dose through photodynamic dose calculation based on online dosimetry of photosensitizer in tissue before treatment. Methods and Materials: Photosensitizer verteporfin was quantified via multiple fluorescence microprobe measurements immediately before treatment. To compensate individual PDT treatments, photodynamic doses were calculated on an individual animal basis, by matching the light delivered to provide an equal photosensitizer dose multiplied by light dose. This was completed for the lower quartile, median, and upper quartile of the photosensitizer distribution. PDT-induced tumor responses were evaluated by the tumor regrowth assay. Results: Verteporfin uptake varied considerably among tumors and within a tumor. The coefficient of variation in the surviving fraction was found significantly decreased in groups compensated to the lower quartile (CL-PDT), the median (CM-PDT), and the upper quartile (CU-PDT) of photosensitizer distribution. The CL-PDT group was significantly less effective compared with NC-PDT (Noncompensated PDT), CM-PDT, and CU-PDT treatments. No significant difference in effectiveness was observed between NC-PDT, CM-PDT, and CU-PDT treatment groups. Conclusions: This research suggests that accurate quantification of tissue photosensitizer levels and subsequent adjustment of light dose will allow for reduced subject variation and improved treatment consistency.     

Photodynamic therapy for lung cancers based on novel photodynamic diagnosis using talaporfin sodium (NPe6) and autofluorescence bronchoscopy

BACKGROUND: We had previously developed the possibility of use of a photodynamic diagnosis (PDD) system using a tumor-selective photosensitizer and laser irradiation for the early detection and photodynamic therapy (PDT) for centrally located early lung cancers. Recently, we established the autofluorescence diagnosis system integrated into a videoendoscope (SAFE-3000) as a very useful technique for the early diagnosis of lung cancer. PATIENTS AND METHODS: Twenty-nine patients (38 lesions) with centrally located early lung cancer received PDD and PDT using the second-generation photosensitizer, talaporfin sodium (NPe6). Just before the PDT, we defined the tumor margin accurately using the novel PDD system SAFE-3000 with NPe6 and a diode laser (408nm). RESULTS: Red fluorescence emitted from the tumor by excitation of the photosensitizer by the diode laser (408nm) from SAFE-3000 allowed accurate determination of the tumor margin just before the PDT. The complete remission (CR) rate following NPe6-PDT in the cases with early lung cancer was 92.1% (35/38 lesions). We also confirmed the loss of red fluorescence from the tumors immediately after the PDT using SAFE-3000. We confirmed that all the NPe6 in the tumor had been excited and photobleached by the laser irradiation (664nm) and that no additional laser irradiation was needed for curative treatment. CONCLUSIONS: This novel PDD system using SAFE-3000 and NPe6 improved the quality and efficacy of PDT and avoided misjudgement of the dose of the photosensitizer or laser irradiation in PDT. PDT using NPe6 will become a standard option of treatments for centrally located early lung cancer.     

光动力疗法联合相关治疗研究进展

目的:总结可增强光动力疗法(PDT)抗肿瘤免疫效应的相关治疗研究进展.方法:应用PubMed和CNKI数据库,以“光动力疗法、肿瘤、免疫疗法、靶向光敏剂、肿瘤疫苗和免疫抑制”等为关键词,检索2000-01—2011-08的相关文献.纳入标准:1)PDT联合的免疫疗法;2)靶向光敏剂;3)PDT光照剂量;4)PDT免疫抑制效应; 5)PDT肿瘤疫苗.排除重复研究与本研究无关的文献,符合分析的文献32篇.结果:PDT处理后对宿主免疫系统的影响极为复杂,既能促进抗肿瘤免疫,在某些情况下又能抑制免疫;PDT联合免疫佐剂、细胞因子或免疫细胞可有效增强PDT抗肿瘤免疫反应;疫抑制效应具有条件性,可通过制备靶向光敏剂、调节光照剂量和去除负向调节性免疫效应分子及调节性T细胞等多种途径减弱甚至去除这种生物效应;PDT处理后肿瘤细胞裂解物具有免疫刺激作用,可用作肿瘤疫苗.结论:联合免疫疗法或其他方法来增强PDT抗肿瘤免疫效应、拮抗免疫抑制效应,可显著提高PDT抗肿瘤疗效.     

ABCG2-mediated transport of photosensitizers: potential impact on photodynamic therapy

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered followed by activation of the photosensitizer by exposure to a light source of a given wavelength. This, in turn, generates reactive oxygen species that induce cellular apoptosis and necrosis in tumor tissue. Based on our earlier finding that the photosensitizer pheophorbide a is an ABCG2 substrate, we explored the ability of ABCG2 to transport photosensitizers with a structure similar to that of pheophorbide a. ABCG2-overexpressing NCI-H1650 MX50 bronchoalveolar carcinoma cells were found to have reduced intracellular accumulation of pyropheophorbide a methyl ester and chlorin e6 compared to parental cells as measured by flow cytometry. The ABCG2 inhibitor fumitremorgin C was found to abrogate ABCG2-mediated transport. Intracellular fluorescence of hematoporphyrin IX, meso-tetra(3-hydroxyphenyl)porphyrin, and meso-tetra(3-hydroxyphenyl)chlorin was not substantially affected by ABCG2. ABCG2-overexpressing cells also displayed decreased intracellular fluorescence of protoporphyrin IX generated by exogenous application of 5-aminolevulinic acid. Mutations at amino acid 482 in the ABCG2 protein known to affect substrate specificity were not found to impact transport of the photosensitizers. In cytotoxicity assays, ABCG2-transfected HEK-293 cells were 11-fold, 30-fold, 4-fold, and >7-fold resistant to PDT with pheophorbide a, pyropheophorbide a methyl ester, chlorin e6, and 5-aminolevulinic acid, respectively. ABCG2-transfected cells were not resistant to PDT with meso-tetra(3-hydroxyphenyl) chlorin. Neither multidrug resistance-associated protein 1 expression nor P-glycoprotein expression appreciably decreased the intracellular fluorescence of any of the photosensitizers examined as determined by flow cytometry. The results presented here implicate ABCG2 as a possible cause for cellular resistance to photodynamic therapy.     

Use of multiple photosensitizers and wavelengths during photodynamic therapy: a new approach to enhance tumor eradication

Several studies have examined the synergism of hyperthermia or chemotherapy agents in combination with photodynamic therapy (PDT) to enhance tumor eradication. In our unique approach to treatment, multiple photosensitizers and wavelengths were used: two photosensitizers, Photofrin II and meso-tetra-(4-sulfonatophenyl)-porphine (TPPS4), irradiated at the appropriate therapeutic wavelength for each photosensitizer. EMT-6 mammary tumors were induced in the flanks of BALB/c mice. The mice were assigned to a control group (50 mice) or treatment group (150 mice). All treatment animals and some control animals received photosensitizing drug (5 mg/kg of TPPS4, 5 mg/kg of Photofrin II, or 2.5 mg/kg of both TPPS4 and Photofrin II). All treatment animals and some control animals also received light treatment (630 nm for TPPS4 and/or 658 nm for Photofrin II). The results show that the approach using both drugs and the corresponding therapeutic wavelengths enhanced the effectiveness of PDT. This approach achieved a cure rate of up to 100%, which was, depending on the light intensity used, as much as 40% greater than the rate achieved by the approach using one drug and one wavelength. The results also show that lesser amounts of drug and/or light may be required if both drugs and wavelengths are used, thus lowering the chances of side effects common to PDT. Furthermore, the results indicate that the increased tumor kill is due to a synergistic effect of the two photosensitizers that was tested on the tumor microvasculature in the first few hours after PDT.     

Photodynamic-induced vascular damage of the chick chorioallantoic membrane model using perylenequinones

The chorioallantoic membrane (CAM) assay is a widely used bioassay in early in vivo cancer research. The CAM allows non-invasive study of in vivo microvasculature and blood circulation. This report describes the first topical application investigation of photodynamic response in the CAM model using Hypericin (HY) and Hypocrellin B (HB) that belongs to the perylenequinone family. Briefly, cultivated carcinoma of the human bladder cell line (MGH), were inoculated on the CAM of fertilized eggs of embryo age (EA) 9. Tumor growth was evaluated by digital stereomicroscopy. Photodynamic therapy (PDT) was performed following topical application of the photosensitizers. We were able to demonstrate that these perylenequinones localized selectively in the xenografted bladder tumor and in the vasculature of the CAM. Photodynamic treatments were performed using a custom-made non-laser light source coupled into a flexible fiber bundle to selectively excite the photosensitizers in order to induce photodamage to the tumor and vasculature. The vascular damage induced was quantitatively measured following topical application of the photosensitizers. Both photosensitizers exhibited very similar degrees of photodamage to the CAM. The CAM model offers an exciting avenue for the study of PDT induced effect on the vasculature. Our preliminary results support that the CAM model could potentially serve as a customized model to study photodynamic therapy effects of various photosensitizers on specific tumor models.     

Biodistribution and photodynamic therapy with hypericin in a human NPC murine tumor model

The use of photodynamic therapy (PDT) for the treatment of recurrent and residual nasopharyngeal carcinoma (NPC) has been encouraging. To determine the potential of hypericin as a PDT tool in the treatment of NPC, we investigated the effect of hypericin-mediated PDT on subcutaneously implanted NPC/HK1 tumor cells and the relationship between the biodistribution of hypercin and photodynamic effects. The plasma hypericin level increased rapidly and reached its peak concentration at 1 h after injection. The uptake of hypercin in tumor tissue was maximal 6 h after hypericin administration, at which time the drug concentration in the circulation was low. The efficacy of hypericin-mediated PDT was maximal when light irradiation was performed at 6 h after hypericin administration. Tumor relative regression percentage (RRP) induced by PDT at 1-h interval was comparable to that at 6-h interval, whereas light treatment performed at other time intervals induced less tumor RRP, albeit significant when compared to the control group. Hypericin appears to be an effective photosensitizer for the treatment of NPC. It is likely that hypericin-mediated PDT induces both vascular damage and direct tumor cell killing, thereby bringing about tumor necrosis and shrinkage.     

Potent and specific antitumor effect of CEA‐targeted photoimmunotherapy

Conventional photodynamic therapy (PDT) for cancer is limited by the insufficient efficacy and specificity of photosensitizers. We herein describe a highly effective and selective tumor‐targeted PDT using a near‐infrared (NIR) photosensitizer, IRDye700DX, conjugated to a human monoclonal antibody (Ab) specific for carcinoembryonic antigen (CEA). The antitumor effects of this Ab‐assisted PDT, called photoimmunotherapy (PIT), were investigated in vitro and in vivo. The Ab‐IRDye conjugate induced potent cytotoxicity against CEA‐positive tumor cells after NIR‐irradiation, whereas CEA‐negative cells were not affected at all, even in the presence of excess photoimmunoconjugate. We found an equivalent phototoxicity and a predominant plasma membrane localization of Ab‐IRDye after both one and six hours of incubation. Either no or little caspase activation and membrane peroxidation were observed in PIT‐treated cells and a panel of scavengers for reactive oxygen species showed only partial inhibition of the phototoxic effect. Strikingly, Ab‐IRDye retained significant phototoxicity even under hypoxia. We established a xenograft model, which allowed us to sensitively investigate the therapeutic efficacy of PIT by non‐invasive bioluminescence imaging. Luciferase‐expressing MKN‐45‐luc human gastric carcinoma cells were subcutaneously implanted into both flanks of nude mice. NIR‐irradiation was performed for only the tumor on one side. In vivo imaging and measurement of the tumor size revealed that a single PIT treatment, with intraperitoneal administration of Ab‐IRDye and subsequent NIR‐irradiation, caused rapid cell death and significant inhibition of tumor growth, but only on the irradiated side. Together, these data suggest that Ab‐IRDye‐mediated PIT has great potential as an anticancer therapeutics targeting CEA‐positive tumors.     

纳米粒载体在光动力治疗肿瘤中的应用

 近年来,纳米粒载体技术被广泛应用到光动力治疗肿瘤中,可通过多种途径增强光敏剂的稳定性,提高光动力治疗的肿瘤靶向性、作用深度、单态氧产出率。依靠纳米粒,光动力治疗还可与其他治疗方法联合应用。纳米粒载体技术应用到光动力治疗中可以提高治疗肿瘤的效果、减少不良反应、扩大适用范围,是一项极具发展前景的新技术。     

A mechanism-based combination therapy reduces local tumor growth and metastasis in an orthotopic model of prostate cancer

Therapy-induced stimulation of angiogenic molecules can promote tumor angiogenesis leading to enhanced tumor growth and cancer metastasis. Several standard and emerging therapies, such as radiation and photodynamic therapy (PDT), can induce angiogenic molecules, thus limiting their effectiveness. PDT is approved for the treatment of several cancers; however, its induction of vascular endothelial growth factor (VEGF) creates conditions favorable to enhanced tumor growth and metastasis, therefore mitigating its cytotoxic and antivascular effects. This is the first report showing that subcurative PDT in an orthotopic model of prostate cancer (LNCaP) increases not only VEGF secretion (2.1-fold) but also the fraction of animals with lymph node metastases. PDT followed by administration of an antiangiogenic agent, TNP-470, abolished this increase and reduced local tumor growth. On the other hand, administration of TNP-470 before PDT was less effective at local tumor control. In addition, animals in all groups, except in the PDT + TNP-470 group, had a weight loss of >3 g at the time of sacrifice; the weight of the animals in the PDT + TNP-470 group did not change. The significant reduction (P < 0.05) in tumor weight and volume observed between the PDT + TNP-470 group and the control group suggests that the combination of PDT and antiangiogenic treatment administered in the appropriate sequence was not only more effective at controlling local tumor growth and metastases but also reduced disease-related toxicities. Such molecular response-based combinations merit further investigations as they enhance both monotherapies and lead to improved treatment outcomes.     

NF - κB 在光动力治疗肿瘤中的研究进展简

传统的抗癌治疗模式给癌症患者带来众多副作用,而光动力疗法作为一种新型的癌症治疗方法,以其独特的靶向性、无耐药性等,引起越来越多学者的关注。以往研究表明,光动力杀伤肿瘤细胞是由多种机制介导的,除了已知的坏死和凋亡机制外,光动力诱导产生的炎性反应也能够间接有利于肿瘤细胞的清除。NF—κB转录因子是调控炎性因子（如细胞因子、趋化因子和细胞粘性因子）的主要因素。此外,NF—κB还调节抗凋亡基因、环氧化酶（COXs）和金属蛋白酶（MMPs）的表达。本文就近年来NF—κB在光动力治疗肿瘤细胞中的研究进展做～综述。     

空腔脏器肿瘤的光动力治疗进展

光动力治疗是一种有前景的治疗肿瘤的方法,光敏剂注入肿瘤患者体内很快富集于肿瘤组织,采用特定波长可见光照射肿瘤组织,光敏剂会产生致死性细胞毒剂,杀死肿瘤细胞,引起肿瘤组织的微血管完全封闭,营养枯竭,进而肿瘤组织坏死。近年来多种肿瘤的光动力治疗体现出良好的临床疗效,并取得了很大的进步。随着新型光敏剂及光源的开发应用,PDT将会成为治疗多种肿瘤更加切实可行的方法。由于空腔脏器的解剖学特点,光动力治疗在该类肿瘤的治疗中具有独特的优势。光动力正逐渐整合到空腔脏器肿瘤的治疗中,以提高治疗效果,现就空腔脏器的光动力治疗做一综述。     

Polymeric iron chelators for enhancing 5-aminolevulinic acid-induced photodynamic therapy

5-Aminolevulinic acid (5-ALA) is an amino acid that can be metabolized into a photosensitizer, protoporphyrin IX (PpIX) selectively in a tumor cell, permitting minimally invasive photodynamic diagnosis/therapy. However, some malignant tumor cells have excess intracellular labile iron and facilitate the conversion of PpIX into heme, which compromises the therapeutic potency of 5-ALA. Here, we examined the potential of chelation of such unfavorable intratumoral labile iron in photodynamic therapy (PDT) with 5-ALA hydrochloride, using polymeric iron chelators that we recently developed. The polymeric iron chelator efficiently inactivated the intracellular labile iron in cultured cancer cells and importantly enhanced the accumulation of PpIX, thereby improving the cytotoxicity upon photoirradiation. Even in in vivo study with subcutaneous tumor models, the polymeric iron chelator augmented the intratumoral accumulation of PpIX and the PDT effect. This study suggests that our polymeric iron chelator could be a tool for boosting the effect of 5-ALA-induced PDT by modulating tumor microenvironment.     

Fluorescence resonance energy transfer reveals a binding site of a photosensitizer for photodynamic therapy

Phthalocyanine (Pc) 4, like many photosensitizers for photodynamic therapy (PDT), localizes to intracellular membranes, especially mitochondria. Pc 4-PDT photodamages Bcl-2 and Bcl-xL, antiapoptotic proteins interacting with the permeability transition pore complex that forms at contact sites between the inner and outer mitochondrial membranes. These complexes and the inner membrane are unique in containing the phospholipid cardiolipin. Nonyl-acridine orange (NAO) is a specific probe of cardiolipin. Here we show evidence for fluorescence resonance energy transfer from NAO to Pc 4, defining a binding site for the photosensitizer. This observation establishes an innovative tool for exploring the localization of other photosensitizers and additional fluorescent, mitochondrion-localizing drugs having appropriate spectral properties.     

Photodynamic therapy for experimental tumors using ATX-S10(Na), a hydrophilic chlorin photosensitizer, and diode laser.

ATX-S10(Na), a hydrophilic chlorin photosensitizer having an absorption maximum at 670 nm, is a candidate second-generation photosensitizer for use in photodynamic therapy (PDT) for cancer treatment. The effectiveness of PDT using ATX-S10(Na) and a diode laser for experimental tumors was evaluated in vitro and in vivo. In-vitro PDT using ATX-S10(Na) and the diode laser showed drug concentration-, laser dose- and drug exposure time-dependent cytotoxicity to various human and mouse tumor cell lines. In Meth-A sarcoma-implanted mice, optimal PDT conditions were found where tumors were completely eliminated without any toxicity. Against human tumor xenografts in nude mice, the combined use of 5 mg / kg ATX-S10(Na) and 200 J / cm(2) laser irradiation 3 h after ATX-S10(Na) administration showed excellent anti-tumor activity, and its efficacy was almost the same as that of PDT using 20 mg / kg porfimer sodium and a 100 J / cm(2) excimer dye laser 48 h after porfimer sodium injection. Microscopic observation of tumor tissues revealed that PDT using ATX-S10(Na) and the diode laser induced congestion, thrombus and degeneration of endothelial cells in tumor vessels, indicating that a vascular shutdown effect plays an important role in the anti-tumor activity of PDT using ATX-S10(Na) and the diode laser.