Photochemical Internalization of Gemcitabine Is Safe and Effective in Locally Advanced Inoperable Cholangiocarcinoma.

BACKGROUND: Photochemical internalization (PCI) is a novel technology for light-induced enhancement of the local therapeutic effect of cancer drugs, utilizing a specially designed photosensitizing molecule (fimaporfin). The photosensitizing molecules are trapped in endosomes along with macromolecules or drugs. Photoactivation of fimaporfin disrupts the endosomal membranes so that drug molecules are released from endosomes inside cells and can reach their therapeutic target in the cell cytosol or nucleus. Compared with photodynamic therapy, the main cytotoxic effect with PCI is disruption of the endosomal membrane resulting in delivery of chemotherapy drug, and not to the photochemical reactions per se. In this study we investigated the effect of PCI with gemcitabine in patients with inoperable perihilar cholangiocarcinoma (CCA). METHODS: The in vitro cytotoxic effect of PCI with gemcitabine was studied on two CCA-derived cell lines. In a fimaporfin dose-escalation phase I clinical study, we administered PCI with gemcitabine in patients with perihilar CCA (n = 16) to establish a safe and tolerable fimaporfin dose and to get early signals of efficacy. The patients enrolled in the study had tumors in which the whole length of the tumor could be illuminated from the inside of the bile duct, using an optical fiber inserted via an endoscope (Fig. 1). Fimaporfin was administered intravenously at day 0; gemcitabine (i.v.) and intraluminal biliary endoscopic laser light application on day 4; followed by standard gemcitabine/cisplatin chemotherapy. RESULTS: Preclinical experiments showed that PCI enhanced the effect of gemcitabine. In patients with CCA, PCI with gemcitabine was well tolerated with no dose-limiting toxicities, and no unexpected safety signals. Disease control was achieved in 10 of 11 evaluable patients, with a clearly superior effect in the two highest dose groups. The objective response rate (ORR) was 42%, including two complete responses, while ORR at the highest dose was 60%. Progression-free survival at 6 months was 75%, and median overall survival (mOS) was 15.4 months, with 22.8 months at the highest fimaporfin dose. CONCLUSION: Photochemical internalization with gemcitabine was found to be safe and resulted in encouraging response and survival rates in patients with unresectable perihilar CCA.

Drug-Loaded Photosensitizer-Chitosan Nanoparticles for Combinatorial Chemo- and Photodynamic-Therapy of Cancer.

In this study we have developed biodegradable polymeric nanoparticles (NPs) containing the cytostatic drugs mertansine (MRT) or cabazitaxel (CBZ). The NPs are based on chitosan (CS) conjugate polymers synthesized with different amounts of the photosensitizer tetraphenylchlorin (TPC). These TPC-CS NPs have high loading capacity and strong drug retention due to π-π stacking interactions between the drugs and the aromatic photosensitizer groups of the polymers. CS polymers with 10% of the side chains containing TPC were found to be optimal in terms of drug loading capacity and NP stability. The TPC-CS NPs loaded with MRT or CBZ displayed higher cytotoxicity than the free form of these drugs in the breast cancer cell lines MDA-MB-231 and MDA-MB-468. Furthermore, light-induced photochemical activation of the NPs elicited a strong photodynamic therapy effect on these breast cancer cells. Biodistribution studies in mice showed that most of the TPC-CS NPs accumulated in liver and lungs, but they were also found to be localized in tumors derived from HCT-116 cells. These data suggest that the drug-loaded TPC-CS NPs have a potential in combinatory anticancer therapy and as contrast agents.

Highly efficient destruction of squamous carcinoma cells of the head and neck by photochemical internalization of Ranpirnase.

INTRODUCTION: Photochemical Internalization is a novel drug delivery technology for cancer treatment based on the principle of Photodynamic Treatment. Using a photosensitizer that locates in endocytic vesicles membranes of tumor cells, Photochemical internalization enables cytosolic release of endocytosed antitumor agents in a site-specific manner. The purpose of the present in-vitro study was to explore whether Photochemical Internalization is able to enhance the efficacy of Ranpirnase, a cytotoxic amphibian ribonuclease, for eradication of squamous cell carcinoma of the head and neck. METHODS: Cell viability was measured in 8 primary human cell lines of squamous cell carcinoma of the head and neck after treatment with Ranpirnase and Photochemical Internalization. For Photochemical Internalization the photosensitizer disulfonated tetraphenyl porphine was incubated with tumor cells followed by exposure to blue light (435 nm). RESULTS: Our study demonstrates significant enhancement of antitumor activity of Ranpirnase by Photochemical Internalization. Treatment responses were heterogeneous between the primary cancer cell lines. Combining Photochemical Internalization with Ranpirnase resulted in 4.6 to 1,940-fold increased cytotoxicity when compared with the ribonuclease alone (P < 0.05). CONCLUSION: Cytotoxicity of Ranpirnase can be markedly enhanced by Photochemical Internalization in squamous cell carcinoma of the head and neck.

Endosome Targeting meso-Tetraphenylchlorin-Chitosan Nanoconjugates for Photochemical Internalization.

Four amphiphilic covalently linked meso-tetraphenylchlorin-chitosan nanoconjugates were synthesized and evaluated for use in photochemical internalization (PCI) in vitro and in vivo. The synthetic protocol for the preparation of two different hydrophobic chlorin photosensitizers, 5-(4-aminophenyl)-10,15,20-triphenylchlorin and 5-(4-carboxyphenyl)-10,15,20-triphenylchlorin, was optimized. These monofunctional photosensitizers were covalently attached to carrier chitosan via silyl-protected 3,6-di-O-tert-butyldimethylsilyl-chitosan (Di-TBDMS-chitosan) with 0.10 degree of substitution per glucosamine (DS). Hydrophilic moieties such as trimethylamine and/or 1-methylpiperazine were incorporated with 0.9 DS to give fully water-soluble conjugates after removal of the TBDMS groups. A dynamic light scattering (DLS) study confirmed the formation of nanoparticles with a 140-200 nm diameter. These nanoconjugates could be activated at 650 nm (red region) light, with a fluorescence quantum yield (ΦF) of 0.43-0.45, and are thus suitable candidates for use in PCI. These nanoconjugates were taken up and localized in the endocytic vesicles of HCT116/LUC human colon carcinoma cells, and upon illumination they substantially enhanced plasmid DNA transfection. The nanoconjugates were also evaluated in preliminary in vivo experiments in tumor-bearing mice, showing that the nanoconjugates could induce a strong photodynamic therapy (PDT) and also PCI effects in treatment with bleomycin.

Photochemical internalization in bladder cancer - development of an orthotopic in vivo model.

The possibility of using photochemical internalization (PCI) to enhance the effects of the cytotoxic drug bleomycin is investigated, together with photophysical determination and outlines of a possible treatment for intravesical therapy of bladder cancer. In vitro experiments indicated that the employment of PCI technology using the novel photosensitizer TPCS2a® can enhance the cytotoxic effect of bleomycin in bladder cancer cells. Furthermore, experiments in an orthotopic in vivo bladder cancer model show an effective reduction in both the necrotic area and the bladder weight after TPCS2a based photodynamic therapy (PDT). The tumor selectivity and PDT effects may be sufficient to destroy tumors without damaging the detrusor muscle layer. Our results present a possible new treatment strategy for non-muscle invasive bladder cancer, with the intravesical instillation of the photosensitizer and bleomycin followed by illumination through an optic fiber by using a catheter.

Disulfonated tetraphenyl chlorin (TPCS2a)-induced photochemical internalisation of bleomycin in patients with solid malignancies: a phase 1, dose-escalation, first-in-man trial.

BACKGROUND: Photochemical internalisation, a novel minimally invasive treatment, has shown promising preclinical results in enhancing and site-directing the effect of anticancer drugs by illumination, which initiates localised chemotherapy release. We assessed the safety and tolerability of a newly developed photosensitiser, disulfonated tetraphenyl chlorin (TPCS2a), in mediating photochemical internalisation of bleomycin in patients with advanced and recurrent solid malignancies. METHODS: In this phase 1, dose-escalation, first-in-man trial, we recruited patients (aged ≥18 to <85 years) with local recurrent, advanced, or metastatic cutaneous or subcutaneous malignancies who were clinically assessed as eligible for bleomycin chemotherapy from a single centre in the UK. Patients were given TPCS2a on day 0 by slow intravenous injection, followed by a fixed dose of 15 000 IU/m(2) bleomycin by intravenous infusion on day 4. After 3 h, the surface of the target tumour was illuminated with 652 nm laser light (fixed at 60 J/cm(2)). The TPCS2a starting dose was 0·25 mg/kg and was then escalated in successive dose cohorts of three patients (0·5, 1·0, and 1·5 mg/kg). The primary endpoints were safety and tolerability of TPCS2a; other co-primary endpoints were dose-limiting toxicity and maximum tolerated dose. The primary analysis was per protocol. This study is registered with ClinicalTrials.gov, number NCT00993512, and has been completed. FINDINGS: Between Oct 3, 2009, and Jan 14, 2014, we recruited 22 patients into the trial. 12 patients completed the 3-month follow-up period. Adverse events related to photochemical internalisation were either local, resulting from the local inflammatory process, or systemic, mostly as a result of the skin-photosensitising effect of TPCS2a. The most common grade 3 or worse adverse events were unexpected higher transient pain response (grade 3) localised to the treatment site recorded in nine patients, and respiratory failure (grade 4) noted in two patients. One dose-limiting toxicity was reported in the 1·0 mg/kg cohort (skin photosensitivity [grade 2]). Dose-limiting toxicities were reported in two of three patients at a TPCS2a dose of 1·5 mg/kg (skin photosensitivity [grade 3] and wound infection [grade 3]); thus, the maximum tolerated dose of TPCS2a was 1·0 mg/kg. Administration of TPCS2a was found to be safe and tolerable by all patients. No deaths related to photochemical internalisation treatment occurred. INTERPRETATION: TPCS2a-mediated photochemical internalisation of bleomycin is safe and tolerable. We identified TPCS2a 0·25 mg/kg as the recommended treatment dose for future trials. FUNDING: PCI Biotech.

Photochemical internalization of bleomycin and temozolomide--in vitro studies on the glioma cell line F98.

Here we evaluate the photosensitizer meso-tetraphenyl chlorin disulphonate (TPCS2a) in survival studies of rat glioma cancer cells in combination with the novel photochemical internalization (PCI) technique. The tested anticancer drugs were bleomycin (BLM) and temozolomide (TMZ). Glioma cells were incubated with TPCS2a (0.2 µg ml(-1), 18 h, 37 °C) before BLM or TMZ stimulation (4 h) prior to red light illumination (652 nm, 50 mW cm(-2)). The cell survival after BLM (0.5 µm)-PCI (40 s light) quantified using the MTT assay was reduced to about 25% after 24 h relative to controls, and to 31% after TMZ-PCI. The supplementing quantification by clonogenic assays, using BLM (0.1 µm), indicated a long-term cytotoxic effect: the surviving fraction of clonogenic cells was reduced to 5% after light exposure (80 s) with PCI, compared to 70% in the case of PDT. In parallel, structural and morphological changes within the cells upon light treatment were examined using fluorescence microscopy techniques. The present study demonstrates that PCI of BLM is an effective method for killing F98 glioma cells, but smaller effects were observed using TMZ following the "light after" strategy. The results are the basis for further in vivo studies on our rat glioma cancer model using PDT and PCI.

Photochemical internalisation, a minimally invasive strategy for light-controlled endosomal escape of cancer stem cell-targeting therapeutics.

Despite progress in radio-, chemo- and photodynamic-therapy (PDT) of cancer, treatment resistance still remains a major problem for patients with aggressive tumours. Cancer stem cells (CSCs) or tumour-initiating cells are intrinsically and notoriously resistant to conventional cancer therapies and are proposed to be responsible for the recurrence of tumours after therapy. According to the CSC hypothesis, it is imperative to develop novel anticancer agents or therapeutic strategies that take into account the biology and role of CSCs. The present review outlines our recent study on photochemical internalisation (PCI) using the clinically relevant photosensitiser TPCS2a/Amphinex® as a rational, non-invasive strategy for the light-controlled endosomal escape of CSC-targeting drugs. PCI is an intracellular drug delivery method based on light-induced ROS-generation and a subsequent membrane-disruption of endocytic vesicles, leading to cytosolic release of the entrapped drugs of interest. In different proof-of-concept studies we have demonstrated that PCI of CSC-directed immunotoxins targeting CD133, CD44, CSPG4 and EpCAM is a highly specific and effective strategy for killing cancer cells and CSCs. CSCs overexpressing CD133 are PDT-resistant; however, this is circumvented by PCI of CD133-targeting immunotoxins. In view of the fact that TPCS2a is not a substrate of the efflux pumps ABCG2 and P-glycoprotein (ABCB1), the PCI-method is a promising anti-CSC therapeutic strategy. Due to a laser-controlled exposure, PCI of CSC-targeting drugs will be confined exclusively to the tumour tissue, suggesting that this drug delivery method has the potential to spare distant normal stem cells.

Photochemical internalization of CD133-targeting immunotoxins efficiently depletes sarcoma cells with stem-like properties and reduces tumorigenicity.

BACKGROUND: The normal stem cell marker CD133 is also a putative marker of cancer stem cells (CSCs) in different types of cancers. Hence, a major challenge when targeting CD133-expressing CSCs is to prevent depletion of the normal stem cell pool. We hypothesized that the site-specific and light-controlled drug delivery method photochemical internalization (PCI) may have the potential to enhance selectivity and endosomal escape of CD133-targeting immunotoxins in stem-like sarcoma cells. METHODS: We have used a sarcoma model, SW872 cells isolated from xenografts harboring CSCs within a ~2% CD133(high) subpopulation to investigate the potential of PCI of CD133-targeting toxin as a novel strategy to kill CSCs. Model immunotoxins were generated by binding the ribosome-inactivating protein toxin saporin to each of the monoclonal antibodies CD133/1 (AC133) or CD133/2 (293C), specific for individual CD133-epitopes. Cellular targeting, intracellular co-localization with the PCI photosensitizer, disulfonated meso-tetraphenylchlorin (TPCS2a), and cytotoxic efficacy of PCI of the CD133-targeting toxins were evaluated. RESULTS: PCI of CD133-saporin efficiently targets CD133-expressing SW872 and HT1080 sarcoma cells and results in loss of cell viability. Following sub-toxic treatment, surviving SW872 cells, depleted of the CD133-expressing population, display reduced proliferative capacity and attenuated CSC properties, such as reduced colony-forming ability and tumorigenicity. CONCLUSION: Here we present a proof-of-concept study, where PCI enables light-triggered delivery of CD133-targeting antibody-drug conjugates, resulting in decreased sarcoma tumor-initiating capacity. GENERAL SIGNIFICANCE: PCI of CD133-targeting toxins may be used as a minimal invasive strategy in the treatment of sarcomas, and potentially as a therapeutic for other solid tumors expressing CD133.

Light-triggered, efficient cytosolic release of IM7-saporin targeting the putative cancer stem cell marker CD44 by photochemical internalization.

We have used the site specific and light-depended drug delivery method photochemical internalization (PCI) to release an immunotoxin (IT), targeting the CD44 receptor, into the cytosol of target cells. The IT consisted of a pan CD44 mAb (clone IM7) bound to the ribosome inactivating protein (RIP) saporin by a biotin-streptavidin linker named IM7-saporin. PCI is based upon photosensitizing compounds localized in the membrane of endosomes and lysosomes causing membrane rupture upon illumination followed by release of the IT into the cytosol. In this in vitro study, we have used 7 different human cancer cell lines of various origins to investigate the cytotoxic effect of PCI-based targeting of the cancer stem cell (CSC) marker CD44. Epi-fluorescence microscopy shows both specific binding and uptake of the IM7-Alexa488, after 30 min and 18 h of incubation, and colocalization with the PCI-photosensitizer TPCS2a prior to light-triggered cytosolic release of the CD44-targeting IT. PCI of IM7-saporin resulted in efficient and specific cytotoxicity in CD44-expressing but not in CD44-negative cancer cells. A higher level of reactive oxygen species (ROS) was found in untreated and photodynamic therapy (PDT)-treated LNCaP (CD44(neg)) compared to that of DU145 (CD44(pos)) prostate cancer (PC) cells. This may explain the PDT-resistance observed in the DU145 cells. PCI-based targeting of CD44-expressing cancer cells gives very potent and specific cytotoxic effects and may represent a rational strategy for achieving site-selective elimination of CSCs in aggressive androgen-independent and treatment-resistant PC cells preventing cytotoxic effects on distant normal stem cells.

Photophysical and photobiological properties of a sulfonated chlorin photosensitiser TPCS(2a) for photochemical internalisation (PCI).

This study investigated the photophysical and photobiological properties of a new amphiphilic chlorin photosensitiser, disulfonated tetraphenylchlorin (TPCS(2a)), for photochemical internalisation (PCI). The absorption and fluorescence spectra of TPCS(2a) were examined in a range of solvents together with fluorescence lifetime measurements. The fluorescence lifetime of TPCS(2a) was found to be 8.5 ns in methanol, whereas non-exponential decays were observed in distilled water due to sensitiser dimerisation. The singlet oxygen quantum yield of TPCS(2a) was determined as 0.62 in deuterated methanol by direct observation of singlet oxygen phosphorescence. In a human oral squamous carcinoma (HN5) cell line, intracellular co-localisation of TPCS(2a) and Alexa488-labelled saporin, a macromolecular toxin, was observed corresponding predominantly to a lysosomal distribution. Intracellular fluorescence redistribution of TPCS(2a) and Alexa488-saporin was observed after 405 nm irradiation. Using two-photon confocal microscopy at 840 nm, and fluorescence lifetime imaging (FLIM), the lifetime was measured as 6 ns in HN5 cells. PCI using TPCS(2a) was shown to be very effective, and a synergistic increase in saporin toxicity was achieved in HN5 cells where viability was significantly reduced after light exposure compared to saporin (25 nM) treatment alone. The results demonstrate the favourable photophysical and photobiological properties of TPCS(2a) for PCI, which induces the relocalisation of a macromolecular anti-cancer toxin inside cells and significantly enhances cell death.

Proteomic analysis reveals mechanisms underlying increased efficacy of bleomycin by photochemical internalization in bladder cancer cells.

Photochemical internalization (PCI) is a promising new technology for site-specific drug delivery, developed from photodynamic therapy (PDT). In PCI, light-induced activation of a photosensitizer trapped inside endosomes together with e.g. chemotherapeutics, nucleic acids or immunotoxins, allows cytosolic delivery and enhanced local therapeutic effect. Here we have evaluated the photosensitizer meso-tetraphenyl chlorine disulphonate (TPCS2a/fimaporfin) in a proteome analysis of AY-27 rat bladder cancer cells in combination with the chemotherapeutic drug bleomycin (BML). We find that BLMPCI attenuates oxidative stress responses induced by BLM alone, while concomitantly increasing transcriptional repression and DNA damage responses. BLMPCI also mediates downregulation of bleomycin hydrolase (Blmh), which is responsible for cellular degradation of BLM, as well as several factors known to be involved in fibrotic responses. PCI-mediated delivery might thus allow reduced dosage of BLM and alleviate unwanted side effects from treatment, including pulmonary fibrosis.

Photochemically-Mediated Inflammation and Cross-Presentation of Mycobacterium bovis BCG Proteins Stimulates Strong CD4 and CD8 T-Cell Responses in Mice.

Conventional vaccines are very efficient in the prevention of bacterial infections caused by extracellular pathogens due to effective stimulation of pathogen-specific antibodies. In contrast, considering that intracellular surveillance by antibodies is not possible, they are typically less effective in preventing or treating infections caused by intracellular pathogens such as Mycobacterium tuberculosis. The objective of the current study was to use so-called photochemical internalization (PCI) to deliver a live bacterial vaccine to the cytosol of antigen-presenting cells (APCs) for the purpose of stimulating major histocompatibility complex (MHC) I-restricted CD8 T-cell responses. For this purpose, Mycobacterium bovis BCG (BCG) was combined with the photosensitiser tetraphenyl chlorine disulfonate (TPCS2a) and injected intradermally into mice. TPCS2a was then activated by illumination of the injection site with light of defined energy. Antigen-specific CD4 and CD8 T-cell responses were monitored in blood, spleen, and lymph nodes at different time points thereafter using flow cytometry, ELISA and ELISPOT. Finally, APCs were infected and PCI-treated in vitro for analysis of their activation of T cells in vitro or in vivo after autologous vaccination of mice. Combination of BCG with PCI induced stronger BCG-specific CD4 and CD8 T-cell responses than treatment with BCG only or with BCG and TPCS2a without light. The overall T-cell responses were multifunctional as characterized by the production of IFN-γ, TNF-α, IL-2 and IL-17. Importantly, PCI induced cross-presentation of BCG proteins for stimulation of antigen-specific CD8 T-cells that were particularly producing IFN-γ and TNF-α. PCI further facilitated antigen presentation by causing up-regulation of MHC and co-stimulatory proteins on the surface of APCs as well as their production of TNF-α and IL-1ß in vivo. Furthermore, PCI-based vaccination also caused local inflammation at the site of vaccination, showing strong infiltration of immune cells, which could contribute to the stimulation of antigen-specific immune responses. This study is the first to demonstrate that a live microbial vaccine can be combined with a photochemical compound and light for cross presentation of antigens to CD8 T cells. Moreover, the results revealed that PCI treatment strongly improved the immunogenicity of M. bovis BCG.

Photochemical activation of endosomal escape of MRI-Gd-agents in tumor cells.

Endocytosis is a common internalization pathway for cellular labeling with MRI contrast agents. However, the entrapment of the Gd(III) complexes into endosomes results in a "quenching" of the attainable relaxivity when the number of Gd(III) complexes reaches the number of ca. 1 × 10(9)/cell. Herein we show that the use of the newly developed photochemical internalization technique provides an efficient method for attaining the endosomal escape of GdHPDO3A molecules entrapped by pinocytosis into different kind of cells. Furthermore, it has been found that a new "quenching" limit is observed when the number of Gd-HPDO3A complexes is ca. five times higher than the value observed for the endosome entrapped conditions. The observed behavior is explained in terms of the attainment of the conditions in which the difference in proton relaxation rates between the cytoplasmic and the extracellular compartment is higher than the exchange rate of water molecules across the cellular membrane. The experimental data points have been reproduced by using a properly designed theoretical compartment T(1)-relaxation model.

Disulfonated tetraphenyl chlorin (TPCS2a), a novel photosensitizer developed for clinical utilization of photochemical internalization.

Photochemical internalisation (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers that locate in endocytic vesicles, and that upon activation by light induce a release of macromolecules from the endocytic vesicles. PCI has been shown to stimulate delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane. The preclinical evaluation of PCI has been performed with aluminum phthalocyanine disulfonate (AlPcS(2a)) as photosensitizer. AlPcS(2a), due to its large number of isomers potentially with batch-to-batch ratio variations, is not an optimal photosenstizer for clinical use. Disulfonated tetraphenyl chlorin (TPCS(2a)) has therefore been developed by di-imide reduction of disulfonated tetraphenyl porphine (TPPS(2a)). The synthesized TPCS(2a) contains 3 isomers as shown by HPLC with low (<4%) inter-batch variation with respect to isomer formation, less than 0.5% (w/w) of the starting material TPPS(2a) and absorbs light at 652 nm. As prerequisites for a PCI photosensitizer TPCS(2a) was found to localize in intracellular granules assumed to be endocytic vesicles. In cells in culture TPCS(2a)-PCI induced activation of gelonin as seen by enhanced cytotoxicity, increased transfection efficacy by an enhanced green fluorescence protein (EGFP)-encoding plasmid, induced gene silencing by siRNA towards EGFP and induced in a synergistic manner tumor growth delay by TPCS(2a)-mediated PCI of bleomycin in CT26.CL25 carcinomas growing subcutaneously in athymic mice. TPCS(2a)-PCI of bleomycin was found superior to meso-tetraphenyl chlorin-based photodynamic therapy (mTHPC-PDT) with respect to inhibition of tumor growth. The tumor growth delay by PCI of bleomycin was independent of the time of bleomycin administration between 3 h prior to light to immediately after light, while bleomycin administered 24 h prior to or 24 h after the light exposure induced suboptimal or only additive effects on tumor growth delay respectively. TPCS(2a)-PDT and -PCI induced indistinguishably strong edema the first 3-4 days after TPCS(2a)-administration and only weak erythema the first day after TPCS(2a) administration. In contrast, mTHPC-PDT induced moderate edema the first 7 days after mTHPC administration, but strong erythema resulting in open wounds and escar formation the first 2-3 days after mTHPC administration. The pharmacokinetic properties of TPCS(2a) were evaluated in athymic mice. The plasma pharmacokinetics was best fit to a 2-compartment model with half-lives of 0.78 and 36 hrs. TPCS(2a) was found to be a clinically suitable PCI photosensitizer for photochemical activation of molecules that do not readily penetrate the cellular plasma membrane.

Squamous cell carcinoma of the ear treated by photochemical internalization.

A 77-year-old Caucasian male was diagnosed with squamous cell cancer of the right ear. The patient elected to take part in the first-in-man phase I TPCS2a based bleomycin photochemical internalization (PCI). On Day 0, The patient received the photosensitiser [Amphinex (TPCS2a)], by slow intravenous injection. Four days later, surface illumination based (PCI) was implemented 3 h after the slow infusion of Bleomycin. Four weeks following the infusion of the photosensitiser, the cancerous area turned into black rigid mass with clear demarcation from the macroscopically normal skin. The size of the treated area has been substantially reduced. Histopathologic assessment of the excised necrotic mass revealed no viable tumour and the excised margins (PCI-treated margins) were tumour-free. This case was a clear indication that PCI is a clinically relevant technique that has potential in the treatment of such cancers to avoid radical intervention.

Photochemical internalization (PCI)-mediated activation of CD8 T cells involves antigen uptake and CCR7-mediated transport by migratory dendritic cells to draining lymph nodes.

Antigen cross-presentation to cytotoxic CD8+ T cells is crucial for the induction of anti-tumor and anti-viral immune responses. Recently, co-encapsulation of photosensitizers and antigens into microspheres and subsequent photochemical internalization (PCI) of antigens in antigen presenting cells has emerged as a promising new strategy for inducing antigen-specific CD8+ T cell responses in vitro and in vivo. However, the exact cellular mechanisms have hardly been investigated in vivo, i.e., which cell types take up antigen-loaded microspheres at the site of injection, or in which secondary lymphoid organ does T cell priming occur? We used spray-dried poly(lactic-co-glycolic acid) (PLGA) microspheres loaded with ovalbumin and the photosensitizer tetraphenyl chlorine disulfonate (TPCS2a) to investigate these processes in vivo. Intravital microscopy and flow cytometric analysis of the murine ear skin revealed that dendritic cells (DCs) take up PLGA microspheres in peripheral tissues. Illumination then caused photoactivation of TPCS2a and induced local tissue inflammation that enhanced CCR7-dependent migration of microsphere-containing DCs to tissue-draining lymph nodes (LNs), i.e., the site of CD8+ T cell priming. The results contribute to a better understanding of the functional mechanism of PCI-mediated vaccination and highlight the importance of an active transport of vaccine microspheres by antigen presenting cells to draining LNs.

Photochemical internalization: a new tool for gene and oligonucleotide delivery.

Photochemical internalization (PCI) is a novel technology for release of endocytosed macromolecules into the cytosol. The technology is based on the use of photosensitizers located in endocytic vesicles. Upon activation by light such photosensitizers induce a release of macromolecules from their compartmentalization in endocytic vesicles. PCI has been shown to increase the biological activity of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins, immunotoxins, plasmids, adenovirus, various oligonucleotides, dendrimer-based delivery of chemotherapeutica and unconjugated chemotherapeutica such as bleomycin and doxorubicin. This review will present the basis for the PCI concept and the most recent significant developments.

Apparent Complete Response of a Treatment Refractory and Recurrent Squamous Cell Carcinoma Lesion to Photochemical Internalization: A Clinical Case Study.

Photochemical internalization (PCI) depends on the delivery of sublethal photodynamic reaction to facilitate the work of a chemotherapeutic agent. We discuss our experience in managing a patient with extensive squamous cell carcinoma of the right face and scalp under the TPCS2a -based bleomycin PCI treatment protocol. In this case, an 84-year-old Caucasian received 0.25 mg kg-1 of TPCS2a (Amphinex® , PCI Biotech AS, Oslo, Norway). Surface illumination photochemical internalization was carried out after 4 days, which was preceded by the chemotherapeutic agent infusion (Bleomycin). After one week from the illumination time, tissue necrosis was evident and tumor shrinkage was most noticeable at day 14 postillumination. Follow-up at 6 weeks continued to show tissue healing and regeneration with no clinical evidence of recurrence. Multiple surgical biopsies were taken at 1 and 3 months postillumination and found to be tumor free. PCI's depth of effect has been very significant with negligible damage to the collateral tissues. This technology has a role in interventional oncology especially when managing challenging cases.

Photochemical Internalization for Intracellular Drug Delivery. From Basic Mechanisms to Clinical Research.

Photochemical internalisation (PCI) is a unique intervention which involves the release of endocytosed macromolecules into the cytoplasmic matrix. PCI is based on the use of photosensitizers placed in endocytic vesicles that, following light activation, lead to rupture of the endocytic vesicles and the release of the macromolecules into the cytoplasmic matrix. This technology has been shown to improve the biological activity of a number of macromolecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), gene-encoding plasmids, adenovirus and oligonucleotides and certain chemotherapeutics, such as bleomycin. This new intervention has also been found appealing for intracellular delivery of drugs incorporated into nanocarriers and for cancer vaccination. PCI is currently being evaluated in clinical trials. Data from the first-in-human phase I clinical trial as well as an update on the development of the PCI technology towards clinical practice is presented here.