Purpose: Photochemical internalization is under development for improving macromolecular therapy by inducing photochemical damage
to endocytic vesicles. This damage leads to the release of ...therapeutic macromolecules entrapped in endocytic vesicles into
the cytosol. The macromolecules may in this way be able to interact with therapeutic targets instead of being degraded by
lysosomal hydrolases. Bleomycin is used in several standard cancer chemotherapy regimens. Its hydrophilic and relatively large
chemical structure limits its ability to penetrate membrane structures, which causes the accumulation of bleomycin in endocytic
vesicles. The purpose of this study was to evaluate the therapeutic potential of aluminum phthalocyanine disulfonate (AlPcS 2a )–based photochemical delivery of bleomycin.
Experimental Design: Three tumors of different origin were grown s.c. in BALB/c ( nu/nu ) mice. The photosensitizer AlPcS 2a and bleomycin were systemically administered and the tumor area was exposed to red light when the tumor volume had reached
100 mm 3 . The tumor volume was measured frequently after treatment and the time for the tumor volume to reach 800 to 1,000 mm 3 was selected as the end point.
Results: The photochemical delivery of bleomycin induced a delayed tumor regrowth, and in two out of three tumor models, lead to 60%
complete response, whereas no complete responses were seen after treatment with bleomycin alone. A statistical model to assess
synergism was established. Combination of the photochemical treatment and bleomycin was found to induce a synergistic delay
in tumor growth.
Conclusion: AlPcS 2a -based photochemical internalization of bleomycin induces a synergistic inhibition of tumor growth in three different tumor
models. This treatment combination should be further considered for clinical utilization.
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.
A successful cure of cancer by biopharmaceuticals with intracellular targets is dependent on both specific and sufficient delivery of the drug to the cytosol or nuclei of malignant cells. However, ...cytosolic delivery and efficacy of membrane-impermeable cancer therapeutics are often hampered by the sequestration and degradation of the drugs in the endolysosomal compartments. Hence, we developed photochemical internalization (PCI) as a site-specific drug delivery technology, which bursts the membrane of endocytic vesicles inducing release of entrapped drugs to the cytosol of light exposed cells. The principle of PCI has been demonstrated in >80 different cell lines and 10 different xenograft models of various cancers in different laboratories demonstrating its broad application potential. PCI-induced endosomal escape of protein- or nucleic acid-based therapeutics and some chemotherapeutics will be presented in this review. With a joint effort by life scientists the PCI technology is currently in a Phase I/II clinical trial with very promising initial results in the treatment of solid tumors.
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Summary 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/m2 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/cm2 ). 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.
The cancer stem cell (CSC) marker CD133 is an attractive target to improve antitumor therapy. We have used photochemical internalization (PCI) for the endosomal escape of the novel CD133-targeting ...immunotoxin AC133–saporin (PCIAC133–saporin). PCI employs an endocytic vesicle-localizing photosensitizer, which generates reactive oxygen species upon light-activation causing a rupture of the vesicle membranes and endosomal escape of entrapped drugs. Here we show that AC133–saporin co-localizes with the PCI-photosensitizer TPCS2a, which upon light exposure induces cytosolic release of AC133–saporin. PCI of picomolar levels of AC133–saporin in colorectal adenocarcinoma WiDr cells blocked cell proliferation and induced 100% inhibition of cell viability and colony forming ability at the highest light doses, whereas no cytotoxicity was obtained in the absence of light. Efficient PCI-based CD133-targeting was in addition demonstrated in the stem-cell-like, triple negative breast cancer cell line MDA-MB-231 and in the aggressive malignant melanoma cell line FEMX-1, whereas no enhanced targeting was obtained in the CD133-negative breast cancer cell line MCF-7. PCIAC133–saporin induced mainly necrosis and a minimal apoptotic response based on assessing cleavage of caspase-3 and PARP, and the TUNEL assay. PCIAC133–saporin resulted in S phase arrest and reduced LC3-II conversion compared to control treatments. Notably, co-treatment with Bafilomycin A1 and PCIAC133–saporin blocked LC3-II conversion, indicating a termination of the autophagic flux in WiDr cells. For the first time, we demonstrate laser-controlled targeting of CD133 in vivo. After only one systemic injection of AC133–saporin and TPCS2a, a strong anti-tumor response was observed after PCIAC133–saporin. The present PCI-based endosomal escape technology represents a minimally invasive strategy for spatio-temporal, light-controlled targeting of CD133+ cells in localized primary tumors or metastasis.
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•The compound N1-(4-(4-(Benzyl(methyl)amino)thieno2,3-dpyrimidin-6-yl)benzyl)-N2,N2-dimethylethane-1,2-diamine (compound 1) may be a novel class of photosensitizer.•Compound 1 shows cytotoxicity ...toward F98 rat glioma cells in vitro.•Compound 1 produce singlet oxygen under UV-radiation.
Glioblastoma multiforme is one of the most aggressive cancer forms in humans, and has low recovery rates after surgery, ionizing radiation and chemotherapy. Therefore, there is a high interest in the development of new treatment methods, as for instance photodynamic therapy (PDT). It is here presented results of the cytotoxic properties of the novel compound N1-(4-(4-(benzyl(methyl)amino)thieno2,3-dpyrimidin-6-yl)benzyl)-N2,N2-dimethylethane-1,2-diamine (1), which was found ten-fold more active on the rat glioma cell model F98 than the reference drug Temozolomide (TMZ). Further cell survival studies showed a profound increase in F98 cell death on UVA-radiation (330 nm, 0.5 mW/cm2). Photochemical internalization induced delivery of compound 1, but in contrast to the cytostatic drug Bleomycin, a higher cytotoxicity was not observed. Localization studies using fluorescence microscopy revealed that compound 1 readily internalized into the cytosol but did not enter the cell nucleus. The compound was shown to be a relatively weak epidermal growth factor receptor inhibitor, which is not likely to explain its cytotoxicity. However, the quantum efficiency for generation of singlet oxygen was 23%, suggesting generation of reactive oxygen species as one possible mechanism. Although more studies are needed to reveal detailed mode of action, compound 1 is a promising photosensitizer candidate for further development in tests of animal models.
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.
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Here we report on the induction of resistance to photodynamic therapy (PDT) in the ABCG2-high human breast cancer cell line MA11 after repetitive PDT, using either Pheophorbide A ...(PhA) or di-sulphonated meso-tetraphenylchlorin (TPCS2a) as photosensitizer. Resistance to PhA-PDT was associated with enhanced expression of the efflux pump ABCG2. TPCS2a-PDT-resistance was neither found to correspond with lower TPCS2a-accumulation nor reduced generation of reactive oxygen species (ROS). Cross-resistance to chemotherapy (doxorubicin) or radiotherapy was not observed. TPCS2a-PDT-resistant cells acquired a higher proliferation capacity and an enhanced expression of EGFR and ERK1/2. p38 MAPK was found to be a death-signalling pathway in the MA11 cells post TPCS2a-PDT, contrasting the MA11/TR cells in which PDT generated a sustained phosphorylation of p38 that had lost its death-mediated signalling, and an abrogated activation of its downstream effector MAPKAPK2. No difference in apoptosis, necrosis or autophagy responses was found between the treated cell lines. Development of TPCS2a-PDT resistance in the MDA-MB-231 cell line was also established, however, p38 MAPK did not play a role in the PDT-resistance. MCF-7 cells did not develop TPCS2a-PDT-resistance. Photochemical internalisation (PCI) of 1 pM of EGF-saporin induced equal strong cytotoxicity in both MA11 and MA11/TR cells. In conclusion, loss of p38 MAPK-inducing death signalling is the main mechanism of resistance to TPCS2a-PDT in the MA11/TR cell line. This work provides mechanistic knowledge of intrinsic and acquired PDT-resistance which is dependent on choice of photosensitizer, and suggests PCI as a rational therapeutic intervention for the elimination of PDT-resistant cells.
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.
The photochemical internalisation (PCI) intervention was safe and tolerable by all patients when applied in a phase I first‐in‐human trial. The study dealt with a very difficult‐to‐treat group of patients who exhausted all the available treatment options of surgery, radiotherapy and chemotherapy. PCI showed excellent anti‐tumour effect in patients with life expectancy not exceeding few months, yet some of them still lived 4 years after the end of the trial. The PCI‐related adverse events were negligible. The current evidence highlighted the uniform PCI effect causing tumour death on a number of patients with very advanced and recurrent malignancies.
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.
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