PCI Biotech (Norway)

IntroductionMarine oils and fatty fish rich in long-chain n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (C20:5n-3, EPA) and docosahexaenoic acid (C22:6n-3, DHA), have been reported to enhance fatty acid (FA) oxidation and reduce plasma triacylglycerol and cholesterol levels. In addition to n-3 PUFAs, herring oil contains long-chain monounsaturated fatty acids (MUFAs), including cetoleic acid (C22:1n-11). This study aimed to investigate the effect of consuming CETO3® oil—derived from herring and naturally rich in n-3 PUFAs and cetoleic acid—on plasma lipid levels, FA composition, mitochondrial oxidation, and inflammation in rats.MethodsRats were fed low-fat diets supplemented with 5% CETO3® oil (experimental) or soy oil (control) for 10 weeks. Plasma lipid profile (triglycerides (TG), total cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL)) and FA composition in both liver and plasma were analyzed. In vitro substrate oxidation was assessed using ¹⁴CO₂-trapping in human liver and human myotubes. Safety parameters, including blood hematology, glucose tolerance, and organ weights, were also measured.ResultsCETO3® supplementation decreased plasma levels of total fat (−58%), TG (−55%), total cholesterol (−41%), and LDL cholesterol (−45%), while increasing the ratio of HDL to LDL cholesterol (46%). Supplementation also increased hepatic and plasma levels of long-chain n-3, n-9, and n-11 MUFAs, including C22:1n-11, and decreased n-6 FA accumulation. The reduction in saturated long-chain FAs in both the liver and plasma indicated increased hepatic peroxisomal and mitochondrial activity. Furthermore, increased oleic acid oxidation was observed in human myotubes in the presence of C20:1n-11 and C20:1n-9.DiscussionThese findings suggest that intake of CETO3® oil lowers plasma lipids, potentially through enhanced peroxisomal and mitochondrial FA oxidation. The shift in FA composition, with reduced n-6 FAs and increased n-3 and n-11 MUFAs, indicates an anti-inflammatory effect. CETO3® oil also appears safe, as hematological parameters, glucose tolerance, and organ weights remained unaffected.

BackgroundPhotochemical 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).MethodsThe 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.ResultsPreclinical 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.ConclusionPhotochemical internalization with gemcitabine was found to be safe and resulted in encouraging response and survival rates in patients with unresectable perihilar CCA.

Triple-negative breast cancer (TNBC) and malignant melanoma are highly aggressive cancers that widely express the cell surface chondroitin sulfate proteoglycan 4 (CSPG4/NG2). CSPG4 plays an important role in tumor cell growth and survival and promotes chemo- and radiotherapy resistance, suggesting that CSPG4 is an attractive target in cancer therapy. In the present work, we applied the drug delivery technology photochemical internalization (PCI) in combination with the novel CSPG4-targeting immunotoxin 225.28-saporin as an efficient and specific strategy to kill aggressive TNBC and amelanotic melanoma cells. Light-activation of the clinically relevant photosensitizer TPCS2a (fimaporfin) and 225.28-saporin was found to act in a synergistic manner, and was superior to both PCI of saporin and PCI-no-drug (TPCS2a + light only) in three TNBC cell lines (MDA-MB-231, MDA-MB-435 and SUM149) and two BRAFV600E mutated malignant melanoma cell lines (Melmet 1 and Melmet 5). The cytotoxic effect was highly dependent on the light dose and expression of CSPG4 since no enhanced cytotoxicity of PCI of 225.28-saporin compared to PCI of saporin was observed in the CSPG4-negative MCF-7 cells. The PCI of a smaller, and clinically relevant CSPG4-targeting toxin (scFvMEL-rGel) validated the CSPG4-targeting concept in vitro and induced a strong inhibition of tumor growth in the amelanotic melanoma xenograft A-375 model. In conclusion, the combination of the drug delivery technology PCI and CSPG4-targeting immunotoxins is an efficient, specific and light-controlled strategy for the elimination of aggressive cells of TNBC and malignant melanoma origin. This study lays the foundation for further preclinical evaluation of PCI in combination with CSPG4-targeting.

BackgroundDevelopment of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma.Methods5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin.ResultsThe 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS2a, altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105-expressing 5-FUR cells, whereas little effect was seen in the CD105-negative non-resistant parental cancer cell lines. Strikingly, using the intracellular drug delivery method photochemical internalization (PCI) by combining light-controlled activation of the TPCS2a with nanomolar levels of CD105-saporin resulted in strong cytotoxic effects in the 5-FUR cell population.ConclusionOur findings suggested that autophagy is an important resistance mechanism against the chemotherapeutic drug 5-FU in pancreatic cancer cells, and that inhibition of the autophagy process, either by CQ or lysosomal photodamage, can contribute to increased sensitivity to 5-FU. For the first time, we demonstrate the promise of PCI-based targeting of CD105 in site-specific elimination of 5-FU resistant pancreatic cancer cells in vitro. In conclusion, PCI-based targeting of CD105 may represent a potent anticancer strategy and should be further evaluated in pre-clinical models.

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. 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). 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). Cytotoxicity of Ranpirnase can be markedly enhanced by Photochemical Internalization in squamous cell carcinoma of the head and neck.

Objective. Activation of membrane receptor guanylate cyclase-C (GC-C) is implicated in gastrointestinal fluid and electrolyte balance, preservation of intestinal barrier integrity, anti-trophic effects and inhibition of pain sensation. To evaluate GC-C signaling, we examined the regulation of GC-C (GUCY2C/Gucy2c) and its endogenous ligands guanylin (GN/GUCA2A/Guca2a) and uroguanylin (UGN/GUCA2B/Guca2b) in colonic Crohn’s disease (CD), ulcerative colitis (UC) and in rats with 2,4,6-Trinitrobenzene sulphonic acid (TNBS) colitis. Correlation analyses between expression of GUCA2A and GUCY2C and expression of inflammatory cytokines (IL1A, IL1B, TNFA and IFNG) were conducted. Additionally, expression of transcription factors for GUCA2A and GUCY2C, and the GC-C signaling pathway, were examined. Material and methods. Biopsies from active UC/CD, un-inflamed UC/CD and healthy controls, and inflamed and healthy rat colon were investigated with gene expression microarray, immunohistochemistry (IHC) and in situ hybridization (ISH). Results. GUCA2A/Guca2a, GUCA2B, GUCY2C/Gucy2c, transcription factors, as well as several cyclic guanosine-3′,5′-monophosphate downstream mediators were all significantly down-regulated in both inflamed colonic inflammatory bowel disease (IBD) mucosa and TNBS colitis. Expression of GUCA2A and GUCY2C negatively correlated to expression of inflammatory cytokines. IHC and ISH confirmed microarray results for GUCA2A/Guca2a and GUCY2C/Gucy2c in inflamed samples. We identified a highly significant positive correlation between the expression of the transcription factor caudal type homeobox 2 (CDX2) and the expression of the downstream target gene GUCY2C. Conclusions. GUCA2A, GUCA2B and GUCY2C as well as several steps of the GC-C signaling pathway are down-regulated in IBD. This may have implications in IBD pathogenesis.

Abstract The successful therapeutic application of small interfering RNA (siRNA) largely relies on the development of safe and effective delivery systems that are able to guide the siRNA therapeutics to the cytoplasm of the target cell. In this report, biodegradable cationic dextran nanogels are engineered by inverse emulsion photopolymerization and their potential as siRNA carriers is evaluated. The nanogels are able to entrap siRNA with a high loading capacity, based on electrostatic interaction. Confocal microscopy and flow cytometry analysis reveal that large amounts of siRNA‐loaded nanogels can be internalized by HuH‐7 human hepatoma cells without significant cytotoxicity. Following their cellular uptake, it is found that the nanogels are mainly trafficked towards the endolysosomes. The influence of two different strategies to enhance endosomal escape on the extent of gene silencing is investigated. It is found that both the application of photochemical internalization (PCI) and the use of an influenza‐derived fusogenic peptide (diINF‐7) can significantly improve the silencing efficiency of siRNA‐loaded nanogels. Furthermore, it is shown that an efficient gene silencing requires the degradation of the nanogels. As the degradation kinetics of the nanogels can easily be tailored, these particles show potential for intracellular controlled release of short interfering RNA.

CataCleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)-ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Förster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs.

Photochemical internalization (PCI) is a targeting technique that facilitates endosomal escape of macromolecules, such as transgenes, in response to photochemical treatment with endosome/lysosome-localized photosensitizers, such as disulfonated meso-tetraphenylporphine (TPPS(2a)). In gene therapy this leads to enhanced transgene expression. Moreover, photochemical treatment generally activates transcription of stress-response genes, such as heat-shock proteins (HSPs), via stimulation of corresponding promoters. Therefore, we used HSP70 (HSPp; a promoter from the HSP family gene) and investigated whether the PCI stimulus could also activate HSPp and thereby stimulate transcription (expression) of the HSPp-controlled transgene internalized via PCI. Using human colorectal carcinoma and hepatoma cell lines in vitro, we showed that TPPS(2a)-based photochemical treatment enhances expression of cellular HSP70, which correlated with a photochemically enhanced expression (approximately 2-fold, at PCI-optimal doses) of the HSPp-controlled transgene integrated in the genome. Furthermore, PCI enhanced expression of the HSPp-controlled episomal transgene delivered as a plasmid. However, in plasmid-based transfection, PCI-mediated enhancement with HSPp did not exceed the enhancement achieved with the constitutive active CMV promoter. In conclusion, we demonstrated that the PCI-relevant treatment initiates HSP70 response and that the HSP70 promoter can be used in combination with PCI, leading to PCI-enhanced expression of the HSPp-controlled transgene.