Castration resistant prostate cancer
A technology for castration resistance and prostate cancer, which is applied in the direction of antineoplastic drugs, medical preparations with non-active ingredients, containing abnormal peptide bonds, etc., and can solve the problems of immature clinical tests
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Embodiment 1
[0295] Example 1 - Preparation of polyplexes
[0296] Chemicals for LPEI Synthesis and PEGylation
[0297] NHS-PEG-OPSS (o-pyridyl disulfide-polyethylene glycol-N-hydroxysuccinimide ester), also known as PDP-PEG-NHS (PDP: pyridyl dithiopropionate), with a molecular weight of Approximately 2 kDa, purchased from Creative PEGworks (Winston, USA). Poly(2-ethyl-2-oxazoline), average molecular weight (Mn) about 50 kDa, and anhydrous dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (Israel). Absolute ethanol was purchased from Romical (Israel). All solvents were used without further purification.
[0298] Synthesis of LPEI (free base form)
[0299] Synthesis of LPEI and PEGylation of LPEI were performed as previously described (WO 2015 / 173824; Joubran et al., 2014, Optimization of liganded polyethyleneimine polyethylene glycol vector fornucleic acid delivery, Bioconjug Chem 2014, 25(9):1644-1654).
[0300] Briefly, 8.0 g (0.16 mmol) of poly(2-ethyl-2-oxazoline) were hy...
Embodiment 3
[0328] Example 3 - In vitro and in vivo tests
[0329] In vitro analysis of the bystander effect induced by PPD / polyIC treatment
[0330] The co-culture system was used to analyze the PPD / polyIC bystander effect in vitro. Treated PSMA-overexpressing cells were co-cultured with PBMCs alone or with PBMCs and cells not expressing PSMA (to represent adjacent cancer cells). Bystander effects were assessed by cell lines stably expressing luciferase (LNCaP-Luc / GFP, PC3-Luc / GFP or MCF7-Luc / GFP). The viability (luciferase activity) of these cells was measured (Luciferase Assay System, Promega).
[0331] Co-culture system of LNCaP-Luc / GFP-PBMC: Inoculate LNCaP-Luc / GFP cells (10,000 cells / well, 96-well plate pre-coated with polylysine, in triplicate), culture for one day and mix with PPD / polyIC Incubation. After 24 hours, freshly isolated PBMCs (1×10 per well 5 cells) were added to the culture, and the survival (luciferase activity) of LNCaP-Luc / GFP cells was detected after 24 hours...
Embodiment 4
[0336] Example 4 - Results of In Vitro Tests
[0337] Specific binding and uptake of DUPA coupled to Dylight680 via a specific peptide linker into PSMA-overexpressing cells
[0338] This example demonstrates that the PSMA ligand DUPA coupled via a peptide linker to another compound, such as a fluorescent dye or a multimeric complex consisting of PEI-PEG / polyIC, selectively targets PSMA-overexpressing cancer cells and will The polyplexes of the invention are delivered to these compound cells.
[0339] DUPA was coupled to the fluorescent dye Dylight680 (Thermo Scientific) via a specially designed peptide linker consisting of a hydrocarbon chain of 8-aminooctanoic acid, while the short peptide part avoided the steric hindrance of the PSMA ligand DUPA. The peptide linker used in this example included the peptide moiety of SEQ ID NO: 1 (Cys-Gly-Trp-Trp-Gly-Phe, see Figure 1A ).
[0340] Binding of DUPA-linker-Dylight680 conjugates to cancer cells was measured by confocal fluore...
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