Development of porphyrin-antiangiogenic antibody immunoconjugates for photodynamic therapy

Photodynamic therapy is a novel cancer treatment, involving the combination of light, photosensitiser and molecular oxygen to produce cytotoxic species. Currently, commercially available photosensitisers, including Photofrin and Foscan, only exhibit a passive selectively towards cancerous tissue. Therapeutically, this results in a variety of unfavourable characteristics, obstructing PDT from becoming a more viable cancer treatment. Research has been directed towards improving the pharmacokinetic and biodistribution profiles of photosensitisers, which can be achieved by conjugating the photosensitiser moiety to a biomolecule which has affinity for the diseased tissue; these 3rd generation photosensitisers include photoimmunoconjugates.Three synthetic routes were developed, with the aim of producing novel bioconjugatable porphyrins, which could be subsequently conjugated to cysteine residues expressed by a suitable monoclonal antibody (SIP(L19)). Two cationic, thiol reactive photosensitisers were synthesised and successfully conjugated to SIP(L19) in a reproducible manner, affording well defined immunoconjugates, which retained both the immunoreactivity of the antibody moiety and the photoactivity of the photosensitiser. The effect of the length and hydrophilicity of the linker connecting the photosensitiser and bioconjugatable group was investigated, and it was observed that the photoactivity of the immunoconjugate was enhanced using a longer, hydrophilic chain. Synthetic routes leading to the production of bioconjugatable porphyrin dimers, and photosensitisers from symmetrical porphyrin, were developed, but failed to yield final products

New routes to acyltetramic acids and analogues

3-acyltetramic acids, such as reutericyclin, belong to a group of natural products which contain a 5-membered pyrrolidine-2,4-dione heterocycle with an acyl group at the 3-position. Molecules containing this motif have been shown to contain a wealth of desirable bio-activity such as antibiotic, antitumor, antiviral, antiulcerative, fungicidal and cytotoxic properties. The motivation for synthetic efforts towards reutericyclin and analogues is that it has been shown to have potential as an antibiotic treatment against superbug C. difficile. Our synthetic approach used a pyrroloisoxazole bicyclic system as a masked form of the acyltetramic acid core structure, which enables us to make selective modifications towards these bio-active products and produce more analogues suitable for biological testing. We report the synthesis of several novel compounds closely related to a masked reutericyclin as well as elaborations at the C-3 methyl group through aldol chemistry. The route began with a naturally occurring amino acid that underwent N-protection, carboxyl reduction and conversion to an oxime. This oxime is precursor to a nitrile oxide used in a 1,3-dipolar cycloaddition to achieve a substituted isoxazole that was deprotected and, through an intramolecular peptide coupling reaction, provided the pyrroloisoxazole core as the masked acyltetramic acid. Acylation reactions were completed upon this pyrroloisoxazole using butyllithium as a base and a range of acyl chlorides. Other developments made in this synthesis were the isolation of the chlorinated oxime and its use in a solvent study and significant improvements in the peptide coupling reaction. Also, synthetic efforts were made to produce an analogue of the natural product laccarin A using this methodology

Cancer targeting with biomolecules: a comparative study of photodynamic therapy efficacy using antibody or lectin conjugated phthalocyanine-PEG gold nanoparticles

The functionalisation of therapeutic nanoparticle constructs with cancer-specific biomolecules can enable selective tumour accumulation and targeted treatment. Water soluble gold nanoparticles (ca. 4 nm) stabilised by a mixed monolayer of a hydrophobic zinc phthalocyanine photosensitiser (C11Pc) and hydrophilic polyethylene glycol (PEG) have been prepared. The C11Pc-PEG gold nanoparticle constructs were further functionalised with jacalin, a lectin specific for the cancer-associated Thomsen–Friedenreich (T) carbohydrate antigen, or with monoclonal antibodies specific for the human epidermal growth factor receptor-2 (HER-2). The two biofunctionalised nanoparticle conjugates produced similar levels of singlet oxygen upon irradiation at 633 nm. Importantly, both nanoparticle conjugates demonstrated extensive, yet comparable, phototoxicity in HT-29 colorectal adenocarcinoma cells (80–90%) and in SK-BR-3 breast adenocarcinoma cells (>99%). Non-conjugated C11Pc-PEG gold nanoparticles were only minimally phototoxic. Lysosomal colocalisation studies performed with the HT-29 colon cancer cells and the SK-BR-3 breast cancer cells revealed that both nanoparticle conjugates were partially localised within acidic organelles, which is typical of receptor-mediated endocytosis. The similarity of the targeted PDT efficacy of the two biofunctionalised C11Pc-PEG gold nanoparticles is discussed with respect to targeting ligand binding affinity and cell surface antigen density as key determinants of targeting efficiency. This study highlights how targeting small cell-surface molecules, such as the T antigen, can mediate a selective photodynamic treatment response which is similar to that achieved when targeting overexpressed protein receptors, such as HER-2. The high prevalence of the T antigen present on the cellular surface of primary tumours emphasises the broad potential applications for lectin-targeted therapies

New routes towards reutericyclin analogues

A range of N-acylpyrrolo[3,4-c]isoxazoles and derived N-5 acyltetramides has been prepared via a nitrile oxide dipolar cycloaddition approach, as analogues of the acyltetramic acid metabolite reutericyclin, of interest for their antibiotic potential against Gram-positive bacteria including hospital-acquired infections of resistant Clostridium difficile

New routes towards reutericyclin analogues

A range of N-acylpyrrolo[3,4-c]isoxazoles and derived N-5 acyltetramides has been prepared via a nitrile oxide dipolar cycloaddition approach, as analogues of the acyltetramic acid metabolite reutericyclin, of interest for their antibiotic potential against Gram-positive bacteria including hospital-acquired infections of resistant Clostridium difficile

Development of porphyrin-antiangiogenic antibody immunoconjugates for photodynamic therapy

Photodynamic therapy is a novel cancer treatment, involving the combination of light, photosensitiser and molecular oxygen to produce cytotoxic species. Currently, commercially available photosensitisers, including Photofrin and Foscan, only exhibit a passive selectively towards cancerous tissue. Therapeutically, this results in a variety of unfavourable characteristics, obstructing PDT from becoming a more viable cancer treatment. Research has been directed towards improving the pharmacokinetic and biodistribution profiles of photosensitisers, which can be achieved by conjugating the photosensitiser moiety to a biomolecule which has affinity for the diseased tissue; these 3rd generation photosensitisers include photoimmunoconjugates. Three synthetic routes were developed, with the aim of producing novel bioconjugatable porphyrins, which could be subsequently conjugated to cysteine residues expressed by a suitable monoclonal antibody (SIP(L19)). Two cationic, thiol reactive photosensitisers were synthesised and successfully conjugated to SIP(L19) in a reproducible manner, affording well defined immunoconjugates, which retained both the immunoreactivity of the antibody moiety and the photoactivity of the photosensitiser. The effect of the length and hydrophilicity of the linker connecting the photosensitiser and bioconjugatable group was investigated, and it was observed that the photoactivity of the immunoconjugate was enhanced using a longer, hydrophilic chain. Synthetic routes leading to the production of bioconjugatable porphyrin dimers, and photosensitisers from symmetrical porphyrin, were developed, but failed to yield final products