A formulation of pancreatic pro-enzymes provides potent anti-tumour efficacy: a pilot study focused on pancreatic and ovarian cancer.

Proteolytic enzymes have shown efficacy in cancer therapy. We present a combination of the two pro-enzymes Trypsinogen and Chymotrypsinogen A with potent in vitro and in vivo anti-tumour efficacy. A synergetic anti-tumour effect for Trypsinogen and Chymotrypsinogen A was determined at a ratio 1:6 (named PRP) using 24 human cancer cell lines. The antiangiogenic effect of PRP was analysed by matrigel-based tube formation and by fibrous capsule formation assays. Furthermore, cell invasion and wound healing assays together with qRT-PCR determination of epithelial-to-mesenchymal transition (EMT) markers were performed on human cancer cells treated with PRP. Additionally, in vivo pharmacokinetic studies were implemented and the PRP's anti-tumour efficacy was explored against orthotopic pancreatic and ovarian cancer tumours. PRP formulation was proven to inhibit in vitro angiogenesis, tumour growth, cancer cell migration and invasiveness; and to be an effective and well tolerated in vivo anti-tumour treatment. Finally, the clinical efficacy of a suppository formulation containing both pancreatic pro-enzymes in the context of a UK Pharmaceuticals Special Scheme was evaluated in advanced cancer patients. Consequently, PRP could have relevant oncological clinical applications for the treatment of advanced or metastatic pancreatic adenocarcinoma and advanced epithelial ovarian cancer.

Prevention of benzyl alcohol-induced aggregation of chymotrypsinogen by PEGylation.

OBJECTIVES: Addition of the antimicrobial preservative benzyl alcohol to reconstitution buffer promotes the formation of undesirable aggregates in multidose protein formulations. Herein we investigated the efficiency of PEGylation (attachment of poly(ethylene glycol)) to prevent benzyl alcohol-induced aggregation of the model protein α-chymotrypsinogen A (aCTgn). METHODS: Various PEG-aCTgn conjugates were prepared using PEG with a molecular weight of either 700 or 5000 Da by varying the PEG-to-protein ratio during synthesis and the formation of insoluble aggregates was studied. The effect of benzyl alcohol on the thermodynamic stability and tertiary structure of aCTgn was also examined. KEY FINDINGS: When the model protein was reconstituted in buffer containing 0.9% benzyl alcohol, copious amounts of buffer-insoluble aggregates formed within 24 h (>10%). Benzyl alcohol-induced aggregation was completely prevented when two or five molecules of PEG with a molecular weight of 5000 Da were attached to the protein, whereas two or four molecules of bound 700 Da PEG were completely inefficient in preventing aggregation. Mechanistic investigations excluded prevention of structural perturbations or increased thermodynamic stability by PEGylation from being responsible for the prevention of aggregation. Simple addition of PEG to the buffer was also inefficient and PEG had to be covalently linked to the protein to be efficient. CONCLUSIONS: The most likely explanation for the protective effect of the 5000 Da PEG is shielding of exposed hydrophobic protein surface area and prevention of protein-protein contacts (molecular spacer effect).