ABSTRACT
Branched poly(ethylene glycols) (PEG2) are nowadays widely used for protein and peptides bioconjugation, for their favourable properties (such as the ability to protect the protein surface in an 'umbrella like' fashion). The discovery that mPEG(2)-LysMetbeta AlaOEt lost one mPEG chain during standard base-catalysed ester hydrolysis conditions prompted us to investigate the hydrolytic stability of such systems and the mechanism involved in the PEG chain loss. A series of branched PEGs, substituted with different aminoacids and dipeptides, have been prepared to test the influence of steric hindrance, chain lengths, ramification and Lys-AA amide substitution on hydrolysis. Unexpected results reveal an anchimeric assistance of the Lys-AaA amide proton to the hydrolysis of the carbamoyl moiety joining mPEG to the alpha-amino group of lysine through the formation of an hydantoin system.
Subject(s)
Lysine/chemistry , Polyethylene Glycols/chemistry , Amino Acids/chemistry , Dipeptides/chemistry , Drug Stability , HydrolysisABSTRACT
A new poly(ethylene glycol) (PEG) conjugate of 10-amino-7-ethyl camptothecin, a potent antitumor analogue of camptothecin, has been synthesized and preliminary in vivo tests have been performed. Successful chemoselective N-acylation of 10-amino-7-ethyl camptothecin was accomplished using phenyl dichlorophosphate, a coupling reagent used in esterification of alcohols, while other coupling methods failed, due to the low nucleophilicity of the amino group in position 10. The conjugate was tested against P388 murine leukemia cell lines and resulted equipotent to CPT-11, a camptothecin analogue already in clinical use.
Subject(s)
Camptothecin/analogs & derivatives , Camptothecin/chemistry , Camptothecin/metabolism , Polyethylene Glycols/chemistry , Polyethylene Glycols/metabolism , Acylation , StereoisomerismABSTRACT
Despite the high antitumor activity of camptothecins, few derivatives have been developed and tested for human treatment of solid tumors, due to unpredictable toxicity mainly connected to their poor water solubility. We report the conjugation of the antitumor agent 10-amino-7-hydroxy camptothecin (SN-392) to linear or branched poly(ethylene glycol)s (PEGs) of different loading capacity through a tri- or tetrapeptide spacer selectively cleaved by lysosomal enzymes (cathepsins). A synthetic strategy based on the chemoselective acylation of the aromatic amino group in the presence of the unprotected C20 tertiary alcohol allowed high overall yields. Two conjugates demonstrated good stability at physiological pH and in mouse plasma (nonspecific proteases) but slowly released the drug payload in the presence of the lysosomal enzyme cathepsin B1. Compound 3, selected for in vivo experiments, was very active against P388, P388/ADM leukaemia, and Meth A fibrosarcoma cell lines, scoring T/C% values comparable with the camptothecin derivative CPT-11. Pharmacokinetic studies indicated that 3 acts as a reservoir of 10-amino-7-ethylcamptothecin, as the mean residence time (MRT) is about 3-fold higher than that of the free drug.
Subject(s)
Antineoplastic Agents/chemical synthesis , Camptothecin/chemical synthesis , Oligopeptides/chemical synthesis , Polyethylene Glycols/chemistry , Prodrugs/chemical synthesis , Animals , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Camptothecin/analogs & derivatives , Camptothecin/pharmacokinetics , Camptothecin/pharmacology , Cathepsin B/chemistry , Cell Line, Tumor , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Hydrolysis , Male , Mice , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
Nisin is a natural antimicrobial peptide produced by Lactococcus lactis and widely employed as food preservative. Its low solubility in neutral aqueous solutions, its instability at physiological pH and its rapid breakdown by proteolytic enzymes has limited its use for processed foods (processed cheese, milk and derivatives, canned vegetables). The conjugation to poly(ethylene glycol) (PEG) could improve its solubility and protect it towards enzymes present in non optimally processed food. We report the synthesis of a PEG-nisin conjugate, and the microbiology assays against some bacterial cell lines.
