In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "The invention is based on novel Y-shaped PEGylated granulocyte colony stimulating factor (YPEG-G-CSF) with single site modification. Particularly, the invention relates to YPEG modified G-CSF and its preparation method, the pharmaceutical composition comprising YPEG-G-CSF and use thereof. Particularly, YPEG-G-CSF of the present invention is modified on the 17th lysine (K17) as a singe site modification. The use of K17 single site modified YPEG-G-CSF achieves good therapeutic effects in animals. In addition, K17 single site modified YPEG-G-CSF has significantly prolonged pharmacokinetic half-life in serum.
"In one aspect, this invention relates to Y-shaped PEG derivatives (also recorded as YPEG) modified G-CSF (also recorded as YPEG-G-CSF or PEG-G-CSF), wherein its molecular composition is as follows
"##STR00005## wherein, Pa and Pb are same or different PEGs;
"j is an integer from 1 to 12;
"Ri is H, a substituted or unsubstituted C.sub.1-C.sub.12 alkyl group, a substituted aryl, an aralkyl or a heteroalkyl;
"X.sub.1 and X.sub.2 are independently a linking group, wherein X.sub.1 is (CH.sub.2).sub.n, and X.sub.2 is selected from the group consisting of (CH.sub.2).sub.n, (CH.sub.2).sub.nOCO, (CH.sub.2).sub.nNHCO, and (CH.sub.2).sub.nCO; n is an integer from 1 to 10; and
"F is a terminal group selected from the group consisting of a hydroxyl group, a carboxyl group, an ester group, acyl chloride, hydrazide, maleimide, pyridine disulfide, capable of reacting with amino, hydroxyl or mercapto group of a therapeutic agent or a substrate to form a covalent bond.
"In one embodiment, the structural formula of the Y-shaped PEG-G-CSF has the following structure:
"Wherein R and R' are independently a low molecular weight alkyl, preferably methyl; j is an integer of 1-12; m and m' denote the degree of polymerization and can be any integer; preferably m=m' and m+m' is from 600 to 1500. In formula (II), the Y-shaped branched PEG binds to G-CSF through an amide bond at a single site.
"In a preferred embodiment, in said Y-shaped PEG-G-CSF, G-CSF binds Y-PEG through the amide bond formed by the .epsilon.-amino group on the side chain of lysine in G-CSF corresponding to position 17 of SEQ ID NO: 1 and the terminal carboxyl group of Y-PEG.
"Alternatively, the G-CSF of the present invention can be extracted from natural sources or obtained through recombinant biotechnology. Preferably, G-CSF extracted from natural sources or obtained through recombinant biotechnology is human G-CSF (hG-CSF) shown in SEQ ID NO: 1. More preferably, said human G-CSF is recombinant human G-CSF (rhG-CSF). rhG-CSF can be synthesized, expressed by prokaryotic systems such as E. coli, expressed by eukaryotic systems such as Pichia pastoris, or expressed by other insect cell systems or mammalian cell systems such as CHO cells. The method for preparation of natural or recombinant G-CSF and the method to detect the activities of G-CSF and its YPEG modified products are conventional to one in the art.
"In another aspect, the present invention relates to a method for preparation of YPEG modified G-CSF. In one embodiment, activated derivatives of YPEG such as polyethylene glycol succinimide (YPEG-NHS) shown in formula III can be used to covalently bind PEG with the .epsilon.-amino group on lysine corresponding to position 17 in G-CSF through nucleophilic substitution reaction:
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