US 7,524,813 B2
Selectively conjugated peptides and methods of making the same
Magali A. Zundel, Søborg (Denmark); Bernd Peschke, Maalov (Denmark); Florencio Zaragoza Dörwald, Ballerup (Denmark); Niels Peter Fiil, Frederiksberg (Denmark); Nils Langeland Johansen, Copenhagen Ø (Denmark); and Henning Ralf Stennicke, Kokkedal (Denmark)
Assigned to Novo Nordisk Health Care AG, Zurich (Switzerland)
Filed on Mar. 31, 2006, as Appl. No. 11/395,784.
Application 11/395784 is a continuation of application No. PCT/DK2004/000685, filed on Oct. 08, 2004.
Claims priority of provisional application 60/510892, filed on Oct. 14, 2003.
Claims priority of application No. 2003 01496 (DK), filed on Oct. 10, 2003.
Prior Publication US 2007/0111926 A1, May 17, 2007
Int. Cl. A61K 38/00 (2006.01); A61K 38/16 (2006.01); A61K 38/26 (2006.01); A61K 38/27 (2006.01); A61K 38/28 (2006.01); C07C 251/00 (2006.01); C07C 251/32 (2006.01); C07C 251/72 (2006.01); C07C 281/00 (2006.01); C07K 14/605 (2006.01); C07K 14/61 (2006.01); C07K 14/62 (2006.01)
U.S. Cl. 514—3  [514/2; 514/12; 530/300; 530/303; 530/308; 530/324; 570/113] 3 Claims
 
1. A conjugated peptide obtainable by a method comprising the steps of
i) reacting in one or more steps a peptide with a first compound bearing one or more functional groups, which are not accessible in any of the amino acids residues constituting said peptide, in the presence of an enzyme capable of catalyzing the incorporation of said first compound into the C-terminal of said peptide to form a transacylated peptide, and
ii) reacting in one or more steps said transacylated peptide with a second compound comprising one or more functional groups, wherein said functional group(s) do not react with functional groups accessible in the amino acid residues constituting said peptide, and wherein said functional group(s) in said second compound is capable of reacting with said functional group(s) in said first compound so that one or more covalent bond between said transacylated peptide and said second compound is formed, and wherein said peptide P is reacted in one or more steps with a first compound, which is an α-amino acid amide represented by the formula

OG Complex Work Unit Drawing
in the presence of carboxypeptidase to form a transacylated peptide of the formula

OG Complex Work Unit Drawing
said transacylated peptide being further reacted in one or more steps with a second compound of the formula
Y-E-Z
to form a conjugated peptide of the formula

OG Complex Work Unit Drawing
wherein R represents a linker or a bond;
P′ represents a peptide formed by removing the C-terminal amino acid from the peptide P, P selected from the group consisting of: human insulin, GLP-1, GLP-2, human growth hormone (GH) and factor VII;
X is selected from amongst keto-, aldehyde-, —NH—NH2, —O—C(O)—NH—NH2, —NH—C(O)—NH—NH2, —NH—C(S)—NH—NH2, —NHC(O)—NH—NH—C(O)—NH—NH2, —NH—NH—C(O)—NH—NH2, —NH—NH—C(S)—NH—NH2, —NH—C(O)—C6H4—NH—NH2, —C(O)—NH—NH2, —O—NH2, —C(O)—O—NH2, —NH—C(S)—O—NH2, alkyne, nitril-oxide and azide;
Y is selected from amongst keto-, aldehyde-, —NH—NH2, —O—C(O)—NH—NH2, —NH—C(O)—NH—NH2, —NH—C(S)—NH—NH2, —NHC(O)—NH—NH—C(O)—NH—NH2, —NH—NH—C(O)—NH—NH2, —NH—NH—C(S)—NH—NH2, —NH—C(O)—C6H4—NH—NH2, —C(O)—NH—NH2, —O—NH2, —C(O)—O—NH2, —NH—C(O)—O—NH, —NH—C(S)—O—NH2, alkyne, nitril-oxide and azide;
E represents a linker or a bond;
A represents oxime, hydrazones, phenylhydrazone, semicarbazone or triazole moieties; and
Z comprises the moiety to be conjugated to the peptide where said moiety decreases the clearance of compound of the formula

OG Complex Work Unit Drawing
in comparison with the clearance of peptide P and where the moiety is selected from the group consisting of: PEG; mPEG; straight, branched and/or cyclic C2-22alkyl, C2-22alkenyl, C2-22alkynyl, C1-22heteroalkyl, C2-22heteroalkenyl, C2-22heteroalkynyl, wherein each may be optionally substituted with one or more substituents selected from hydroxyl, halogen, carboxyl, heteroaryl and aryl;
steroid;
lipids;
polysaccharides;
ethylene/maleic anhydride polymer; and
peptides.