Claims for Patent: 5,747,447

Summary for Patent: 5,747,447

Title:	Stable polypeptide composition
Abstract:	A (injectable biologically active) polypeptide is stabilized by dissolving said polypeptide forming a liquid solution in citrate buffer of about pH 5.0-5.5.
Inventor(s):	Robert L. Swift, Charles P. Du Mee, Anne Randolph
Assignee:	COR Therapeutics Inc , Millennium Pharmaceuticals Inc
Application Number:	US08/462,661
Patent Claims:	1. A method of storage-stabilizing a substantially pure platelet aggregation inhibitor (PAI) polypeptide comprising preparing a liquid solution consisting essentially of said polypeptide in a citrate buffer by dissolving said polypeptide in said citrate buffer to form a storage-stable solution having a pH of from about 5.0 to about 5.5. 2. A method according to claim 1 wherein said polypeptide has the formula ##STR6## wherein K* has the formula R.sup.1.sub.2 N(CH.sub.2).sub.4 CH(NH)CO-- wherein (G/Sar) is selected from the group consisting of G and Sar; wherein each R1 is independently H, alkyl (1-6 C), or at most one R1 is R2 --C═NR3, wherein R2 is H, alkyl (1-6 C) or is a substituted or unsubstituted phenyl or benzyl residue, or is NR4 2 in which each R4 is independently H or alkyl (1-6 C), and R3 is H, alkyl (1-6 C), phenyl or benzyl, or R2 --C═NR3 is a radical selected from the group consisting of ##STR7## where m is an integer of 2-3, and each R5 is independently H or alkyl (1-6 C); and where one or two (CH2) is optionally replaced by O or S provided said O or S is not adjacent to another heteroatom; AA1, is a small, neutral (polar or nonpolar) amino acid and nl is an integer of 0-3; AA2 is a neutral, nonpolar large (aromatic or nonaromatic) or a polar aromatic amino acid and n2 is an integer of 0-3; AA3 is a proline residue or a modified proline residue of the formula ##STR8## wherein one or two of the methylenes of said proline or modified proline residue is optionally replaced by --NR--, --S--, or --O-- wherein R is hydrogen or alkyl (1-6 C) and n3 is an integer of 0-1; AA4 is a neutral, small amino acid or the N-alkylated form thereof and n4 is an integer of 0-3; each of X1, and X2 is independently a residue capable of forming a bond between X1, and X2 to obtain a cyclic compound as shown; and each of Y1 and Y2 is independently a noninterfering substituent or is absent; wherein one or more peptide linkages may optionally be replaced by linkage selected from the group consisting of --CH2 NH--, --CH2 S--, --CH2 CH2 --, --CH═CH-- (cis and trans), --COCH2 --, --CH(OH)CH2 -- and --CH2 SO--. 3. The method of claim 2 wherein said Y1 is H, acyl, or a peptide residue or derivatized form thereof or is absent and Y2 is OH, NH2, --A--NH2 or a peptide residue or derivatized form thereof or is absent, X1 and X2 are selected from the group consisting of cysteine (C), mercaptovaleryl (Mvl), mercaptoproprionyl (Mpr) and penicillamine (Pen), AA1, is G and n1 is 0 or 1, AA2 is selected from the group consisting of W, F, L, Y, and V and K* is K, Har, acetimidyl-Lys or phenylimidyl-Lys. 4. The method of claim 3 wherein the polypeptide is selected from the group consisting ______________________________________ (SEQ ID NO:53) Mpr-K-G-D-W(Formyl)-P-C-NH.sub.2 (SEQ ID NO:54) Mvl-K-G-D-W-P-C-NH.sub.2 (SEQ ID NO:55) Mpr-K-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:63) Mpr-(Har)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:66) Mpr-(Har)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:67) Mpr(Acetimidyl-Lys)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:68) Mpr(Acetimidyl-Lys)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:73) Mpr(Phenylimidyl-Lys)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:75) Mpr(Phenylimidyl-Lys)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:81) Mpr-Ala-(Har)-G-D-W-P-C-NH.sub.2 Mpr-L-homoarginine-G-D-W-P-C-NH.sub.2 ! and (SEQ ID NO:30) Mpr-K-G-D-W-P-C-NH.sub.2 ______________________________________ 5. A method according to claim 2 wherein said solution is stable at about 70° C. for at least 49 days. 6. The method according to claim 1 wherein said polypeptide is biologically active for inhibiting thrombus formation, preventing platelet loss during extracorporeal circulation of blood or for treating a patient suspected of having a platelet-associated ischemic syndrome. 7. The method according to claim 1 wherein the pH is about 5.25. 8. The method according to claim 7 wherein the stabilizing is at about -15° C. to about 30° C. 9. The method according to claim 1 wherein the stability is at about 5 to about 30° C. 10. The method according to claim 1 wherein said polypeptide is a cyclic polypeptide containing up to 10 amino acid residues and is stabilized in a solution having a pH of about 5.25, in which the citrate buffer solution includes sodium hydroxide. 11. A storage-stable composition comprising a substantially pure platelet aggregation inhibitor polypeptide dissolved in a liquid solution in which additives that provide storage stability consist essentially of a stabilizing effective amount of a citrate buffer, said composition having a pH of from about 5.0 to about 5.5. 12. A storage-stable composition according to claim 11, wherein said polypeptide has the formula ##STR9## wherein K* has the formula R.sup.1.sub.2 N(CH.sub.2).sub.4 CH(NH)CO-- wherein (G/Sar) is selected from the group consisting of G and Sar; wherein each R1 is independently H, alkyl(1-6 C), or at most one R1 is R2 --C═NR3, wherein R2 is H, alkyl or is a substituted or unsubstituted phenyl or benzyl residue, or is NR4 2 in which each R4 is independently H or alkyl(1-6 C), and R3 is H, alkyl(1-6 C), phenyl or benzyl, or R2 --C═NR3 is a radical selected from the group consisting of ##STR10## where m is an integer of 2-3, and each R5 is independently H or alkyl (1-6 C); and wherein one or two (CH2) is optionally replaced by O or S provided said O or S is not adjacent to another heteroatom; AA1, is a small, neutral (polar or nonpolar) amino acid and n1 is an integer of 0-3; AA2 is a neutral, nonpolar large (aromatic or nonaromatic) or a polar aromatic amino acid and n2 is an integer of 0-3; AA3 is a proline residue or a modified proline residue of the formula ##STR11## wherein one or two of the methylenes of said proline or modified proline residue is optionally replaced by --NR--, --S--, or --O-- wherein R is hydrogen or alkyl (1-6 C) and n3 is an integer of 0-1; AA4 is a neutral, small amino acid of the N-alkylated form thereof and n4 is an integer of 0-3; each of X1 and X2 is independently a residue capable of forming a bond between X1 and X2 to obtain a cyclic compound as shown; and each of Y1 and Y2 is independently a noninterfering substituent or is absent; wherein one or more peptide linkages is optionally be replaced by a linkage selected from the group consisting of --CH2 NH--, --CH2 S--, --CH2 CH2 --, --CH═CH-- (cis and trans), --COCH2 --, --CH(OH)CH2 -- and --CH2 SO--. 13. The composition of claim 12 wherein said Y1 is H, acyl, or a peptide residue or derivatized form thereof or is absent and Y2 is OH, NH2, --A--NH2 or a peptide residue or derivatized form thereof or is absent, X1 and X2 are selected from the group consisting of cysteine (C), mercaptovaleryl (Mvl), mercaptoproprionyl (Mpr) and penicillamine (Pen), AA1 is G and n1 is 0 or 1, AA2 is selected from the group consisting of W, F, L, Y, and V and K* is K, Har, acetimdyl-Lys or phenylimidyl-Lys. 14. The composition of claim 12 wherein the polypeptide is selected from the group consisting of ______________________________________ (SEQ ID NO:53) Mpr-K-G-D-W(Formyl)-P-C-NH.sub.2 (SEQ ID NO:54) Mvl-K-G-D-W-P-C-NH.sub.2 (SEQ ID NO:55) Mpr-K-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:63) Mpr-(Har)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:66) Mpr-(Har)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:67) Mpr(Acetimidyl-Lys)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:68) Mpr(Acetimidyl-Lys)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:73) Mpr(Phenylimidyl-Lys)-G-D-W-P-C-NH.sub.2 (SEQ ID NO:75) Mpr(Phenylimidyl-Lys)-G-D-W-P-Pen-NH.sub.2 (SEQ ID NO:81) Mpr-Ala-(Har)-G-D-W-P-C-NH.sub.2 Mpr-L-homoarginine-G-D-W-P-C-NH.sub.2 ! and (SEQ ID NO:30) Mpr-K-G-D-W-P-C-NH.sub.2. ______________________________________ 15. A composition of claim 12 stabilized at about 70° C. or less for at least 49 days. 16. A storage-stable therapeutic liquid composition comprising an injectable, biologically effective amount of a substantially pure platelet aggregation inhibitor polypeptide dissolved in a liquid solution in which additives that provide storage stability consist essentially of a citrate buffer, said solution having a pH of from about 5.0 to about 5.5. 17. The liquid composition according to claim 16 wherein said polypeptide is biologically active for inhibiting thrombus formation, preventing platelet loss during extracorporeal circulation of blood or for treating a patient suspected of having a platelet-associated ischemic syndrome. 18. The composition according to claim 16 wherein said pH is about 5.25. 19. The liquid composition according to claim 16 wherein said polypeptide is a cyclic polypeptide containing up to 10 amino acid residues and is stabilized in a solution having a pH of about 5.25, in which the citrate buffer solution includes sodium hydroxide. 20. An article of manufacture comprising a sterile delivery vial, bag or bottle filled with a liquid composition of claim 16 in injectable form.