Details for Patent: 5,424,394

Title:	Synthetic preparation of amylin and amylin analogues
Abstract:	Synthetic amylin and amylin analogs which have high biological activity and which are substantially free from deletion peptides and other contaminating peptides are provided. Also provided are methods for the solid phase peptide synthesis of amylin and amylin analogs.
Inventor(s):	Lehman de Gaeta; Laura S. (La Jolla, CA), Albrecht; Elisabeth (San Diego, CA)
Assignee:
Filing Date:	Jul 08, 1993
Application Number:	08/090,361
Claims:	1. Synthetic human amylin produced by solid phase peptide synthesis having an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less according to an assay of insulin-stimulated incorporation of labelled glucose into glycogen in isolated rat soleus muscle. 2. Synthetic human amylin according to claim 1 which is substantially free of deletion and other contaminating peptides. 3. A method for solid phase synthesis of amylin or an amylin analog which has an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less and is substantially free of deletion and other contaminating peptides which comprises (a) successive synthesis cycles wherein, in each cycle, a protected designated amino acid is covalently coupled to a growing peptide chain covalently linked to an insoluble solid resin support to give a peptide having an additional amino acid, under coupling conditions, wherein said coupling conditions include use of a N-methyl pyrrolidone solvent system for said coupling cycles which maximizes swelling of said resin support, minimizes secondary structure elements of the peptide chain during synthesis, and minimizes intrapeptide and interpeptide hydrogen bonding, and wherein, if initial coupling of amino acid to each peptide chain in a synthesis cycle is less than about 97%, a second coupling step is performed in the synthesis cycle; (b) a capping step after each synthesis cycle and before the next synthesis cycle wherein unreacted deprotected .alpha.-amino groups on the growing peptide chain are reacted to block further chain extension; (c) selectively deprotecting cysteine residues and forming an intramolecular disulfide bond between cysteine residues on the peptide chain before cleaving the peptide chain from the solid support; and (d) cleaving said amylin or amylin analog from said solid support. 4. A method according to claim 3 wherein said second coupling step is performed if initial coupling is less than about 99%. 5. A method for solid phase synthesis of a peptide which comprises amylin or an amylin analog which has an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less and is substantially free of deletion and other contaminating peptides wherein said peptide is synthesized using successive synthesis cycles whereby in each such synthesis cycle a designated amino acid is added to a growing peptide chain attached to an insoluble resin support by formation of a peptide linkage between an .alpha.-amino group of the peptide chain and an .alpha.-carboxyl of the designated amino acid wherein each synthesis cycle comprises: (a) treating the growing peptide chain under .alpha.-amino deprotecting conditions to remove an .alpha.-amino protecting group from the .alpha.-amino group of the terminal amino acid and generate a free .alpha.-amino group; (b) activating the .alpha.-carboxyl group of the .alpha.-amino protected designated amino acid; (c) contacting said growing peptide chain and said designated amino acid under coupling conditions to form a peptide linkage between the free .alpha.-amino of the peptide chain and the activated .alpha.-carboxyl of the designated amino acid wherein said coupling conditions include use of a N-methyl pyrrolidone solvent system which maximizes swelling of the resin support, minimizes formation of secondary structure of the peptide chain during synthesis and minimizes intrapeptide and interpeptide hydrogen bonding; (d) measuring the coupling efficiency of step (c); (e) if the measured coupling efficiency of step (c) is less than about 97%, repeating steps (b) and (c); (f) capping unreacted free .alpha.-amino groups of the peptide chain to render them unreactive; (g) repeating said synthesis cycle of steps (a) to (f) using the appropriate designated amino acids to give amylin or any amylin analog of a specific sequence; (h) upon completion of said successive synthesis cycles selectively deprotecting cysteine residues of the peptide and forming an intramolecular disulfide bond between the cysteine residues before cleaving the peptide from the support; and (i) cleaving said amylin or amylin analog from said solid support. 6. A method according to claim 5 wherein steps (b) and (c) are repeated if the coupling efficiency of step (c) is less than about 99%. 7. A method for solid phase synthesis of a peptide which comprises amylin or an amylin analog which has an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less and is substantially free of deletion and other contaminating peptides wherein said peptide is synthesized using successive synthesis cycles, whereby in each such synthesis cycle, a designated amino acid is added to a growing peptide chain attached to an insoluble resin support by formation of a peptide linkage between an .alpha.-amino group of the growing peptide chain and an .alpha.-carboxyl of the designated amino acid; and wherein each synthesis cycle comprises: (a) treating the growing peptide chain under .alpha.-amino deprotecting conditions to remove an .alpha.-amino group of the terminal amino acid and generate a free .alpha.-amino group; (b) activating the .alpha.-carboxyl group of the .alpha.-amino protected designated amino acid; (c) contacting said growing peptide chain and said designated amino acid under coupling conditions to form a peptide linkage between the free .alpha.-amino of the peptide chain and the activated .alpha.-carboxyl of the designated amino acid wherein said coupling conditions include use of a N-methyl pyrrolidone solvent system which maximizes swelling of said resin support, minimizes secondary structure elements of the peptide chain during synthesis cycles and minimizes intrapeptide and interpeptide hydrogen bonding; (d) if coupling efficiency of step (c) is less than about 97%, repeating steps (b) and (c); (e) capping unreacted free .alpha.-amino groups of the peptide chain to render them unreactive; (f) repeating said synthesis cycle of steps (a) to (e) using appropriate designated amino acids to give amylin or an amylin analog of specified sequence; (g) upon completion of all said synthesis cycles selectively deprotecting cysteine residues of the peptide chain and forming an intramolecular disulfide bond between the cysteine residues before cleaving the peptide chain from the support; and (h) cleaving said amylin or amylin analog from said solid support. 8. A method according to claim 7 wherein steps (b) and (c) are repeated if the coupling efficiency of step (c) is less than about 99%. 9. A method for solid phase synthesis of a peptide which comprises amylin or an amylin analog which has an EC.sub.50 of about 2.5 nanomoles per liter.+-.0.3 log units or less and is substantially free of deletion and other contaminating peptides wherein said peptide is synthesized using successive synthesis cycles, a designated amino acid is added to a growing peptide chain attached to an insoluble resin support by formation of a peptide linkage between an .alpha.-amino group of the growing peptide chain and .alpha.-carboxyl of the designated amino acid; and wherein each synthesis cycle comprises: (a) treating the growing peptide chain under .alpha.-amino deprotecting conditions to remove an .alpha.-amino group of the terminal amino acid and generate a free .alpha.-amino group; (b) activating the .alpha.-carboxyl group of the .alpha.-amino protected designated amino acid; (c) contacting said growing peptide chain and said designated amino acid under coupling conditions to forma a peptide linkages between the free .alpha.-amino of the peptide chain and the activated .alpha.-carboxyl of the designated amino acid; (d) repeating steps (b) and (c); and (e) capping unreacted free .alpha.-amino groups of the peptide chain to render them unreactive; and after completion of all synthesis cycles; (f) selectively deprotecting cysteine residues of the peptide and forming an intramolecular disulfide bond between the cysteine residues before cleaving the peptide from the support; and (g) cleaving said amylin or amylin analog from said solid support. 10. A method for the solid phase synthesis of amylin or an amylin analog having an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less which comprises successively coupling protected amino acids in a predetermined order to a growing peptide chain covalently attached to a solid support to give a peptide, under coupling conditions which include N-methyl pyrrolidone solvent conditions substantially disruptive of secondary structure of the growing peptide chain during synthesis; upon completion of successive coupling, forming an intramolecular disulfide bond between cysteine residues on the peptide before cleaving the peptide from the solid support; and cleaving said amylin or said amylin analog from said solid support. 11. Amylin or an amylin analog produced according to the process of any of claims 3, 5, 7, 9 or 10. 12. Amylin or an amylin analog according to claim 11 having an EC.sub.50 of about 2.5 nanomoles/liter.+-.0.3 log units or less according to an assay of insulin stimulated incorporation of labelled glucose into glycogen in isolated rat soleus muscle.