Long acting proteins and peptides and methods of making and using the same

Abstract

Disclosed is a method for refolding a protein or peptide that does not contain essential disulfides and that contains at least one free cysteine residue. Also disclosed are polymer IFN-γ conjugates that have been created by the chemical coupling of polymers such as polyethylene glycol moieties to IFN-γ, particularly via a free cysteine in the protein. Also disclosed are analogs of bioactive peptides that may be used to create longer acting versions of the peptides, including analogs of glucagon, glucagon-like peptide-1 (GLP-1), GLP-2, Gastric inhibitory peptide (GIP), PYY, exendin, ghrelin, gastrin, amylin, and oxyntomodulin.

1 .- 64 . (canceled) 
     
     
         65 . A cysteine analog of ghrelin (SEQ ID NO:13), wherein a cysteine residue is added to the peptide either by substitution for a native amino acid in ghrelin or by insertion preceding the first amino acid of ghrelin or following the last amino acid of ghrelin, wherein the cysteine residue substituted for the native amino acid is selected from the group consisting of G1, S2, S3, F4, L5, S6, P7, E8, H9, Q10, R11, V12, Q13, Q14, R15, K16, E17, S18, K19, K20, P21, P22, A23, K24, L25, Q26, P27, and R28. 
     
     
         66 . (canceled) 
     
     
         67 . The cysteine analog of  claim 65 , wherein a cysteine residue is inserted preceding the first amino acid of ghrelin. 
     
     
         68 . The cysteine analog of  claim 65 , wherein S3 of ghrelin is acetylated. 
     
     
         69 .- 125 . (canceled) 
     
     
         126 . The cysteine analog of  claim 65 , wherein the added cysteine residue is modified with a cysteine reactive moiety selected from the group consisting of: a polyethylene glycol, a polyvinyl pyrolidone, a carbohydrate, a dextran, a sugar, a starch, a peptide, a lipid, a label, a dye, a polymer, a chromophore, and a radionucleotide. 
     
     
         127 . The cysteine analog of  claim 126 , wherein the reactive group on the cysteine-reactive moiety is selected from the group consisting of a maleimide group, a vinylsulfone group, and an iodoacetamide group. 
     
     
         128 . The cysteine analog of  claim 65 , wherein the added cysteine residue is modified with a polyethylene glycol. 
     
     
         129 . The cysteine analog of  claim 128 , wherein the polyethylene glycol moiety is selected from the group consisting of a linear polyethylene glycol and a branched polyethylene glycol. 
     
     
         130 . The cysteine analog of  claim 65 , wherein the added cysteine residue is modified with a lipid. 
     
     
         131 . The cysteine analog of  claim 65 , wherein the added cysteine residue is modified with a sugar. 
     
     
         132 . The cysteine analog of  claim 65 , wherein the cysteine analog of ghrelin is synthesized by chemical methods. 
     
     
         133 . The cysteine analog of  claim 65 , wherein the cysteine analog of ghrelin is a recombinant protein. 
     
     
         134 . The cysteine analog of  claim 126 , wherein the cysteine analog is exposed to a cysteine-reactive moiety to obtain a cysteine-modified protein, wherein the step of exposing is conducted in the presence of a reducing agent. 
     
     
         135 . The cysteine analog of  claim 65 , wherein the cysteine analog is selected from the group consisting of a homologue of ghrelin and a truncated form of ghrelin. 
     
     
         136 . The cysteine analog of  claim 65 , wherein a cysteine residue is inserted following the last amino acid of ghrelin. 
     
     
         136 . A composition comprising the cysteine analog of  claim 65  and a pharmaceutical accepted carrier. 
     
     
         137 . A method for treating a disease treatable with ghrelin in a patient, the method comprising administering to the patient a therapeutically effective amount of the ghrelin cysteine analog of  claim 65 . 
     
     
         138 . The method of  claim 137 , wherein the disease treatable with ghrelin is obesity. 
     
     
         139 . A method for treating a disease treatable with acylated ghrelin in a patient, the method comprising administering to the patient a therapeutically effective amount of the acylated ghrelin cysteine analog of  claim 68 . 
     
     
         140 . The method of  claim 138 , wherein the acylated ghrelin stimulates appetite in the patient. 
     
     
         141 . The method of  claim 138  wherein the disease treatable with acylated ghrelin is selected from the group consisting of anorexia and cachexia.