| Method for producing acylated peptides -> Monitor Keywords |
|
|
 
Method for producing acylated peptidesRelated Patent Categories: Chemistry: Natural Resins Or Derivatives; Peptides Or Proteins; Lignins Or Reaction Products Thereof, Peptides Of 3 To 100 Amino Acid Residues, 25 Or More Amino Acid Residues In Defined SequenceMethod for producing acylated peptides description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20080033147, Method for producing acylated peptides. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation of U.S. application Ser. No. 10/671,260 filed Sep. 25, 2003, which claimed priority under 35 U.S.C. 119 of Danish application no. PA 2002 01421 filed Sep. 25, 2002, and U.S. provisional application No. 60/413,684 filed Sep. 26, 2002, the contents of which are fully incorporated herein by reference. FIELD OF THE INVENTION [0002] The present invention relates to methods for acylating peptides and proteins. More specifically, the invention relates to a method of introducing one or more acyl groups into a peptide or a protein. BACKGROUND OF THE INVENTION [0003] A large number of peptides have been approved for use in medical practice, and the peptides may be produced in suitable host cells by recombinant DNA technology or they may be produced synthetically by well established peptide synthesis technology. However, native peptides as well as analogues thereof tend to exhibit high clearance rates which are unacceptable for many clinical indication where a high plasma concentration of the peptide is required over a pro-longed period of time. Examples of peptides which in their native form have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease. [0004] A variety of derivatizations of peptides and peptide analogs have been found to influence the clearance rate of the peptides in a favorable direction. One such derivatization is the introduction of a lipophilic acyl group into the therapeutic peptide causing a desirable protracted profile of action relative to the non-acylated peptide. Hence, less frequent administration of the therapeutic protein improves the patients compliance to the prescribed therapy, and it reduces the amount of peptide to be administered. This has been described and demonstrated in WO98/08871, which i.a. discloses acylation of GLP-1 and analogs thereof, in WO98/08872, which i.a. discloses acylation of GLP-2 and analogs thereof, and WO99/43708, which i.a. discloses acylation of exendin and analogs thereof. Mono- or dipeptide spacers such as aspartic acid and glutamic acid, between the peptide and the acyl-group was demonstrated to be desirable. Spacers including a free carboxylic acid group must be protected before acylation and subsequently deprotected. [0005] EP 1227107 discloses the acylation of E-amino groups of human insulin. [0006] WO00/55119 discloses a method for acylating peptides (e.g. GLP-1) and novel acylating agents. [0007] In order for therapeutic peptides to be economically viable the cost of producing the peptides as well as the therapeutic dosage of the peptide are pivotal. A major cost during production of therapeutic peptides is the purification steps required to separate the target protein from impurities which are closely related to the target protein, e.g. isomers, desamido forms etc. These purification steps are usually performed by chromatography implying expensive chromatography matrices and solvents as well as reduced overall yield. [0008] It is the aim of the present invention to provide an efficient and economic method for the introduction of lipophilic groups into peptides via .alpha.-amino-.alpha.,.omega.-dicarboxylic acid spacers. The method is more specific, and thus results in higher yields and reduced formation of closely related impurities. A significant reduction of the cost of producing the acylated peptides are achieved. Less expensive acylated peptides are highly desirable for maximizing the number of patients for whom the treatment is available as well as for exploiting the advantages of alternative delivery routes which have lower bioavailability than subcutaneous injection, e.g. transdermal and pulmonal delivery. SUMMARY OF THE INVENTION [0009] The present invention provides a method for acylating one or more amino groups of a peptide or protein, the method comprising the steps: [0010] a) reacting the peptide having at least one free amino group with an acylating agent of the general formula I [0011] wherein [0012] n is 0-8; [0013] R.sup.1 is COOR.sup.4; [0014] R.sup.2 is a lipophilic moiety; [0015] R.sup.3 together with the carboxyl group to which R.sup.3 is attached designate a reactive ester or a reactive N-hydroxy imide ester; and [0016] R.sup.4 is selected from hydrogen, C.sub.1-12-alkyl and benzyl, [0017] under basic conditions in an aqueous mixture; [0018] b) if R.sup.4 is not hydrogen, saponifying the acylated peptide ester group (COOR.sup.4) under basic conditions; [0019] c) isolating the N-acylated peptide, characterized by said aqueous mixture in step a) containing less than 10% w/w aprotic polar solvent. [0020] In one embodiment of the method, said reaction in step a) takes place in an aqueous mixture containing less than 8% w/w aprotic polar solvent, preferably less than 5% w/w aprotic polar solvent and even more preferable less than 3% w/w aprotic polar solvent. [0021] In another embodiment of the method, the acylating agent is added to the reaction mixture as a solid. [0022] In another embodiment of the method, the acylating agent is added to the reaction mixture as a solution in a aprotic polar solvent which is stabilized by adding an acid. DESCRIPTION OF THE INVENTION [0023] Peptides and Proteins [0024] The present invention is useful for the introduction of lipophilic acyl groups into any peptide (or protein) in order to reduce the in vivo clearance rate. Examples of such peptides and proteins are ACTH, corticotropin-releasing factor, angiotensin, calcitonin, exendin and analogues thereof, insulin and analogues thereof, glucagon and analogues thereof, glucagon-like peptide-1 and analogues thereof, glucagon-like peptide-2 and analogues thereof, insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease. [0025] It should be understood that the peptide (or protein) should carry at least one free amino group, such an amino group being the N-terminal amino group or a side chain amino group. The peptides or protein may comprise amino acids which are not encoded by the genetic code, such as D-amino acids, 3-hydroxyproline, ornithine and pentylglycine. Particularly interesting are amino groups of lysine and ornithine amino acid residues. The method is particular relevant for the N-acylation of the .epsilon.-amino group of lysine residues. It should also be understood that the peptide or protein in question may comprise two or more pendant amino groups which all may be N-acylated according to the present invention. [0026] The present invention is especially suitable for the acylation of GLP-1 and analogues thereof. Examples of GLP-1 and analogues which can be N-acylated according to the present invention are GLP-1 and truncated analogues, such as Arg.sup.26-GLP-1(7-37); Arg.sup.34-GLP-1(7-37); Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.38GLP-1(7-38); Arg.sup.26,34Lys.sup.39-GLP-1(7-39); Arg.sup.26,34Lys.sup.40-GLP-1(7-40); Arg.sup.34Lys.sup.36-GLP-1(7-37); Arg.sup.26Lys.sup.39-GLP-1(7-39); Arg.sup.34Lys.sup.40-GLP-1(7-40); Arg.sup.26,34Lys.sup.36,39-GLP-1(7-39); Arg.sup.26,34Lys.sup.36,40-GLP-1(7-40); Gly.sup.8Arg.sup.26-GLP-1(7-37); Gly.sup.8Arg.sup.34-GLP-1(7-37); Gly.sup.8Lys.sup.36-GLP-1(7-37); Gly.sup.8Arg.sup.26,34Lys.sup.36-GLP-1(7-37); Gly.sup.8Arg.sup.26,34Lys.sup.39-GLP-1(7-39); Gly.sup.8Arg.sup.26,34Lys.sup.40-GLP-1(7-40); Gly.sup.8Arg.sup.26Lys.sup.36-GLP-1(7-37); Gly.sup.8Arg.sup.34Lys.sup.36-GLP-1(7-37); Lys.sup.36-GLP-1(7-37); Arg.sup.26,34Lys.sup.36-GLP-1(7-37); Arg.sup.26,34-GLP-1(7-37); Arg.sup.26,34Lys.sup.40-GLP-1(7-37); Arg.sup.26Lys.sup.36-GLP-1(7-37); Arg.sup.34Lys.sup.36-GLP-1(7-37); Val.sup.8Arg.sup.22-GLP-1(7-37); Met.sup.8Arg.sup.22-GLP-1(7-37); Gly.sup.8His.sup.22-GLP-1(7-37); Val.sup.8His.sup.22-GLP-1(7-37); Met.sup.8His.sup.22-GLP-1(7-37); His.sup.37-GLP-1(7-37); Gly.sup.8-GLP-1(7-37); Val.sup.8-GLP-1(7-37); Met.sup.8-GLP-1(7-37); Gly.sup.8Asp.sup.22-GLP-1(7-37); Val.sup.8Asp.sup.22-GLP-1(7-37); Met.sup.8Asp.sup.22-GLP-1(7-37); Gly.sup.8Glu.sup.22-GLP-1(7-37); Val.sup.8Glu.sup.22-GLP-1(7-37); Met.sup.8Glu.sup.22-GLP-1(7-37); Gly.sup.8Lys.sup.22-GLP-1(7-37); Val.sup.8Lys.sup.22-GLP-1(7-37); Met.sup.8Lys.sup.22-GLP-1(7-37); Gly.sup.8Arg.sup.22-GLP-1(7-37); Val.sup.8Lys.sup.22His.sup.37-GLP-1(7-37); Gly.sup.8Glu.sup.22His.sup.37-GLP-1(7-37); Val.sup.8Glu.sup.22His.sup.37-GLP-1(7-37); Met.sup.8Glu.sup.22His.sup.37-GLP-1(7-37); Gly.sup.8Lys.sup.22 His.sup.37-GLP-1(7-37); Met.sup.8Lys.sup.22His.sup.37-GLP-1(7-37); Gly.sup.8Arg.sup.22His.sup.37-GLP-1(7-37); Val.sup.8Arg.sup.22His.sup.37-GLP-1(7-37); Met.sup.8Arg.sup.22His.sup.37-GLP-1(7-37); Gly.sup.8His.sup.22His.sup.37-GLP-1(7-37); Val.sup.8His.sup.22His.sup.37-GLP-1(7-37); Met.sup.8His.sup.22His.sup.37-GLP-1(7-37); Gly.sup.8His.sup.37-GLP-1(7-37); Val.sup.8His.sup.37-GLP-1(7-37); Met.sup.8His.sup.37-GLP-1(7-37); Gly.sup.8Asp.sup.22 His.sup.37-GLP-1(7-37); Val.sup.8Asp.sup.22His.sup.37-GLP-1(7-37); Met.sup.8Asp.sup.22His.sup.37-GLP-1(7-37); Arg.sup.26-GLP-1(7-36)-amide; Arg.sup.34-GLP-1(7-36)-amide; Lys.sup.36-GLP-1(7-36)-amide; Arg.sup.26,34Lys.sup.36-GLP-1(7-36)-amide; Arg.sup.26,34-GLP-1(7-36)-amide; Arg.sup.26,34Lys.sup.40-GLP-1(7-36)-amide; Arg.sup.26Lys.sup.36-GLP-1(7-36)-amide; Arg.sup.34Lys.sup.36-GLP-1(7-36)-amide; Gly.sup.8-GLP-1(7-36)-amide; Val.sup.8-GLP-1(7-36)-amide; Met.sup.8-GLP-1(7-36)-amide; Gly.sup.8Asp.sup.22-GLP-1(7-36)-amide; Gly.sup.8Glu.sup.22His.sup.37-GLP-1(7-36)-amide; Val.sup.8Asp.sup.22-GLP-1(7-36)-amide; Met.sup.8Asp.sup.22-GLP-1(7-36)-amide; Gly.sup.8Glu.sup.22-GLP-1(7-36)-amide; Val.sup.8Glu.sup.22-GLP-1(7-36)-amide; Met.sup.8Glu.sup.22-GLP-1(7-36)-amide; Gly.sup.8Lys.sup.22-GLP-1(7-36)-amide; Val.sup.8Lys.sup.22-GLP-1(7-36)-amide; Met.sup.8Lys.sup.22-GLP-1(7-36)-amide; Gly.sup.8His.sup.22His.sup.37-GLP-1(7-36)-amide; Gly.sup.8Arg.sup.22-GLP-1(7-36)-amide; Val.sup.8Arg.sup.22-GLP-1(7-36)-amide; Met.sup.8Arg.sup.22-GLP-1(7-36)-amide; Gly.sup.8His.sup.22-GLP-1(7-36)-amide; Val.sup.8His.sup.22-GLP-1(7-36)-amide; Met.sup.8His.sup.22-GLP-1(7-36)-amide; His.sup.37-GLP-1(7-36)-amide; Val.sup.8Arg.sup.22His.sup.37-GLP-1(7-36)-amide; Met.sup.8Arg.sup.22His.sup.37-GLP-1(7-36)-amide; Gly.sup.8His.sup.37-GLP-1(7-36)-amide; Val.sup.8His.sup.37-GLP-1(7-36)-amide; Met.sup.8His.sup.37-GLP-1(7-36)-amide; Gly.sup.8Asp.sup.22 His.sup.37-GLP-1(7-36)-amide; Val.sup.8Asp.sup.22His.sup.37-GLP-1(7-36)-amide; Met.sup.8Asp.sup.22His.sup.37-GLP-1(7-36)-amide; Val.sup.8Glu.sup.22His.sup.37-GLP-1(7-36)-amide; Met.sup.8Glu.sup.22His.sup.37-GLP-1(7-36)-amide; Gly.sup.8Lys.sup.22 His.sup.37-GLP-1(7-36)-amide; Val.sup.8Lys.sup.22His.sup.37-GLP-1(7-36)-amide; Met.sup.8Lys.sup.22His.sup.37-GLP-1(7-36)-amide; Gly.sup.8Arg.sup.22His.sup.37-GLP-1(7-36)-amide; Val.sup.8His.sup.22His.sup.37-GLP-1(7-36)-amide; Met.sup.8His.sup.22His.sup.37-GLP-1(7-36)-amide; and derivatives thereof. [0027] Each of these GLP-1 analogues and truncated analogues constitutes alternative embodiments of the present invention. [0028] The present invention is also especially suitable for the acylation of GLP-2 and analogues thereof. Examples of GLP-2 and analogues which can be N-acylated according to the present invention are GLP-2 analogues and truncated analogues, such as Lys.sup.20GLP-2(1-33); Lys.sup.20Arg.sup.30GLP-2(1-33); Arg.sup.30Lys.sup.34GLP-2(1-34); Arg.sup.30Lys.sup.35GLP-2(1-35); Arg.sup.30,35Lys.sup.20GLP-2(1-35); and Arg.sup.35GLP-2(1-35). Each of these GLP-2 analogues and truncated analogues constitutes alternative embodiments of the present invention. [0029] The present invention is also especially suitable for the acylation of exendin-3 and exendin-4 and analogues thereof. Examples of exendin analogues which can be N-acylated according to the present invention are disclosed in e.g. WO99/43708. Each of these exendin analogues and truncated analogues constitutes alternative embodiments of the present invention. Continue reading about Method for producing acylated peptides... Full patent description for Method for producing acylated peptides Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Method for producing acylated peptides patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Method for producing acylated peptides or other areas of interest. ### Previous Patent Application: Methods for manufacturing aryl-aryl coupled polymers Next Patent Application: Anti-il-tif antibodies and methods of using in inflammation Industry Class: Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof ### FreshPatents.com Support Thank you for viewing the Method for producing acylated peptides patent info. IP-related news and info Results in 0.79676 seconds Other interesting Feshpatents.com categories: Novartis , Pfizer , Philips , Polaroid , Procter & Gamble , 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
