(12) Patent Application Publication (10) Pub. No.: US 2009/0192073 A1 HABERMANN Et Al
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US 20090192073A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2009/0192073 A1 HABERMANN et al. (43) Pub. Date: Jul. 30, 2009 (54) METHOD FOR PRODUCING INSULIN (30) Foreign Application Priority Data ANALOGS HAVINGADBASICB CHAN TERMINUS Jul. 11, 2006 (DE) ......................... 102OO6031955.9 (75) Inventors: to thisETErt an Publication Classification Frankfurt am Main (DE) (51) Int. Cl. A638/28 (2006.01) Correspondence Address: CI2P 2L/06 (2006.01) ANDREA Q. RYAN C07K I4/62 (2006.01) SANOF-AVENTIS U.S. LLC 104.1 ROUTE 202-206, MAIL CODE: D303A BRIDGEWATER, NJ 08807 (US) (52) U.S. Cl. ............................. 514/3; 435/68.1:530/303 (73) Assignee: SANOF-AVENTS DEUTSCHLAND GMBH, Frankfurtr am Main (DE)(DE (57) ABSTRACT (21) Appl. No.: 12/349,854 1-1. The invention relates to a method for producing a type of (22) Filed: Jan. 7, 2009 insulin by genetically engineering a precursor thereof and Related U.S. Application Data converting said precursor to the respective insulin in an .S. App enzyme-catalyzed ligation reaction with lysine amide or argi (63) Continuation of application No. PCT/EP2007/ nine amide, or by lysine or arginine which is modified by 005933, filed on Jul. 5, 2007. protective groups, and optionally Subsequent hydrolysis. US 2009/0192073 A1 Jul. 30, 2009 METHOD FOR PRODUCING INSULIN acids and the B chain with 30 amino acids. The chains are ANALOGS HAVING ADIBASIC B CHAIN linked together by 2 disulfide bridges. Insulin preparations TERMINUS have been employed for many years for the therapy of diabe tes. Moreover, not only are naturally occurring insulins used, but more recently also insulin derivatives and analogs. 0001. This application is a continuation of International 0006 Insulin analogs are analogs of naturally occurring application No. PCT/EP2007/005,933, filed Jul. 5, 2007, insulins, namely human insulin or animal insulins which which is incorporated herein by reference in its entirety: differ by replacement of at least one naturally occurring which claims the benefit of priority of German Patent Appli amino acid residue by other amino acid residues and/or addi cation No. 10 2006 031955.9, filed Jul. 11, 2006. tion/deletion of at least one amino acid residue from the corresponding, otherwise identical, naturally occurring insu DESCRIPTION lin. U.S. Pat. No. 5,656,722 for example describes des-Phe '-insulin derivatives. The amino acid residues which have 0002. The invention relates to a method for preparing an been added and/or replaced may also be ones which do not insulin with dibasic chain end by biotechnological prepara occur naturally. tion of a precursor thereof and Subsequent conversion in an enzyme-catalyzed ligation reaction with lysinamide or 0007 Insulin derivatives are derivatives of naturally argininamide, or lysine or arginine modified by protective occurring insulins or insulin analogs in which one or more groups, and optionally subsequent hydrolysis, to give this amino acid residues and/or the N or C termini of the A and/or insulin. B chain are replaced by functional groups. The functional 0003. About 177 million people around the world suffer groups are selected from a group comprising amide residues, from diabetes mellitus. These include about 17 million type I amine residues, carboxyl residues, alkyl residues, alcohol diabetics for whom replacement of the lacking endocrine residues and alkoxy residues. insulin secretion is the only possible therapy at present. Those 0008. An efficient insulin therapy makes use of so-called affected are dependent on insulin injections, usually several basal insulins. By these are meant formulations which make times a day, throughout life. Type II diabetes contrasts with slow, continuous release of exogenously administered insulin type I diabetes in that there is not always a deficiency of possible. In this way, a baseline insulin concentration in the insulin, but in a large number of cases, especially in the body which has advantageous effects on the physiological advanced stage, treatment with insulin, where appropriate condition of the person suffering from diabetes is achieved combined with an oral antidiabetic, is regarded as the most over a lengthy period. favorable type of therapy. 0009. The recombinant insulin analog Gly(A21), Arg 0004. In healthy people, insulin release is strictly coupled (B31), Arg(B32) human insulin (insulin glargine) is in this to the blood glucose concentration. Elevated blood glucose connection notable for needing to be supplied to the body levels like those occurring after meals are rapidly compen only every 24 hours i.e. only one a day—in order to achieve sated by a corresponding rise in insulin secretion. In the a basal effect. The once-a-day administration leads to an fasting state, the plasma insulin level falls to a baseline value improved quality of life. The improved physiology leads for which suffices to ensure a continuous supply of glucose to example to a reduction in the Hbalc level and it can be insulin-sensitive organs and tissues and to keep hepatic glu expected that, owing to this improvement, the late sequelae of cose production low during the night. Replacement of the diabetes will appear if at all—considerably later, thus mak endogenous insulin secretion by exogenous, usually subcu ing it possible to prolong the life expectancy of the relevant taneous administration of insulinusually does not come close diabetic. to the quality of the physiological regulation of blood glucose 0010. The demand for this insulin analog is correspond described above. Upward or downward rearrangements of the ingly high. Since the number of diabetics is continually blood glucose level are frequent and may, in their most severe increasing, it is moreover of economic interest to minimize forms, be life-threatening. However, in addition, elevated the costs for preparing corresponding analogs. U.S. Pat. No. blood glucose levels lasting for years represent, even without 5,656,722 describes the possible preparation of insulin ana initial symptoms, a considerable health risk. The large-scale logs via a preproinsulin fusion protein which consists of a DCCT study in the USA (The Diabetes Control and Compli fusion portion (pre portion) and of a monkey proinsulin cations Trial Research Group (1993) N. Engl. J. Med. 329, variant. One of the analogs described comprises glycine 977-986) unambiguously proved that chronically elevated instead of asparagine in position A(21). The corresponding blood glucose levels are substantially responsible for the fusion protein is a peptide precursor variant for preparing development of late diabetic complications. Late diabetic insulin glargine. The method provides for deletion of the pre complications and micro- and macrovascular damage which portion and the C peptide from this fusion protein by reaction in some circumstances becomes manifest as retinopathy, with trypsin. EP-A 0668292 describes a fusion protein which nephropathy or neuropathy and leads to blindness, renal fail follows the same principle but allows insulin glargine to be ure and loss of extremities and, in addition, is associated with prepared by a method which is an improvement over U.S. Pat. an increased risk of cardiovascular disorders. It is to be No. 5,656,722. It is clear to the skilled worker in this connec inferred therefrom that an improved therapy of diabetes must tion that a partial cleavage is possible in particular at the primarily aim at keeping blood glucose as closely as possible boundary of the insulin B chain and C chain, which is defined within the physiological range. The intensive insulin therapy by the dibasic structure Arg-Arg, and leads to a B31 mono-arg policy intends to achieve this by injections several times a day human insulin analog. This faulty product must be removed of fast- and slow-acting insulin preparations. Fast-acting for from the actual compound of interest. This leads to a marked mulations are given at meal times in order to compensate the impairment of the yield. The problem can be avoided by postprandial rise in blood glucose. Slow-acting basal insulins recombinant preparation of proinsulins and reaction thereof are intended to ensure the basic supply of insulin especially with a specific endoprotease such as, for example, lysyl during the night without leading to hypoglycemia. endopeptidase, and reacting the resulting des-B30 human 0005 Insulin is a polypeptide composed of 51 amino acids insulin (analog) in a semisynthetic peptide chemistry divided into 2 amino acid chains: the A chain with 21 amino approach with the tripeptide Thr-Arg-Arg. EP-A 0 132 769 US 2009/O 192073 A1 Jul. 30, 2009 and WO 2003/044210 describe the need to protect the reac endoprotease which does not cleave the insulin derivative is tive groups of the tripeptide during the reaction. The protec incorporated in order to eliminate the pre or fusion portion tive groups are eliminated Subsequent to the reaction. This appropriately. Enterokinase (DDDDK) or factor Xa (IEGR) route is associated with costs arising from the preparation of are mentioned by way of example. The invention also relates the tripeptide by chemical synthesis and the introduction of thereto. It is moreover clear to the skilled worker that the two protective groups. Thus, it would be desirable to have a cleavage reactions can proceed in a one-pot reaction. A fur method allowing Arg(B31), Arg(B32)-insulin analogs to be ther possibility is to eliminate the fusion portion only in a prepared from the Arg(B31) human insulin precursor. following step. The fusion protein portion can in this case be 0011 German patent application No. 10 2005 046 113.1 chosen to be derivatives of a large number of efficiently (not published) describes a method including the trypsin secreted proteins. Examples which may be mentioned for catalyzed ligation of amino acids which have C-terminal ami bacteria are DHFR (dihydrofolate reductase), glutathione dation to peptides whose C-terminal amino acid consists of S-transferase and hirudin.