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CTP-based insulin analogs for treatment of diabetes

An analog, single-chain insulin technology, used in insulin, hormone peptides, metabolic diseases, etc.

Inactive Publication Date: 2014-10-22
INDIANA UNIV RES & TECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Native insulin has approximately 100-fold selective affinity for the insulin receptor relative to the related insulin-like growth factor 1 receptor, but little selectivity for two different insulin receptor isoforms (named A and B)

Method used

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  • CTP-based insulin analogs for treatment of diabetes
  • CTP-based insulin analogs for treatment of diabetes
  • CTP-based insulin analogs for treatment of diabetes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment approach

[0147] According to one embodiment, provided herein are insulin analogs modified to introduce one or more glycosylation sites. Glycosylation of peptide-based drugs can confer additional benefits on the base peptide, including increased serum half-life; increased functional in vivo half-life; and reduced degradation. Introduction of a glycosylation site into an insulin analog provides a site for attaching a sugar moiety to an insulin agonist such that when the insulin agonist is produced in a eukaryotic cell capable of glycosylation, the insulin agonist is glycosylated . In one embodiment, insulin analogs are provided wherein the peptide sequence has been modified by addition and / or substitution of amino acids to add new glycosylation sites not present in native insulin. In one embodiment, a glycosylation site is introduced at the amino- or carboxy-terminus of the B chain, or in the case of a single-chain analog, a glycosylation site may be introduced into the linking peptide ...

Embodiment 1

[1159] Synthesis of insulin A chain and B chain

[1160] Insulin A and B chains were synthesized on 4-methylbenzhydrylamine (MBHA) resin or 4-hydroxymethyl-phenylacetamidomethyl (PAM) resin using Boc chemistry. Peptides were cleaved from the resin using HF / p-cresol 95:5 for 1 hour at 0°C. After removal of HF and ether precipitation, the peptide was dissolved in 50% aqueous acetic acid and lyophilized. Alternatively, peptides were synthesized using Fmoc chemistry. Using trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / H 2 O (95:2.5:2.5) cleaves the peptide from the resin for 2 hours at room temperature. The peptide was precipitated by adding excess diethyl ether, and the precipitate was dissolved in aqueous acidic buffer. The quality of the peptides was monitored by RP-HPLC and confirmed by mass spectrometry (ESI or MALDI).

[1161] Synthesized with a single free cysteine ​​at amino acid 7, and all other cysteines are protected as acetamidomethyl A-(SH) 7 (Acm) 6,11,2...

Embodiment 2

[1168] PEGylation of amine groups (N-terminal and lysine) via reductive alkylation

[1169] a. Synthesis

[1170]Mix insulin (or insulin analogs), mPEG20k-formaldehyde (Aldyhyde) and NaBH at a molar ratio of 1:2:30 3 CN is soluble in acetate buffer at pH 4.1-4.4. The reaction solution consisted of 0.1 N NaCl, 0.2 N acetic acid and 0.1 N Na 2 CO 3 composition. The insulin peptide concentration was about 0.5 mg / ml. The reaction was carried out at room temperature for 6 hours. The extent of the reaction was monitored by RP-HPLC and the yield of the reaction was about 50%.

[1171] b. Purification

[1172] The reaction mixture was diluted 2-5 times with 0.1% TFA and applied to a preparative RP-HPLC column. HPLC conditions: C4 column; flow rate 10 ml / min; A buffer: 10% ACN and 0.1% TFA in water; B buffer: ACN containing 0.1% TFA; linear gradient B% 0-40% (0-80 minutes ); PEG-insulin or analog elutes at approximately 35% buffer B. After chemical modification by sulftolysis...

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Abstract

Insulin analogs comprising a non-native glycosylation site sequence are provided having high potency and specificity for the insulin receptor. In one embodiment a peptide sequence of greater than 18 amino acids is used as a linking moiety to link human insulin A and B chains, or analogs or derivatives thereof, to provide high potency single chain insulin analogs. In one embodiment the linking moiety comprises one or more glycosylation sites. Also disclosed are prodrug and conjugate derivatives of the insulin analogs.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application No. 61 / 578,052, filed December 20, 2011, the disclosure of which is expressly incorporated herein by reference in its entirety. Background of the invention [0003] Insulin is a proven therapy for juvenile-onset diabetes and later stages of adult-onset diabetes. The peptide is biosynthesized as a larger linear precursor of low potency (approximately 2%-9% of native insulin), called proinsulin. Proinsulin is proteolytically converted to insulin by selective removal of a 35-residue linking peptide (C-peptide). A heteroduplex with a total of 51 amino acids formed by disulfide bond formation between the insulin "A chain" (SEQ ID NO: 1) and "B chain" (SEQ ID NO: 2) chains has High potency (nM range). Native insulin has approximately 100-fold selective affinity for the insulin receptor relative to the related insulin-like growth factor 1 receptor, but lit...

Claims

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Application Information

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IPC IPC(8): A61K38/28A61K38/27
CPCA61K47/48246A61K38/00A61K47/60A61K47/64A61K38/28A61P3/10C07K14/00C07K14/62C07K2319/31C07K2319/91
Inventor R.D.迪马基P.李
Owner INDIANA UNIV RES & TECH CORP
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