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PEGylated bioactive peptides and uses thereof

A technology of drugs and analogues, applied in the field of PEGylated bioactive peptides and their uses, can solve problems such as inactivity and low efficacy

Active Publication Date: 2019-01-04
GRIFFON PHARMA INT SA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The least bioactive conformation of the GHRH peptide was determined to be present in the first 29 amino acids of the full-length peptide (Frohman et al., 1986a), but low potency of bioactivity could be detected in GHRH(1-21) (Ling et al. 1984a); finding , GHRH peptides shorter than 21 amino acids are inactive

Method used

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  • PEGylated bioactive peptides and uses thereof
  • PEGylated bioactive peptides and uses thereof
  • PEGylated bioactive peptides and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0201] Example 1: Materials and methods

[0202] Synthesis and Preparation of GHRH Analogs

[0203] Using fluorenylmethoxycarbonyl-protected α-amino acids with appropriate side chain protection and benzhydrylamine (BHA) resin (Bachem AG) at a loading of 0.75 mmol / g, using manual or automated solid-phase peptide synthesis methods Preparation of GHRH analogs described herein. Prior to amino acid coupling, 6-aminocaproic acid and Rink linkers were coupled to the resin, followed by [2-(6-chloro-1H-benzotriazol-1-yl)-1,1,3,3 -tetramethylammonium hexafluorophosphate] (HCTU) or (1-cyano-2-ethoxy-2-oxoethyleneaminooxy)dimethylamino-morpholino-carbon Coupling of hexafluorophosphate (COMU) and diisopropylethylamine (DIEA) in N,N-dimethylformamide (DMF) to Fmoc-[9H-fluoren-9-ylmethoxycarbonyl] protected Amino acid about 1 hour. Fmoc deprotection was performed using 20% ​​(v / v) piperidine in DMF for about 0.5 hours. A general procedure for N-capping the peptides described herein wit...

Embodiment 2

[0207] Example 2: PEGylated GHRH Peptides in Cell-Based Hit (DiscoveRx TM ) Determination of Agonist Potency in Assay

[0208] DiscoverRx TM A panel of cell lines stably expressing an untagged GPCR that signals through cAMP was developed. Hit cAMP assay using DiscoverRx TM A technique called Enzyme Fragment Complementation (EFC), which has β-galactosidase (β-Gal) as a functional reporter, was developed to signal via Gs second messenger in a uniform non-imaging assay format Activation of GPCRs is monitored.

[0209] Amplify cAMP Hunter from frozen stocks according to standard protocols TM cell line. Cells were seeded into white-walled 384-well microplates in a total volume of 20 μL and incubated at 37°C for an appropriate time prior to testing. Using DiscoverRx TM Hit Hunter TM The cAMP XS+ assay determines cAMP regulation. For agonist assays, cells are incubated with samples to induce a response. Media was aspirated from the cells and replaced with 15 μL of 2:1...

Embodiment 3

[0218] Example 3: GH Release Kinetics in Sprague-Dawley Rats in Response to Subcutaneous Injection of Selected GHRH Analogs

[0219]Protocol: Sprague-Dawley rats (female, body weight 250 to 300 g) were obtained from Charles River Inc. Animals were used according to the protocols of the Committee on Animal Care and the principles of the Canadian Council for Animal Care's Guide for the Care and Use of Laboratory Animals. Animals were maintained on standard laboratory chow on a 12:12 light:dark cycle. They were maintained in groups of 4 rats per cage. Anesthetize the animal with isoflurane 2.5%. A midsection opening is made in the neck to expose the carotid artery. The carotid artery was cannulated with polypropylene tubing (PE-50) to allow blood draw. Following surgical preparation, rats received subcutaneous injections of GHRH analogs (10 to 100 μg / kg) dissolved in 20 mM AcONa + 5% mannitol (pH 5). Blood samples (400 μL / time point) were collected from 2 to 4 animals per gr...

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Abstract

Described herein are bioactive peptides that are modified at one or more positions with a PEG moiety. An example of such a PEGylated bioactive peptide is a GHRH analog that is modified at one or morepositions with a PEG moiety. Also described are pharmaceutically acceptable salts thereof and pharmaceutical compositions comprising such analogs or salts thereof, as well as methods, kits and uses thereof, for example for inducing or stimulating growth hormone secretion in a subject and for diagnosing, preventing or treating GH-deficient conditions in a subject.

Description

[0001] Cross References to Related Applications [0002] This application claims the benefit of US Provisional Application No. 62 / 324,600, filed April 19, 2016, which is hereby incorporated by reference in its entirety. technical field [0003] The present disclosure relates to biologically active peptides conjugated to polyethylene glycol and uses thereof. [0004] Sequence Listing Citation [0005] Pursuant to 37 C.F.R. 1.821(c), the Sequence Listing is hereby submitted as an ASCII compatible text file named "Sequence_Listing.txt," created April 18, 2017, and having a size of approximately 26,000 bytes. The contents of the aforementioned file named "Sequence_Listing.txt" are hereby incorporated by reference in their entirety. Background technique [0006] Growth hormone releasing hormone (GHRH), also known as growth hormone releasing factor (GRF or GHRF), is a 44-amino acid peptide secreted by neurons innervating the anterior pituitary Somatotroph cells secrete growth h...

Claims

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Application Information

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IPC IPC(8): C07K14/60A61K47/60A61P5/06
CPCA61K38/25C07K14/60A61P5/06A61K47/60A61K38/00
Inventor 克里什纳·G·佩里
Owner GRIFFON PHARMA INT SA
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