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Optimization of enzyme replacement therapy for treatment of homocystinuria

A cysteine ​​and amino acid technology, applied in the field of cystathionine β synthase conjugates, can solve the problems of poor dietary compliance of HCU patients

Pending Publication Date: 2020-02-04
UNIV OF COLORADO THE REGENTS OF +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Met restriction is generally effective in reducing plasma Hcy levels, HCU patients are poorly compliant with their diet and thus symptomatic

Method used

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  • Optimization of enzyme replacement therapy for treatment of homocystinuria
  • Optimization of enzyme replacement therapy for treatment of homocystinuria
  • Optimization of enzyme replacement therapy for treatment of homocystinuria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0100] Embodiment 1. Experimental procedure

[0101] A. Chemicals

[0102] Unless otherwise stated, all materials were purchased from or FISHERSCIENTIFIC TM . L-[ 14 C]-serine was obtained from PERKIN Life Sciences.

[0103] b.animals

[0104] All animal procedures are approved by the University of Colorado Denver Institutional Animal Care and Use Committee (IACUC) under Animal Procedure #B-49414(03)1E, which is AAALAC - Accredited (#00235), Public Health Service-covered (#A 3269-01) and USDA-licensed (#84-R-0059) facility. Such as Maclean et al., Mol. Genet. Metab. 2010, 101, (2-3), 153-62; Bublil et al., J Clin Invest 2016, 126, (6), 2372-84 and international application PCT / 2016 / 061050 ( The entire contents of which are incorporated herein by reference), "human-only" (HO) mice have previously been generated, bred and genotyped. Import animals as products from TEKLAD GLOBAL RODENT (Harlan, Livermore, CA, USA) standard extruded diets 2918, 2919 or 2920X were f...

Embodiment 2

[0121] Example 2. Optimizing PEGylation of htCBS C15S

[0122] To understand the PEGylation pattern and optimize the conjugation reaction to produce more uniform modifications, individual PEG-htCBSC15S species were isolated and the residues targeted with the ME-400MA maleimide PEG molecule were subsequently identified ( figure 2 ). First, the PEGylation reaction was loaded onto a DEAESepharose column equilibrated in low conductivity and higher pH (10mM TRIS. Protein elution was performed under a salt gradient (5%, 10-110 mM NaCl). figure 2 The chromatogram of one such run and the recovery of the two pools, which were subsequently analyzed on SDS-PAGE and native PAGE together with unmodified htCBS C15S and 400MA PEG-htCBS C15S reaction mixtures, are shown. Gel analysis of the pool provided evidence that the 400MA PEG-htCBS C15S consisted of at least 3 different PEG-htCBS C15S species.

[0123] The form enriched in pool 1 (P1) has a weaker negative charge than the form re...

Embodiment 3

[0128] Example 3. Reproducibility of PEGylation of NHS ester PEG molecules with htCBS C15S

[0129] Problems with the reproducibility of PEGylation of htCBS C15S with maleimide PEG led to the analysis of other PEGylation chemistries, especially using NHS ester-activated PEG. First, modifications of htCBS C15S were evaluated with three NHS ester PEG molecules to determine their effect on enzymatic activity. In PEGylation with 5, 10 and 20 kDa linear NHS ester PEG molecules, respectively, four, three and two different PEG:CBS molar ratios were tested. Unlike site-directed selective maleimide PEGylation, NHS ester PEG molecules yield a reproducible mixture of highly modified isomers that are virtually inseparable using SDS-PAGE as well as native PAGE or SEC-HPLC.

[0130] For example, on SDS-PAGE ( 10% TGX gel) or native PAGE ( 4-15% htCBS C15S modified by three linear 5, 10 and 20 kDa NHS ester PEG molecules (5NHS, 10NHS and 20NHS) (different molar excess relative to ...

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Abstract

The present invention provides a method of PEGylating a human truncated cystathionine -synthase protein containing a mutation of a cysteine to a serine at amino acid position 15 (htCBS C15S). The htCBS C15S was PEGylated with one of 5 kDa, 10 kDa, or 20 kDa NHS ester PEG molecules. In-process monitoring of the PEGylation process was used in the method to reduce levels of unPEGylated htCBS C15S andhtCBS C15S with insufficient PEGylation. Administration of the PEGylated htCBS C15S had efficacy throughout the course of treatment for homocystinuria.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to US Provisional Patent Application 62 / 486,246, filed April 17, 2017, which is hereby incorporated by reference in its entirety. [0003] sequence listing [0004] This application is filed with a sequence listing in electronic format. The sequence listing file is named 20891004PCT_SL.txt, created on April 17, 2018, and has a size of 10,794 bytes. The information in electronic format of the Sequence Listing is hereby incorporated by reference in its entirety. [0005] field of invention [0006] The present invention relates to the characterization of a PEGylated human truncated cystathionine beta synthase (htCBS) conjugate to identify its suitability for reproducible production and further clinical development as an enzyme replacement therapy for classical homocystinuria . [0007] Background of the invention [0008] Human cystathionine β synthase (CBS, KEGG enzyme identification c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/51A61K47/14C12N9/88C12N9/96
CPCA61K38/51C12N9/88C12N9/96C12Y402/01022A61K47/60
Inventor T·马伊坦J·P·克劳斯E·M·巴布利F·格拉文M·塞洛斯-莫拉
Owner UNIV OF COLORADO THE REGENTS OF
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