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Preparation and/or formulation of proteins cross-linked with polysaccharides

A protein and protein matrix technology, applied in skin care preparations, animal/human proteins, albumin peptides, etc., can solve the problems of destroying the natural structure of molecules, toxicity, carcinogenicity, etc.

Active Publication Date: 2013-11-06
APTALIS PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chemical cross-linking methods often result in a large number of intramolecular cross-links, which disrupt the natural structure of the molecule; micronization or homogenization techniques that enable the product to be injected do not help preserve full-length or substantially full-length protein molecular structure
Additionally, chemical crosslinkers used to crosslink hyaluronic acid and proteins are known to be toxic and can cause irritation, inflammation, or pose a cancer risk

Method used

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  • Preparation and/or formulation of proteins cross-linked with polysaccharides
  • Preparation and/or formulation of proteins cross-linked with polysaccharides
  • Preparation and/or formulation of proteins cross-linked with polysaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0198] Schematic representation of the preparation of soluble hyaluronic acid crosslinkers using carbodiimide and N-succinimide (NHS):

[0199] Hyaluronic acid (HA) is a polysaccharide containing β-D-glucuronic acid-[1-3]-β-D-N-acetylglucosamine disaccharide units. The ideal structure of HA is shown in figure 1 middle.

[0200] As can be seen, HA contains one carboxyl / disaccharide unit, which is a functional group that can be used in at least one of the crosslinking methods disclosed herein. In this method, a covalent chemical bond is formed between the carboxyl groups of HA and the free amino groups of the protein. This can be done by reacting HA with a carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) to form a reactive o-acylisourea ester. When this compound is reacted with a nucleophile such as a primary amino group, it is also unstable and hydrolyzes rapidly in water without any suitable reactive group. Therefore, the formation of more stable i...

Embodiment 2

[0203] Schematic representation of the preparation of soluble hyaluronic acid crosslinkers using the heterobifunctional reagent allyl glycidyl ether (AGE)

[0204] The method is based on derivatizing hyaluronic acid (HA) with heterobifunctional reagents, which is a two-step method of cross-linking proteins with HA. The method chosen in this example uses allyl glycidyl ether (AGE), where the initial incorporation of AGE occurs when the oxirane group reacts with the hydroxyl group of HA under strongly basic conditions to form a stable ether linkage . The allyl group thus incorporated can then be converted to a halohydrin by reaction with a halide such as bromine. These halohydrins can then react with proteins to form stable secondary amine linkages through primary amino groups. The reaction between halohydrins and primary amino groups occurs more efficiently at high pH, ​​but some reactions begin to occur above pH 8.5-9.

[0205]

[0206] 2.HA-R-CH=CH 2 +Br 2 +H 2 O→HA-...

Embodiment 3

[0210] Preparation of polysaccharide cross-linking agent A:

[0211] In this example, 5ml of 1% low molecular weight hyaluronic acid solution, 1ml of H 2O. 100 mg of NHS (Sigma) and 100 mg of EDC (Sigma) were mixed together and reacted at room temperature for 1 hour. Derivatized hyaluronic acid was then precipitated with two volumes of IPA, briefly pressed to reduce the water and solvent content of the precipitate, rinsed with 66% ethanol, and then redissolved in 4 ml of phosphate-buffered saline (Sigma) at room temperature. The derivatized HA dissolved completely within 1 hour. The concentration of derivatized HA was measured using a temperature analyzer and then diluted to a 2% solution prior to protein crosslinking. All preparations were sterile and experiments were performed in a laminar flow hood when possible.

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Abstract

Therapeutic compositions and / or formulations are provided, comprising: at least one cross-linked protein matrix, wherein the at least one cross-linked protein matrix comprises at least one protein residue and at least one saccharide-containing residue, and methods of producing the same. The cross-linked protein matrix may be derived from cross-linking a full length or substantially full length protein, such as tropoelastin, elastin, albumin, collagen, collagen monomers, immunoglobulins, insulin, and / or derivatives or combinations thereof, with a saccharide containing cross-linking agent, such as a polysaccharide cross-linking agent derived from, for example, hyaluronic acid or a cellulose derivative. The therapeutic compositions may be administered topically or by injection. The present disclosure also provides methods, systems, and / or kits for the preparation and / or formulation of the compositions disclosed herein.

Description

[0001] References to related applications [0002] This application is related to U.S. Provisional Application U.S. 61 / 3444,940, filed November 23, 2010; incorporated herein by reference in its entirety. technical field [0003] The present disclosure relates to and can be applied to the preparation and / or formulation of proteins cross-linked with polysaccharides. Background technique [0004] Injectable implants are currently used to bulk or augment tissue in medical applications for vocal cord reconstruction, incontinence, and cosmetic treatment of wrinkles. Current implants are composed of a variety of materials, including hyaluronic acid, proteins such as collagen, polymers such as polylactic acid, and biomaterials such as hydroxyapatite. [0005] For example, hyaluronic acid ("HA"), sometimes called hyaluronan or hyaluronate, is a naturally occurring mucopolysaccharide found in, for example, synovial fluid, vitreous humor, blood vessel walls, and umbilical cord and in...

Claims

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Application Information

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IPC IPC(8): A61L27/24A61L27/20C08H1/00A61K8/64A61L27/22
CPCC08L1/286A61L27/22C08L5/02C08H1/06A61Q19/08C08L1/28C08H1/00C08L89/06C08L89/00C08L5/00A61K2800/91C08B37/0063C08B37/0033C08L3/14C08L1/284C08B37/0024C08B37/0096C08L5/10C08B11/08A61L2400/06C08B37/0069C08B37/0009C08L5/08C08B37/0021C08B11/12A61L2430/34A61K8/64C08B37/0075C07K14/78A61L27/20C08B37/0072C08L5/04A61L27/26C08B37/0084C08B33/04A61P17/00A61P17/02A61P19/00A61P19/02A61P27/02A61P9/00A61F2/0059A61K8/735A61L27/227A61L27/58C07K14/765A61L27/50
Inventor 简斯·萨默尔-克努森
Owner APTALIS PHARMA
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