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Process for Preparing Cardiodilatin Fragments; Highly Purified Cardiodilatin Fragments and Intermediate Products for the Preparation of Same

a technology of cardiodilatin and fragments, which is applied in the field of preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates, and can solve the problems of high risk of therapeutic application, inability to achieve synthesis at only a small scale, and insufficient purity of cardiodilatin fragments prepared according to the procedures described in literatur

Inactive Publication Date: 2010-12-16
IMMER HANSUELI +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This process achieves cardiodilatin fragments with yields of 15-20% and purities of up to 99.9%, significantly reducing peptide impurities and enhancing biological activity and scalability.

Problems solved by technology

However, the cardiodilatin fragments prepared according to the procedures described in literature did not have the purity necessary for clinical studies and for the authorization as medicinal product because, due to the synthesis, peptide impurities had been introduced into the final product which could not be removed even by subsequent purification processes.
Due to their immunogenic properties, the impurities may give rise to undesirable side-effects when administered to the patient, so that therapeutic application involved risk.
Moreover, the synthesis could be accomplished at only a small scale under reasonable technical input and was not economically suitable for a larger production scale.
Furthermore, another drawback of known processes for synthesis was the existing potential risk of racemization due to which the urodilatin was obtained with lower purity, lower biological activity and in insufficient yield.
Racemization of the product which frequently occurs with existing syntheses often resulted in insufficient optical purity of the final product, and these impurities frequently cannot be removed or only with exceedingly high technical input.

Method used

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  • Process for Preparing Cardiodilatin Fragments; Highly Purified Cardiodilatin Fragments and Intermediate Products for the Preparation of Same
  • Process for Preparing Cardiodilatin Fragments; Highly Purified Cardiodilatin Fragments and Intermediate Products for the Preparation of Same
  • Process for Preparing Cardiodilatin Fragments; Highly Purified Cardiodilatin Fragments and Intermediate Products for the Preparation of Same

Examples

Experimental program
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example 1

General Procedures of Solid-Phase Synthesis According to the Merrifield Process

a) Solid-Phase Synthesis on a Support Resin

[0049]Starting from the C-terminus of the peptide to be synthesized, the first amino acid (AA) protected by the Fmoc group at the N-terminal end, is bound to the support resin (Fmoc-AA-OHMPB-support resin). With a standard batch of 6.66 mmoles, the Fmoc protecting group is subsequently removed by adding 100 ml of a solvent mixture of piperidine and N-methylpyrrolidine (1:4 v / v). Then, the resin suspension is stirred for 10 minutes, subsequently filtrated, and again, 100 ml of the piperidine and NMP solvent mixture is added. Then, the suspension is stirred for 10 minutes, filtrated and subsequently washed with NMP an isopropanol, and completeness of the reaction is checked using the Kaiser test.

[0050]Thereafter, the next amino acid is coupled to the resin. Initially, 20 mmoles of a 0.5 M solution of diisopropylethanylamine (DIPEA) in NMP is added to the resin, the...

example 2

Preparation of Fragment ANP(109-120)

[0053]Following the general procedures of Example 1, and starting from 273 g of Fmoc-Gly-OHMPB-support resin (corresponding to 130 mmoles), 170.3 g of the fully protected cardiodilatin fragment ANP(109-120) is obtained.

example 3

Preparation of Fragment ANP(121-126)

[0054]Following the general procedures of Example 1, and starting from 264 g of Fmoc-Tyr-OHMPB-support resin (corresponding to 115 mmoles), 150.7 g of the fully protected cardiodilatin fragment ANP(121-126) is obtained. Here, the N-terminal end of the fragment is protected by the Fmoc group.

[0055]Subsequently, the terminal hydroxy group at the C-terminal end of the fragment is converted to the OtBu protecting group. For esterification, 149 g of the fully protected fragment is dissolved in 500 ml of trifluoroethanol and 4.1 l of chloroform. This is followed by addition of 141 ml of TBTA (t-butyl-2,2,2-trichloroacetimidate), and the solution is heated at reflux for one hour. After the reaction is completed, the solution is concentrated to give a crystalline-oily residue, 6.8 l of diisopropyl ether is added, and the suspension is stirred at room temperature for 14 hours. The product is filtrated off and dried to constant weight. 136.7 g of fragment 3...

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Abstract

The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS[0001]This application is a continuation application of application Ser. No. 11 / 084,190, filed Mar. 21, 2005, now abandoned, which is a continuation application of application Ser. No. 09 / 027,777, filed Feb. 23, 1998, now abandoned, which is a divisional application of patent application Ser. No. 08 / 737,927, filed Dec. 2, 1996, which issued as patent number 5,767,239 on Jun. 16, 1998. Application Ser. No. 08 / 737, 927 is a national stage entry of PCT / EP95 / 02050, filed May 30, 1995, now expired. The disclosures of the prior applications are hereby incorporated by reference in their entireties.STATEMENT REGARDING SEQUENCE LISTING[0002]Submitted herewith, in both computer-readable and written form, are sequence listings for each sequence contained herein. The information contained in the computer-readable copy is identical to the written sequence listings.FIELD OF THE INVENTION[0003]The invention relates to a process for the preparation of cardiod...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/16C07K1/107C07K1/06C07K1/20C07K14/00C07K7/08A61K38/10C07K7/06C07K5/10C07K5/08A61K38/00A61K38/22A61P7/10C07K14/47C07K14/58
CPCC07K14/58A61K38/00A61P7/10
Inventor IMMER, HANSUELIFORSSMANN, WOLF-GEORGADERMANN, KNUTKLESSEN, CHRISTIAN
Owner IMMER HANSUELI