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Interleukin-6 polyethylene glycol conjugate and its preparing method and use

A technology of interleukin and polyethylene glycol, which is applied in the field of preparation of interleukin-6 polyethylene glycol conjugates, can solve the problems of short half-life, difficult selection of reagents, difficult modification, etc., and achieves the dosage and frequency of use. Reduce and improve the effect of safety in use and clinical drug safety

Inactive Publication Date: 2011-09-07
CHENGDU INST OF BIOLOGICAL PROD
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Many other problems were found during the PEGylation of polypeptides: for example (1) amidation of tyrosine residues in proteins also resulted in reduced biological activity of modified products; (2) some PEGylated proteins were extremely unstable , has no medicinal value at all; (3) the reagents in the modification process are also difficult to choose, and some reagents used in the reaction process cannot properly complete the reaction, which takes a long time, so that the protein denatures before and after modification , or no reaction at all; (4) Hydrolysis of proteins in aqueous solutions at physiological pH tends to prevent PEGylation of proteins, resulting in many modification difficulties
IL-6 was still modified with PEG-5000. In this study, a single modified product component Fr3 with a molecular weight of 26,500 was obtained. Although this component retained a lot of in vitro activity, it was found in vivo when it was subcutaneously injected into mice. The half-life is extremely short (the half-life of the elimination phase is about 12 hours), and the residence time in the body is about 25 hours, which is not much different from that of unmodified IL-6. Its in vivo activity is also significantly lower than that of highly modified components, and its in vivo activity is less than 50 times Dose of IL-6
However, the single-modified DmPEG-IL-6 prepared with DMMAn as the amino-protecting agent not only has no significant difference in pharmacokinetic parameters and in vivo activity from the single-modified PEG-IL-6, but also requires three steps of reaction to obtain , the preparation process is complex
[0009] Most of the PEG-modified products of IL-6 disclosed above are mixtures of products with different modification sites and degrees of modification. Due to the inhomogeneity of the products, effective quality control cannot be carried out, and it is difficult to ensure safety, effectiveness, and quality controllable, and it is difficult to invest Practical application; however, the polyethylene glycol conjugates of monomodified IL-6 obtained in rare cases have defects such as too short half-life, low activity, poor curative effect, and low yield

Method used

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  • Interleukin-6 polyethylene glycol conjugate and its preparing method and use
  • Interleukin-6 polyethylene glycol conjugate and its preparing method and use
  • Interleukin-6 polyethylene glycol conjugate and its preparing method and use

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] [Example 1] Preparation of interleukin-6 polyethylene glycol conjugate (PEG-IL-6)

[0088] The chemical modification reaction formula for preparing IL-6 polyethylene glycol conjugate is as follows:

[0089]

[0090] Wherein i and j are integers of 100-1000, the sum of i and j makes the molecular weight of the mPEG part of the conjugate be 15000-30000, preferably 20000, and the amino group of -NH-IL-6 in the reaction formula structure is a Lys residue side chain amino group.

[0091] The modification process is as follows:

[0092] Sample: pure human IL-6 (Sigma company), SDS-PAGE purity greater than 95%, protein concentration between 0.5-1mg / ml, pH 9.0, buffer solution is PB, and cannot contain other amino-containing compounds

[0093] Modification reagent: mPEG2-NHS MW20kDa, stored in a dry place at -20°C Modification process:

[0094] a. Heat IL-6 in a water bath to 25°C, weigh mPEG2-NHS 1 to 2 times the total amount of IL-6, and put it into a dry, clean, steril...

Embodiment 2

[0098] [Example 2] Purification of PEG-IL-6

[0099] First, the three batches of products obtained by implementing the method 1 are uniformly mixed, and the G-25 gel column that is balanced to pH 5.0 through 10mM acetate buffer is used for desalting and changing the liquid, and the sample buffer (buffer formula: Na2HPO 12H2O, 15.04 g / L; NaH2PO4 2H2O, 1.25g / L; NaCl 8.77g / L. pH 9.0) the pH was adjusted from 9.0 to 5.0; the mixture of the modification reaction was separated by one-step SP Sepharose High Performance cation exchange chromatography (eluent Formula: Sodium acetate 0.82g / L in liquid A, adjust the pH to 5.0 with acetic acid; 0.82g / L sodium acetate in liquid B, 29.25g / L sodium chloride, adjust the pH to 5.0 with acetic acid). Under this condition, the hydrolyzed mPEG molecules cannot be adsorbed on the column because they are uncharged or negatively charged, and then the multi-modified mPEG-IL-6 is eluted first under the gradually increasing salt ion gradient. down, fo...

Embodiment 3

[0102] [Example 3] Preparation prescription and technical process

[0103] 1 preparation prescription

[0104] 1.1 Preparation prescription (based on 1000 bottles)

[0105] PEG-rhIL-6 was prepared according to the aforementioned method

[0106] Main drug PEG-rhIL-6 15mg

[0107] Human albumin 10g

[0108] Glycine 25g

[0109] Na2HPO412H2O 0.619g

[0110] KH2PO4 0.103g

[0111] NaCl 3.440g

[0112] KCl 0.086g

[0113] 1.2 Types of preparations

[0114] It belongs to the lyophilized preparation for injection stipulated in Appendix I "General Rules of Preparations" of the 2005 edition of "Pharmacopoeia of the People's Republic of China".

[0115] 1.3 Preparation specifications

[0116] 15μg / 0.5ml / bottle (200,000 active units)

[0117] 2 For preparation process, see image 3 .

[0118] Pharmacodynamics test, safety test and pharmacokinetic test

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Abstract

A interleukin-6 polyethylene glycol conjugate and its preparing method and medical compositions comprising the conjugate and pharmaceutically acceptable excipients. The conjugate according to the invention is used for producing medicines treating thrombocytopenia, chemotherapy adjuvants or medicines enhancing immunity. The mono-PEG-modified IL-6 enhances markedly biostability, has longer in vivo half life and lower plasma clearance compared with unmodified IL-6, resulting in a great decrease in frequency and dose of administration as well as side effects. The mono-PEG-modified IL-6 according to the present invention can reach medicinal standards due to its good uniformity.

Description

technical field [0001] The invention relates to an interleukin-6 polyethylene glycol conjugate; [0002] The invention also relates to the preparation method and application of the interleukin-6 polyethylene glycol conjugate. Background technique [0003] Interleukin-6 (Interleukin 6, IL-6 for short), also known as interferon β2, is a multifunctional cytokine. IL-6 mainly acts on the immune system, can promote the differentiation of T cells and nerve cells, stimulate the growth of T cells and stem cells, induce the differentiation of multinucleated cells, and thus promote the production of platelets. The medical use of IL-6 mainly has three aspects: (1) It has a certain curative effect on the thrombocytopenia caused by chemotherapy and radiotherapy. It was once used as a substitute for platelet preparations. Great expectations have been placed on it; (2) It can enhance the immunity of cancer patients, remove the residual tumor cells in the body of cancer patients after sur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/54C07K14/565C07K17/08C07K1/14A61K38/20A61K47/48A61P7/00A61P37/02
CPCC07K1/1077A61K47/48215C07K14/5412A61K47/60A61P7/00A61P37/02
Inventor 张雪梅袁涛张珂饶海林邓杰王智杰
Owner CHENGDU INST OF BIOLOGICAL PROD
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