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Compound containing pigment epidermal derived factors and preparation method and application thereof

A technology of deriving factors and pigment epithelium, which is applied in the direction of non-active ingredients of polymer compounds, medical preparations containing active ingredients, biological testing, etc., can solve the problems of patient inconvenience, increased drug costs, and short half-life, and achieve high inhibition and small Effects of mouse tumor growth, slowing metabolism, and prolonging half-life

Inactive Publication Date: 2009-09-30
焦春 +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, PEDF protein drugs have a short half-life in vivo after administration. In addition to protein degradation in vivo, they will be eliminated from the body through the glomerular filtration pathway. In some cases, they will be cleared or inactivated by binding to specific antibodies.
In order to maintain an effective therapeutic concentration in the body, frequent injections are required. Although this treatment method can achieve the therapeutic purpose, it brings inconvenience and pain to the patient, and increases the cost of the drug.

Method used

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  • Compound containing pigment epidermal derived factors and preparation method and application thereof
  • Compound containing pigment epidermal derived factors and preparation method and application thereof
  • Compound containing pigment epidermal derived factors and preparation method and application thereof

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preparation example Construction

[0043] The preparation method of the complex containing pigment epithelium-derived factor of the present invention comprises the following steps:

[0044] The activated polyenol compound is mixed with the pigment epithelium-derived factor at a molar ratio of 1:5 to 5:1, and reacted at a pH of 3-10 and a temperature of 4-30°C. Preferably, the polyenol compound is polyethylene glycol.

[0045] Preferably, different modified pigment epithelium-derived factors obtained after the reaction are separated by a cationic column or molecular sieve to obtain a complex of PEG modified at a single site at the N-terminal of the pigment epithelium-derived factor.

[0046] The present invention also provides a slow-release preparation comprising the pigment epithelium-derived factor complex, and the slow-release material of the slow-release preparation is selected from microcapsules, hydrogels, microspheres, micro-osmotic pumps or liposomes. The sustained-release preparation can further prolo...

Embodiment 1

[0048] Example 1 Preparation of recombinant pigment epithelium-derived factor

[0049] (1) Construction of strains

[0050] A DNA fragment encoding the polypeptide shown in SEQ NO.2 is cloned from a human cDNA library, constructed into a PET30a plasmid, transformed into a BL21(DE3) host bacterium, and expressed in the polypeptide shown in SEQ NO.2.

[0051] (2) Protein expression

[0052] Inoculate PEDF strains into 30ml LB liquid medium, culture on a shaker at 37°C and 200rpm for 5 hours, then transfer to 500ml LB liquid medium, and the culture conditions are as above. Put this culture solution into a 5L fermenter, the culture conditions are 37°C, pH 7.0, dissolved oxygen 20-30%, at OD 600 Before reaching 10, control residual sugar to 3g / L, at OD 600 After reaching 10, control the residual sugar within 1g / L. Sampling every hour to detect residual sugar and OD 600 , OD 600 Nitrogen source was added at 6 o'clock. OD 600 When it reaches about 20, add IPTG with a final co...

Embodiment 2

[0059] Example 2 N-terminal coupling of 20KD polyethylene glycol to recombinant pigment epithelium-derived factor

[0060] The recombinant human pigment epithelium-derived factor involved in this embodiment includes the recombinant human pigment epithelium-derived factor with the sequence of SEQ NO.2. The specific method is to dialyze the recombinant human pigment epithelium-derived factor into 30mM sodium acetate solution with pH 5.5, measure the protein concentration, and adjust the concentration to 3-8mg / ml. According to the molar ratio of target protein and polyethylene glycol 1 to 2, the molecular weight that needs to be added is the quality of monomethoxypolyethylene glycol propionaldehyde of 20KD, calculates the consumption of reductant CH according to final solution volume simultaneously, its concentration is 20mM. Finally, adjust the pH value of the solution to 5.1-5.3. The reaction solution was stirred evenly and left to stand at room temperature for 4-6 hours or 4...

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Abstract

The invention discloses a compound containing pigment epidermal derived factors, wherein the pigment epidermal derived factors are coupled with polyvinyl alcohol compound or Fc fragments of immunoglobulin. The invention also discloses a method for preparing the compound containing the pigment epidermal derived factors and application of the compound containing pigment epidermal derived factors in preparing medicines for preventing, diagnosing or treating tumor and abnormal hyperplasia disease of novel vessels. The compound containing the pigment epidermal derived factors can obviously prolong the intravital half-life period of the pigment epidermal derived factors and simultaneously has the same activity for inhibiting the growth of the novel vessels and the growth of tumor with unmodified pigment epidermal derived factors and even higher activity than the unmodified pigment epidermal derived factors.

Description

technical field [0001] The invention relates to a compound containing pigment epithelium-derived factor (Pigment Epithelium-Derived Factor, PEDF), in particular to a compound capable of prolonging the half-life of PEDF in vivo, its preparation method and application. Background technique [0002] Neovascularization is the creation of new capillaries on top of existing blood vessels. The growth and migration of tumors depend on the formation of new blood vessels, so inhibiting the formation of new blood vessels has become a new means of anti-tumor therapy. (Folkman J.N Engl J Med 1971;285:1182-1186). [0003] Pigment epithelium-derived factor (PEDF) is a glycoprotein naturally occurring in the eyes of humans and animals, with a molecular weight of 46KD and two structural domains: the N-terminal neurotrophic domain and the C-terminal serine protease inhibitory supergene family reaction loop. DNA sequence analysis showed that PEDF belongs to the serine protease inhibitor gene...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K17/08C07K19/00A61K9/16A61K47/34A61K38/17A61K47/48A61P35/00A61P9/10A61P27/02G01N33/68A61K47/42A61K47/60A61K47/66
Inventor 于昊燕焦春柯静婷
Owner 焦春
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