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Conjugate capable of penetrating blood-labyrinth barrier and preparation method thereof

A conjugate and conjugation technology, applied in the field of conjugates that can pass through the blood labyrinth barrier and its preparation, can solve the problems of high risk, surgical preventive administration, and inconvenience of multiple medications, and achieve the goal of treating neurological disorders. Effects of Deafness, Good Inner Ear Targeting, and Enrichment Function

Active Publication Date: 2021-09-24
PEKING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this barrier system necessary to ensure the special functions of the inner ear has also become an important obstacle to restrict the entry of drug molecules into the inner ear and treat related diseases. Therefore, a series of inner ear diseases related to hearing and balance medicine, including deafness, tinnitus, and vertigo, are worldwide. Difficult diseases, currently there is no practical and effective inner ear drug delivery method except surgery, and surgery has many inconveniences in preventive drug administration and multiple drug use, and the risk is relatively high

Method used

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  • Conjugate capable of penetrating blood-labyrinth barrier and preparation method thereof
  • Conjugate capable of penetrating blood-labyrinth barrier and preparation method thereof
  • Conjugate capable of penetrating blood-labyrinth barrier and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 Building a database

[0073] 1. Based on the sequence analysis of the kunitz region of the active center, the following central skeleton sequence was found, and the construction of the phage-displayed polypeptide library kun-M was designed and completed.

[0074] Build method:

[0075] Library 1: TFYGGRXKRNNF XXXXX (SEQ ID NO: 1)

[0076] X is a random amino acid.

[0077] Long Primer:

[0078] Library1

[0079] 5'-cccaggtgcag ctgcag ACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTTNNKNNKNNKNNKNNK tctaga ggggacccaggtc-3' (SEQ ID NO: 2)

[0080] Said N is A, T, C or G; said K is G or T

[0081] The restriction sites are underlined, and the uppercase letters are the nucleic acid sequences encoding the peptide library.

[0082] Design the first pair of primers NAG-F: GCCCAGGTGCAGCTG (Tm 57.24) (SEQ ID NO: 3)

[0083] The second pair of primers NAG-R: GACCTGGGTCCCCTTAG (Tm 57.29) (SEQ ID NO: 4)

[0084] The primers were synthesized by Primer Synthesis Company.

...

Embodiment 3

[0124] Example 3 In vivo verification in animals

[0125] 1. Peptide synthesis and fluorescent labeling

[0126] Select M1 for in vivo verification in animals, and send the peptide sequence TFYGGRPKRNNFLRGIR (MW: 2053) to Peptide Synthesis Company for synthetic peptide synthesis and labeled fluorescent purification. A polypeptide with a purity of more than 95% is obtained.

[0127] The detection results of unlabeled peptide mass spectrometry are as follows: image 3 shown. The molecular weight of the peptide is 1027.86*2-2=2053.6, which is consistent with the molecular weight.

[0128] The mass spectrometry detection results of the labeled CY5.5 fluorescent peptide are as follows: Figure 4 Show. The fluorescent molecular weight on the CY5.5 marker is 873.22*3-3-2053=564, which is the molecular weight after the reaction of the cy5.5 fluorescent reagent. It shows that the M1 short body is connected with a fluorescent molecule.

[0129] 2. In vivo detection of fluorescent...

Embodiment 4

[0133] Embodiment 4 prepares curcumin-glutaric acid-NHS active ester

[0134]

[0135] Using glutaric acid as a linker, the phenolic hydroxyl group of glutaric anhydride and curcumin (Curcumin) is condensed to form an ester. Figure 8 shown. Then DCC (dicyclohexylcarbodiimide) is used to activate the carboxyl group, and the NHS (N-hydroxysuccinimide) active ester is coupled with the carboxyl group to obtain curcumin-glutaric acid-NHS active ester, which can be directly combined with protein / The lysine residues of the short peptides are linked. Confirmed by mass spectrometry, curcumin-glutaric acid-NHS active ester product was obtained. Theoretical molecular weight: 579, mass spectrum peak 1: 580 (M+H + ), mass spectrum peak 2: 602M+Na + .

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Abstract

The invention relates to a conjugate capable of penetrating a blood-labyrinth barrier and a preparation method thereof. The conjugate is formed by coupling a peptide and an active molecule with inner ear diagnosis or treatment activity, and the peptide has the following structural general formula: TFYGGRX1KRNNFX2X3X4X5X6X7. The conjugate can be administrated through intravenous drip, can carry active molecules to enter the inner ear noninvasively, and breaks through the technical bottleneck that in the prior art, drugs are difficult to enter the inner ear. The invention further specifically provides a peptide-curcumin compound, the inner ear targeting function of the peptide-curcumin compound is verified on the animal experiment level, the peptide-curcumin compound can effectively cross the mouse inner ear labyrinth barrier, nerve deafness is treated, and the auditory protection effect after noise exposure is achieved.

Description

technical field [0001] The invention relates to the technical fields of bioengineering and medicine, in particular to a conjugate capable of passing through the blood labyrinth barrier and a preparation method thereof. Background technique [0002] The anatomical structure of the human ear is mainly divided into the outer ear, the middle ear and the inner ear. The inner ear is a relatively closed and extremely complex structure of the acoustic-electric conversion and balance sensing organ. It is one of the most precise sensory organs in the human body. It is filled with two kinds of lymph fluids that contain different ion concentrations and circulate independently, so as to provide the necessary potential difference for the hair cells to generate action potentials when they undergo acoustic-electric conversion. In order to ensure the normal operation of this mechanism, the material exchange and ion balance between the inner ear lymphatic system and the peripheral blood circu...

Claims

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Application Information

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IPC IPC(8): A61K47/64A61K31/12A61K45/00A61K49/14A61P27/16C07K7/08C07K14/00C07K1/107
CPCA61K47/64A61K31/12A61K45/00A61K49/14A61P27/16C07K7/08C07K14/001
Inventor 林坚钟武时晰王子豪陈龙
Owner PEKING UNIV
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