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Coupling object between CB and biological active peptide or immunoglobhulin or immunological activity original as well as medication usage

A technology of biological activity and conjugates, applied in the direction of non-active ingredient medical preparations, active ingredient-containing medical preparations, pharmaceutical formulations, etc., can solve the problem of unobserved CB-GM1 receptor-mediated intracellular or nervous system Diseases, side effects, lack of targeting, etc.

Inactive Publication Date: 2005-02-16
万选才 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Disadvantages of this technology: First, the systemic menstrual blood administration is not targeted, and it involves the whole brain and spinal cord, and the multifunctional neurotrophic factor is likely to have side effects in this case (such as: someone reported that BDNF may induce epilepsy) and second, antibodies against the transporter block the normal function of the transporter, potentially affecting brain function
All three types have relatively large surgical trauma and cannot be used for long-term or repeated use
But he has never been involved nor observed that CB-GM1 receptors mediate cellular or neurological disorders

Method used

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  • Coupling object between CB and biological active peptide or immunoglobhulin or immunological activity original as well as medication usage
  • Coupling object between CB and biological active peptide or immunoglobhulin or immunological activity original as well as medication usage
  • Coupling object between CB and biological active peptide or immunoglobhulin or immunological activity original as well as medication usage

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0061] Glutaraldehyde GA Conjugation of CB-IgG [Rabbit Anti-Substance P]

[0062] CB [pentamer molecular weight 45-50kDa, List Ltd., US] 3.0 mg dissolved in 1.25% GA [Sigma, used for electron microscope specimens] 1 ml, 0.1 M sodium phosphate (pH6.5) at room temperature overnight [compared Low pH can prevent self-coupling of lysine amino group in CB]; separated activated CB and unlinked glutaraldehyde ( Or pass through the column and dialyze in 0.15M NaCl to remove unlinked glutaraldehyde); use a microporous membrane to concentrate the CB component (molecular weight 45-50kDa) of the column solution to about 1ml.

[0063] Use 1M carbonate buffer (pH9.5) to adjust the pH of the concentrated activated CB to 9.5, add 9.0mg of rabbit anti-substance P IgG (molecular weight 150kDa) dissolved in 1M carbonate buffer (pH9.5) 1ml , reacted with activated CB for 24 hours at a low temperature of 4 degrees; the Sephadex G-200 gel column (1.6 × 90cm) was used to separate the combined state ...

preparation Embodiment 2

[0065] GA coupling of CB-NGF

[0066] CB [molecular weight 45-50kDa, List Ltd., US] 4 mg dissolved in 1.25% GA [Sigma, used for electron microscope specimens] 1 ml, 0.1 M sodium phosphate (pH6.5) at room temperature overnight [lower pH can prevent The amino group of lysine in CB is self-coupling]; the activated CB is separated from unconnected glutaraldehyde (or not passed through the column) by Sephadex G-25 column gel column (40×0.9cm, passed through the column with 0.15M NaCl solution). Dialyze in 0.15M NaCl to remove unlinked glutaraldehyde); use a microporous membrane to concentrate the CB component (molecular weight 45-50kDa) of the column solution to about 1ml.

[0067] Adjust the pH of the concentrated activated CB to 9.5 with 1M carbonate buffer (pH9.5), add NGF (betaNGF, 4mg two chains, molecular weight 26.0kDa, Sigma, N 8133) 2.0mg dissolved in 1M carbonate Buffer (pH9.5) 1ml, react with activated CB for 24 hours at a low temperature of 4 degrees; separate bound (m...

preparation Embodiment 3

[0069] Disulfide Bond S-S Coupling Process of CB-NGF

[0070] Dissolve 2.0mg of NGF (beta NGF, two chains, molecular weight 26.0kDa, Sigma, N 8133) in 1ml, 1M carbonate buffer (pH8.5), slowly add 6.0mg / ml of citraconic anhydride (lemon Anhydride, Sigma, US), placed at room temperature for 1 hour to block free amino groups; G10 gel column was used to separate citric anhydride and NGF peptide. Add 20 mg of EDC to 2.0 mg / 2 ml of NGF to act on the carboxyl group, first adjust the pH to 5, then adjust to 8, and place at room temperature for 10 minutes; pass through a G10 gel column to remove EDC; concentrate the NGF solution to 2 ml; add dropwise 20 mM of PDP solution ( Sigma, US) 0.5ml in NGF solution, stirred at room temperature for 30 minutes; passed through SephadexG-25 (0.1M, PBS elute) gel column to remove excess PDP, and concentrated NGF solution to 2ml by microporous membrane.

[0071] Dissolve 4mg of CB (List, US) in 1ml of 1M carbonate buffer (pH8.5), add 0.5ml of 20mM S...

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Abstract

A choleragen subunit B (CB) used as ligand / carrier, the conjugate of the bioactive polypeptide and immunoglobulin or immunoreactive source, which has the therapeutic action to encephalomyelopathy or cerebrorachidian injury, the compound composition containing said conjugate and medicinal additive, and the preparing process able to keep the bioactivity of said conjugate are disclosed. A delivery system (DS) with nerve receptor mediated endocytosis (RME) is created.

Description

technical field [0001] The invention relates to a conjugate of cholerogen B subunit as ligand / carrier and biologically active polypeptide, immunoglobulin or immune active agent, its medical use and preparation method, and the composition containing it. Background technique [0002] The traditional pharmaceutical drug delivery system is basically the 'blood routed delivery system' (Blood routed delivery system): whether it is the 'gastrointestinal blood circulation' that was first taken orally in history, or various injections (i.m, i.v, etc.) 'into the blood circulation through intramuscular microvessels or directly into blood vessels'. Traditional research on pharmaceutical preparations that cannot penetrate the blood-brain barrier (BBB) ​​also focuses on the 'menstrual blood route', focuses on the BBB itself, and tries to change the preparation to make it easier to pass through the BBB of brain vascular endothelial cells. For example: changing the preparation to make it m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/16A61K38/18A61K39/12A61K39/395A61K47/46A61P25/00A61P25/06A61P25/16A61P25/28
Inventor 万选才李小银
Owner 万选才
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