Oxaliplatin conjugate as well as preparation method and application thereof

An oxaliplatin and conjugate technology, applied in the field of pharmaceutical preparations, can solve problems such as small volume limitation, and achieve the effects of high drug loading, mild reaction conditions and low cost

Active Publication Date: 2021-03-23
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although single-domain antibodies have great advantages in application, their small size still has certain limitations in their clinical application.

Method used

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  • Oxaliplatin conjugate as well as preparation method and application thereof
  • Oxaliplatin conjugate as well as preparation method and application thereof
  • Oxaliplatin conjugate as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1: Preparation of tetravalent platinum prodrug

[0068] According to the present invention, we specifically provide the preparation steps of the tetravalent oxaliplatin prodrug in the examples.

[0069] Disperse 300mg oxaliplatin (Shandong Boyuan Pharmaceutical Co., Ltd.) in 12mL 30% H 2 o 2 reaction at 75° C. in the dark for 2 h to obtain a light yellow solution, and the solvent was evaporated on a rotary evaporator. The product was then precipitated three times with a small amount of acetone and a large amount of cold diethyl ether. Vacuum drying gave a light yellow hydroxyoxaliplatin complex (compound 1).

[0070] In order to prepare the tetravalent platinum prodrug of maleimide group functionalization, compound 1 and 6-(maleimide group) hexanoic acid succinimide ester were mixed in a certain ratio of substances (1:0.9) Dissolved in 5 mL of anhydrous DMF, stirred at 50 °C in the dark for 16 h to obtain a yellow solution, and evaporated the solvent on a...

Embodiment 2

[0072] Example 2: Transformation of plasmids targeting EGFR-targeting single-domain antibodies

[0073] According to the method provided by the present invention, the single domain antibody used in this example is an anti-EGFR single domain antibody named 7D12. First, using the polymerase chain reaction (PCR), add "-linker 1-C tag-linker 2-Q tag" to the C-terminus of the 7D12 cDNA sequence synthesized by the whole gene (Sangon Bioengineering Co., Ltd.), and named it 7D12 -C 3 Q. Where connector 1 is 3 (G 4S) (glycine-glycine-glycine-glycine-serine), linker 2 is 3 (GS) (glycine-serine), C tag is C-C-C (cysteine-cysteine-cysteine), Q The label is L-L-Q-S (leucine-leucine-glutamine-serine). After the PCR reaction, the target sequence fragments were recovered by agarose gel electrophoresis.

[0074] Secondly, the recovered target sequence fragments were digested with restriction endonucleases NdeI and XhoI. At the same time, the expression vector pET-22b was digested with th...

Embodiment 3

[0080] Example 3: The modified single domain antibody 7D12-C in Example 2 3 Expression and purification of Q

[0081] 1. 7D12-C 3 Q overexpression in E. coli:

[0082] Transfer the correctly sequenced plasmid obtained in Example 2 into the Rosetta-gami prokaryotic expression strain, spread the bacterial solution, pick a single clone, and expand the culture to 200mL-1000mL LB medium (containing 100 μg / mL ampicillin antibiotic) cultivated in. Strains were grown to OD 600 When =0.8-1.0, add isopropylthiogalactoside (0.2-1mM) and induce at 25°C for 10-15h. After the cultivation, the bacterial cells were collected by centrifugation at 4000 rpm for 20 min at 4°C.

[0083] 2. Use Ni-NTA affinity chromatography resin for 7D12-C 3 Q preliminary purification:

[0084] Resuspend the cells (cells obtained in 20 mL buffer / L cell solution) with binding buffer (50 mM Tris-HCl pH 8.0, 200 mM NaCl). The resuspended bacteria were ultrasonically disrupted (20KHz, 15min). The cell lysate...

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Abstract

The invention discloses an oxaliplatin conjugate as well as a preparation method and application thereof. The oxaliplatin conjugate has a structure as shown in the following formula: oxaliplatin prodrug single-domain antibody-polyethylene glycol, wherein the oxaliplatin prodrug is an oxaliplatin tetravalent platinum prodrug, and the polyethylene glycol is polyethylene glycol with the molecular weight of 5-40 kDa. Compared with micromolecular oxaliplatin, the conjugate disclosed by the invention has the advantages of low cost, high drug loading capacity, high tumor targeting and permeability and long-acting blood circulation.

Description

technical field [0001] The invention belongs to the field of pharmaceutical preparations, and relates to a method capable of improving the tumor targeting ability of oxaliplatin and prolonging its blood circulation time in vivo and its application. Background technique [0002] Cancer is currently one of the most serious diseases causing high mortality among patients. Due to various inducing factors such as environmental pollution and bad living habits, the incidence and mortality of cancer are showing a rapid upward trend. Chemotherapy, radiotherapy and surgical resection are the three most commonly used treatment strategies in clinical tumor treatment, among which chemotherapy, whether it is chemotherapy alone or adjuvant chemotherapy, is considered to be the most effective and promising treatment strategy. The most commonly used clinical small-molecule anticancer drugs play a central role in chemotherapy, and currently three platinum-based small-molecule drugs (cisplatin,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K47/68A61K47/60A61K31/555A61P35/00
CPCA61K31/555A61K47/60A61K47/6851A61P35/00
Inventor 李丽刘扬中
Owner UNIV OF SCI & TECH OF CHINA
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