Method for improving substitution rate and/or substitution efficiency of hyaluronan-drug conjugates
Inventors
LEE, Szu-Yuan • LAI, Ping-Shan • Lin, Chih-An
Assignees
National Chung Hsing University • Holy Stone Healthcare Co Ltd • Aihol Corp
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Publication Number
US-11458204-B2
Publication Date
2022-10-04
Expiration Date
Abstract
Disclosed herein is a method for preparing a hyaluronan-drug conjugate. The method uses the ethyl cyano(hydroxyimino)acetate/diisopropylcarbodiimide coupling system in a homogeneous reaction phase, which unexpectedly improves the substitution rate and substitution efficiency of hyaluronan-drug conjugates for various drugs.
Core Innovation
The invention relates to a method for synthesizing a hyaluronan-drug conjugate by using diisopropylcarbodiimide (DIC) in combination with ethyl cyano(hydroxyimino)acetate as an additive. The method prepares a drug solution in dimethyl sulfoxide (DMSO) and separately prepares an additive solution in DMSO containing ethyl cyano(hydroxyimino)acetate. A hyaluronan solution is prepared by dissolving hyaluronan molecules in water and DMSO.
The drug solution, the additive solution, and the hyaluronan solution are mixed with diisopropylcarbodiimide (DIC) to obtain a reaction mixture. Components in the reaction mixture are then allowed to react to form the hyaluronan-drug conjugate. The synthesis is conducted under a homogeneous water/DMSO reaction system using DMSO as a co-solvent and DIC as the coupling system.
The approach is described as improving HA-drug conjugation substitution rate and substitution efficiency compared with conventional heterogeneous coupling systems such as EDC/NHS and alternatives including DIC/HOBt and PyBOP. The document also describes conjugates characterized by substitution measures such as degree of substitution (DS) and substitution efficiency (DS/reaction equivalent), and illustrates linear relationships between drug equivalents and DS.
Claims Coverage
The document contains one independent claim (clm-00001). It is supported by multiple dependent claims that refine the method by specifying quantitative ranges, hyaluronan parameters, linker type, drug/linker chemistry options, and additional operational constraints.
DMSO drug solution with ethyl cyano(hydroxyimino)acetate additive
A method for synthesizing a hyaluronan-drug conjugate comprising preparing a drug solution by dissolving a plurality of drug molecules in dimethyl sulfoxide (DMSO), and preparing an additive solution by dissolving an additive in DMSO, wherein the additive is ethyl cyano(hydroxyimino)acetate.
HA solution in water and DMSO
Preparing a hyaluronan solution by dissolving a plurality of hyaluronan molecules in water and DMSO.
DIC mixing and reaction to form the conjugate
Mixing the drug solution, the additive solution, the hyaluronan solution, and diisopropylcarbodiimide (DIC) to obtain a reaction mixture, and allowing components in the reaction mixture to react, thereby forming the hyaluronan-drug conjugate.
HA molecular weight constraint
The hyaluronan has a weight-average molecular weight (Mw) of about 5 kDa to 500 kDa.
Degree of substitution range
The conjugate has a drug-molecule degree of substitution of 0.5% to 70%.
Specified drug therapeutic classes
The drug is one of a steroid drug, a nucleoside or nucleotide analog, a non-steroid anti-inflammatory drug (NSAID), a cytotoxic drug, or an immunomodulator.
Dihydrazide linker for the conjugate
The conjugate uses a dihydrazide linker.
Alternative mixing sequence with delayed DIC addition
Preparing a hyaluronan/additive solution, mixing the drug solution with that hyaluronan/additive solution, and adding DIC to the hyaluronan/additive solution to form a reaction mixture.
Overall claim coverage focuses on a DIC-mediated conjugation method using an ethyl cyano(hydroxyimino)acetate additive in a DMSO-based drug solution and a water/DMSO hyaluronan solution, with formation of a hyaluronan-drug conjugate by reacting components in a reaction mixture. Dependent claims further narrow to HA molecular weight and degree of substitution ranges, specify therapeutic drug classes, define a dihydrazide linker option, and include an alternative mixing sequence in which DIC is added after combining the drug solution with a hyaluronan/additive solution.
Stated Advantages
Improves HA-drug conjugation substitution rate and substitution efficiency versus conventional heterogeneous coupling.
Provides substantially higher degree of substitution and substitution efficiency versus comparison coupling systems including EDC/NHS, DIC/HOBt, and PyBOP.
Demonstrates a linear relationship between drug equivalents and degree of substitution (DS).
Documented Applications
Synthesis of hyaluronan-drug conjugates using DIC with ethyl cyano(hydroxyimino)acetate, with representative conjugates including prednisolone hemisuccinate, HA4-nimesulide, β-Ala-lenalidomide, β-Ala-gemcitabine, and β-Ala-estradiol.
Use of the conjugation approach across multiple described drug classes, including steroids, nucleoside/nucleotide analogs, NSAIDs, cytotoxic drugs, and immunomodulators.
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