Polyethylene glycol chemical modification is to chemically couple polyethylene glycol polymer chains to protein molecules. It can effectively extend the half-life of the drug in the body by increasing the volume of the drug molecule, and at the same time mask its immune site to significantly reduce the immunogenicity. It is internationally recognized as one of the most effective long-acting protein formulation technologies.
Polyethylene glycol (PEG) is a neutral, non-toxic polymer with unique physical and chemical properties and good biocompatibility. It is also one of the very few synthetic polymers approved by the FDA that can be used as injectable medicine. one. Polyethylene glycol (PEG) is highly hydrophilic, has a large hydrodynamic volume in aqueous solution, and has no immunogenicity. When coupled to drug molecules or drug surfaces, its excellent properties can be imparted to the modified drug molecules, changing their biodistribution behavior and solubility in aqueous solutions, creating a spatial barrier around the modified drugs, and reducing the enzyme activity of the drugs. solution to avoid rapid elimination in kidney metabolism and enable the drug to be recognized by cells of the immune system; the PEG modifications introduced by Zhongpeptide include:
(1) PEG2, PEG4, PEG8, PEG12, PEG24
(2) PEG2000, PEG5000, PEG3
(3) PEG20K, PEG40K
1. The origin of polyethylene glycol modification
How to make proteins from animals, plants and microorganisms into drugs suitable for human use?
In the 1970s, biochemistry professor Frank F. Davis could often be seen in the library of Rutgers University in the United States. There were no computers then, so he had to search through rows of bookshelves. Fortunately, there was already a copy machine, and he took full advantage of this condition and copied a large number of documents. At that time, Professor Davis was preparing to apply for a grant from the National Science Foundation, but needed to find an innovative topic. The topic he was thinking hard about and researching was how to make proteins from animals, plants and microorganisms into drugs suitable for human use. There was no recombinant DNA technology at that time, and animals, plants, and microorganisms became the main sources of protein drugs. However, the direct use of these proteins produced an immune response in the human body. Professor Davis believes that the way to solve this problem is to connect a hydrophilic macromolecule to a protein molecule and use the shielding effect of the hydrophilic macromolecule to avoid or reduce the immune response generated by foreign proteins.
What kind of macromolecules can satisfy this condition? Professor Davis focused his attention on polysaccharide biological macromolecules through extensive literature research. Some polysaccharide molecules not only have good water solubility, but also have good bioaffinity and are not likely to cause immune reactions. However, most polysaccharide molecules have complex structures and too many reaction sites, making it difficult to achieve uniform and controllable reaction products. Moreover, previous research in this area has been carried out in the literature, and further research is not novel and innovative. sex, and the results of previous studies have not solved the fundamental problem.
One day, he was flipping through a medical journal and suddenly found a paper on infusing a polyethylene glycol and polypropylene glycol block copolymer solution into human blood, with the purpose of avoiding fat embolism during organ surgery. This paper is very interesting because polyethylene glycol and polypropylene glycol are chemical products in people’s minds and have little to do with drug treatment. But now doctors are using it for infusion and have achieved preliminary success, indicating the biological safety of this compound. Therefore, Professor Davis immediately focused on polyethylene glycol, a hydrophilic compound, and began new research. As his research deepened, he became more and more interested in polyethylene glycol molecules.
Polyethylene glycol (PEG) is a linear polymer polymerized from ethylene oxide. The abbreviation of the molecular formula is HOCH2(CH2OCH2)nCH2OH
where n represents the number of ethoxy units. PEG is not only soluble in water, but also soluble in some organic solvents, and when other organic solvents are added, it can precipitate out of the original solution.
Unlike polysaccharides, only the terminal hydroxyl groups of PEG can be activated and react with proteins, which can avoid the complexity of reactions and products. However, PEG also has two hydroxyl groups. If it reacts with protein, it may also form “PEG-protein-PEG-protein” conjugates with different degrees of cross-linking, and even precipitation may occur. Is it possible to find a polyethylene glycol with only one terminal hydroxyl group? Professor Davis conducted another investigation. He read through a large amount of literature, but to no avail; he also searched for product catalogs of chemical plants, and finally his eyes lit up: Monomethoxypolyethylene glycol (mPEG) was listed in a factory’s product catalog. The difference between mPEG and ordinary PEG is that one end is a monomethoxy group and the other end is a hydroxyl group. The molecular formula is CH3O(CH2CH2O)nH, n=22～33
By activating this hydroxyl group, introducing an activated group and reacting with the protein, only a PEG-protein conjugate can be formed at the hydroxyl end, but no cross-linked product will be formed. The professor quickly contacted the manufacturer and obtained monomethoxypolyethylene glycol. He rushed back to the laboratory excitedly from the library and led his students to carry out experimental research.
The 1970s was a period of rapid rise for Professor Davis’ research group. They received funding from the National Science Foundation (NSF). The experiment achieved very ideal results, proving that after modification with PEG, the immunogenicity of bovine serum albumin and bovine liver catalase in animals was greatly reduced, and their half-life in the body was extended. Subsequently, more proteins were used by them in PEG modifications all show the results of decreased immunogenicity and increased half-life in vivo. Their continuous academic papers published in the international journal J. Biological Chem. have attracted the attention of the academic community.
In 1977, Professor Davis applied for a U.S. invention patent for the modification of proteins and peptides with polyethylene glycol or polypropylene glycol. The molecular weight range of the polyethylene glycol or polypropylene glycol used was 500 to 20 000. The modified products were injected into animals There is no immunogenicity.
In 1983, Abuchowski, a doctoral graduate of Professor Davis, found investors and established Enzon, the world’s first biotechnology company focusing on polyethylene glycol modification technology. After a bumpy road to industrialization, Enzon has become an internationally renowned polyethylene glycol modification technology company, with more than a dozen drugs on the market or in clinical stages, and total assets reaching US$600 million.
2. Benefits brought by polyethylene glycol modification
Chemical modification of proteins has a long history. Since the beginning of studying protein structure, biochemists have used various chemical methods to modify the amino acid side chain groups of proteins, such as thiol groups, amino groups, imidazolyl groups, guanidine groups, and indole groups, and connected various chemical groups to investigate protein structure and Functional changes. Therefore, when Professor Davis’s research on PEG-modified proteins was published, it did not cause much repercussions in the biochemistry community. Moreover, when the enzyme is modified, the measured catalytic activity decreases or even becomes very large. From this point of view, some people have a negative attitude towards PEG modification. Although modifications can reduce the immunogenicity of proteins, the substantial decrease in biological activity often causes researchers to give up further exploration. However, when people introduced the modified protein into animals and measured its biological activity in the body, they found that the biological activity in the body was unexpectedly high and lasted for a long time. The discovery is exciting for biotechnology companies because they can use this technology to develop long-acting genetically engineered protein drugs. Low in vitro activity but high in vivo activity has become a common phenomenon of PEG-modified proteins, which many people have not thought of before.
Through further research, it was found that the increase in in vivo activity is related to the molecular weight of PEG: an increase in molecular weight can usually increase the half-life (or half-life) of drugs in the body, thus promoting polyethylene glycol manufacturers to synthesize higher molecular weight mPEG, from 10,000 increased to 20,000, 30,000 or even 40,000. Branched PEGs have also been synthesized and have been shown to extend half-life in vivo better than linear PEGs of the same molecular weight. So far, the half-life of polyethylene glycol-modified protein drugs on the market is several times longer than that of the original drug. The drug use has been changed from one injection per day to one per week, which greatly facilitates patients and also improves the efficacy of the drug. effect. For example, PEG-modified interferon is used to treat hepatitis C, and its effect is better than that of unmodified interferon. Therefore, polyethylene glycol modification has evolved from its original intention of overcoming immunogenicity to a major means of extending drug efficacy and improving therapeutic effects.
3. Is polyethylene glycol modification safe?
Are PEG modified drugs safe? People often ask this question. Although this technology has a history of more than 30 years and the drug has been on the market for more than 20 years, like any drug, it cannot be answered with “no problem” from a rigorous and scientific perspective. The side effects of a drug, especially hidden side effects of long-term use, often do not become known until many years have passed. What can be said so far is that there have been no reports of major accidents involving PEG-modified drugs, but people, especially experts in the medical field, are paying attention to and studying the possible side effects of these drugs.
Polyethylene glycol (PEG) has a longer history as a medical excipient. The early human blood gamma globulin freeze-dried powder preparation approved by the US FDA included PEG as one of the formulas. However, the PEG added here is polyethylene glycol with hydroxyl groups at both ends, which is conventional PEG. Regarding conventional PEG, animal safety tests were conducted in the 1940s (Smyth et al., 1947), and its metabolism was discussed (Schaffer et al., 1950). Tests have shown that PEG with a smaller molecular weight, such as a few hundred, has certain nephrotoxicity; when the molecular weight is greater than 1,000, the toxicity is not obvious enough. For example, PEG with a molecular weight of 1,450, 3,350, and 6,000 was administered intravenously to rabbits, with a total dose of up to 10g. /kg, no toxic reactions were found in animals. Experiments on metabolism show that renal elimination is the main way for low molecular weight PEG to be cleared from the body. As the molecular weight increases, fecal elimination is also a way to clear it from the body.
Since monomethoxypolyethylene glycol (mPEG) is very similar to PEG, except that one hydroxyl group at the end has been replaced, people initially deduced from early PEG animal test data that mPEG also had biosafety. There are also articles showing that mPEG-modified interferon with a molecular weight of 40,000 is excreted in the feces through liver metabolism (Modi et al., 2000). However, in recent years, studies have found that mPEG-modified protein drugs can cause the body to produce antibodies against PEG, thereby accelerating the clearance of the drugs. The production of this antibody may be related to the monomethoxy group at its terminal (Armstrong et al., 2007; Sherman et al., 2012). So far, the discovery of PEG antibodies has been limited to reported PEG-modified protein drugs such as urate oxidase, which affects drug efficacy to some extent, but whether it will have a negative impact on the human body requires further research.
4. Prospects of polyethylene glycol modification
Research on drug side effects or potential problems indicates that PEG-modified drug technology and its applications are becoming stable and mature. Today, almost all biopharmaceutical companies have people studying PEG modification technology: extending from PEG modification to other modifications, such as albumin modification, fatty acid modification, polysaccharide modification, etc.
As the most typical and most valued biomodification technology, which has achieved huge economic benefits and plays an outstanding role in the treatment of certain diseases such as hepatitis C, polyethylene glycol modification is still an important tool for us to develop new drugs, remove drug side effects, and improve existing ones. important method of drug efficacy.
Research on the effects or potential problems indicates that PEG-modified drug technology and its applications are becoming stable and mature. Today, almost all biopharmaceutical companies have people studying PEG modification technology: extending from PEG modification to other modifications, such as albumin modification, fatty acid modification, polysaccharide modification, etc.
As the most typical and most valued biomodification technology, which has achieved huge economic benefits and plays an outstanding role in the treatment of certain diseases such as hepatitis C, polyethylene glycol modification is still an important tool for us to develop new drugs, remove drug side effects, and improve existing ones. important method of drug efficacy.
