Small molecules are more and more difficult to do, and macromolecules are developing strongly, which seems to be one of the important performances of drug R & D market in recent years. Many companies, boss and researchers have also started to focus their R & D on macromolecular biopharmaceuticals. But objectively, the development mode of small molecule chemical drugs is obviously different from that of biomacromolecule drugs in many aspects. Rapid "cross-border" is really difficult for researchers. Therefore, the "polypeptide" drugs between small molecules and large molecules can be said to be a good transition turning point. Of course, peptide drugs are not chicken ribs. They have been the focus of life scientists, chemists and pharmacists ever since they were found. At present, more than 80 drugs have been on the market, and some varieties have also developed into blockbusters with annual sales of billions of dollars. Therefore, whether it is the transformation of research and development direction or the transition of technology, the understanding of peptide drugs Digging deeply is one of the tasks that drug researchers must do!

Properties and characteristics of polypeptide drugs

Polypeptides, which can be said to exist widely in biological systems, are signal molecules, transport molecules and digestive molecules. As signal molecules, peptides control biological functions of organisms, such as cell division, mating, chemotaxis, pain, growth and immunity. Peptide synthesis has become one of the most powerful tools in biochemistry, pharmacology, immunology and biophysics. As transport molecules, peptides can promote ions to pass through cell membrane channels;As digestive molecules, peptides play an important role in the nutrient absorption of cells and complete organisms. In addition, peptides can also be used as protective agents, such as antibiotics with excellent antibacterial and antiviral properties. Now, polypeptides are important commercial entities, which have been used in different treatment fields, such as diabetes, allergy, anti infection, obesity, diagnosis, tumor, arthritis and cardiovascular diseases.

Because peptide drugs mainly come from endogenous polypeptides or other natural polypeptides, in general, their structure is clear and their mechanism of action is clear. Many properties are often between small molecule chemicals and large molecule protein / antibody drugs. For example, compared with small molecule chemicals, the half-life is generally short, unstable, easy to be rapidly degraded in vivo, unstable preparations must be preserved at low temperature Compared with macromolecular protein, it has better stability, less dosage, higher unit activity and lower cost, and the chemical synthesis technology of polypeptide is mature, easy to separate with impurities or by-products and high purity.

The above shortcomings, such as stability problems, some polypeptides can be modified or composed of stable compounds with other materials; cost problems, with the progress of science and technology, equipment update and process improvement, the cost can be better controlled. The biggest problem of polypeptide drugs is that they can not be taken orally, mainly because they are easy to be degraded and difficult to pass through the intestinal mucosa. However, there are various alternative routes of drug delivery, such as subcutaneous injection, nasal spray, etc.

Table 1: simple comparison of polypeptide, traditional medicine and protein medicine

Classification of polypeptidesAccording to the source classification, polypeptides can be divided into bioactive polypeptides and synthetic polypeptides; according to the size classification, polypeptides can be divided into small peptide, medium peptide and large peptide. Small peptide refers to polypeptides with the number of amino acid residues less than 15, medium peptide refers to polypeptides with the number of amino acid residues between 15 and 50, and polypeptides with the number of amino acid residues greater than 50 are called large peptides; according to the structure classification, polypeptides can be divided into the same There are two kinds of polypeptides: homopolypeptides including straight chain peptide and circular peptide, heteropolypeptides including pigment peptide, glycopeptide, lipopeptide and lipopeptide; according to functional classification, polypeptides can be divided into exercise protein, hormone protein, toxin protein, polypeptide hormone, enzyme protein, polypeptide antibiotic and toxin peptide, etc.

Peptide R & D - representative events

In the early 20th century, Fischer synthesized peptides with specific sequences for the first time

In 1932, Bergmann et al. Used benzyloxycarbonyl to protect α - amino group, and peptide synthesis has developed to a certain extent.

In 1953, Vigneaud successfully synthesized the first peptide drug oxytocin

In 1963, Merrifield successfully synthesized peptide sequence with resin as solid-phase carrier, and established solid-phase peptide synthesis

In the late 1960s, Merrifield invented the first automatic peptide synthesizer and synthesized biological protease (124aa) for the first time

In the 1970s, the successive discovery of enkephalin and other opioid peptides in the brain made the research of neuropeptide enter a climax

In 1972, Lou carpino first used Fmoc to protect α - amino group, and it was quickly widely used

In 1984, Merrifield won the Nobel Prize in Chemistry for inventing solid-phase peptide synthesis technology

Research and development of peptide drugs

At present, more than 80 kinds of peptide drugs have been approved for marketing in the world, and the number of drugs is mainly distributed in tumor, diabetes, infection, immunity, cardiovascular, urinary and so on.

At first, before the 1970s, less than one peptide drug entered the clinical trials every year on average; in the 1970s, only one peptide drug entered the clinical trials every year on average; in the 1980s and 1990s, the average number of peptide drugs entering the clinical trials was about 5 and 10 respectively; in the first 10 years of the 21st century, the number of peptide drugs entering the clinical trials increased, about 17 on average every year.

Peptide drug market

According to the transparency market research market report, the global peptide drug market scale in 2015 is nearly 20 billion US dollars, and will reach 23.7 billion US dollars in 2020, with a compound growth rate of about 2.8% from 2014 to 2020. Peptide drugs are mainly used in the treatment of cancer, metabolic disorders related major diseases, which have a very important global market. At present, there are more than 80 kinds of peptide drugs in the global drug market, 200-300 kinds of peptide drugs are in clinical trials, 500-600 kinds are in pre clinical trials, and more peptide drugs are in the laboratory research stage. It can be predicted that more and more peptide drugs will be approved by FDA to enter the drug market in the future. Some existing peptide drugs have a considerable market. In terms of drug varieties, in 2016, glatirine acetate was still the top selling polypeptide drug, with sales of more than 4 billion US dollars for consecutive years, followed by lilalutide and growth hormone, with global sales of more than 2 billion US dollars, followed by octreotide, leuprorelin, tripatide, exenatide, goserelin, etc.

Methods for obtaining polypeptide drugs (the following are mainly technical aspects)

There are three main sources: natural extraction, biology and chemistry.

1. Extraction of active components from natural products

2. There are active components in some natural products, which can be extracted from simple mixtures by solvent extraction. If the components of natural products are complex and the content of active components is very small, it is necessary to use enzymatic hydrolysis or microwave-assisted digestion to degrade the protein precursor. The active segments can be separated and detected by detection equipment, which can be quickly and effectively found in mixtures Active peptide.

3. Biosynthesis (gene recombination technology)

Gene recombination technology is to construct the gene sequence of polypeptide on the vector to form recombinant DNA expression vector, and to express, extract and purify polypeptide molecules in prokaryotic or eukaryotic cells. This method is suitable for the preparation of target peptides with more than 50 amino acids and is easy to obtain. With the improvement of the technology of peptide production by genetic engineering, the development and clinical application of peptide drugs by genetic engineering have been accelerated.

3. Chemical synthesis (including liquid-phase and solid-phase methods)

Liquid phase method is the first developed peptide synthesis method: the process is to prepare the required amino acid or short peptide into a solution, in which the amino end of one amino acid and the carboxyl end of another amino acid or short peptide and the side chain group not involved in the reaction are protected by chemical groups, while the carboxyl end of the amino acid or short peptide involved in the reaction needs to be activated, and then coupling occurs in the solution 。 After the reaction, the raw materials and activators which were not involved in the reaction were separated and removed by various methods to obtain the purified peptide products. The synthesis of polypeptide in liquid phase is mainly divided into natural chemical connection and stoodinger connection.Natural chemical linkage is the basic method of peptide segmented synthesis, and its limitation is that the synthesized peptide must contain Cys residues, which limits the application scope of natural chemical linkage method. The extension of natural chemical linkage method includes chemical region selective linkage, separable auxiliary base linkage and light sensitive auxiliary base linkage. Stoodinger linkage method is another basic chip The method of segment connection opens up a wider way for peptide segment connection. The orthogonal chemical connection method is the extension of the stoodinger connection method, which can improve the condensation rate between segments by simplifying the phosphine thioester auxiliary group.

SPPs (very important! ）The principle is to fix the carboxyl group of the first amino acid at the C-terminal of the peptide sequence on the insoluble carrier (resin + linker) through esterification, then take the amino acid as the amino component, remove the amino protection group and react with the excessive activated carboxyl group to form the peptide bond, and then repeat deprotection, condensation and washing The operation is carried out to extend the length of the peptide chain so as to obtain the required length of the peptide chain. Finally, the peptide chain is cut off from the resin by acid (such as trifluoroacetic acid) and all protective groups are removed at the same time. After purification by high performance liquid phase, the required peptide can be obtained. After years of research and development, it not only overcomes the time-consuming and cumbersome of liquid-phase synthesis, but also reduces the loss caused by operation. The biggest advantage is that all purification steps in synthesis are completed by simple washing and filtration, greatly reducing the difficulty of purification,It has the advantages of convenient and quick operation and high yield. Its advantages are easy to realize automation, making it the preferred method of peptide chemical synthesis.
Peptide synthesizer

The first real peptide synthesizer in the world appeared from the late 1960s to the early 1970s. It uses nitrogen bubbles to stir the reactants, and uses computer program control to realize limited automatic synthesis. It represents Beckman 990 peptide synthesizer launched by Beckman company and Vega's 296 peptide synthesizer launched by Vega's biotechnology company The second generation peptide synthesizer was born in the 1980s, representing PS3 peptide synthesizer launched by protein technologies and act peptide synthesizer model 90 launched by advanced Chemtech; the third generation peptide synthesizer was born in the 1990s, representing abi433 peptide synthesizer and CS bio of Applied Biosystems in the United States Cs336 non dead angle peptide synthesizer.

Figure 4: representative products of generation 3 peptide synthesizer

Quality control of polypeptide drugs

The quality control of peptide drugs comes from the synthesis of peptide drugs. The quality research can refer to the general research ideas of chemical drugs. Quality control mainly includes properties, identification and inspection, including general impurities, related substances, residual solvents, crystal form, particle size, clarity and color of solution, loss on drying and water, isomers, in addition, there are special inspection items for peptide drugs, such as amino acid ratio, biological activity inspection, related peptide, anti ion content, peptide diagram analysis, polymers, antihypertensive substances Inspection and other items, content and titer determination. For the synthesized peptides, the physical and chemical properties need to pay attention to the isoelectric point, specific rotation and the solubility of buffer besides the general conventional items.
Because peptide drugs are easy to be enzymolysis, short half-life and poor fat solubility, they are mostly administered by injection. The main dosage forms are freeze-dried powder injection and injection. For general injection and freeze-dried powder injection, due to the unstable and easy degradation of polypeptide substances, the quality control project focuses on the inspection of related substances. The related substances for peptide synthesis can be divided into four categories, mainly including:

1. Process impurities, such as missing peptide, breaking peptide, inserted peptide, unprotected peptide and other peptide related substances brought in during synthesis;

2. Degradation products produced by unstable factors such as deamidation of degradation impurity ~ peptide, oxidation, reduction, hydrolysis, disulfide bond mismatch, β - elimination, etc;

3. Polymer dimer and polymer;

4. Optical impurity ~ racemized, non enantiomeric impurity.

Peptide drugs are mainly injection and injection powder, which are easy to degrade and produce degradation impurities in the process of production and storage. The main detection methods of polypeptide drugs are: reversed phase high performance liquid chromatography, capillary electrophoresis, high performance liquid chromatography-mass spectrometry, high performance molecular exclusion chromatography.

reference:

1. R Ismail，I Csóka.Novel strategies in the oral delivery of antidiabetic peptide drugs-insulin，GLP1 and its analogs[J]. European Journal of Pharmaceutics & Biopharmaceutics, 2017.

2. S Eskandari，T Guerin，I Tot，et al.Recent advances in self-assembled peptides: Implications for targeted drug delivery and vaccine engineering[J].Advanced Drug Delivery Reviews，2016.

3. S Shi，PKNguyen，HJ Cabral et al. Development of peptide inhibitors of HIV transmission[J].Bioactive Materials，2016，1 (2) :109-121

4. 4. Qu Peng, Song Li, Zhao Haodong, et al. Research progress in peptide synthesis [J]. Modern Chinese medicine, 2015

5. Tian Wenjing, Ren Xue, Liao Haiming, et al. Research progress of peptide drug quality control [J]. Journal of drug analysis, 2013

6. Nie Caihui, Xu Hanmei. Development status of polypeptide drugs [J]. Pharmaceutical progress, 2014

7. Wang kequan, Xu Hanmei. Research progress of polypeptide drugs [J]. Pharmaceutical progress, 2015

8. Part of the data comes from Thomson Reuters

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Chengdu Shengnuo Biotechnology Co., Ltd. has "Chengdu polypeptide drug engineering technology research center" in Chengdu, mainly engaged in polypeptide, polypeptide drug and beauty peptide research. Our zero defect has passed the FDA certification, and now it has become the first-class professional peptide drug and product development, technology transfer, technical service and peptide drug industry in the scale production and export of China's parks.