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Drug Development Preclinical Stage Process

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Whether it is new chemical entities (NCEs) representing small molecule compound drugs or new molecular entities (NMEs) or new biological entities (NBEs) representing biomacromolecule drugs, the basic criteria for evaluating drugs are safe, effective, and controllable. The research and development and production of drugs not only involve a series of internal issues such as complex science, technology, technology, quality standards, etc., but also external control issues such as review standards, procedures, regulations, and ethics. Therefore, drug development is a highly complex system engineering. Compared with small molecule compound drugs, biomacromolecule drugs are more complex in structure, slight changes in production often cause huge differences in drug properties, complex quality control standards, and sophisticated production processes. Therefore, their research has the same common features as small molecule drugs. In addition, there are some unique characteristics.

Many links of new drug development can be outsourced to professional contract research organizations, such as efficacy, toxicological evaluation, pharmacokinetic analysis and clinical trials according to GLP requirements. However, the identification and verification of disease-related targets (also known as targets, target molecules), the design and optimization of biologically active NEEs/NMEs and other core issues need to be solved by themselves, which are also two important bottlenecks in the development of new drugs. The reason for the failure of most drug development is the lack of research on the mechanism of drug action and the inability to make better predictions about the safety of human metabolism.

The discovery and selection of drug targets

Once a potential treatable disease is selected as the research target, it is necessary to identify the target molecule related to the disease, that is, to identify the molecule (usually protein) with special biological activity and potential therapeutic effect. The general process of drug target discovery and confirmation includes: ①Finding biomolecular clues related to disease; ②Performing functional research on related biomolecules to determine the target of drug candidates; ③Targeting drug candidates in molecules, cells and Perform pharmacological studies at the overall animal level to verify the effectiveness of the target. In addition, it is also possible to perform receptor fishing based on compounds that have been confirmed to be active to find their targets.

The remarkable achievements of molecular biology and biotechnology have enabled people to identify a variety of proteins that play a key role in ordinary and special cells, thereby forming a hypothesis of how to regulate the function of specific disease-related proteins. The basis of these hypotheses may be a scientific theory, or it may be information obtained from genetic analysis of specific disease organizations. The process of establishing a hypothesis is often referred to as “target confirmation.” Although the entire human genetic map has been completed, it is not easy to find possible drug targets only from the sequenced genes. At present, people have discovered about 500 drug targets with pharmacological significance. According to the research results of the Human Genomics Project, it is estimated that there are more than 5000 possible drug targets in the human body, and more drug targets are needed. Dig further. The technologies currently used in the discovery of new drug targets include genomics, proteomics, and bioinformatics. Genomics technology includes differential gene expression (DGE), expressed sequence tag (EST) and other technologies. Proteomics technology studies disease states and protein differences in cells or tissues under normal conditions at the protein level, and can discover potential drug target proteins. It is also called chemical genomics as a bridge between proteomics and disease treatment. Regardless of whether it is a target gene or a target protein, the relationship between it and the disease is not clear, but as a potential drug target, it does not affect its affinity selection for small molecule ligands, and its activity detection in disease cells or animal models. Clinical research can further understand the relationship between the target and the disease, realize the functional analysis of the target gene or protein, and reveal the disease mechanism and its treatment mechanism at the molecular level. Gene knockout models are highly valuable in the discovery of new targets for gene function and drug action, and also help to determine the adverse effects of drugs on specific targets. At present, we are systematically knocking out mouse inherent genes and determining their functions in the body according to the physiological characteristics of mammals. This research is sufficient to cover almost all proteins and gene families that can be used for drug research, such as ion channels, nuclear hormone receptors, and proteases. , Phosphodiesterase, kinase, phosphatase and other key enzymes. After the gene knockout mouse model is made, a primary phenotypic screen can be used to determine the target of the drug in the heart, lipid metabolism, immunity, neurology, psychiatry, ophthalmology, orthopedics, reproduction, and oncology.

The target selection process in drug discovery is determined by a complex balance between science, medicine, finance, market, regulations, and strategic thinking. One of the main reasons for drug failure is the insufficient or misunderstanding of the biological function of the drug target in the early stage of development. Therefore, “determining” the target, identifying and confirming its physiology and pathology is an extremely important part of the drug development stage. Generally, two aspects should be considered when choosing a target for a drug: firstly, the effectiveness of the target, that is, the target is indeed related to the disease, and the physiological activity of the target can be adjusted to effectively improve the symptoms of the disease; secondly, the side effects of the target, no matter how much the disease is Related, if the regulation of the physiological activity of the target inevitably produces serious toxic side effects, it is also inappropriate to select it as the target of drug action. In the selection of specific targets, full consideration should be given to multiple factors such as old and new targets, existing drugs and no drugs, single target effect and complex target effect, target effect in the upstream and target effect in the downstream, etc. Influence and pros and cons.

Screening of new molecular entities

Once the biological function of the target is clarified, it is necessary to find a drug (NMEs/NBEs) that accurately binds to the target, and conduct biological effect experiments with the target molecule. Designing and screening NMEs/NBEs is a very difficult task, requiring a combination of knowledge, technology, information, and even art, and sometimes patience and luck. In theory, the computer can not only predict the match between the drug and the target with a known structure, but also tailor the drug for the target from the beginning. The more complex the structure of the target, the more difficult it will be to simulate drug interactions. The most promising NMEs/NBEs will be selected and optimized into a form that meets the characteristics of the drug. These optimizations conform to the form of drug properties. These optimized NMEs/NBEs are called “lead compounds/molecules”. Then it is necessary to conduct pharmacological studies at the molecular, cellular and overall animal levels, observe the efficacy in animal models of human diseases, and conduct safety evaluations. Verify the effectiveness of the target. If everything goes well, it will eventually enter human clinical trials. If NMEs/NBEs are too toxic, even the most promising drug candidates will be eliminated. An important part of the analysis of lead compounds is to use ADMET (referring to drug absorption, distribution, metabolism, excretion and toxicity) to clarify the process of drug action in the body. Early ADMET studies can be carried out using animals, but due to the limitations of animal models, especially recombinant protein drugs used for humans are macromolecules with strong antigenicity for animals, so most results are only available in human clinical trials. Will become clear. Ideally, toxicity studies should be completed at an early stage of development, but for macromolecular drugs, it has been difficult to do so far.

The choice of NBEs as drug candidates has a greater impact on the success rate of drug development. Hormones and therapeutic enzyme preparations mostly have clear functional activities, clear and limited use sites, and are irreplaceable for special disease groups that meet the indications (including orphan drugs for rare diseases), and even some require lifelong dependence. . Although with the in-depth research of functional genomics, proteomics and metabolomics, more and more proteins and diseases will be revealed, but there will be fewer and fewer candidate protein molecules that can be developed as such drugs. Cytokines and growth factors have the characteristics of small amounts of potency, rapid action, multiple short-term use, etc., which can meet clinical needs such as enhancing hematopoiesis and immune function, hemostasis and anticoagulation, balancing endocrine disorders, regulating fertility and growth processes, and have significant clinical effects . However, due to the wide range of biological activities of most cytokines and growth factors in the body and multiple targets, the extension of their pharmaceutical effects can cause complex biological effects and even toxic side effects, which will limit their potential as drugs. Most of the currently known molecules with a single site of action (such as cytokines that stimulate red blood cell production and promote leukocyte proliferation, etc.) have been developed into drugs. It can be expected that the development of such protein drugs will become more and more difficult. However, since the patents of such drugs have basically expired, with the unification and improvement of biosimilar/biosimilar drug regulations, the imitation and modification of such drugs will have greater development potential.

Humans have been discussing the use of naturally occurring non-human proteins to treat human diseases in practice. This group of drugs refers to protein drugs that do not exist in the human body or even if they exist but do not normally perform protein functions, including certain foreign proteins with new functions. Source proteins and endogenous proteins that only function at a specific time or in a specific part of the body. For example, collagenase and hyaluronidase for enzymatic degradation of macromolecules, pegylated asparaginase for enzymatic degradation of small molecular metabolites, streptokinase that degrades plasminogen into plasma enzymes, thrombin inhibits Recombinant hirudin and so on. The future development of such drugs mainly depends on the further understanding of the physiological and pharmacological functions of various new foreign proteins. In addition, due to the high immunogenicity of foreign macromolecular proteins, the development and application of such drugs will be largely restricted.

Monoclonal antibodies and antibody fusion proteins (also known as immunoglobulin-related molecules) can directly interfere with the functions of target molecules or target tissues. They have a wide range of targets and have definite therapeutic effects. They have been involved in tumors and related diseases, autoimmune diseases, and infectious diseases. Diseases, organ transplants, allergic diseases, blood diseases, respiratory diseases, and other disease fields are the group with the largest market share and the most research in the future.

In order to obtain better pharmacokinetics, make up for the defects of certain functional proteins in the body or increase the function of the protein in the human body, it is an effective way to transform or modify the structure of natural proteins. Various modifications mostly start from changing the properties of recombinant proteins, such as increasing molecular weight, slowing down protease degradation, reducing immunogenicity, improving biological and chemical stability, etc., thereby improving its pharmacokinetic properties in vivo, extending half-life in vivo or accelerating in vivo Release, reduce the production rate of neutralizing antibodies, improve patient adaptability and therapeutic effects. In view of the fact that restructured or modified proteins have many advantages over unrestructured or modified proteins, the restructuring and modification of recombinant proteins will surely be more and more widely used.

Production platform of recombinant protein drugs

The production system of small molecule chemicals relies on the supply of standardized chemical intermediates and APIs. As the concept of biotechnology drug raw materials has just emerged, its quality standards and product specifications have not yet been formed. So far, most recombinant protein drugs are produced from scratch by biopharmaceutical companies themselves.

The expression, post-translational modification and secretion of recombinant proteins are highly complex. The number of genes involved in synthesis and whether there is glycosylation can affect the activity of the protein. For example, there are both a single amino acid chain (such as growth hormone) produced by a single gene, and two identical amino acid chains (homodimers) (such as α-galactosidase) formed by “connecting peptides” after translation from a single gene A) Recombinant human protein derivatives. The protein may also be produced from two different genes (such as human insulin, pituitary hormone, thyroid stimulating hormone, luteinizing hormone, and follicle stimulating hormone), and it can also be reproduced by numerous human gene fragments through the process of “somatic recombination”. Row to obtain the hypervariable region of the recombinant therapeutic antibody. Therefore, for a specific recombinant protein, a corresponding specific expression system (for example, bacterial and mammalian cell systems) must be selected as the production platform.

At present, all recombinant proteins approved as drugs are basically produced by the following four production systems: Escherichia coli, Saccharomyces cerevisiae, CHO and BHK cells, but most of the newly approved recombinant protein drugs in the past 5 years use mammalian cells System, where CHO has more applications. Under normal circumstances, a specific therapeutic protein can be produced using two different expression systems. For example, insulin and human growth hormone can be expressed in E. coli or Saccharomyces cerevisiae; IFN-β can be expressed in E. coli and CHO cells. The structure of the IFN-β protein expressed in CHO cells is the same as that of the human natural protein. The IFN-β protein expressed in E. coli has the N-terminal deletion, and a cysteine is replaced by a serine without any glycosylation.

The mammalian cell production system has very high requirements for software and hardware. In the industrialized large-scale expression, separation, purification and storage, the stability, folding, and aggregation of the protein can affect the biological activity of the protein. In addition, high production costs and insufficient yields are also bottlenecks, causing the treatment costs of recombinant protein drugs to far exceed those of ordinary small molecule drugs. Therefore, improving the expression level of large-scale mammalian cells, optimizing the production process and technology, improving product quality and productivity, and reducing production costs will become the key development direction for the production of recombinant protein drugs.

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