The Development of Mesenchymal Stem Cell Drugs

Clinical research in the field of stem cells is advancing steadily at a rapid pace, which is the general trend. Understanding the key elements of stem cell drug development can accelerate its industrialization.

Stem cell therapy refers to the use of stem cells or their derived cells, transplanted into the body with special techniques, to replace damaged cells of the patient or to generate new tissues by recruiting endogenous tissue-specific stem cells, or to play a positive role in immune regulation . However, after stem cells enter the body, we need to know where they go and what they do?

Stem cell drugs challenge the basic ideology of traditional medical technology and drug development, but we still have the ability to start with ethics, safety, effectiveness, quality controllability, and technical and economic feasibility. Screening, observing, manipulating, simulating and controlling stem cells in vitro before they are implanted into recipients is expected to break through the technical bottleneck restricting the development of stem cell drugs.

The starting point for the development of stem cell preparations

Although many laboratories have published their own clinical research results in the field of stem cells, in fact, most of the research results published in top journals are difficult to reproduce. We exclude objective factors such as reagents, equipment, and operations. Stem cells alone are the subject of research, and the influence of differences in their sources on the judgment of research results cannot be ignored.

1. Stem cell donors have complex genetic backgrounds

The family history, past medical history, gender, age, race, blood type, histocompatibility antigen (HLA), physical condition, living habits, and living environment of the stem cell donor are dynamically affected by various physical, chemical, and biological factors. There are no two lines of stem cells with the same genetic background in the world.

2. The biological characteristics of stem cells from different tissue sources are different

More than 260 kinds of tissues in the human body are differentiated from stem cells. Stem cells are everywhere, and wherever there are cell activities, there are stem cells. Stem cells are very rich in tissue sources, including embryos, adult tissues (bone marrow, dental pulp, hair follicles, epithelium, muscle, blood vessels, fat, nerves, etc.), perinatal tissues (umbilical cord, amniotic membrane, placenta, cord blood, etc.) stem cell. However, the biological characteristics of the same cell from different tissue sources are heterogeneous.

3. Stem cells are dynamic living cells

Currently, drugs including antibiotics, small chemical molecules, and biological macromolecules are relatively stable systems with clear structures and components. Stem cells are living cells with heterogeneity, with certain differences in size and shape, as well as in functions, behaviors, and states. The same kind of stem cells can play different roles in different microenvironments.

The selection of stem cell types (tissue sources) for clinical use may consider the following aspects:

1) No ethical issues;

2) Have a stable source of organization;

3) Have clear screening indicators and theoretical basis;

4) Meet the requirements for large-scale preparation;

5) Controllability of in vitro process;

6) Safety is within acceptable range.

Compared with stem cells from other sources, perinatal tissue-derived stem cells are 0-year-old cells, which are healthier, more vigorous, and have low immunogenicity. They belong to the reuse of medical waste tissues, have no ethical barriers, are easy to standardize and prepare, and are well resolved. Tissue sources for stem cell drug development.

• The placental stem cell drug developed by an Israeli institution has been approved by EMA and FDA to carry out human clinical trials, and has entered the phase III clinical stage.
• Several R&D institutions in my country have developed umbilical cord mesenchymal stem cell products, established quality standards according to the new drug research norms and the characteristics of stem cells, and obtained complete and standardized preclinical pharmacy, safety and effectiveness evaluation data, and have obtained drug regulatory approval. FDA implied permission to enter the clinical trial stage.

Effective evaluation techniques are key

1. Quality by design (QbD) of stem cell drugs:The quality of stem cell products depends on the choice of process route and product form

Aiming at the outstanding contradiction between the characteristics of stem cells( "heterogeneity", "dynamics" )and the requirements of clinical products("uniformity", "stability"), by means of "standardization, automation, and scale", pre-defined Systematic development of good quality objectives and process control points.For example, the process design of cell products needs to consider: mechanical or enzymatic cell separation, open operation or closed workstation, manual passage or continuous automatic expansion, 2D or 3D culture, low seeding rate, high generation and low limit For the passage, consider heterologous serum or animal-free medium, whether the preparation is in the form of free cells or combined with a matrix, whether the product is fresh cells or frozen products, whether DMSO is used for protection or a new cryopreservation agent, etc.In short, the development of new drugs needs to consider the technology replacement and competition in the next 30 years. The whole new drug development process has the characteristics of risk conservation, and the risk of failure and repetition will bring the overall cost of capital and time. It was pointed out that the harm will be magnified by 20 times for each step of the risk .

2. Quality controllability is the foundation and premise:

Stem cells come from various sources, and the donors have complex genetic backgrounds. The preparation process of stem cells is long ( in vitro separation, purification, expansion, induction, etc. ) , and it is necessary to introduce a variety of foreign components ( antibiotics, growth factors, antibodies, collagenase, protease, animal serum, etc. ) ; there may be contaminated foreign factors ( Bacteria, mycoplasma, viruses, etc. ) ; in vitro operations may cause risks (due to the proliferation potential, differentiation potential, biological efficacy of stem cells, histocompatibility antigens, telomerase, karyotype, proto-cancer tumor suppressor genes, etc.) brought about by changes ); in other processes ( preservation, transportation, resuscitation, preparation ) , the survival rate and biological efficacy of cell products may change.

In the absence of effective quality control methods and preparation standards for cell products, the safety and effectiveness of stem cell clinical applications cannot be achieved.

3. Lack of effectiveness evaluation indicators for stem cell preparations:

Currently, live cell counting is generally used clinically to determine the number of infused stem cells. Stem cell therapy seems to have a "magic" effect on individual patients, but it is ineffective in more patients.

In fact, the in vitro manipulation of stem cells is a dynamic process, and the number of living cells cannot effectively reflect changes in the physiological status of stem cells. Some people even infused sacrificed stem cells into animals and obtained experimental results comparable to infusion of living cells. How is the effectiveness of stem cells related to their differentiation potential, doubling time, secreted factors, number of passages, homing properties, etc.? Here it is necessary to introduce a quantifiable unit of definition of stem cells—biological potency .

Stem Cell Biological Potency

Biological potency, or "potency", is an important indicator for evaluating the effectiveness of biopharmaceuticals, and is used to measure the concentration or quantity of drugs that produce specific physiological functions; the physical quantity ( quality)of biopharmaceuticalsoften cannot directly represent its biological Efficacy, such as protein inactivation after the content remains unchanged and the efficacy decreases, generally need to redefine the potency unit as the gold standard to measure its biological efficacy.

For example: Antibiotic potency units are usually measured by the highest dilution that inhibits microbial growth by half of the antibacterial test, and the highest dilution that can inhibit 50% cytopathic or 50% viral plaque formation effects is measured by the cytopathic inhibition method (CPE). Measures units of interferon (IFN) activity.

(1) Biological potency requirements for stem cells

a. To set the efficacy index, it is necessary to find out the key mechanism pathways related to the clinical treatment effect, which can truly reflect the dose-effect relationship, and it is necessary to consider that stem cells are multi-target and multi-pathway;

b. The efficacy index setting needs to be able to represent the consistency of the cell product in the preparation process and clinical use;

c. The efficacy index setting should be optimized. It is best to use in vitro tests instead of animal models. It is required to be easy to operate and can become the product release standard;

d. The effectiveness index setting should be verified by analytical methodology: linearity, range, accuracy, precision (including repeatability and reproducibility) , detection limit/quantitative limit and durability, etc.;

Cell surface markers can be used to identify the type of stem cells and evaluate the purity of stem cells, but they cannot reflect the biological functions of the cells, and cannot even distinguish whether the cells are dead or alive. Although the differentiation induction test is relatively direct, it is difficult to quantify, and the cycle is long and the variability is large. It is difficult to directly measure the expression of miRNA and factors to reflect the situation in vivo.

(2) Development of stem cell biological efficacy assay method

a. Consider placing stem cells in an "activated" state, rather than under simple in vitro culture or storage conditions, such as simulating a tissue injury microenvironment;

b. Observe the difference in stem cell behavior before and after "activation", and study the relationship between these differences and stem cell differentiation, anti-inflammation, and anti-apoptosis potential;

c. Establish a stimulation system for evaluating biological activity, and screen key factors related to the treatment of target diseases;

d. Finally, develop a stem cell biological efficacy assay method and establish a release standard.

Animal Model Establishment Principles

Experimental animal models plague preclinical safety and effectiveness evaluation of stem cells: experimental animals are often used to evaluate the in vivo distribution, metabolism, effectiveness, acute toxicity, special toxicity, long-term toxicity, etc. of new drugs, providing basic data for human clinical observation of new drugs.

General principles for animal model establishment: a. Animal model construction should be as close as possible to human disease conditions; b. Animal model should be reproducible and can be standardized; c. Animal model construction should specifically and reliably reflect Disease function, metabolism, structural changes and main symptoms and signs; d. The construction of animal models should take into account the clinical application and the development of the disease; e. The methods used in the construction of animal models should conform to the principles of feasibility and economy.

At present, whether animal models can provide an effective reference for clinical research is still questionable. The reasons are as follows: a. Stem cells can proliferate, differentiate, and be rejected, which does not conform to the general law of drug metabolism; b. Stem cells are species-specific, and it is difficult to explain the problem with heterogeneous stem cell implantation. Inconsistent experiments; c. Stem cell tissue matching (incompatible, haploidentical, and compatible) tests are difficult to achieve; d. Live cell tracking tests may cause changes in stem cell biological characteristics, and difficulties in colonization, differentiation, outcome, and tracking; e . What is the law of tissue homing and pathological homing after stem cells enter the body? How is it colonized and differentiated? f. The animal life cycle is short enough to reflect the consequences of long-term engraftment of human stem cells.

Risk control plan is the guarantee of success

Stem cell drug development is very different from molecular drugs with clear chemical structures ( chemical drugs, antibiotics, protein drugs, etc. ) , which challenges some basic concepts and laws of traditional drug development. Human understanding of stem cells is still at a relatively early stage .

Mechanism

The mechanism of action of stem cells is complex and not very clear. For example: Do stem cells directly colonize and differentiate to participate in tissue repair, or do they play a regulating role in the body by secreting cytokines to change the microenvironment? Does the host's endogenous stem cells activate for tissue repair, or does it interact with host cells to change the state of the body's cells? Are the characteristics of stem cells cultured in vitro consistent with their biological behavior in vivo? Can the results of animal experiments be used to predict the role of stem cells in the human body? What is the difference between autologous stem cells and allogeneic stem cell implantation?

In vivo fate after transplantation

Taking MSCs as an example, what are the rules of tissue homing and pathological homing after stem cells enter the body, how do they colonize, differentiate, or are they eliminated by immunity? If stem cells "homing" in non-target organs and target tissues in the body, "differentiate" unintentionally, produce unwanted biological effects, and cause persistent adverse consequences, how to stop?

At present, it is believed that MSCs cannot survive in the body for a long time, and clinical trials have shown that they perform well in terms of safety.

Uncertain validity

What is the relationship between the curative effect of stem cell therapy/transplantation and the patient's disease type, disease course, physical condition, tissue type, age, race, gender, habit, experience, and the number of transplanted cells or the route of infusion? How to determine the dose-response relationship?

Adverse reactions

The most reported adverse reactions after the use of cells are a series of fever and chills, most of which do not require special treatment; there are also a few people who have symptoms such as lethargy, excitement, nausea, and vomiting; very few patients may have rashes and other allergies react . GVHD will appear after hematopoietic stem cell transplantation, and the application of MSCs can treat GVHD. Cysts may appear in nerve cells differentiated from iPSCs or ESCs, and it has also been reported that the use of cell therapy derived from aborted embryos has led to tumor formation.

Indications and Population Selection Recommendations

Suggestions for indications and population selection, including: a. Those who have no effective means to control the disease process; b. Those who have a theoretical basis to break through the limitations of existing means; c. The possible benefits of stem cell therapy obviously outweigh the potential risks; d. Cancer patients or High-risk groups of tumors; e. Whether the HLA type is clear (identical, haploidentical) ; f. Special groups such as pregnant women; g. Whether long-term follow-up can be achieved.

Summary at the end of the article

Stem cell technology has rekindled hope for patients whose traditional medical methods are ineffective, but stem cell therapy is like opening a "Pandora's box", and people are full of curiosity, expectation and fear. Fear stems from the uncertainty of the outcome. Only when we fully estimate and prepare for the worst situation, and control the risks within an acceptable range, can people fully enjoy the benefits brought by new technologies. It must be admitted that in some special cases, the clinical application of stem cell technology may be completed before the development of new drugs. This is the so-called "new medical technology". There are many such examples in the history of medicine.

How to cooperate with the scientific community and regulatory agencies to supervise the standardization of stem cell therapy is a question worthy of serious consideration . We must be soberly aware that there is still a long process of development from stem cell research to clinical application.

Faced with the unlimited potential of stem cell research and application prospects, the scientific community, clinical workers, industry circles, and the majority of subjects all need to maintain a realistic attitude, patience and understanding, as well as their respective responsibilities and mutual cooperation to achieve To ensure that scientific research and clinical application can be carried out in the most favorable environment.

The generation selection of stem cell reinfusion

Looking back at the history of human development over the past few hundred years, the 19th century was the century of the industrial revolution represented by steam engines, the 20th century was the age of information technology represented by computers and communications, and the 21st century will be the age of life sciences represented by biotechnology. century with technology.

Stem cell technology is one of the core contents of biotechnology. Regenerative medicine or tissue regeneration treatment based on stem cells is another revolution in medical technology after drug treatment and surgical treatment. Stem cell treatment has more important significance in the recovery stage of the disease. With the continuous exploration of scientific researchers, there is new hope in the treatment of refractory diseases such as diabetes, heart disease, and Alzheimer's disease.

When using stem cells to treat diseases, attention should be paid to various issues such as treatment timing, route of administration, cell type selection, and quantity used. Only by finding the best feasible solution among multiple factors can we help this technology to be implemented faster.

When applying stem cell therapy clinically, it is often easy to ask which generation of stem cell therapy is more appropriate. These are questions that should be thought about and yet to be solved. We all know that people are divided into men, women, old and young, and stem cells are also divided into young and old. Therefore, the generation of cells is an important factor affecting the activity, clinical effectiveness and safety of stem cells.

The main methods of cell culture are primary and subculture. Theoretically, the higher the cell generation, the stronger the stemness of the stem cells. However, during the process of stem cell culture in vitro, stem cells undergo an adjustment and adaptation process from the tissue microenvironment to the in vitro culture environment. Some cells that do not adapt will be eliminated. Therefore, in the first two or three generations, the stem cell genome has unstable factors, which may Not suitable for clinical application.

Studies have shown that through karyotype analysis, stem cells have a higher proportion of abnormal karyotypes in the first three generations of cells. For mesenchymal stem cells (MSCs) , the optimal number of passages for clinical application should be 4 to 6 passages.

In current clinical research, the most common sources of MSC are bone marrow, fat, umbilical cord, placenta, etc. This article summarizes the three common sources of umbilical cord, fat, and placenta. For mesenchymal stem cells from different sources, the differences after in vitro subculture are different. Changes in some biological characteristics of each generation ( such as cell morphology, cell cycle detection, surface marker detection, karyotype analysis, related gene expression and quantitative analysis of cytokines), due to limited reference materials, there are shortcomings Please understand.

umbilical cord

Among various types of mesenchymal stem cells from different sources, human umbilical cord mesenchymal stem cells (hUC-MSCs), as the "younger" adult mesenchymal stem cells, have prenatal and postnatal capabilities. The comprehensive advantages of MSC have attracted more and more widespread attention.

After long-term in vitro subculture of human umbilical cord mesenchymal stem cells, their basic stem cell biological properties, such as cell surface immune markers and induction of multilineage differentiation potential, did not change significantly. After long-term culture up to 20 generations, no pathogenic or tumorigenic gene mutations were found. There was no significant difference in cell proliferation activity and telomerase activity between high passage (P20) hUC-MSCs and early low passage (P5) cells. As the number of culture generations increases, the cells begin to age, especially in the 20th generation cells.

After in vitro subculture, hUC-MSCs showed exactly the same cell morphology and growth arrangement at the 5th and 10th passage, showing long spindle-like adherent growth and mild spiral flow-like arrangement. The 20th generation cells showed very similar long spindle-like adherent growth, but the cell body was slightly enlarged and the local arrangement was disordered after fusion.

Studies have shown that although the karyotype of hUC-MSCs cultured at high passages remains stable, their telomerase activity will decrease by the 7th passage. We detected the expression of the human telomerase reverse transcriptase activity subunit gene and showed that there was no significant difference between the telomerase activities of hUC-MSCs at passages 5, 10 and 20; and cells of different passages showed similar growth. The proliferation curves showed that there was no difference in their cell proliferation activity.

Detection of proliferation activity of human umbilical cord mesenchymal stem cells of different generations

hUC-MSCs of different passages ( 5th, 10th, and 20th passage) were seeded in 6-well culture plates at the same density, and hUC-MSCs of different passages were collected from 3 wells every day to count and draw the growth curve. The illustration represents three replicate experiments.

In addition, due to the strong professionalism and limited space, similar procedures (conventional karyotype analysis, chromosomal karyotype analysis, surface marker detection, gene expression and cytokine quantitative analysis) will not be described again. In short, although hUC-MSCs at the 20th passage still maintain stem cell biological properties, gene stability and growth characteristics similar to those of lower-passage cells, as the passage and culture time increases, the cells begin to show a certain degree of aging. In view of the fact that excessive subculture will lead to signs of aging in hUC-MSCs, it is recommended that the in vitro culture of hUC-MSCs used for stem cell clinical treatment should not exceed the 10th passage (most laboratories can basically achieve this within 3 passages now, of course. Maybe it's just a mouthful) .

placenta

Mesenchymal stem cells are rich in sources, and placental mesenchymal stem cells also have the advantage of being easy to obtain. The full-term placenta is a waste product after fetal delivery. It is easy to obtain and large in volume. The cost of scientific research is low and does not involve social and ethical issues. Moreover, placental mesenchymal stem cells have functions and characteristics similar to bone marrow mesenchymal stem cells, and have a better proliferation ability than bone marrow mesenchymal stem cells, and can greatly expand the number of mesenchymal stem cells.

Some studies have tested the proliferation ability of P5, P10 and P15 generation cells respectively. The results show that the proliferation ability of P5 generation mesenchymal stem cells is significantly higher than that of P10 and P15 generation. The experimental results show that when placental mesenchymal stem cells are cultured in vitro, , as the number of cell generations increases, cells will gradually age. Since the proliferation ability of placental stem mesenchymal cells will gradually decrease after the P5 generation, it is best to choose mesenchymal stem cells below the P5 generation for in vitro culture for clinical application.

Legend: A in the picture shows that the cells have a uniform shape after adhesion, taking on a long spindle shape and growing parallel or swirling (×100); B shows that the morphology of the cells in the 25th generation changes like paving stones (40×4).

Growth curve, proliferation ability and passage results of placental mesenchymal stem cells

Note: Figure A shows that the doubling time of P5 generation cells is significantly shorter than that of P10 and P15 generation cells; Figure B shows that the proliferation abilities of placental mesenchymal stem cells of different generations are different, and the proliferation ability of P5 generation is significantly higher than that of P10 cells. and P15 generation, compared with P5 generation, aP < 0.01, bP < 0.001, compared with P10 generation, c < 0.05; C is placental mesenchymal stem cell specimens from 5 cases passed to 25th generation, with no adherent cells or weak adhesion. , but the proliferation rate is fast.

In this study, placental mesenchymal stem cells can be passaged for a long time to the 25th generation. Placental mesenchymal stem cells of different generations have different proliferation abilities. The proliferation ability of the P5 generation is significantly higher than that of the P10 and P15 generations, revealing that clinical application should be based on Preferable before P5 generation.

Fat

Adipose derived mesenchymal stem cells (ADSCs) are a type of stem cells that exist in adipose tissue and have multi-lineage differentiation potential. Cell sources are more abundant and materials are more convenient to obtain. Moreover, its immunogenicity is very low and it is easier to amplify in vitro. These advantages make it have better application prospects in tissue engineering, wound repair and gene therapy.

Studies have cultured ADSCs in vitro to passages 1, 3, 5, 7, 10, 14, and 15. It was found that after passage of ADSCs to the 7th passage, the cell proliferation rate began to slow down, and the cell proliferation rate slowed down significantly after the 10th passage. The cell morphology is long spindle-shaped and remains stable.

There are no obvious changes in the karyotype of chromosomes such as translocations and deletions, the expression levels of related genes are slightly different, and the cytokines secreted in the culture medium remain stable.

Studies have shown that during the passage of adipose mesenchymal stem cells (cultured in serum-free medium) to 5 generations, the biological and genetic characteristics are basically stable without significant changes. It has been initially revealed that adipose mesenchymal stem cells can be safely used up to the 5th passage in vitro.

From the above studies, it is easy to see that mesenchymal stem cells can theoretically be cultured for a long time, but it is not recommended to select batches of stem cells with higher passages in clinical applications. MSC cells that have not been expanded in vitro are safe and harmless unless they have some genetic defects. The biggest risk in in vitro expansion is genetic mutation. A research team found that when bone marrow MSCs were cultured in vitro for 18 generations, gene mutations and chromosomal abnormalities appeared. A large number of other studies have confirmed that chromosomal abnormalities will only occur when MSC are cultured in vitro for too high a number of generations (more than 10 generations).

As mentioned in previous articles, another issue that affects the safety of stem cell reinfusion is additives, such as using serum-free medium instead of animal serum culture; avoiding the use of antibiotics through strict aseptic operation. However, for irreplaceable components of cell culture such as trypsin, it is also crucial to strictly inspect the quality during reinfusion to ensure that no components remain.

As the effective component of cell preparations, mesenchymal stem cells have their own safety issues that cannot be ignored; the quality control system of cell preparations during the production and preparation process also affects the safety of their reinfusion.

Drug development strategy of mesenchymal stem cells

Clinical research in the field of stem cells is advancing steadily at a rapid pace, which is the general trend. Understanding the development strategy of mesenchymal stem cell drugs will help accelerate its industrialization.

The purpose of stem cell drug development is to shift from "one-to-one" individualized stem cell clinical application to "one-to-many" clinical treatment plan, and provide standardized stem cell products for clinical practice.

Drug development needs to follow the routine drug development process, including : pharmaceutical research, non-clinical research, clinical trial application, early clinical trial, confirmatory clinical trial, new drug application and marketing approval process .

Overview of key points in the development of stem cell preparations

Pharmaceutical research on stem cell preparations, mainly including process development and quality evaluation:

1. Establishment of stem cell bank and working cell bank: Donor screening, tissue collection, cell separation, purification, culture, preservation, identification, efficacy testing, biological characteristics, genetic stability research, establishment of stem cell bank technical standards and working standards;
2. Stem cell preparation process development and pharmaceutical research: Large-scale cell expansion, cell preparation process, dosage form selection, packaging selection, prescription screening, preparation cryopreservation/recovery process, the impact of in vitro operations on the biological characteristics of stem cells, process quality control, and establishment of stem cell drug release standards;
3. Study on cold chain transportation and stability of stem cell preparations: Product cryopreservation and resuscitation, cold chain transportation technology, clinical rapid test, stability research, etc. Stability research includes test of factors affecting preparations under cryopreservation conditions, long-term test, test of simulated clinical application conditions, etc. The drugs obtained through these experiments Stability information is used to determine the storage and transportation conditions, packaging, and expiration date of the drug.

Principles of Stem Cell Preparation Development

The development process of stem cell preparations is subject to GMP principles, but the difference between living drugs and traditional drugs should be fully considered:

1. Reasonability and screening of donor cells;
2. Production materials should be considered, donor cells, cell classification and grading management in the production process, risk assessment of raw materials for production, removal of exogenous factors, restriction of animal and human-sourced materials, genetic modification/transformation are controlled according to high risks, and the use of excipients considers the necessity, safety and reasonableness;
3. The preparation process and process control should prove the feasibility and robustness, the design of the production process (QbD) should avoid unexpected or abnormal changes in cells, monitor the whole process, continuously improve, and reduce pollution by continuous automation and fully enclosed means;
4. Emphasis on process control and product release complementarity, high-risk operation assessment, conversion verification of cell preparations and reinfusion preparations;
5. Quality research replaces analysis of characteristics, functionality, purity and safety of cells in representative batches and production stages;
6. Quality control is based on process understanding, taking into account cognition, gradually improving, and consistent with confirmation and commercialization;
7. Clinical quality approval, method verification and pharmacopoeia applicability verification, mutual verification of rapid trace methods;
8. Stability research should adopt a continuous process, suitable for packaging sealing research, frozen storage adaptability research, etc., transportation conditions, and simulated use;
9. The safety assessment and compatibility research should be carried out on the direct container and the process container, and the light-shielding, sealing and mechanical pressure resistance of the secondary packaging should be investigated;

Preclinical research

Non-clinical research, including preclinical pharmacodynamics, drug mechanism of action, general pharmacology, toxicology, drug interaction, pharmacokinetics, etc.Non-clinical studies are studies conducted on animals to capture the signals of drug safety and effectiveness, to understand the absorption, distribution, metabolic excretion (ADME) and drug pharmacokinetics (PK)/pharmacodynamics of drugs in animals (PD) . In relevant animal populations, identify, identify, and quantify local and systemic toxic reactions, identify the target organs and sites of toxicity, reversibility (acute or chronic toxicity) , and dose-effect relationship.Recommendations for safe doses and dose escalation regimens, identification of potential target organs for toxicity/activity, identification of clinical monitoring parameters, patient eligibility criteria, termination of potentially unsuccessful development programs if applicable in humans; optimization of dosing Scheme/category justification.

Pharmacology and Toxicology of Stem Cell Preparations

The basic content of pharmacology and toxicology research on stem cell preparations includes:

1. Stem Cell Drug Distribution and Metabolism: Distribution, colonization, differentiation and fate of allogeneic/heterogeneic stem cells in animals;
2. Preclinical effectiveness evaluation of stem cell drugs: Evaluation of the effectiveness of xenogeneic/ allogeneic stem cells in animal (mice and large animals, etc.) disease models;
3. Preclinical Safety Evaluation of Stem Cell Drugs: General toxicity, special toxicity (tumor-causing, tumor-promoting, immunotoxicity, reproductive toxicity, etc.) , long-term toxicity evaluation tests;

Laboratory and animal studies conducted by new drug development institutions to observe the dose-effect relationship of stem cells against target diseases, and at the same time conduct safety assessments on stem cells, which must be completed in GLP experiments.

Stem Cell Clinical Trial Considerations

New drug clinical trial application stage

New drug clinical trial (Investigational New Drug, referred to as IND) application: After the preclinical trial is completed and before the new drug registration is accepted, a communication meeting between the Drug Evaluation Center and the registration applicant is held, and then an IND is submitted to the CDE.

The proposed IND needs to include the following contents: preliminary in vitro test results, in vivo mechanism of action, pharmacology and efficacy research results, safety research results, indications for follow-up research and clinical programs, etc. Clinical evaluation includes safety and effectiveness evaluation , Clinical research risk and emergency control plan .

At the same time, according to the needs, the stem cell seed bank, semi-finished products and finished products used will be rechecked and inspected by the relevant certification institutions. The IND needs to be reviewed and approved by the review department. After 60 working days from the date of acceptance, if the review agency does not give a negative or questionable review opinion, it is deemed to have agreed, and the applicant can carry out clinical trials according to the submitted plan.

New drug clinical trials need to follow the ethical principles of the Declaration of Helsinki, "New Drug Approval Measures", "New Drug Approval Measures", "Drug Registration Management Measures" China/WHO/ICH GCP guidelines, CDE registration requirements/new drug clinical research guidelines.

clinical trial stage

Clinical trials are experiments conducted on humans, on healthy volunteers and diseased patients, with the aim of establishing the safety and effectiveness of a pharmaceutical entity in humans. The clinical trials of cell products are divided into early clinical trials and confirmatory clinical trials, which replace traditional phase I-III clinical trials of drugs.

The main purpose of early clinical trials is safety, the secondary purpose is effectiveness, pharmacokinetic studies of single and multiple doses of cell viability, proliferation, differentiation, distribution, migration, expected survival time and biological functions, whether it exists for a long time or persists effects, etc .; Proof-of-concept ( POC ) trials should consider short-term effects and long-term outcomes, and dose finding trials include initial dose, effective dose, maximum tolerated dose, etc.;

Confirmatory clinical trials may include multiple test items due to different research purposes and research questions. Considering the duration of cell activity and delayed safety, long-term follow-up is required to continuously monitor safety and pharmacological activity. Long-term effectiveness and sufficient Exposure safety, long-term safety monitoring failure, infection, immunogenicity, immunosuppression, malignant transformation and other risks.

Through two phases of clinical trials, the new drug development agency will analyze all the test data, and if the data can successfully prove the safety and effectiveness of the drug, the agency will submit a new drug application to the drug regulatory agency. Once the drug regulatory agency approves the new stem cell drug application, the new stem cell drug can be prescribed by doctors.

New drug launch and post-market re-evaluation: GMP facility construction, product production verification, new drug clinical application safety data tracking and analysis.

Stem Cell Product Development Process

Stem cell drugs include products that do not need to be expanded and those that need to be expanded after isolation .

For products that do not need to be expanded after separation ( such as umbilical cord blood hematopoietic stem cells ) , the final product can be obtained directly after cell separation, and this type of product is currently managed by the state as a third-class medical technology. Such products have already been declared as drugs in foreign countries, and have been approved by the FDA for NDA.

For stem cell drugs that need to be expanded, due to the need for more in vitro operations, the safety evaluation will be the focus. There is no approved stem cell drug in China. The following is the drug development process of mesenchymal stem cells that need to be expanded.

Clinical grade stem cell bank and standard establishment

Referring to the international stem cell guidelines and relevant national standards and regulations, standardize donor screening, sample collection, isolation, purification, induction, storage screening and the personnel, equipment and facilities, raw and auxiliary materials, environment, and operating procedures involved, and establish stem cell Library technical standards, providing clinical-grade seed sources for the preparation of stem cell test products.

Reference quality control indicators include: cell identification, individual DNA fingerprints, cell HLA typing, multilineage differentiation potential, cell purity, karyotype analysis, telomerase activity, and tumorigenicity. Among them, biological activity detection includes total cell number and living cell number, cell viability and potency detection. Exogenous factors are detected for bacteria, fungi, mycoplasma, viruses (HIV-1, hepatitis B, hepatitis C, cytomegalovirus, Epstein-Barr virus, etc.) , and related retroviruses. If antibiotic residues are used, they should also be tested.

Preparation of Standard Stem Cell Test Articles

Establish large-scale stem cell culture, induced differentiation, preparation of cell products, frozen storage, cold chain transmission, clinical preparation and other processes.

In accordance with GMP requirements, quality assurance will run through the entire process of cell preparation, and standard operating procedures (SOPs) will be formulated to regulate the personnel, equipment and facilities, raw and auxiliary materials, environment, and operating procedures involved. After the process is stable, carry out process verification, and prepare more than 10 consecutive batches of test products that meet the standards. The preparation capacity of the test product is dozens of clinical doses per batch, providing a uniform and stable test product for the quality evaluation of cell products.

Quality evaluation of standard stem cell test products

For target diseases treated with mesenchymal stem cells, carry out studies at the molecular level, cellular level, and animal level, set up quality control points, safety evaluation indicators, and functional testing indicators for the entire process from donor screening to clinical reinfusion release, and establish Realize the evaluation of cell morphology, biomarkers, cell purity, cell viability, doubling time limit, cell generation, passage stability, migration ability, differentiation ability, abnormal immune response, tumorigenesis ability, biological efficacy, and establish analysis and test methods and verify.

Evaluate the impact of the in vitro process on biological function, immunogenicity, genome stability, etc., and determine the limited passage number.

Refer to the "Chinese Pharmacopoeia" (Part III) to formulate the "Mesenchymal Stem Cell Manufacturing and Verification Regulations for Clinical Use (Draft)" to realize stem cell donor screening, storage screening, process quality control, intermediate product testing, and semi-finished products Testing, testing of cell products and testing of raw and auxiliary materials.

Preclinical animal test evaluation

Establish animal evaluation models based on stem cell preparation types, indications, and administration routes, and carry out tissue distribution, migration, homing, colonization, differentiation, and outcome of low/high-passage and limited-passage stem cell preparations in combination with in vitro cytology research data , Inducing/promoting/inhibiting tumor test, immunogenicity and immunotoxicity test, hemolysis test, allergy test, toxicity test, special safety test, effectiveness test, etc., to verify and evaluate safety indicators and biological efficacy parameters.

In relevant animal populations, identify, identify, quantify the target organs and parts of the toxicity/activity of local and systemic toxicity, confirm the reversibility and dose-effect relationship of acute/chronic toxicity, study DNA interaction, tissue compatibility, secretion Substances, viral replication ability, insertion mutations, oncogenes, etc.

For human application, recommendations for safe doses and dose escalation schemes, identification of clinical monitoring parameters, recommendations for patient inclusion criteria, and recommendations for primary and secondary clinical endpoints.

Clinical Trials

Early clinical trials are the first clinical trials conducted in humans . The purpose of the study is to understand the safety of the drug, determine the effective dose range/minimum effective dose(MED), determine the indication/optimum dose of the drug, conduct a preliminary evaluation of the efficacy of the drug, and establish a comparison with the placebo or control The effectiveness and safety of the drug, the observation of the human body's tolerance to the new drug, and the study of the pharmacokinetics provide a basis for formulating the drug regimen.

The minimum number of cases in early clinical trials is 20-30 cases, and the overall number generally does not exceed 100 cases . Escalation studies are often used to estimate the maximum dose (MTD)before unacceptable toxicity is reached.

Confirmatory clinical trials, usually randomized, double-blind, controlled (placebo/positive control) multicenter, large-scale confirmatory studies. The purpose of the research is to confirm the efficacy and safety of the new drug on patients with target indications. The total number of drug trials must be more than 300, and evidence of statistically significant efficacy must be provided . After the product is launched, it is still necessary to continue to evaluate the risk and safety of new stem cell drugs as well as their risk-benefit in the "real world" environment.

Summary at the end of the article

New drugs enter the clinical trial stage from the laboratory, which is often called the "valley of death" of new drugs. The development of new drugs is a long-term and complicated process, which is capital-intensive, talent-intensive, with a low success rate and huge risks. At present, no stem cell drug has been approved for marketing in my country, but some institutions have applied for stem cell drugs and been approved to enter clinical trials.

With the promulgation of documents such as "Administrative Measures for Clinical Stem Cell Clinical Research (Trial)" and "Guiding Principles for Quality Control and Preclinical Research of Stem Cell Preparations (Trial)", the National Stem Cell Clinical Research Expert Committee was established, and stem cell clinical research institutions and research projects started filing Work. Standardizing stem cell clinical research helps to provide a proof of concept (PoC) for the selection of stem cell drug indications and reduce the risk of drug development.