Animal Models For In Vivo Testing (1): Establishment And Classification

The significant role ofin vitro screening models in drug discovery is undeniable. By simulating the interactions between drugs and biomolecules, these models provide preliminary insights into the physicochemical properties, metabolic pathways, drug interactions, and transmembrane permeability of candidate molecules. These predictions notonly contribute to a deeper understanding of drug mechanisms of action but also offer valuable guidance for subsequent in vivo testing.

However, it is crucial to acknowledge the inherent differences between the controlled environment ofin vitro models and the complex biochemical environment of a living organism. These discrepancies can arise from various factors, including the complexity of biological systems, intercellular interactions, and differing physiological conditions betweenin vitroandin vivo settings.

Due to these differences, the results ofin vitro activity assays may not fully reflect the true efficacy of a drug in vivo. Therefore, while in vitro screening models hold an indispensable position in early-stage drug development, they cannot replace in vivo testing. Regulatory agencies worldwide mandate the submission of comprehensive in vivo data before a new drug can be approved for market release, ensuring its safety and efficacy.

Directly utilizing human subjects for drug screening is clearly impractical. From an ethical standpoint, administering insufficiently validated drugs to humans poses potential health risks and could erode public trust in the pharmaceutical development industry. From an economic perspective, human trials require substantial resource investment andare subject to considerable uncertainty, placing significant financial strain on research projects from the outset.

Consequently, establishing effective and feasible animal models to evaluate the effects of candidate drugs on the human body and disease progression has become an indispensable step in drug development. Animal models can simulate the pathogenesis and pathophysiological processes of human diseases, providing a test environment that more closely approximates the human condition. Through animal models, a comprehensive assessment of the safety, efficacy, and pharmacokinetic properties of candidate drugs can be conducted, providing robust support for subsequent clinical trials.

This article will focus on exploring the applications and advantages of animal models in drug development, with the aim of providing valuable insights for future research endeavors. (Due to length constraints, this topic will be covered in two parts. Part 1 will discuss the establishment and classification of animal models, while Part 2 will delve into classic animal models.)

2.1 Chemical Intervention

Establishing animal models through chemical intervention involves administering specific chemical substances to animals, either through injection or feeding, to induce specific pathophysiological changes. The critical aspects of this methodare selecting the appropriate chemical substance and the correct dosage to ensure the stable and reproducible induction of pathological alterations within the animal.

The MPTP administration model for Parkinson's disease serves as a prime example. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a neurotoxin that selectively damages dopaminergic neurons in the substantia nigra, to a decrease in dopamine levels and mimicking the pathological features of Parkinson's disease.

The specificprocedure involves selecting the appropriate animal strain and age, such as commonly used C57BL/6 mice. Subsequently, the MPTP dosage is calculated and determined based on the experimental objective and animal body weight. Typically, MPTP is administered via intraperitoneal injection, once daily, for several days to weeks.

During the administration period, close observation of the animal's behavioral changes, such as motor coordination and postural balance, is necessary to assess model establishment. Furthermore, biochemical assays and histological analyses can be employed to further validate the pathophysiological characteristics of the model.

The MPTP-induced Parkinson's disease model offers several advantages: it effectively replicates the typical pathological hallmarks of Parkinson's disease, such as the loss of dopaminergic neurons in the substantia nigra and decreased dopamine levels; the model establishment process is relatively straightforward, operationally convenient, and cost-effective; and due to the selective and specific toxic effects of MPTP, the model exhibits high stability and reproducibility.

However, the MPTP-induced Parkinson's disease model also has certain limitations. For instance, it may not fully recapitulate the complex pathogenesis and pathological progression of human Parkinson's disease, and the administration process may induce certain side effects and toxic reactions. Therefore, careful evaluation of its applicability and limitations is necessary when using this model for disease research and drug development.

2.2 Physical Intervention

Consider the scenario where we aim to study diseases caused by external physical factors, such as fractures, sprains, or exercise-induced injuries. Directly experimenting on humans is infeasible due to ethical concerns and significant risks. This is where animal models become invaluable.

Physical intervention methods for establishing animal models are diverse, with surgical or mechanical force application being the most common. For example, in the study of bone fracture healing, researchers may create a fracture model in the legs of mice or rats. They utilize specialized surgical instruments to simulate the trauma of human fractures and subsequently observe the healing process in the animals. This can be viewed as a miniature simulation of an "accident" on a small stage, allowing observation of how the "injured" subject recovers.

Another example involves the study of heart valve diseases. Researchers might employ catheterization techniques to simulate valve stenosis or regurgitation in animal hearts. Through precise surgical manipulation, catheters are inserted into the animal's heart to mimic valvular lesions, and subsequent changes in cardiac functionare observed. This is akin to intentionally introducing a malfunction in a complex machine and observing its response.

These physical intervention methods notonly aid in simulating various disease states but also facilitate the evaluation ofnew drug efficacy and safety. For instance, in a fracture model, administering a newly developed drug and observing the rate and quality of bone healing can provide valuable insights. Similarly, in a heart valve disease model, the ability of a new drug to improve cardiac functionand reduce complications can be assessed.

Furthermore, physical intervention methods are often combined with other techniques, such as gene editing and drug induction, to more comprehensively simulate human diseases. For example, in cancer research, researchers might first use gene editing to induce specific gene mutations in animals andthen employ physical interventions (such as radiation or chemical induction) totrigger tumor formation.

2.3 Genetic Intervention

Genetic intervention involves modifying an animal's genes to mimic human diseases. The core of this method lies in leveraging advanced gene editing technologies, such as CRISPR/Cas9, to precisely alter the animal's genome. This technology acts like "molecular scissors," capable of precisely cutting and replacing DNA sequences, thereby creating animal models withspecific genetic mutations.

For instance, if the goal isto study a hereditary disease caused by a specific gene mutation, such as autism spectrum disorder, CRISPR/Cas9 technology can be used to introduce the identical mutation into the genome of an animal (e.g., mice or dogs). These animals will then exhibit disease characteristics similarto human patients, providing an ideal platform for research.

The canine model of autism spectrum disorder is a compelling example of a successful genetic intervention animal model. The research team led by Professor Yongqing Zhang in China successfully introduced the Shank3 gene mutation into dogs using CRISPR/Cas9 technology, creating an autism spectrum disorder canine model. These mutant dogs effectively replicated the core clinical manifestations of autism in humans, such as social deficits, offering scientists a novel research tool forin-depth exploration of the pathogenesis and treatment strategies for autism.

In establishing animal models through genetic intervention, combining it with other techniques like transgenesis and chemical induction can further simulate the complexity of human diseases. For example, a specific gene mutation can be introduced via gene editing, followed by the use of chemical agents to induce specific physiological or pathological changes, to a more comprehensive recapitulation of the human disease state.

3.1 Homologous Animal Models

Homologous animal models are those that exhibit a high degree of similarity to human disease mechanisms. They arenotonlysimilarin gene expression and physiological characteristics but, more importantly, in the etiology, progression, and drug response of the disease, mirroring human conditions remarkably closely. This allows scientists to accurately simulate the pathophysiological processes of human diseases in animals, providing invaluable experimental data for drug development. Key characteristics include:

• High Fidelity Simulation: Homologous animal models accurately replicate the causes, symptoms, and treatment responses of human diseases, making the drug development process more relevant and efficient.
• Predictive Power: Due to their high similarity to human disease mechanisms and drug responses, homologous animal models can predict drug efficacy and side effects in humans with reasonable accuracy, providing strong support for clinical trials.

Bacterial infection models, established by simulating the human process of bacterial infection, are animal models withsimilar pathophysiological features. These models are crucial for understanding the pathogenesis of bacterial infections and evaluating the efficacy of antibacterial drugs. For example, in the development of antibiotics, scientists use bacterial infection models to assess the inhibitory and bactericidal effects of different antibiotics, providing a scientific basis for clinical medication.

3.2 Isomorphic Animal Models

Isomorphic animal models refer to those that share similar symptoms with human diseases andare amenable to the same treatment approaches. However, unlike homologous models, the underlying causes of the disease in isomorphic models may differ from those in humans. Consider arthritis-related degenerative damage: while injecting iodoacetate into the joints of an animal model can induce arthritis and be used to study the anti-degenerative effects of compounds, the formation mechanism of human osteoarthritis is considerably more complex andnot solely caused by iodoacetate. Key features of isomorphic animal models include:

• Symptom Similarity: Isomorphic animal models exhibit symptoms highly similarto human diseases, allowing for the observation of analogous pathophysiological changes in animals, providing intuitive referencesfor drug development.
• Treatment Parallels: Due to the symptomatic similarities, the same treatment strategies can often be applied toboth isomorphic animal models and human diseases. This facilitates the evaluation of drug efficacy and safety.
• Etiological Differences: Despite similarities in symptoms and treatment approaches, the fundamental causes of the disease in isomorphic animal models may differ from those in humans. This difference necessitates a more cautious analysis of experimental results whenusing isomorphic models for drug development to avoid directly extrapolating animal model findings to humans.

Using arthritis-related degenerative damage as an example, intra-articular injection of iodoacetate can induce arthritis in animal models. This model shares symptomatic similarities with human osteoarthritis, such as joint swelling, pain, and limited mobility. Consequently, this model can be used to assess the anti-degenerative potential of drugs. However, the development of human osteoarthritis is more intricate, involving various factors like genetics, environment, and age. Therefore, whenusing isomorphic animal models for drug development, a comprehensive consideration of these factors is essential to ensure the accuracy and reliability of experimental results.

3.3 Predictive Animal Models

Predictive animal models are employed as tools for drug development when there isn't a direct animal model that corresponds to a human disease. These models simulate the symptoms and responses of the disease using various methods. This can be likened to using a hand-drawn sketch to plan a route when a real map is unavailable.

Consider mental illnesses. These conditions are exceptionally complex, not only due to their diverse etiologies but also because directly observing and understanding an animal's mental state is challenging. Therefore, definitively determining whether an animal truly suffers from a mental illness is difficult. Fortunately, scientists have developed an approach: inducing or exacerbating mental symptoms in animals by administering certain drugs.

For instance, imagine developing a new drug to treat depression. In the absence of a suitable animal model, researchers might inject mice with a substance known to induce depressive symptoms andthen observe changes in their behavior.

If the new drug can alleviate the depressive symptoms in the mice, it provides preliminary evidence suggesting the drug might also be effective in treating depression in humans. Naturally, this is a preliminary prediction, and the actual efficacy needs to be validated through human clinical trials.

4.1 Species Selection

When considering species selection for model development, it is akin to carefully selecting the right resources for a complex project. Choosing the appropriate animal model isas crucial as selecting the finest materials.

It is important to recognize that notall animals are suitable as models for drug development. Just asnotall materials are fit for a particular purpose, several factors must be considered, including the animal's physiological structure, metabolic system, and susceptibility to the disease of interest.

When selecting animal models, priority is generally given to species that are physiologically and pathologically most similar to humans. For instance, mice and rats are frequently used in drug development due to their rapid breeding cycles, ease of genetic manipulation, and low cost.

However, for specific diseases, more specialized animal models may be necessary. For example, rabbits and monkeys are preferred models for studying ophthalmological diseases due to their ocular structures being similar to those of humans.

Non-human primates (NHPs), as social animals with clear hierarchical structures and complex behaviors, are phylogenetically, anatomically, physiologically, and biomedically more similar to humans than commonly used rodent models. They serve as advanced experimental animals in medical and life science research, playing an irreplaceable role in vaccine development for human disease prevention and control, and in the study of human brain function and neurological disorders. As research into human medicine and health deepens, lower-order model animals are proving insufficient in certain areas, creating an urgent need for animal models that are evolutionarily closer to humans, such as monkeys.

In practical applications, scientists have achieved remarkable results by selecting appropriate animal models. For example, in cancer treatment research, mouse models have aided in the screening of numerous effective anti-tumor drugs. In the study of neurodegenerative diseases, fruit fly and nematode models have provided insights into the molecular mechanisms underlying disease onset. These successful examples underscore the importance of selecting suitable animal models in new drug development.

4.2 Sample Size Requirements

Determining the appropriate number of experimental animals hinges on five key factors: statistical principles, experimental objectives, animal characteristics, prior experience, and relevant regulations.

Statistical Principles

In drug development, the goal is to obtain results that are not only effective but also reliable and reproducible. This necessitates the application of statistical principles to estimate the required sample size based on the anticipated effect size, experimental error, and desired level of confidence. In essence, much like flipping a coin more times leads to a more accurate prediction of the probability of heads or tails.

Experimental Objectives

The experimental objective is another crucial determinant of the required number of experimental animals. Different experimental objectives necessitate varying sample sizes. For instance, drug toxicity studies typically require larger sample sizes to ensure the reliability and safety of the results, whereas smaller sample sizes may suffice during the preliminary drug screening phase.

Animal Characteristics

Factors such as the species, age, sex, and health status of the animals can influence sample size determination. Similar to how different ingredients require different cooking times and heat levels, animals with varying characteristics may exhibit different responses in experiments. Therefore, adjusting the sample size based on the characteristics of the experimental animals is necessary.

Prior Experience

Previous experience and relevant regulations can also inform the determination of animal numbers. For example, studies on certain diseases or drugs may have established animal models and experimental protocols, providing a basis for determining the number of animals required.

Relevant Regulations

Different countries and regions have specific regulations and guidelines regarding the number and conditions of animals used in experiments. Adherence to these regulations is mandatory when setting the experimental animal numbers.