Rodents in Drug Discovery

Abstract

Animals, especially rodents, are an integral part of any drug discovery and development program. Once initial in silico and in vitro experiments are completed, a new chemical is tested for its pharmacokinetic profile, efficacy, and safety in animals, rodents being the most commonly used animals. Millions of rodents (rats and mice) are being used annually to understand the properties of new chemicals. Apart from wild types, genetically modified rats and mice such as knock-out or knock-in animals are very popular nowadays in understanding the biology behind diseases. Though the emergence of advanced technologies undermines the use of rodents in research, replacing animal use in research now seems to be a dream.

1. Introduction

Thinking of rodents, rat or mouse, chills run down anyone’s spine. These small creatures can be a source of lots of nuisance in our daily life like damaging household items, spoiling food in granaries, spreading diseases like plague and so on. However, there is always another side of the coin; rodents are very useful animals, especially in biomedical research. One cannot think of bioresearch sans rodents. They are considered as an integral part of biomedical research [1]. Animal research and testing are behind almost every prescription medicine available today. Moreover, animal research saves animals, too. It has resulted in many remarkable lifesaving and life-extending treatments for cats, dogs, farm animals, wildlife, and endangered species [2].

Successful and efficient development of a new drug revolves around – chemistry, manufacturing and controls (CMC), non-clinical studies (distribution, metabolism, and pharmacokinetic [DMPK], pharmacology and toxicology), and clinical trials. The use of animal models in research aided in the advancement of knowledge about the pathobiology of several diseases of animals and humans, which led to the discovery and development of new therapies for the prevention and/or treatment of many diseases symptomatic or disease-modifying. Animal testing is a vital part of drug development. Animal testing is necessary for understanding the safety and proper dosages of new medicines and treatments. A new chemical is initially tested in isolated cells, tissue slices or organs. The next step is testing in living animals to show whether the chemical works the same way inside the body as it did in the artificial environment of the laboratory. Animal testing also sheds light on how the chemical alters the interactions between different cells and organs of the body.

The significance of animal use in biomedical research is emphasized from time to time by various scientific groups around the globe. For example, in 1993, the NIH office released a position statement on the use of animals in research which stated, “The development of knowledge necessary for the improvement of the health and well-being of humans, as well as other animals, requires in vivo experimentation with a wide variety of animal species.” “Whole animals are essential in research and testing because they best reflect the dynamic interactions between the various cells, tissues, and organs comprising the human body” [3].

Among animals, rodents play a crucial role in biomedical research. More than 95% of animals used in biomedical research are mice and rats. Their close resemblance with humans in term of physiology and genetic makeup makes them a natural choice for biomedical research. Though there are certain differences between people and rodents, the similarities outweigh them and provided researchers with an enormously powerful and versatile tool to investigate human diseases [4].

Why are rodents (rats and mice) most commonly used in biomedical research? The answer lies in the fact that they reproduce quickly, they are social, they are adaptable, and they are omnivores. Additionally, the rodent’s diminutive size allows relatively easy storage in labs, and their shared evolutionary roots with humans mean the similarity of their genetic makeup to human DNA. Rodents have become the animal model of choice for biomedical researchers because their physiology and genetic makeup closely resemble that of people. Table 1 depicts how much rats and mice genome is similar to the humans compared to other animals [5, 6]. With the evolution of genetic engineering, research started using genetically modified or humanized rodents for the identification of gene/s responsible for various diseases, exploring the mechanism of diseases, and understanding how to circumvent and find a solution/treatment for many diseases. Rodents are also used for testing the biocompatibility of medical devices used in humans. Therefore, the use of rodents becomes an integral/indispensable part of drug discovery [4, 7].

Species	Genetic similarity (%)
Humans and Humans	99.9
Humans and Chimpanzee	>99
Humans and Mice	> 98
Humans and Rats	≈ 97–99
Humans and Pigs	98
Humans and Dogs	94
Humans and Cats	90
Humans and Cows	80
Humans and Fruit flies	60

Table 1.

Genetic correlation between Humans and laboratory animals.

2. History of rodents use in biomedical research

There is no verifiable evidence available of when and where people research started using animals in biomedical research. Based on the writings of Aristotle, Diocles, Praxagoras, Erasistratus and Herophilusit, it can be estimated that the use of animals in biomedical research might have started as early as the third-century BC [8].

Mice and rats have been used for research since the seventeenth century, but with the rise of biological and genetic experiments in the 1900s, they have become the most widely used animal in research. The first publication using mice in research dates to 1902, with French biologist Lucien Cuénot. Early in the twentieth century, mouse breeding started scientifically, resulting in some of the first inbred mouse lines in biomedical research. In 1929, Clarence Cook Little (1888–1971) founded the Jackson Lab in Bar Harbor Maine, which became the first American institution to supply laboratory animals as a tool for genetics and medical research across the globe [9].

The first evidence of the use of Albino rats was dated back to 1828, when these rats were used to study the quality of proteins present in the body. Similar rats continued to be used until 1906, when an institute in Philadelphia created rats by selecting inbreeding to be used as a model for present-day biomedical research. Most present-day rats originated from that original colony maintained at Wistar institute [9].

During the last three decades, due to emergence of sequencing technologies and platforms led to significant advancements in the rat genome assembly. This has led to the discovery of genome variants in rats, which have been widely used to detect quantitative trait loci underlying complex phenotypes based on gene, haplotype, and sweep association analyses [10].

3. Usage of animals in research

Tens of millions animals are used in biomedical research worldwide; rodents outnumber others. The exact number is difficult to arrive as many organizations never disclose how many animals they use. In one report [11], about 1.1 million rodents were used for scientific procedures in Great Britain alone in the year 2021 [12]. In the United States of America, an estimated 100 million mice and rats were held captive in laboratories or used in experiments in 2019 [13].

4. Commonly used rodent strains in biomedical research

The most commonly used rodents strains include but are not limited to the following:

• Mice: C57BL/6, BALB/c, Swiss, CD-1

• Rat: Wistar, Sprague–Dawley, Brown Norway, Long-Evans, F344

• Hamster: Golden or Syrian hamster

• Gerbil: Mongolian Gerbil

5. Rodents in drug discovery

5.1 Process of drug discovery and development

A successful drug needs about 15 years and hundreds of millions of dollars, from the conception of the idea to marketing approval. For a chemical to be declared drug and launch in the market, several thousands of chemicals are tested for their efficacy and safety. It is estimated that 1 in 10,000 chemicals reach their final destination. Therefore, a major chunk of the research and development budget is spent developing candidate drugs that are not approved. Furthermore, there are instances where drugs are withdrawn from the market even after approval, mostly due to either lack of sufficient efficacy or unanticipated toxicity that appeared during post-market surveillance [14].

A typical drug discovery and development process consists of several stages. The preliminary basic research on clinical disease and biomarker identification leads to a better understanding of disease diagnosis, progression, and outcomes, followed by the involvement of chemists and biologists collaborating to develop candidate therapeutic agents. These are typically evaluated for efficacy in cell culture-based high throughput screening assays. For new drug development, small molecules, therapeutic proteins/antibodies, antisense oligonucleotides, and off-target effects of currently available pharmaceuticals are considered.

Animal experiments are a very important milestone of drug discovery which generally starts as soon as the in-vitro data is available and continues till phase 3 studies or even post-marketing. The major areas where animals are used include pharmacokinetics (the action of the body on the medicine), efficacy testing, and safety evaluation. Apart from the above said, animals are also used to prove the mechanism of action of the proposed drug.

Animal testing is necessary for understanding the safety and proper dosages of new medicines and treatments. The use of animals in research remains essential to understand the causes, diagnoses, and treatment of disease and suffering in humans and in animals [15]. Experimentation in humans generally begins once the researchers confirm that the drug is safe and effective based on animal studies. Prior to drug approval, human and animal testing is regulated by law, and regulatory agencies (like FDA, EMEA) mandate animal testing before any clinical trials in humans for safety reasons. Animal research and testing is a crucial step in drug discovery and development since testing drugs in humans before assuring their safety would be extremely dangerous and unethical.

There are arguments against use of animals in research. Often times it is believed that animal findings do not translate to humans. This is particularly more relevant to the research in the field of neuropsychiatry as animals do not manifest psychiatric symptoms. It is true that we may not be able to translate rodent or any other non-clinical data completely in humans which may be due to several factors, but still, animals serve as a valuable tool in biomedical research. Since there is no alternative that mimics complex functions of human physiology as of now, the use of animals cannot be substituted completely. Animals will continue to be used in drug discovery to identify new drugs for various unmet medical conditions both of humans and animals [15].

5.2 Pharmacokinetics (the action of body on the drug)

Any drug after administration (with the exception of topically administered drugs) must enter the bloodstream and get distributed to the site of action to show its pharmacological response. The process of movement of drug from the site of administration to site of action is known as pharmacokinetics, which includes absorption, distribution, metabolism and elimination of drug [16].

• Absorption: defined as passage of drug from its site of administration into the blood

• Distribution: defined as the delivery of the drug to the tissues

• Metabolism: defined as the breakdown of drug into metabolites (usually pharmacologically inactive or less active)

• Elimination: defined as excretion of drug from the body

For assessment whether the drug is being absorbed from the intestine and reaches to the systemic circulation, rodents are primarily used for this kind of experiment early in the discovery phase. Similarly, comparative bioavailability of different routes of administration is also carried out in animals, again primarily in rodents. Other animals used in these experiments are dogs, monkeys, rabbits, and non-human primates. Once it is determined that systemic exposures in rodents are sufficient to exert therapeutic benefit, a drug moves to efficacy studies.

Other parts of pharmacokinetics like distribution, metabolism, and elimination of chemicals, are also evaluated later in the discovery phase in a timely manner. Metabolic profiling is a very important aspect of drug development. Initially, in-vitro studies are done to understand the extent of metabolism in different species like rodents, non-rodents and humans using either liver microsomes or primary hepatocytes. Once the extent of metabolism is done, the next step is to see whether there is any unique metabolite form in humans which is not form in any animal species checked. This exercise is important for the selection of the right species for subsequent safety studies.

Tissue distribution and elimination patterns are also a very important aspect of the discovery program. Initially, rats are used to determine the extent of tissue distribution and elimination pattern.

5.3 Pharmacodynamics (the action of drug on the body)

Pharmacodynamics is defined as the physiological and biochemical response of drugs and their mechanism of action at organ system, sub-cellular and molecular levels. It includes binding the drug to the molecular target and eliciting the desired response.

Pharmacology is defined as the branch of science that deals with the interaction of drugs administered with the living system to produce a biological response. It can be divided into the following three categories: [17, 18].

• primary pharmacology/pharmacodynamics

• secondary pharmacology/pharmacodynamics

• safety pharmacology.

5.3.1 Primary pharmacology

Primary pharmacology studies demonstrate the intentional drug-related effects on enzymes, receptors and other targets. The pharmacology of a drug influences pre-clinical species selection for some studies [19]. Efficacy evaluation is one of the most important aspects of drug discovery. The first step is to identify the right target and the right mechanism of the disease in question. Once it is determined, several new entities (either chemicals or biological) are tested for their potential interaction with the target using in-vitro techniques. Out of several entities, only few go to the next level of testing to determine their efficacy in reliable models, either in vitro, ex vivo, or in vivo, most often combination of all.

Different animal models are being used based on the target/disease in question, mostly rodents, to understand the efficacy of chemicals/drugs. The reason for this widespread use of rodents is their similarity with humans. Many genetically engineered mice/rats are developed to mimic human pharmacology/genomics to understand the pathology, efficacy and safety of new drugs, and they are sometimes called as humanized animals (wherein human genes are incorporated in animals).

There are hundreds of animal models developed so far to test chemicals for their efficacy in different diseases. In general, once the chemical proves to be efficacious in animal model/s, it moves to human testing only if considered to be safe to be administered in humans. Examples of some of the most commonly used rodent models are chemically induced diabetes models, diabetic wound models, cancer models, colitis models, etc. (Table 2). Apart from the model developed for many diseases in wild-type rodents, new research focuses on genetically engineered animals to mimic human pathology. The commonly used transgenic/genetically modified models are listed in Table 3 [22].

Animal models for Disease Conditions
Mouse	Cancers and genetic diseases
Rats	Osteoporosis, inflammatory diseases, diabetes, obesity, cardiovascular dysfunctions, neurodegenerative diseases, cancers

Table 2.

Animal models for Disease conditions [20].

System/organ affected	Human genetic disease	Gene/genetically modified rats and mice
Cardiovascular	Pulmonary arterial hypertension	BMPR2/KO
	Primary pulmonary hypertension 4 (PPH4)	Kcnk3/KO
	Atrial fibrillation, familial, 18 (ATFB18)	Myl4/KO
	Familial hypertrophic cardiomyopathy and myocardial genetic diseases	Myh7b/KO
Nervous system	Epileptic encephalopathy, early infantile, 63 (EIEE63)	Cplx1/KO
	Dystonia 25 (DYT25)	Gnal/KO
	Schizophrenia	Drd2/KI reporter
	Amyotrophic lateral Sclerosis (ALS)	Fus/KI point mutation; R521C
	Epileptic encephalopathy, early infantile, 24 (EIEE24)	Hcn1/KO
	Autism spectrum disorder	Cntnap2/KO; Shank2/KO
	Parkinson’s disease	Lrrk2/KO
Gastrointestinal	Hereditary tyrosinemia type I	Fah/KO
	Hirschsprung disease	Ednrb/KO
	Rotor syndrome	OATP1B2 /KO
	Atypical hereditary non-polyposis; colorectal cancer	Msh6/KO
	familial colon cancer	Apc/KO
Muscle	Muscular dystrophy (Duchenne and Becker forms)	Dmd/KO and BigDel
	Myostatin-related muscle hypertrophy	Mstn/KO
Lung	Cystic fibrosis	Cftr/KO and DF508; CFTR/KI and G5551D
Endocrine	Glucocorticoid resistance	Nr3c1/cKO
	Estrogen resistance (ESTRR)	Esr1/KO and Esr2/KO
	Congenital hypothyroidism	Tshr/KO
	Allan-Herndon Dudley-syndrome	Mct8/KO
Metabolic	Congenital leptin deficiency	Lep/KO
	Leptin receptor deficiency	Lepr/KO
	Aceruloplasminemia	Cp/KO
	Diabetes mellitus, non-insulin dependent,; 5 (NIDDM5)	AS160 (TBC1D4)/KO
	Dwarfism	Ghsr/Tg, Ghsr/KO
	Obesity	Mc3RMc4R/DKO
Nephrology	Focal segmental glomerulosclerosis 2; (FSGS2)	Trpc6/KO BigDel
	C3 glomerulopathy	C3/KO
	REN-related kidney disease	Ren/KO
Ophthalmology	Autosomal dominant congenital stationary night blindness and retinitis pigmentosa	Rho s334ter/Tg
	Retinitis pigmentosa 85 (RP85)	Ahr/KO
Cancer	Li-Fraumeni syndrome	Tp53
Immune and hematological systems	Hemophilia A	F8/KO
	SCID	Rag1/KO; Rag2/KO; Prkdc/KO
	X-linked SCID	Il2Rg/KO
Sleep disorders	Narcolepsy	Orexin KO mouse; Orexin/ataxin-3 transgenic mouse or rat

Table 3.

Genetically modified rat and mice models of human genetic diseases (reproduced from [21]).

5.3.2 Secondary pharmacology

Secondary pharmacology studies evaluate the potential off-target or unintentional effects of a drug. These studies are important in predicting potential toxicities and demonstrating safety [19]. This is a cost-effective approach used by many pharmaceutical companies as a safety screen early in drug development. Information on the potency of a drug for a given biological target can be used to determine structure–activity relationships, assess potential liability for off-target effects, and influence early clinical trial design, dose selection and patient monitoring [23].

5.3.3 Safety pharmacology

Safety pharmacology studies are defined as those studies that investigate the potential undesirable pharmacodynamic effects of a substance on physiological functions in relation to exposure in the therapeutic range and above.

The main objectives of these studies are to identify undesirable pharmacodynamic properties of a substance that may have relevance to its human safety, to evaluate adverse pharmacodynamic and/or pathophysiological effects of a substance observed in toxicology and/or clinical studies, and to investigate the mechanism of the adverse pharmacodynamic effects observed and/or suspected.

In the first phase of safety pharmacology studies, functions of the most critical organs or systems such as the cardiovascular, respiratory and central nervous systems, are assessed. Subsequently, other organ systems, such as the renal or gastrointestinal system, the functions of which can be transiently disrupted by adverse pharmacodynamic effects without causing irreversible harm, are of less immediate investigative concern and can be evaluated later in development if the need arises [24, 25]. Functions of these organs/systems are evaluated using various techniques; some are well established, and many are emerging (Table 4).

Safety Pharmacology Studies- Core Battery
Organs/Systems	Established	Emerging
Central Nervous System	Behaviour- (modified) Irwin, FOB; Locomotion- Photo Electric Beam interruption/rotarod; Nociception- hot plate/tail flick/paw pressure; Seizure liability- EEG	Nociception- video automated system; Seizure liability- integrated video EEG, in-vitro hippocampal brain slice; Drug abuse- lever chamber models; Drug dependence- telemetry
Cardiovascular System	In-vitro patch clamp- hERG assay- QT prolongation; Telemetry- blood presssure, heart rate, ECG; Isolated myocardial systems- heart rate, ECG	In-vitro Automated high-throughput patch clamp; External telemetry with high definition oscillometry; In-silico computer modeling; hESC-CM and hiPS-CM models
Respiratory System	Respiratory rate, tidal volume, O2 saturation, airway resistance, compliance- Plethysmography, telemetry	Unrestrained video-assisted plethysmography Biomarker- VQM
Secondary/Follow-up Battery
Gastrointestinal System	Gastric emptying and secretion, intestinal motility, ulcer index, histopathology	Capsule endoscopy, Telemetry, PBPK modeling,; Biomarkers- Citruline, miR-194, Calprotectin
Renal System	Urine- volume, osmolality, pH, Na+, Cl-, K+, Urea, AST, ALT, LDH, GGT, ALP, β-NAG, Serum- Osmolality, BUN, Creatinine, Cystatin C.; GFR, Clearance rate	Biomarkers: β2- Macroglobulin, KIM-1, CLU, TFF3, NGAL, α-GST, μ-GST, RPA-1

Table 4.

Safety Pharmacology studies [26].

ALP- alkaline phosphatase; AST - aspartate aminotransferase; ALT - alanine aminotransferase, ; BUN - blood urea nitrogen; β-NAG - N-acetyl-β-D-glucosaminidase; CLU - clusterin, EEG -electroencephalography; ECG - electrocardiogram; FOB - Functional Observation Battery, GGT - γ-glutamyl transferase; GFR- glomerular filtration rate; GST - glutathione S transferase, hESC-CM- human embryonic stem cell derived cardiomyocytes; hiPS-CM- human inducible pluripotent stem cell derived cardiomyocytes, KIM-1 - kidney injury molecule-1; LDH - lactate dehydrogenase, miR - microRNA, RPA-1 - renal papillary antigen-1; NGAL - neutrophil gelatinase-associated lipocalin; PBPK - physiologically based pharmacokinetics; TFF3- trefoil factor 3VQM - ventilation (V)/perfusion (Q) mismatch (M).

5.4 Toxicology

The non-clinical safety assessment is one of the most important aspects for marketing approval of any drug; a set of studies need to be completed before filing a new drug application for market authorization, including general toxicity studies, genotoxicity, and reproduction and development toxicity studies. Other toxicity studies such as carcinogenicity, phototoxicity, immunotoxicity, juvenile animal toxicity and abuse liability need to be conducted on a case-by-case basis, based on the property of drug, outcome of other studies, intended length of use, target population and so on [27].

This requirement may vary in different countries, but overall several studies are required to assess the toxic potential of any drug. Similarly, timing to conduct these studies may also differ in different regions. For example, in the United States, assessment of embryo-fetal development can be deferred until before phase 3 for women of childbearing potential (WOCBP) using precautions to prevent pregnancy in clinical trials. In the EU and Japan definitive non-clinical developmental toxicity studies should be completed before exposure to WOCBP (with some exceptions).

The ultimate aim of safety assessment is characterization of toxic effects, identification of target organs, exposure-response relationship, and potential reversibility of drug-induced adverse effects. The resultant information is helpful in deciding a dose that can be administered for human safety without any expected adverse events. The information from these studies is also helpful in finding out biomarkers that can be used for clinical monitoring of potential adverse events.

5.4.1 General Toxicity studies

5.4.1.1 Single or repeated dose studies

These studies range from single dose (acute toxicity) to repeated doses up to 12 months or more in duration in two animal species; one rodent and one non-rodent. Information on the acute toxicity of pharmaceutical agents could be useful in predicting the consequences of human overdose situations. The recommended duration of the repeated-dose toxicity studies is usually related to the duration, therapeutic indication, and scope of the proposed clinical trial.

Among rodents, most commonly used species is the rat. Rats are considered the most suitable species for the purpose of being small in size, having a limited life span of about 2 years, having high breeding capacity, having ease in handling, and having availability of robust historical data [28].

5.4.2 Reproductive and developmental toxicity studies

To identify hazard and characterize reproductive risk for human pharmaceuticals, animal testing is a regulatory requirement. The most commonly used species are rats and rabbits. Sometime NHPs are also used especially for biological.

As appropriate, observations through one complete life cycle (i.e., from conception in one generation through conception in the following generation) permit the detection of immediate and latent adverse effects. To evaluate all stages of life cycle, the following studies are generally conducted to support new drugs;

1. Fertility studies

2. Early embryonic development study

3. Embryo-fetal developmental studies

4. Pre and postnatal developmental studies

There are several combinations that can be used to evaluate the effect of any drug on a complete life cycle such as combining fertility studies with early embryonic development studies; combining EFD with prenatal developmental studies, etc. The ultimate goal is to estimate the effects of drug on all stages of human life. That is, pre-mating from conception to birth to sexual maturity [29].

5.4.3 Genotoxicity

The genotoxic potential of all drugs is evaluated early in drug development. Though the initial screening is carried out using in vitro tests like AMES, chromosomal aberration tests in cell lines, mouse lymphoma test, mammalian cell gene mutation test, and many more, at least one test is mandatory using animals. The most commonly used in vivo tests are the rodent micronucleus test, rodent comet assay, and bone marrow chromosomal aberration test in rodents [30].

5.4.4 Carcinogenicity

Carcinogenicity studies are generally required for the drugs that are expected to be used regularly over a substantial part of patient’s life. These studies are generally performed in rodents, particularly rats and mice. The duration of these studies is lifetime exposure of the drug in these species to mimic possible human exposure (24 months for rats and 18 months for mice). Regulators require data from two species, that is, rat and mouse, to understand the potential carcinogenic effect as well as to assess human risk. With the invention of genetically modified mice, 18-month mice study can be replaced with 6-month study in genetically modified mice which is considered enough to explore the tumorigenic potential of drug.

Several factors need to be assessed before taking a call on the necessity of conducting a carcinogenicity study. These are duration of exposure (expected clinical use is continuous for at least 6 months or frequently in an intermittent manner in the treatment of chronic or recurrent conditions), evidence of genotoxicity, structure–activity relationship suggesting carcinogenic risk, evidence of pre-neoplastic lesions in repeated dose toxicity studies, previous demonstration of carcinogenic potential in the product class, etc. [31, 32]. A waiver request can be asked if the sponsor can justify that carcinogenicity studies are not required for drug approval. The requirement of carcinogenicity studies may be deferred to post-marketing with proper justification and in agreement with the regulators. This is generally possible when the drug is intended to be used in patients with severe diseases and very limited or no therapeutic options are available.

5.4.5 Phototoxicity

Phototoxicity is defined as a toxic response that is elicited after the initial exposure of skin to certain chemicals and subsequent exposure to light or that is induced by skin irradiation after systemic administration (oral, intravenous) of a chemical substance [33]. It is a kind of photosensitivity. In general, the phototoxic potential of any drug is evaluated by conducting two tests viz., phototoxicity, also known as photoirradiation and photoallergy. Phototoxicity studies evaluate the effect of an acute light-induced tissue response to a photo-reactive chemical, whereas photoallergy studies evaluate the effect of an immunologically mediated reaction to a chemical.

All drugs need not be evaluated for phototoxicity potential. Only drugs (chemical) that possess the following characteristics are required to undergo extensive evaluation-

• absorbs light within the range of natural sunlight (290–700 nm);

• generates a reactive species following absorption of UV/visible light;

• distributes sufficiently to light-exposed tissues (e.g., skin and eye).

If a chemical does not meet any of these conditions, then phototoxicity evaluation is not mandatory as these chemicals will not have a photo safety concern [34].

5.4.6 Special/mechanistic studies

5.4.6.1 Immunotoxicity studies

Assessment of potential adverse effects on the immune system is an important component of the overall evaluation of drug toxicity. The first assessment of immunotoxicity is generally observed during repeated-dose general toxicity studies where many aspects of the immune system are evaluated. In case of any adverse effect is observed, follow-up studies may be required to understand the toxic effect and its human relevance. In addition, immunosuppression should also be assessed for new drugs. Again, this is generally a part of repeat dose toxicity studies which include detailed clinical and anatomic pathology evaluation such as serum globulin levels, differential leukocytes count, gross pathology, weights of immune system-related organs (thymus and spleen) and their histological examination (thymus, spleen, lymph nodes, and bone marrow) [35].

5.4.7 Juvenile animal toxicity studies

When any drug is intended for pediatric population, depending on the therapeutic indication, and age of the pediatric population, non-clinical safety studies for sufficient duration to support pediatric trials in juvenile animals needs to be conducted. These studies are generally conducted in rodents [36].

5.4.8 Abuse potential studies

Any drug/pharmaceutical agent or its metabolite must be evaluated for abuse potential if it crosses the blood–brain barrier and reaches the brain. A set of studies determined that abuse potential needs to be assessed, which should be carried out to ascertain abuse potential and subsequent label updates. Rodents are the most commonly used animals for these studies (drug discrimination, physical dependence, and self-administration study). These studies are not always necessary to conduct for all chemicals, but the decision should be taken after a thorough review of the properties of chemicals/drugs and a detailed discussion with regulatory agencies about the need for these studies [37].

6. Conclusion

While in vitro and in silico methodologies play an integral role in drug discovery, animal models continue to prove their value. Although animal ethics groups impose many challenges against animal use in research, rodents, especially rats and mice, will continue to play a critical role in biopharmaceutical research. Whole animals are essential in research and testing because they best reflect the dynamic interactions between the various cells, tissues, and organs comprising the human body.

With the advancement of science and the genesis of humanized rodents, the role of rodents increased significantly in understanding disease processes in a better way and facilitating the transition of research from bench to bedside. Therefore, a complete removal of rodents from drug discovery, at present, is impossible, and they will be continued to provide deep insights during the drug discovery process.