INTRODUCTION:

Animals undergo discomfort, suffering, and even death during scientific experiments; this is a contentious topic. Millions of experimental animals are tortured and killed worldwide in order to carry out scientific experiments for research and teaching objectives. ^1,2,3 All of these, nevertheless, are governed and carried out by well-established ethical committees, including institutional ones, which uphold the ethics of using animal models in scientific study. Using animals in research is immoral and excessively harmful to the animals, several social activists and animal lovers are against the practice. From last few decades, numerous worldwide laws, norms, regulations, and Guidelines have been established regarding the use of animals in studies.^4-5 Institutional ethical review boards only authorize the bare minimum of animals necessary for a given experiment.

A redundancy method is one that substitutes an animal experiment by using other testing techniques rather than using animals. It is often referred to as a non-animal approach. Ex- vivo, in- vitro, in- vivo, in- silico, and in chemical reduced or refined procedures are examples of 'testing methods' that fall under this category. Expert system utilization is included as a non-testing approach. The use of experimental animals might be replaced, curtailed, or improved and scientifically supported with much proven techniques that promote animal welfare. It has been discovered that these methods offer significant substitutes for doing different biological research projects.³ The prospect of creating novel treatments is brightened by computer-based drug design, discovery, docking, and simulation research. intrinsic complexity of biological systems, it is typically impractical to replace an animal test one-to-one with a non-animal equivalent. Therefore, a variety of alternative approaches must be used.^3,5To develop a comprehensive, mechanistic knowledge of how a chemical interacts with a biological system to produce a disruption that results in an apical consequence, each approach can offer a unique piece of information (i.e., an observable or quantifiable whole-organism outcome). Complementary information can be provided via in vitro, in-silico (computational), omics, organ-on-a-chip technologies, high-throughput screening (HTS), and mathematical biology. ^6-12

The need for alternative approaches and the emergence of the field of alternatives to animal experimentation.

Due to ethical concerns, limits have been placed on laboratory procedures using higher vertebrate models and including rodents, guinea pigs, dogs, monkeys, etc. As a result, the following animals have been identified to be used:

Microorganisms:

Microbes as a substitute for animal testing is widespread. Their effects on life and a range of biological illnesses have been investigated.

Brewing yeast, Saccharomyces cerevisiae, is regarded as the most important and highly effective model organism.¹³ Numerous neurological illnesses, including Alzheimer's, Parkinson's, and Huntington's, are studied using S. cerevisiae.^12–14 Microorganisms like the bacterium Escherichia coli, the fungus Schizosaccharomyces pombe, and the protozoa Dictyostelium discoideum are useful models for molecular and genetic studies, but Bacillus subtilis may be used to examine cellular development.³

Vertebrates in lower order

Because of their genetic similarities, lower vertebrates are the best substitute for mammals (higher vertebrates). Moreover, there are moral dilemmas when using lesser vertebrates in experiments. With their brief life cycles, lower vertebrates are extensively research.

Types of alternative methods for biological testing of drugs:

Because of advancements in tissue engineering and microfabrication technologies, researchers are keen to generate novel in-vitro disease models. Some of the in-vitro and in-vivo options for using animals are as follows:

1. Culture of cells

Animal experiments can be replaced in many cases by cell culture. Different kinds of organ cultures, tissue cultures, callus cultures, and cell cultures are utilized for different kinds of studies.¹⁵ It has been noted that immortalized primary cell culture is typically used to assess the toxicity of in vitro-grown cells.^16,17 Additionally, there are opportunities in this sector for recombinant DNA technologies, drug screening, cell-based bioassays, and gene therapy as a substitute for animal models.²

2. Assays for cell transformation

Assays for cell transformation are more rapid, costly, and need fewer animals. It serves as a substitute for both the transgenic mouse model and the rodent bioassay in the carcinogenicity assay.^{25, 26} The Balb/c3T3 test and the Syrian hamster embryo (SHE) are two examples.

3. Models of stem cells

Stem cell models are utilized as an alternative to animal experiments for in-vitro toxicological assessment of illnesses.^26–29

The potential application of mesenchymal stem cells (MSC) in the field of regenerative medicine has been the subject of several investigations.^30–33 Stem cells are being employed more and more often these days to check new compounds for potential harm to humans and animals.⁵ Along with their benefits, these techniques also have certain drawbacks.

4. In-vitro pyrogen test

The pyrogen testing is performed on rabbits, which is replaced with the following tests:

a. Amoebocyte Lysate (LAL): LAL is an aqueous extract of blood cells of Limulus polyphemus or horseshoe crab. LAL is used for testing of pharmaceuticals and testing devices for blood or cerebrospinal fluid.¹⁹

b.Monocyte Activation Test (MAT): This test uses cryopreserved human whole blood and produces an interleukin-1β response when pyrogens are identified. It is superior to LAL and rabbit pyrogen tests.²⁰

5. To evaluate Toxicity

Eye Toxicity test

In the past, the Draize test was used to evaluate compounds that elicit irritation by directly administering the drug to rabbits' corneas. Another practice is a number of tests have been created; some of them are listed below .

Test of hen's egg on chorioallantoic membrane
HET-CAM: a method of measuring the irritability of the chorioallantoic membrane, which has a lot of blood vessels, using fertilized chicken eggs. Red blood cell (RBC) hemolysis test: assesses hemolysis and protein denaturation phenomena brought on by the test substance's activity .

Opacity and permeability of the cow cornea (BCOP).
International organizations have thoroughly verified this technique (OECD guideline 437, 2009). The BCOP test was developed as an alternative to the ocular dermal irritation test in rabbits (Draize) .Isolated rabbit or chicken eye test: eyes isolated from dead animals (which were discarded) are tested for edema.^23,24

6. Cell Viability Test:

In cell viability experiments, substances are added to certain cells' growth conditions to evaluate specific elements of their viability (or damage to their membranes at their junction). Cell cultures derived from various tissues can be used, and various chemicals can be subjected to toxicological testing. Certain characteristics, including toxicity, are examined using metrics like cell viability and structural damage. In addition to chicken embryos, fish and amphibians can be employed for the toxicity test at various stages of development and reproduction. One study found that this approach is highly significant.

Guidelines number 129/2009 - Using cytotoxicity tests to determine starting dosages for acute oral systemic toxicity studies is the subject of Guidelines number 129/2009, a guidance document from the OECD that provides recommendations for minimising the use of animals in research. This paper explains how to utilise neutral red uptake (NRU) and in vitro data to calculate baseline drug cytotoxicity in vitro and establish starting dosages for oral toxicity in animals. The NRU concentration, which is 50% lower than the controls (IC50, for example), is found via a dose-response test. Rats are utilised in acute oral toxicity studies to establish a starting dosage, and the value of LD50 by the oral route is estimated using a linear regression equation utilising the IC50 value. The quantity of creatures needed by employing NRU in a method to calculate the starting dosage for acute oral doses, the amount of material needed for testing and the quantity of substances needed to assess toxicity can be reduced. All other quantitative information and data, including the structure activity relationship (QSAR), the LD50 of related substances, the LD50 of related QSAR, and other information needed to estimate the dose that most closely resembles the real value of LD50, should be considered when calculating the initial doses.^36,41

7. Some Technological advancement in alternatives to non- animal models:

a. Computational methods for drug screening and testing method

The use of computer-aided drug discovery (CADD) techniques is growing because they may help address the problems with scale, time, and expense associated with traditional experimental techniques. Computational target identification, virtual screening of vast chemical libraries for promising therapeutic candidates, further optimization of candidate compounds, and in silico evaluation of their potential toxicity are all included in CADD. Following the computer execution of these procedures, in vitro and in vivo studies are performed on candidate compounds to provide confirmation. Therefore, by eliminating ineffective and hazardous chemical compounds from consideration, CADD techniques can decrease the number of chemical compounds that need to be examined experimentally while raising the success rate²¹.

Today, computational prediction tools are included into each step of the drug discovery process, thanks to developments in computational algorithms and knowledge databases.

b. Mathematical models:

By specifying variables and putting hypotheses to the test, mathematical models can aid in experimental work by lowering costs and improving efficiency. Predicting the physical and chemical characteristics of a protein structure using mathematical models is one of the example

Models of QSAR:

The field of in silico pharmacology began in the early 1960s when computational techniques demonstrated quantitative relationships between chemical structure and PD and PK effects in biological systems. Since then, the identification and research of QSAR have been crucial to modern medicinal chemistry and pharmacology. It involves creating a mathematical model that links a molecular structure to a chemical characteristic or biological effect using statistical techniques²².

c. Descriptor-based method:

Utilising molecular descriptors as numerical representations of chemical structures: On the basis of this, one-dimensional descriptors, which encode numerically general features like molecular weight, molar refractivity, and octanol /water partition coefficient, reasonably represent the size, shape, and lipophilicity of molecules. Many of these descriptors are frequently identified as physiologically relevant descriptors in QSAR equations because, in spite of their low dimensionality, some of them have been connected to substances' pharmacological characteristics. The 3D structures of molecules serve as the direct source of 3D descriptors for the so-called 3D-QSAR techniques. The need for the molecules to be aligned before creating the model is a dependence reason for many 3D-QSAR methods, which has partially contributed to the introduction of different approaches and strategies for aligning molecules. The highlight is the application of 3D descriptors to the current molecular conformation. This explains why a lot of 3D-QSAR methods require the molecules to be aligned before creating the model, which has added to the development of a number of different approaches and strategies for molecular alignment. When experimentally confirmed structural information about the target protein is available, these techniques involve docking chemicals in an active region of a protein directly aligning molecules through the flexible superposition of molecules Lemmen Among these alignment-dependent 3D-QSAR techniques, noteworthy are the well-known comparative[34,38,40].

d. Contributions from interatomic distance in ligand-protein complexes are essentially transformed into pair-potential functions for the different types of ligand and protein atoms using a knowledge-based approach. The free energy of interaction between a ligand and a protein is then estimated by adding the contributions from ligand-protein atom pairs within a given distance. Important aspect about this approach is that it implicitly accounts for important but little-known contributions to ligand-protein interaction, such as solvation and entropic terms. The types of ligand and protein atoms that are determined, the kind and size of the experimental complexes used, the range of interatomic distances scanned, and the width of the distance bins are the main distinctions among the different potentials^34,35.

e. Virtual ligand screening

Virtual screening is the act of evaluating and classifying compounds in huge chemical libraries based on how likely they are to have an affinity for a certain target. In this way, virtual screening may be seen as an effort to broaden the scope of QSAR, which was initially restricted to small groups of similar compounds. This expansion is along the chemical dimension, which is characterised by both plausible and already-synthesised molecules. The phrase was first used in the late 1990s, when computer-based procedures were sufficiently developed to provide an option to experimental high-throughput screening (HTS) methods, which were surprisingly performing worse and costing more than expected . The pharmaceutical business has come to terms with the fact that virtual screening²¹.

f. Target-oriented techniques

The nearly exponential increase in the number of experimentally established protein structures has allowed for functional coverage of protein families, which has been leveraged in the development of target-based affinity profiling techniques. Unfortunately, not all of the therapeutically important protein families are now equally covered by 3D structures, mostly due to technological challenges (e.g., membrane-bound proteins). Enzymes, for instance, are by far the architecturally most populated family, with over 20,000 entries. Comparatively, only a small number of GPCR structures have been determined; in comparison, almost 200 structures for nuclear receptors and ligand-gated ion channels are known. In the latter scenario, homology modelling approaches are needed to supplement the limited coverage levels of structures currently discovered by experimentation with computationally derived structural models.^50,51

g. Visualisation of data

Such data analysis would point to the necessity of multidimensional techniques and maybe advanced visualisation tools for data mining. For any molecular structure, computational algorithms may produce predictions for a wide range of pharmacological and physical features. Commercially accessible tools like Diva and Spot fire have been frequently utilised for analysis of ADME and physicochemical property data or incorporated into proprietary decision support systems, even though more current methodologies with equivalent 3D graphing and filtering features are also available. Other methods depend on 2D structural similarity, such as agglomerative hierarchical clustering and recursive partitioning^49–50.

h. 3D human-derived models

The recent development of 3D human-derived models used for translation into non-animal technologies represents a significant advancement. Researchers should be able to obtain more vitally pertinent human data that, by utilising omics and in silico methods to optimise these human-derived models, may be used to clinical trial design and improving effective clinical outcomes⁵⁰.

i. Three-dimensional (3D) bioprinting

Through 3D bioprinting, which is increasingly promising for the creation of customized in vitro models of significant scientific and medical value, 3D cell culture offers itself as an alternative to animal models. In place of using animal models, 3D bioprinting can advance research by lowering expenses, accelerating the pace of research development, and novel treatments will be successful.^{34,37, 38}

In contrast to expensive experimental animal testing, 3D-culture systems offer more accurate in-vivo data and a superior translational model. ³⁷

j. ML ( Machine learning ) and Artificial Intelligence ( A1):

AI models that use reliable datasets and computer vision can potentially save millions of animal lives. This is a viable substitute for animal models that has enormous potential to produce safe and dependable results for drug discovery. With discoveries and experiments, the rise of quantum computing is paving the way significantly. As a result, using several animals for unreliable animal testing is not necessary. Substituting animal testing with the ever-growing capabilities of AI and deep learning could help minimize the need for creatures in scientific discovery. Digital Animal Replacement Technology (DART), an AI-based solution which uses ethically sourced human stem cells, a digital workstation, and artificial intelligence to predict drug safety based on stem cell-drug interactions^34,38.

j. Human-patient simulators

Computerized human patient simulators that bleed, breathe, shake, talk, and even die are used to teach students physiology and pharmacology. These simulators are superior then cutting up animals^42,43.

FUTURE DIRECTIONS AND RESEARCH NEEDS:

Any proposed research inside an institution needs to go through the IACUC review process. The protocol review includes, among other things, an explanation of the study, strategies for using less number of animals used, rationale for their usage, and details on euthanasia techniques, veterinary care, and pain and distress relief. This is all hypothetical, as work cannot start until clearance is received. A necessity for periodic or ongoing evaluation also exists. Furthermore, when a study design changes, an IACUC must be consulted and given permission before any additional work may be done. With a concentration on planned work at the cost of tracking existing research, the process has grown lengthy and onerous^44,45.

CONCLUSION:

Animal ethical issue has equal importance as the human welfare. More efforts need to be undertaken for effective implementation of 3 Rs policy to minimize the usage of laboratory animals in research activities. Various alternatives techniques / methods have been suggested, which need to be implemented. For this integration of various computer models, bioinformatics tools, in vitro cell cultures, enzymatic screens and model organisms are necessary. Use of modern analytical techniques, data acquisition and statistical procedures to assess the results of alternative protocols can provide dependable outcomes. These integrated approaches would result in minimum involvement of animals in scientific procedures³⁸.

CONFLICT OF INTEREST:

The author confirms having no conflict of interest.

ACKNOWLEDMENTS:

The author would like to thank DST for providing financial support and Department of Pharmacy for providing facility.

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Received on 03.05.2024 Modified on 13.06.2024

Research J. Pharm. and Tech 2024; 17(8):4097-4102.

DOI: 10.52711/0974-360X.2024.00635