The presentation provides insight into In-vitro and In-silico technologies as an alternative to animal experimentation.

1. In-vitro & In-silico
Techniques: Alternatives
to Animal Experiments
Dr. Ashwani K. Dhingra
Professor,
Guru Gobind Singh College of Pharmacy

2. • Introduction
• Laws and regulations
• Need for alternative to animals
• Refinement
• Reduction
• Replacement
• In vitro methods
• In silico methods
• Advantages and disadvantages
• Summary
Content

3. Animals are used in education for:
• Undergraduates teaching to learn physiological mechanism, anatomy
and effect of various drugs on human body
• Postgraduate teaching to show effects of various drugs, to find out the
nature of unknown drug and for bioassay.
• Research to understand the working of body and processes of disease
and health, screening for drugs, bioassay and for preclinical testing of
new drug
• Animal models are used to test possibilities that would be difficult or
impossible to test using the target species (Humans)
• It is mandatory to do extensive toxicological studies in animals before
the candidate drug gets approval for clinical trials in humans

4. There is no doubt that the best test
species for humans are humans. It is not
possible to extrapolate animal data
directly to humans due to interspecies
variation in anatomy, physiology and
biochemistry.
• Animals are used for
 Toxicity studies
 Therapeutic efficacy
 Efficacy of Medical devices

5. Laws and regulations
Year Law
1960 Prevention of Cruelty to Animals (PCA) Act 1960, amended 1982
1964 Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)
1972 Wild life protection act
1992 Indian National Science Academy (INSA) “Guidelines for care and use of animals in scientific research”,
revised 2001
1998 “Breeding of and Experiments on Animals (Control and Supervision) Rules, 1998”, amended 2001,
2001 Indian Council of Medical Research (ICMR) “Guidelines for use of Laboratory animals in Medical
Colleges”
2009 MCI amendment-Recommends to use alternatives to replace animal experiments
2012 Ministry of Health & Family Welfare bans use of animals in educational institutes
2013 University Grants Commission (UGC) “Guidelines for discontinuation of dissection and animal
experimentation in zoology/life sciences in a phased manner

6. Toxic
drugs
Psychological
Disturbances
Acquired
Infections
Disturbances
food, water &
sleep pattern
Irreversible
tissue/organ
damage
Physical
Disabilities
Forced activity

7. PAIN, DISTRESS
& UNETHICAL
BEHAVIOUR TO
ANIMALS
INVASIVE
TECHNIQUES
TIME
CONSUMING,
EXPENSIVE
REQUIRED
SKILLED
MANPOWER
LOWER
TRANSLATIONAL
RATES
POOR
METHODOLOGIES
DISADVANTAGES

8. ICCVAM &
ICEATM
• To promote scientific validation and
regulatory acceptance of new alternatives
• More predictive of Human Health &
Ecological Effects
• Improves Public Health by
Better Risk Assessment
Enhanced Risk Management
Impediment of Injury & Disease

9. SCIENTIFIC
SUPPORT FOR
ANIMAL
EXPERIMENTATION
Essential for preventing, curing or
alleviating human diseases
Significant success in drug discovery
achieves only via the Animal Testing
Effectively Mimics the Biochemical
Investigations.
Catastropic Consequences occur if no
Animal Experimentation will done.

10. 3R strategy proposed by Russel and burch in 1959
¤ Refinement- refine experimental methods to decrease
unnecessary pain and trauma to animals
¤ Reduction- reduce the number of animals used in these
experiments
¤ Replacement- replace the animal experiments eg-
computer simulation models, In-vitro methods, cell culture
techniques

11. REFINEMENT

12. REDUCTION

13. REPLACEMENT
IN VITRO MODELS
IN SILICO STUDIES
STEM CELL MODELS

14. • In-vitro assays,
• In-silico modelling,
• High-throughput screening,
• Organ-on-a-chip technology,
• Omics and mathematical biology,
Alternatives to animal
experiments

15. IN VITRO
MODELS
• In vitro biomedical research entails the
Study and maintenance of organ, tissue or
cell outside the body
• Can be grown and Maintained as
independent cell lines or preserve the
architecture of the entire organ as organ
culture and tissue culture
• Stem cells

16. SOURCES
EMBRYOS –
CHICK,
RAT/MICE
NEURONAL
PROGENITOR
CELLS
CORD BLOOD
DERIVED
STEM CELLS
CELL
LINES

17. In vitro Pyrogen Test
Embryonic stem cell test
Neutral Red Uptake assay
Toxicity test
Carcinogenicity test
Local Lymph Node Assay
Skin Patch Test
IN VITRO
METHODS

18. IN VITRO PYROGEN TEST
Rabbit Pyrogen Test can be replace by
 Limulus Amoebocyte Lysate (LAL)
 Monocyte Activation Test
On the basis of release of inflammatory mediators from cells due to
pyrogenic contamination response will be evaluated

19. LIMULUS
AMOEBOCYTE
LYSATE
• Aqueous Extract of Blood
Cells From Limulus
polyphemus.
• Used to test Parenteral
Pharmaceutical products and
medical devices

20. MONOCYTE
ACTIVATION
TEST
• MAT involves the utilization
of mononuclear cells from
human whole blood and
considered to be more specific
and sensitive test.

21. EMBRYONIC
STEM CELL
Used to determine Embryonic
Toxicity of new drug
Also predicts the toxicity of any drug
metabolite in vivo
Results classifies the drug as positive
when likely to cause hazardous
effects.

23. NEUTRAL RED
UPTAKE ASSAY
• Replace Draize Rabbit Eye
Test
• Neutral Red dye concentrates
in lysosomes
• Assay measures the potency
of test drug to inhibit the
uptake of dye.

24. LOCAL
LYMPH
NODE
ASSAY
Employed for Skin
Sensitization
Proliferation rate of
Lymphocytes acts as a
determinant for dose use

25. SKIN PATCH
TEST
Determine Chemical Corrosivity
Test includes a biomembrane and a
chemical detector.
When exposed to corrosive substance
become colored that will be detected by a
detector
Biomembrane used are EPI-DERM & EPI-
SKIN
TEST REPLACED THE
DRAIZE RABBIT TEST FOR IRRITATION

26. INVERTEBRATE
ANIMALS
Drosophilia
melanogaster, a fruit fly
Caenorhabditis elagans,
a nematode worm

27. Drosophilia
melanogaster
Short Life Cycle
Pharmacologically advantageous over Vertebrates
Used for Detecting
Mutagenecity
Teratogenecity
Reproductive Toxicity

28. VERTEBRATE
ANIMAL-Zebra
Fish
Easy to Handle & Maintain
Inexpensive
Molecular resemblance with vertebrates
Used for screening the tissue/organ specific
toxicity
Screening of Psychotropic Drugs

29. IN SILICO
MODELS
Computer aided molecular drug design CADD
Quantitative structure activity relationships QSAR
Computer assisted learning CAL
Computer mathematical analysis CMA
Microfluidic Chips
DNA/Organ Chips

30. The EU regulation concerning the Registration, Evaluation,Authorisation and
restriction of Chemicals (REACH), which came into force in June 2007, aims to
protect humans and the environment from the adverse effects of the use of
chemicals.
A wide range of in silico tools are available that can predict the ADME
characteristics of a chemical (determinants of its internal exposure) as well as
its intrinsic activity (toxicity).
Quantitative structure–activity relationship (QSAR) or quantitative structure–
property relationship (QSPR) models to be developed.
Since 2013, the Cosmetics Regulation (Regulation (EC) No 1223/2009)
has banned the testing of cosmetic ingredients and products on animals,
and has prohibited the marketing of cosmetics for which the ingredients
or products were tested on animals since the introduction of the ban.

31. Target Selection Lead
Discovery
Medicinal
Chemistry
In Vitro
Studies
Preclinical
studies
Clinical
Trials
Library
Development
SAR Studies
In Silico
Screening
Chemical
Synthesis
In silico screening
• Computer simulated screening of chemicals
• Helps in finding structures that are most likely to
bind to drug target.
• Economic than HTS

34. LIGAND BASED
▶ Don’t know receptors
▶ Know ligands
STRUCTURE BASED
▶ Don’t know ligands
▶ Know receptor structures
STRUCTURE BASED AND
LIGAND BASED DRUG DESIGNING

36. STRUCTURE BASED DRUG
DESIGNING
▶ Protein structure determination (HOMOLOGY MODELING,
FOLDING RECOGNITION, Ab initio PROTEIN MODELING)
▶ Docking
▶ Binding free energy
▶ Flexibility of protein-ligand complex
▶ De novo evolution

37. DOCK
 DOCK is a fragment based method using shape and chemical
complementary methods for creating possible orientations for the
ligand.
 These orientations can be scored using scoring functions such as
solvation or hydrophobicity.

38. 1. Get the complex from protein data bank
2. Clean the complex
3. Add the missing hydrogen / side chain atoms and minimize the
complex
4. Clean the minimized complex
5. Separate the minimized complex in macromolecule (lock) and ligand
(key)
6. Prepare the docking suitable files for lock and key
7. Prepare all the needing files for docking
8. Run the docking
9. Analyze the docking results
STEPS INVOLVED IN DOCKINGPROGRAM

39. There are to types of docking that are :-
1. Rigid docking : In rigid docking the molecules are rigid, in 3D space
of one of the molecule which brings it to an optimal fit with other
molecule in terms of scoring function. Also the internal geometry of
both the receptor and ligand are rigid.
2. Flexible docking : In this type of docking the molecules are flexible,
conformations of the receptor and ligand molecules as they appear in
complex.
TYPES OF DOCKING

40. Sr. No. Docking
Program
Year
Published
DockingApproach
1. DOCK 1988 Shape fitting
(sphere sets)
2. Auto Dock 1990 Genetic
Algorithm, Simulated
Annealing
3. Flex X 2001 Incremental
construction
4. FRED 2003 Shape fitting
5. VLifeMDS Protein-ligand based design
6. FLOG 1994 Rigid body docking program
7. HADDOCK 2003 Protein-Protein docking, Protein-
Ligand docking
Common Software's Used for Docking Purpose

42.  It is used in determination of the lowest free energy structures for the
receptor-ligand complex.
 It is also used to calculate the differential binding of a ligand of two
different macro-molecular receptors.
 Study the geometry of a particular complex.
 It can also be used to predict the pollutants that can be degraded by
enzymes.
 De novo design for lead generation.
 To check the specificity of the potential drug against homologous
proteins through docking.
 Docking is widely used as a tool for predicting protein-protein
interaction.
APPLICATIONS OF MOLECULAR DOCKING

43. LIGAND-BASED DRUG DESIGN
▶ Quantitative structure-activity relationship (QSAR)
▶ CoMFA
▶ CoMSIA

44. QUANTITATIVE STRUCTURE-
ACTIVITY RELATIONSHIP
▶ Employs statistics and analytical tools to
investigate the relationship between the
structures of ligands and their corresponding
effects.
▶ Mathematical models are built based on
structural parameters to describe
▶ Earlier 2D-QSAR, but 3D-QSAR have been
adopted
▶ 3D-QSAR methodologies: CoMFA, CoMSIA

45. Molecular Structure ACTIVITIES
Representation Feature Selection & Mapping
Descriptors
Quantitative structure-activity relationships correlate, within
congeneric series of compounds, their chemical or biological
activities, either with certain structural features or with
atomic, group or molecular descriptors.
Quantitative StructureActivity Relationship (QSAR)

46. WHY DO WE NEED DESCRIPTORS?
• Relate structure to activity (QSAR).
• Descriptors act as independent variable.
• Describe different aspects of molecules.
• Compare different molecular structures.
• Compare different conformation of same
molecule.

47. Data
Selection
Descriptor
Evaluation
T
raining and
T
est set
selection
Variable
selection
Statistical
Evaluation
Model
Evaluation
Model
Interpretation
LEAD IDENTIFICATION
AND
MODIFICATION
Typical Work flow of QSAR Studies

48. TYPES OF QSAR
• 1D-QSAR correlating activity with global molecular properties like pKa, log P
,
etc.
• 2D-QSAR correlating activity with structural patterns like connectivity
indices, 2D-pharmacophores, without taking into account the 3D-
representation of these properties.
• 3D-QSAR correlating activity with non-covalent interactionfields surrounding
the molecules.
• 4D-QSAR additionally including ensemble of ligand configurations in 3D-
QSAR.
• 5D-QSAR explicitly representing different induced-fitmodels in 4D-QSAR.
• 6D-QSAR further incorporating different solvationmodels in 5D-QSAR.

49. CoMF
A
▶ Comparative molecular field analysis
▶ Biological activity of a molecule is dependent of
the surrounding molecular fields (Steric and
electrostatic fields)
▶ Has several problems

50. CoMSIA
▶ Comparative molecular similarity index analysis
▶ Includes more additional field properties
▶ Steric
▶ Electrostatic
▶ Hydrophobic
▶ Hydrogen bond donor
▶ Hydrogen bond acceptor
▶ Can offer a more accurate structural-activity
relationship than CoMFA

51. DRUG DISCOVERY & DEVELOPMENT
Identify disease
Isolate protein
involved in
disease (2-5 years)
Preclinical testing
(1-3 years)
Formulation
Human clinical trials
(2-10 years)
Find a drug effective
against disease protein
(2-5 years)
Scale-up
FDA approval
(2-3 years)
Drug Design
- Molecular Modeling
- Virtual Screening

52. Freely available databases for toxicological, physico-chemical and other relevant information for
safety assessment.
database Website details and further information
AMBIT http://cefic-lri.org/toolbox/ambit/
Developed by European Chemical Industry Council’s Long Range Initiative (Cefic-LRI), it contains information on >450,000
chemicals including the European Chemicals Agency’s (ECHA’s) REACH data.
Chemspider http://www.chemspider.com/
Developed by the Royal Society of Chemistry, it provides information on over 83 million chemicals, using 275 data sources;
includes direct links to other relevant resources.
ChemIDplus https://chem.nlm.nih.gov/chemidplus/
Developed by the US National Library of Medicine; contains information relating to >300,000 chemical structures including
physico-chemical property and toxicity data.
Computational
Toxicology
Dashboard
https://comptox.epa.gov/dashboard
Hosted by the US Environmental Protection Agency (US EPA); a repository of data currently for 875,000 chemicals; links out to
additional data sources; integrates data e.g. from ToxCast/Tox21 high-throughput screening initiatives.
PubChem https://pubchem.ncbi.nlm.nih.gov/
Open chemistry database from US National Institutes of Health (NIH) with data on over 102 million chemicals.

53. Freely available databases for toxicological, physico-chemical and other relevant information for
safety assessment.
database Website details and further information
eChemPortal http://www.echemportal.org
Developed in collaboration with the Organisation for Economic Cooperation and Development (OECD), provides links
to information prepared for governmental chemical reviews at national and international levels, including
submissions to the European Chemicals Agency (ECHA); provides exposure and use information.
EMBL-EBI/ChEMBL https://www.ebi.ac.uk/
https://www.ebi.ac.uk/chembl/
European Molecular Biology Laboratory’s European Bioinformatics Institute (EMBL-EBI); source of biological and
biomolecular data incorporating the ChEMBL database of bioactive molecules with drug-like properties (>15 million
values from >1.8 million chemicals).
OCHEM https://ochem.eu/home/show.do
Online chemistry database with modelling environment; 2.9 million records for over 600 properties, based on the
wiki principle.
QSAR Toolbox https://www.qsartoolbox.org/
Developed to help fill data gaps in (eco)toxicity data; version 4.4 contains 57 databases, with 2.6 million data points
for 92,134 chemicals.

54. Databases are usually searchable by using a range of chemical
identifiers, such as:
– Name
– Simplified Molecular Input Line Entry System (SMILES) string
– Hashed code derived from the International Chemical Identifier
(i.e. InChIKey);
– Registry number (e.g. Chemical Abstracts Service (CAS)
– European Inventory of Existing Commercial chemical Substances
(EINECS) number).

55. PubChem: It can be searched by using name, synonyms, molecular
formula, structure, SMILES, InChIKey or registry number. It is also
possible to search for chemicals that are similar with respect to 2-D
fingerprint or physico-chemical properties. The type of information
available is divided into approximately 20 major categories (depending
on the nature of the chemical), and each major category expands into
multiple subcategories providing information on, for example: identifiers;
chemical and physical properties; uses; pharmacology; safety/hazard
data; and toxicity data references.
Chemspider is another comprehensive resource with information on
identifiers, physical properties and chemical properties (experimental
and/or predicted values), with links to predictions from ACD/labs,
EPISuite, Chemaxon and Mcule. Chemspider provides information on
common uses, chemical class, safety information, references, and links to
other sources of information.

56. ChemIDplus is searchable by using a range of
identifiers, and provides chemical classification
codes, physical property and toxicity data (e.g. LD50
data for multiple species and routes) with links to
original references.
The Computational Toxicology (CompTox) Chemicals
Dashboard can be searched by chemical identifiers
(e.g. CAS number), product categories and
assays/genes associated with high-throughput
screening. It provides extensive information on
chemistry, toxicity and exposure data, including
physical and chemical properties, environmental
fate, usage, in vivo toxicity data and results from a
wide range of in vitro assays.

57. – Validity of data can be defined as “evaluating the method used to generate data relative to
accepted guidelines” or “the extent to which the methods used find the truth as a result of
the investigator actually measuring what they intended to measure.”
– Accuracy can be defined as “the closeness of agreement between test method results and
accepted reference values.”
– Reliability of data is linked to the reliability of the experiments carried out. For example,
whether the results can be confirmed by comparison to standards, and whether the
methodology is repeatable.
– Relevance is the relationship between the test that is carried out and the effect that is of
interest (i.e. the meaningfulness of the assay). For example, the highest quality data are
required for the safety assessment of individual chemicals; however, lower quality data may
suffice for general screening or ranking of chemicals in product development.

58. There are numerous examples of software (freely available and commercial) that can generate simple physicochemical
properties for chemicals, apply rules-of-thumb for predicting properties or performing read-across.
Software Website details and further information
ACD/PhysChem
Suite
http://www.acdlabs.com/products/percepta/
Prediction of properties: physico-chemical; ADME; toxicity.
ADMET Predictor http://www.simulations-plus.com/
Prediction of properties: physico-chemical; ADME; toxicity.
AMBIT http://cefic-lri.org/toolbox/ambit/
Freely available: incorporates extensive database, integrates models for toxicity prediction;
provides a workflow to support category formation and read-across.
AutoDock http://autodock.scripps.edu/
Freely available suite of automated docking tools to predict interaction between small molecules
(e.g. substrates or drug candidates) and receptors.
ChemMine Tools https://chemminetools.ucr.edu/
Freely available: tool for similarity analysis or clustering of chemicals based on physico-chemical
or structural similarity.
Cloe PK www.cyprotex.com
Prediction of human pharmacokinetic properties; physiologically-based pharmacokinetic
modelling.

59. There are numerous examples of software (freely available and commercial) that can generate simple physicochemical
properties for chemicals, apply rules-of-thumb for predicting properties or performing read-across.
Software Website details and further information
ToxMatch https://sourceforge.net/projects/toxmatch/
Freely available software for similarity analysis; can be used for grouping chemicals into
categories.
EPISUITE http://www.epa.gov/opptintr/exposure/pubs/episuitedl.htm
Freely available suite of programs from the US EPA; prediction of properties: physico-chemical;
dermal uptake; toxicity to aquatic organisms (fish, Daphnia, algae).
KNIME https://www.knime.com/
Open platform enabling development of nodes for multiple applications, e.g. Indigo, CDK and
RDKit chemoinformatics tools for QSAR descriptor generation, 2-D and 3-D model building,
conversion of chemical identifiers, structure generation, substructure searching, fingerprinting,
etc.
OCHEM https://ochem.eu/home/show.do
Freely available database; operates on the wiki principle; capacity to screen chemicals.against
numerous structural alerts for toxicity (human health and environmental).
VEGA HUB https://www.vegahub.eu/
Free available software; prediction of a range of toxicity endpoints using QSAR models.

60. SwissADME, from the Swiss Institute of
Bioinformatics
(http://www.swissadme.ch/index.php).
Molinspiration
(http://www.molinspiration.com/)
readily identifies chemicals with
potential Lipinski Rule of Fives
violations.
freely available
web-based
application

61. • CAL deals with a range of software packages which simulate the animal experiments
• Two softwares are currently used in india
• Expharm- developed by JIPMER, India Contains programs on Effect of drugs on the rabbit eye
Bio assay of histamine using guinea pig ileum Effect of drugs on the frog heart Effect of drugs on
dog blood pressure and heart rate Effect of drugs on the ciliary movement of frog esophagus •
The user can conduct experiment and collect data • Each program can be run in two modes- a)
tutorial mode , (b) examination mode
• X-cology • video demonstrations of different procedures like isolation and mounting of animal
tissues • Screen interactive interface to study the effects of various drugs on the isolated tissues •
Content is classified into three sections Experimental animals Equipment Experimental
technique – procedure to carry out bioassay and experiments on whole animals
Computer assisted learning
(CAL)

62. Advantages
• Alternative scientific tests are often
more reliable than animal tests.
• The use of human tissue in toxicity
testing is more accurate than the animal
models.
• Cruelty-free products are more
environmentally friendly.
• The development of new technologies,
and the use of all available tools, in
combination, will drive forward the
replacement of animal tests with
scientifically-justified, mechanistically-
interpretable and species-relevant
alternative methods.

63. THANK
YOU!