Pharmacological screening by harikesh maurya

CORRELATIONS BETWEEN

VARIOUS ANIMAL MODELS AND

HUMAN SITUATIONS

By: Harikesh MauryaResearch Scholar

Animal models, especially in pathophysiology and

toxicology, can be used to predict human outcomes.

Whether animals can be used to predict human response

to drugs and other chemicals is apparently a controversial

issue. However, when one empirically analyzes animal

models using scientific tools being able to predict human

responses.

INTRODUCTION

As predictive models of human for investigation of cancer & AIDS.

As predictive models of human for testing drugs/chemicals.

As “spare parts”, e.g. receives an aortic valve from a pig.

In bio-production of insulin/monoclonal antibodies.

In the study of basic physiological principles (tissues).

As educational training for medical/biology students.

As a basic component (model) of science & research.

In research design to benefits other animal of the same species.

ANIMALS IN RESEARCH CATEGORIES

PROBLEMS ASOCIATED WITH ANIMALS

The animal study trying to address three research problems:

(1) Failure of preclinical models to represent human disease

(2) Emerging problem related study of human-specific genes

(3) Poor track record of drug development for interpretation

In fact, clinical trials are essential because animal studies do

not predict with sufficient certainty correlating to human being.

Because

The common causes of reduced external validity of animal studies:

Animals used for experiment are young and healthy, whereas in patients

the disease mainly occurs in elderly people with co-morbidities.

Assessment of treatment efficacy in a homogeneous group of animals vs.

a heterogeneous group of patients.

Insufficient similarity for inducing a disease/injury in animal models .

Differences in outcome measures and assessment timing between

preclinical and clinical study.

POOR VALIDITY OF ANIMAL STUDIES

Decades-old tests that could not be

validated for current purpose.

Least predictive of human responses,

esp. for different patient populations .

Species variation and extrapolation.

Poor Reliability/reproducibility of disease models.

Expensive, time-consuming and slightest agreeable to HTS.

Research data interpretation from animals to humans is not as

efficient as studying humans directly.

RESTRICTION OF ANIMAL STUDIES

According to FDA 92% of drugs that passes preclinical testing, highly

found to be fail in clinical trials.

“Now it must be required to modernize the critical development path that

leads from scientific discovery to the patients” regulating;

Safety Assessment

• Animal toxicology is laborious, time-consuming, requires large quantities

of product, and may fail to predict the specific safety problem.

• ADME testing problems responsible for 60-90% of drug failures.

Efficacy Assessment

• Currently available animal models have limited predictive value.

• Attempting to improve poor animal models is totally wastage .

ANIMAL STUDY- A CRITICAL PATH IN FDA

The multi-constraint theory provides three criteria as follows;

1. Structural consistency: The analogy presents a structural

isomorphism (e.g. Mouse & Patient)

2. Similarity: Corresponding terms are similar in meaning (e.g.

look-for & hope-for are similar and because are identical)

3. Realistic coherence: The analogy successfully relates the

patient’s behavior to the treatment (e.g. medicine & devices).

CRITERIA FOR ASSESSING ANALOGS

Cont…

Comparison between animal & human in PFS

(Porsolt Forced-Swim) test

Animal Human

Mouse Patient

Longer-time Further-extent

Look-for (mouse, safety) Hope-for (patient, goals)

Persist (mouse, longer-time) Persist (patient, further-extent)

Animal tumors are inherently different from human tumors.

Generally grow much more quickly and also regress spontaneously.

Natural animal tumors are generally of different types than human

ones, e.g., mice highly suffer from bone & muscle tumors than human.

Metabolism is significantly divergent between species, impacting both

response to cancer-causing chemicals & chemotherapeutic drugs.

Induction of cancer in animals is highly unnatural and irrelevant to the

real environmental and genetic risks in humans.

Animals did not develop cancer when exposed to tobacco. A similar

situation was true for asbestos, coal tar, and other hazards.

PROSPECTS OF ANIMALS IN CANCER

THERAPIES

In vivo assays are not usually performed after achieving the lead

optimization stage.

The large number of preliminary hits from high-throughput

screening (HTS) requires, which is high cost with low speed of

conventional models.

Preliminary study is required earlier, before performing in vivo

testing of chemicals.

Thus, researchers have developed new model systems using both

vertebrates & invertebrates for drug screening.

NEW MODEL FOR BOTH VERTEBRATES &

INVERTEBRATES

Generally researchers do not use humans for their investigations because of the

obvious risk to life. Instead, they use various animal, fungal, bacterial, and plant

species as model organisms for their studies.

Table 1: Models used to study genetic principles and human diseases

Common Name Research Applications

Yeast Biological studies of cell processes (mitosis) and diseases (cancer)

Pea plant Used by Gregor Mendel to describe patterns of inheritance

Drosophila Gene mapping, recombination technology and mutant screens

Roundworm Development of nervous systems and  aging process

Zebrafish Mapping and identifying genes involved in organ development

House mouse Study genetic principles and human disease

Brown rat Study genetic principles and human disease

NEW MODELS FOR GENETIC DISEASE

Model organisms such as fruit flies, mice, and zebrafish are useful for investigating gene function because they are easy to grow, dissect, and genetically manipulate in the laboratory.

The most popular invertebrate model organisms, Drosophila and

Caenorhabditis elegans, have been used extensively in many areas of

biological research, especially genetics and development.

These models is supported by the existence of highly conserved molecular

pathways between invertebrates and humans, such as the MAP (Mitogen-

activated protein) kinase pathway.

Combined with the powerful genetics, cellular and molecular biology tools,

are very suitable for drug discovery & research.

The large number and stereotypic pattern of the 800 unit eyes (ommatidia)

make it easy for researchers to identify genes interacting with a genetic

diseases or drugs affecting components of the pathway (Figure 1).

Cont…

NEW SCREENING MODEL - ‘THE FLY’

EnVivo Pharmaceuticals has developed models for neurodegenerative diseases based on

the expression of human disease genes in the fly. These models mimic the characteristic

neuropathology and specific symptoms of the diseases, and they can be used for drug

screening.

Figure 1. An eye on flies. Researchers use scanning electron microscope imaging to view differences between wild-type D. melanogaster (A), which has a normal compound eye with accurate arrangement of unit eyes (ommatidia), and a mutant fly (B), which has a small and rough compound eye with dramatically fewer ommatidia. (Photos courtesy of Kevin Moses, Emory University.)

NEW SCREENING MODEL - ‘THE FLY’

The well-characterized invertebrate model C. elegans, contains fewer

than 1000 somatic cells. Its simple structure and transparency allow

for direct observation of cellular phenotypes (Figure 2).

Researchers have extensively studied apoptosis in C. elegans and found

its regulation is highly similar to mammals. Thus, the worm provides a

convenient model for studying genes involved in apoptosis and

screening for developing treatment of cancer and neurodegenerative

diseases.

Researchers have also created imaging algorithms for automated real-

time analysis of living worms, enabling HTS based on large-scale

behavioral analysis.Cont…

NEW SCREENING MODEL - ‘THE WORM’

Figure 2. A worm with a view. Researchers use bright-field imaging (A) to see individual cells in C. elegans or fluorescent imaging (B) to see particular cells expressing green fluorescent protein. (Photos courtesy of Jae Hyung An and Keith Blackwell, Joslin Diabetes Center, Harvard Medical School.)

NemaRx Pharmaceuticals is using the worm to test drugs for disorders

affecting the nervous system, including pain and Alzheimer’s disease. Because

of their small size and simple structure, C. elegans are very suitable for high-

throughput in vivo screenings.

Recently, researchers have created assays based on the zebrafish (Danio rerio), a

small freshwater teleost. Zebrafish embryos are transparent and develop

externally from the mothers, permitting direct assessment of drug effects on

internal organs and tissues in vivo (Figure 3).

Figure 3. Crystal clear analysis. The see-through nature of the zebrafish allows researchers to distinguish a normal specimen (A) stained with an antibody that highlights the kidney (arrow) from a drug-treated individual (B) with smaller kidneys (arrow). (Photos courtesy of Phylonix Pharmaceuticals)

NEW SCREENING MODEL - ‘ZEBRAFISH’

SCREENING MODEL FOR APOPTOSIS

The apoptotic processes in zebrafish and mammals are similar, and most

mammalian apoptosis-related genes have been identified.

Apoptosis can easily detect in live zebrafish embryos by using acridine

orange and fluorescence-conjugated caspase substrate.

Researchers have developed quantitative assays, performed in 96-well

micro-plates in conjunction with a fluorescent plate reader, enabling HTS.

Researchers can induce apoptosis in specific populations of cells to mimic

certain disorders.

Example: Aminoglycoside antibiotics can cause degeneration of hair cells in

zebrafish neuromasts, similar to the adverse effects of these antibiotics in

human hair cells.

SCREENING MODEL FOR TUMORIGENESIS

Zebrafish are responsive to carcinogenic chemicals and develop

neoplasm, similar to human cancers.

Because of the rapid development of the zebrafish embryo, researchers

can use it for testing drugs that affect cell proliferation. E.g., Moon et al.

discovered novel microtubule inhibitors by zebrafish embryo screening.

It is an excellent model for screening of specific cancer therapeutics,

such as angiogenesis and apoptosis modulators. Angiogenesis pathways

in zebrafish and mammals are highly conserved.

Zebrafish homologues of several important mammalian angiogenesis

regulatory genes are expressed in patterns similar to those in mammals.

Human anxiety disorders represent a multi-factorial

phenomenon frequently comorbid with major depression and

other psychiatric problems, while in animal models which is

consistently reflect.

When using experimental models to understand homologies

between animal and human behavior, we have to consider the

circumstance for animal investigation.

Both the functional significance and relevance of the behavioral

parameters that are quantified.

DEPRESSION AND ANXIETY MODEL

The incidence of stroke increases with age and other health problems

that might increase stroke risk, complicated clinical course and affect

functional outcome.

Animals used in stroke models were almost invariably young and

female animals were highly used. Over 95% of the studies were

performed in rodents because of biologically closer to humans.

Functional outcome is the primary measure in clinical efficacy,

whereas preclinical rely on infarct volume.

The assessment time for animal model is 1–3 days, whereas for

human it is 3 months.

STROKE MODEL

Animal models have been developed to study various aspects of

alcohol use and dependence, including alcohol-seeking behavior,

alcohol-related organ damage, tolerance to alcohol, and physical

dependence on alcohol. Because animal models can be genetically

manipulated, they are also valuable for research into the genetic

determinants of alcoholism, because they allow researchers to use

methods that cannot be used with human subjects.

MODELS FOR ALCOHOLISM

Agents Mouse Rat Rabbit Hamster Primate Dog Cat

Butyl benzyl phthalate + + –

Mono benzyl phthalate + – –

Carbaryl ± – ± – – +

Diazinon – + – – +

Dieldrin + ± – + –

Diethylstilbestrol + – – – –

Lead acetate – + – + +

Methyl mercury + + + + – + +

Polychlorinated Biphenyls – – – ± +

* Evidence of harmful effects (+), no evidence of harmful effects (–), or equivocal evidence (±)

Source: Schardein JL. Chemically Induced Birth Defects, 3rd Ed. Rev, pp 1109. Marcel Dekker (2000).

SPECIES DIFFERENCES IN PREDICTIONS OF

CHEMICALLY INDUCED BIRTH DEFECTS*

Target discovery

• Genomics profiles of human tissues

• Proteomics profiles of human tissues

• Epidemiology with genetic analysis

Safety and efficacy testing

• In vitro (tissue cultures, physicochemical)

• Genomics, proteomics & imaging

biomarkers in clinical studies.

HUMAN-BASED DEVELOPMENT MODEL

• Human molecular biology & chemical databases, QSARS, computer

modeling and simulation as predictive model.

Improvement of in vitro methods for measuring human ADME patterns

of chemicals.

Development of guidance on the use of physiologically-based models in

chemical risk assessment.

Development of prototype PB models to integrate exposure,

toxicokinetic & toxicity data for hazard assessment.

Developing databases providing critical parameters to build the in vitro

models.

Bioinformatics tools/algorithms in order to analyse and integrate data.

Faster results, Less expensive, Greater repeatability, reproducibility and

more predictive.

ADVANTAGES OF MODERN HUMAN-BASED

IN VITRO TECHNOLOGIES

When animal models are employed in the study of human disease,

they are frequently selected because of their similarity to humans in

terms of genetics, anatomy, and physiology.

Rodents are the most common type of mammal employed in

experimental studies, and extensive research has been conducted

using rats, mice, gerbils, guinea pigs, and hamsters.

The majority of genetic studies have employed in mice, not only

because their genomes are so similar to that of humans, but also

because of their availability, ease of handling, high reproductive rates.

MODELS FOR STUDY OF HUMAN DISEASE

Despite their genomic similarities to humans, most model organisms

typically do not contract the same genetic diseases as people, so

scientists must alter their genomes to induce human disease states.

Scientists approach this task in two main ways: one that is directed &

disease driven, and the other is non-directed & mutation driven.

The non-directed, mutation-driven method uses radiation and chemicals

to cause mutations.

The directed, disease-driven approach can employ any techniques,

depending on the exact type of mutation involved in the disease.

Common directed techniques include transgenesis, single-gene knock-

outs and knock-ins, conditional gene modifications, and chromosomal

rearrangements.

METHODS OF INDUCING HUMAN DISEASE

Animals are screened in an attempt to determine randomly make

mutations, which ones show phenotypes that are similar to human

diseases. Thus, instead of being driven by the disease mutation.

The most effective ways to generate mutations are by exposing

organisms to X-rays or chemical N-ethyl-N-nitrosourea (ENU).

X-rays often cause large deletion and translocation mutations that

involve multiple genes, whereas ENU treatment is linked to mutations

within single genes, such as point mutations.

ENU can produce mutations with loss and gain of function, and

frequently employed in screening model such as zebrafish.

SCREENING FOR LARGE-SCALE MUTATION

SCREENING MODEL OF TRANSGENESIS

Transgenic animals are generated by adding foreign genetic

information to the nucleus of embryonic cells by inhibiting gene

expression. This can be achieved by either injecting the

foreign DNA directly into the embryo or by using a retroviral vector to

insert the transgene into an organism's DNA.

Recently, scientists have developed a way to increase the size of the

DNA fragments used in transgenesis by cloning them in yeast or

artificial bacterial chromosomes.

The use of transgenic mice has dramatically increased in the past two

decades, and this type of animal model has contributed greatly in

disease development.

Single-Gene Knock-Out and Knock-In

Knock-out mice carry a gene that has been inactivated, which creates

less expression and loss of function

knock-in mice are produced by inserting a transgene into an exact

location where it is over expressed.

In this technology, embryonic stem (ES) cells can contribute to all cell

lineages when injected into blastocysts, and they can be genetically

modified and selected for the desired gene changes. 

Many knock-out and knock-in mice have similar, identical, phenotypes

to human patients and good models for human disease.

CHROMOSOMAL REARRANGEMENT

Mouse models of these disorders can be

created by using indirect approaches, such

as radiation, but their usefulness is restricted

because pathological end points are unpredictable and undefined.

Site-specific recombinase technology necessary to produce accurate

models of defects caused by human chromosomal rearrangements.

These mutations can include chromosome deletions, duplications,

inversions, and translocations, as well as nested chromosome

deletions.

NEW TOOLS FOR DISEASE RESEARCH:

REPROGRAMMED CELLS IN DISEASE MODELLING

Research is often limited by access to patients and availability of diseased tissues

to study. 'Disease models' can help overcome these problems by enabling

scientists to examine diseases in the lab. Stem cells, including reprogrammed or

'iPS' cells, a new source of cells that can be used as models for human diseases.

Animal models, such as mice, are widely used in research as they have

been studied for many years and can develop symptoms similar to

human disease symptoms.

Animals can never fully mirror all aspects of human biology.

Treatments that have been developed and found to be effective in

animal models can give vital clues and information, but do not always

work when transferred to human patients.

Disease models based on human cells can help tackle these problems.

In particular, using cells avoids the difficulty of differences between

animals and humans because scientists can study human cells.

HUMAN CELLS IN DISEASE MODELLING

Cont…

Research on cancer cells contributed significantly to this progress

because cancer cells can grow for a prolonged and even indefinite

period of time if given the right nutrients.

Healthy cells have a limited lifespan and are much more difficult to

maintain and multiply in a dish. While some cells, such as skin cells or

blood cells, can be taken from a patient relatively easily.

Example: Human brain cells are difficult to extract without major

surgery and study a large number of neurodegenerative diseases.

Certain types of stem cells may offer a solution.

HUMAN CELLS IN DISEASE MODELLING

A new type of stem cell – iPS cells are completely new, lab-made type of

stem cell have been widely adopted for disease modelling. Experiment needed

to understand the reprogramming process and its effects on the human cells.

Recreating a disease – iPS cells recreate features of a disease outside the

patient by generating cells with the patient’s genetic code. Diseases are often

fully or partially caused by errors in DNA code & other biological processes.

Diseases often affect many types of cells – iPS disease models allow

researchers to study how specific types of cells are affecting complex tissues

or organs containing several different kinds of cells in many interacting layers.

CHALLENGES FOR  INDUCED PLURIPOTENT

STEM CELLS (iPSCs) DISEASE MODELLING

Disease modelling with iPS cells: Skin cells from a patient can be reprogrammed to behave like embryonic stem cells. Scientists can then watch their development to model what happens in the diseased human body.

Current and future disease modelling with iPS cells

By January 2012, several studies had managed to recreate some

features of the disease in question and had tried to create a cell-based

disease model.

Some of the neurological diseases that have been studied in detail

with iPS cells are spinal muscular atrophy (SMA), Down’s syndrome,

familial dysautonomia, schizophrenia and Alzheimer’s disease.

Disease models using iPS cells are also under development for blood-

related, metabolic, cardiovascular, immunodeficiency and other

conditions.

Recent (February 2012) an Alzheimer’s disease was created by using iPS

cells skin samples taken from Alzheimer’s disease patients and from

healthy human. The researchers then made nerve cells from these iPS

cells in the laboratory over a period of about 8 weeks.

Then purified the nerve cells and compared those from Alzheimer’s

patients with the non-Alzheimer’s samples. The disease is age-related and

affects the human body after many years of life, while the laboratory nerve

cells are only 8 weeks old.

Based on the findings of this study, the Alzheimer’s disease can be

modelled in the lab using human Alzheimer’s-iPS cells. Now research

groups continued the search for the causes of Alzheimer’s and can work

towards developing an effective treatment.

iPS disease modelling for Alzheimer’s

CONCLUSION

Animal models have greatly improved our understanding of the cause

and progression of human genetic diseases and have proven to be a

useful tool for discovering targets for therapeutic drugs.

Most available animal models are made in rodents and they recreate

some aspects of the particular human disease. Perhaps using non-

human primates might alleviate some of these discrepancies because

their physiology is closer to that of humans.

Indeed, because of the tremendous genetic resources that are

currently available, use of stem cell (iPS) models might become more

accessible and may lead us into a new era of disease research

and drug discovery.

Niall Shanks, Ray Greek and Jean Greek. Are animal models predictive for humans? Philosophy, Ethics, and Humanities in Medicine 2009, 4:2

Schardein JL. Chemically Induced Birth Defects, 3rd Ed. Rev, 1109 pp. New York: Marcel Dekker (2000).

Yadid G, Nakash R, Deri I, et al. Elucidation of the neurobiology of depression: insights from a novel genetic animal model. Prog Neurobiol. 2000;62:353-378.

Nestler EJ, Gould E, Manji H, et al. Preclinical models: status of basic research in depression. Biol Psychiatry. 2002;52:503-528.

Cryan JF, A. Markou A, Lucki I. Assessing antidepressant activity in rodents: recent developments and future needs. Trends Pharmacol Sci. 2002;23:238-245.

Amsterdam, A. & Hopkins, N. Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. Trends Genet. 22, 473–478 (2006).

Shepard, J. L. et al. A mutation in separase causes genome instability and increased susceptibility to epithelial cancer. Genes Dev. 21, 55–59 (2007).

Zambrowicz, B. P.; Turner, C. A.; Sands, A. T. Curr. Opin. Pharmacol. 2003, 3, 563–70. Bates, G. P.; Hockly, E. Curr. Opin. Neurol. 2003, 16, 465–470. Ma, C.; et al. Innov. Pharmaceut. Technol., Nov 2003, pp 38–45.

REFERENCES

Animals are sacrificing their life for

human,

But we have nothing to

sacrifice for them!!!

H. Maurya

Thank You