pre-clinical testing Flashcards
Involves experiments conducted on living organisms, typically animals, to observe the effects of a drug in a whole-body system.
In Vivo Testing
Involves experiments conducted on living organisms, typically animals, to observe the effects of a drug in a whole-body system.
In Vivo Testing
Involves experiments performed on isolated cells, tissues, or organs outside of a living organism, providing a simplified model for studying drug effects.
In Vitro Testing
Regulatory Requirements for in vivo Studies
• Global Regulatory Bodies
• Ethical Considerations
• Standardized Protocols
The primary agency responsible for regulating animal use in drug
development in the Philippines
Bureau of Animal Industry (BAI), under the Department of Agriculture (DA)
The BAI oversees and implements the _____, which provides the legal framework for the humane treatment of animals used in research and development.
Animal Welfare Act of 1998 (Republic Act No. 8485)
Other agencies that may be involved
in regulating animal use in drug development in the Philippines
Department of Health (DOH)
Philippine Council for Health Research and Development (PCHRD)
Institutional Animal Care and Use Committees (IACUCs)
They may be involved in setting standards for the use of animals in
biomedical research.
Department of Health
They may provide guidelines and ethical review for research involving animals.
Philippine Council for Health Research and Development (PCHRD)
They are established within research
institutions to review and approve animal use protocols.
Institutional Animal Care and Use Committees (IACUCs)
They play a crucial role in the development and testing of new
drugs.
Animal models
The different rodent models
Mice
Rats
Guinea Pigs
Small size, short lifespan, easy to
breed and handle.
Mice
Larger size, longer lifespan, more
robust physiology.
Rats
Similar to humans in terms of vitamin C requirements.
Guinea Pigs
Non-Rodent Models
Primates
Pigs
Dogs
Rabbits
Closest to humans in terms of physiology and genetics, valuable for studying diseases like HIV and Alzheimer’s
Primates
Used for studying organ transplantation and cardiovascular research, similar size and anatomy to humans.
Pigs
Large animal models for studying cancer, diabetes, and cardiovascular diseases, long lifespan allows for chronic
studies.
Dogs
Used for studying reproductive and developmental toxicology, convenient size and relatively low cost.
Rabbits
They are widely used in drug research due to their physiological similarities
to humans.
Rats
Understanding different rat strains is crucial for selecting the most
appropriate model for specific research questions.
They are are an albino strain characterized by their white fur and pink eyes.
Wistar rats
They are known for their rapid growth and adaptability, making them suitable for various research purposes.
Wistar Rats
They are a popular strain known for their docile nature, rapid growth rate, and high reproductive rate.
These qualities make them suitable for various studies, including toxicology, pharmacology, and safety testing.
Sprague Dawley Rats
Their consistent physiology allows for reliable data collection and interpretation.
Researchers often use these rats for general toxicity testing and to evaluate drug efficacy.
Sprague Dawley
These rats are known for their hooded coat pattern, featuring a darker, pigmented area on the head and shoulders.
Long-Evans
This strain is also recognized for its relatively larger size compared to other commonly used rat strains.
Long-Evans
rats are often used in behavioral research due to their well-documented behavioral patterns and responses.
They are also valuable models for studying neurological disorders
and drug-induced behavioral changes.
Long-Evans
These rats are genetically predisposed to hypertension, characterized by elevated blood pressure.
These rats are commonly used in research on cardiovascular
diseases, including stroke, heart failure, and hypertension.
Spontaneously Hypertensive Rats (SHR)
They are valuable models for evaluating new drugs for hypertension and other cardiovascular disorders.
Their sensitivity to hypertension makes them particularly useful
for testing the efficacy of antihypertensive drugs.
Spontaneously Hypertensive Rats
These rats are genetically predisposed to
obesity and type 2 diabetes.
These rats are characterized by their distinctive white fur and tendency to develop insulin resistance.
Zucker Diabetic Fatty (ZDF) rats
ZDF rats are valuable models for studying diabetes, obesity, and related metabolic disorders.
Researchers utilize ZDF rats to evaluate the efficacy and safety of new drugs for managing diabetes and obesity.
Considerations in Rat Selection
• Genetic Background
• Age and Sex
• Health Status
• Environmental Factors
• Ethical Considerations
Consider the specific genetic makeup of the rat strain, as it can influence susceptibility to diseases and drug responses.
Genetic Background
It can significantly affect physiology and drug metabolism, influencing experimental outcomes.
Age and Sex
Ensure rats are healthy and free from pre-existing conditions that might confound research findings.
Health Status
Controlled environmental conditions, including _____, are crucial for consistent results.
temperature, humidity, and light cycles
It is paramount, ensuring humane treatment and minimizing stress for accurate research outcomes.
Animal welfare
These are essential in evaluating the safety and potential side effects of new pharmaceutical compounds.
These studies are conducted on living organisms, typically animal models, to assess the drug’s impact on various physiological systems.
In vivo drug toxicity studies
Components of Acute Toxicity Studies
• Dose Administration
• Observation Period
• Pathology Analysis
During observation period, animals are closely monitored for signs of toxicity, such as changes in ___, ___, and ____.
behavior
physiology
mortality
Detailed examination of organ tissues to identify any damage or abnormalities.
Pathology Analysis
Components of Sub-chronic Toxicity Studies
• Exposure Duration
• Dose Levels
• Comprehensive Evaluation
Exposure Duration : The drug is administered for a longer
period, typically ____, to assess
medium-term effects.
28-90 days
In acute toxicity studies, ____ of the drug is given to assess immediate, short-term effects.
single, high dose
In sub-chronic toxicity studies, ___ are tested to determine the threshold for toxicity and establish safe dosing ranges.
Multiple dose levels
In-depth analysis of clinical chemistry,
hematology, and histopathology to
identify potential organ-specific toxicity.
Comprehensive Evaluation
Components of Chronic Toxicity Studies
• Long Term Exposure
• Dose Escalation
• Comprehensive Assessment
In chronic toxicity studies, tbe drug is administered for an extended period, typically _____, to assess long-term effects.
6 months to 2 years
Gradually increasing the dose over time to identify the maximum tolerable dose and potential cumulative toxicity.
Dose Escalation
In-depth evaluation of organ function,
histopathology, and any other adverse
effects that may develop.
Comprehensive Assessment
Reproductive Toxicity Studies:
• Fertility
• Embryonic/Fetal Development
• Postnatal Development
• Multi-generation Studies
Assessing the drug’s impact on mating, conception, and sperm/egg production.
Fertility
Evaluating the drug’s effects on the developing embryo and fetus during pregnancy.
Embryonic/Fetal Development
Monitoring the growth and development of offspring exposed to the drug in utero.
Postnatal Development
Examining the drug’s effects over multiple generations to identify any heritable changes.
Multi-generation Studies
Genetic Toxicity Studies
• Mutagenicity
• Clastogenicity
• DNA Damage
Assessing the drug’s potential to cause permanent changes in the genetic material of cells.
Mutagenicity
Evaluating the drug’s ability to induce chromosomal breaks, rearrangements, or other abnormalities.
Clastogenicity
Analyzing the drug’s impact on the integrity and repair of genetic material.
DNA Damage
Pharmacokinetic Studies
• Absorption
• Distribution
• Metabolism
• Excretion
Efficacy Studies
• Proof-of-Concept
• Dose-Response
• Therapeutic Window
• Long-Term Efficacy
Demonstrates the drug’s ability to achieve its intended effect.
Proof-of-Concept
Determines the optimal dosage for efficacy.
Dose-Response
Identifies the range of doses that provide benefit without excessive
toxicity.
Therapeutic Window
Assesses the drug’s effectiveness over extended periods.
Long-term Efficacy
Safety Pharmacology Studies
• Cardiovascular
• Respiratory
• Central Nervous System
• Gastrointestinal
Assess effects on heart rate, blood pressure, and rhythm.
Cardiovascular
Evaluate impact on breathing rate, depth, and lung function.
Respiratory
Examine effects on behavior, cognition, and motor function.
Central Nervous System
Assess impact on digestion, absorption, and bowel movements.
Gastrointestinal
Immunogenicity Studies
• Antibody Response
• Cell-Mediated Immunity
• Cytokine Profile
• Histopathology
Measure the production of antibodies against the drug or its components.
Antibody Response
Analyze the levels of various immune signaling molecules (cytokines) in blood or tissues.
Cytokine Profile
Evaluate the activation of immune cells like T cells, which can directly attack cells
expressing the drug.
Cell-Mediated Immunity
Examine tissue samples under a microscope to assess the presence of immune cells and inflammation.
Histopathology
Applying rigorous statistical methods to identify significant drug-related
effects and trends.
Statistical Analysis
Evaluating how changes in dose affect the magnitude and incidence of observed effects
Dose-Response Relationship
Carefully translating animal data to predict potential safety and efficacy
in human clinical trials.
Extrapolation to Humans
The use of animal models in drug development is a complex issue, balancing the need for scientific progress and the ethical treatment of animals.
Ethical Principles in Animal Research: The 3 Rs
• Replacement
• Reduction
• Refinement
Seek to replace animal models with alternative methods such as in vitro or in
silico approaches whenever possible.
Replacement
Design studies to minimize the number of animals used while maintaining scientific validity.
Reduction
Continually improve procedures to enhance animal welfare and minimize
pain, distress, and suffering.
Refinement
Ensure that the study design and methodology is robust and can yield meaningful, reproducible results.
Scientific Validity
Continually seek and implement
alternative methods that can replace
animal models when feasible.
Alternative Exploration
Carefully weigh the potential benefits
of the research against the risks and
suffering imposed on animals.
Minimizing Harm
Require rigorous ethical review and
approval before conducting any
animal research studies.
Ethical Review
Minimizing Animal Suffering:
• Anesthesia
• Analgesia
• Euthanasia
Utilize appropriate anesthetic agents to eliminate pain and distress during procedures.
Anesthesia
Provide analgesics to manage pain and discomfort before, during, and after experiments.
Analgesia
Ensure humane and painless methods of euthanasia when animals must be sacrificed
Euthanasia
Ensuring Proper Animal Welfare
• Housing
• Nutrition
• Veterinary Care
Provide appropriate, spacious, and enriched environments to support the
animals’ natural behaviors.
Housing
Ensure that animals receive a balanced and nutritious diet to maintain their health and well-being.
Nutrition
Establish comprehensive veterinary oversight to monitor the animals’ health and address any issues promptly.
Veterinary Care
Limitations of In Vivo Studies
• Ethical Considerations
• Species Differences
• High Costs and Time Commitment
• Limited Control Over Variables
Animal welfare is paramount,
necessitating strict ethical guidelines
and regulatory oversight to minimize
animal suffering.
Ethical Considerations
Results from animal models may not
fully translate to humans due to physiological and metabolic variations.
Species Differences
In vivo studies involve significant
financial investments and time to
conduct, especially for long-term
studies.
High Costs and Time Commitment
Individual animal responses can vary,
influencing the reliability of study
results and requiring careful statistical
analysis.
Limited Control over Variables
Balancing Scientific Progress and Ethical
Considerations
• Ongoing Dialogue
• Continual Improvement
• Responsible Research
Maintain an open and constructive dialogue between researchers, ethicists, and the public.
Ongoing Dialogue
Continuously strive to enhance
animal welfare and seek alternatives
to reduce reliance on animal
models
Continual Improvement
Ensure that animal research is
conducted with the highest ethical
standards and scientific rigor.
Responsible Research
Most common animal used in pre-clinical tests
Rodent Models
Most common rodent used in the Philippines
Mice
Rats
A fusion of albino and kitchen rats
- stronger / larger
Long-Evans Rats
Smaller than Wistar Rats
Sprague Dawley Rats
Immunomodulation - stimulates the immune system
Immunosuppression - suppression of the immune system
In Vivo studies assess the potential
for a drug to cause adverse effects,
such as ___,___, and ___.
organ damage
reproductive issues
carcinogenicity
Long ears, tail length shorter than body length and wider head
Wistar Rats
Grows faster than Wistar rats (Gain 400 g BW by 12 weeks and Wistar rats gains-350 g BW)
Sprague Dawley Rat
Black hooded rat. Cross between a female albino and a wild male (Rattus norvegicus)
Long-Evans Rat
Animal model of essential (or primary) hypertension used to study cardiovascular disease
Spontaneous Hypertensive Rats
These are a crucial part of the drug development process, allowing researchers to evaluate the potential efficacy and safety of drug candidates before moving to animal and human trials.
In vitro drug studies
Cell-Based Assays:
• Cell Line Selection
• Functional Readouts
• Toxicity Evaluation
Choosing the appropriate cell line is
crucial, as it should be representative of
the target tissue or disease state.
Cell Line Selection
Cell-based assays can measure various
cellular responses, such as proliferation,
differentiation, or signaling pathway
activation.
Functional Readout
These assays can assess the cytotoxic
effects of drug candidates on cells,
helping identify potential safety concerns.
Toxicity Evaluation
Genotoxicity and Mutagenicity Tests
• Ames Test
• Micronucleus Assay
• Comet Assay
Detecting mutagenic potential using bacterial reverse mutation assays.
Ames Test
Evaluating chromosomal damage and aneuploidy in mammalian cells.
Micronucleus Assay
Measuring DNA strand breaks and alkaline-labile sites in individual cells.
Comet Assay
Receptor Binding Assays
• Receptor Specificity
• Binding Affinity
• Functional Activation
These assays determine the selectivity of a drug candidate for its intended receptor target, versus off-target receptors.
Receptor Specificity
Receptor binding assays quantify the strength of the interaction between a drug candidate and its target receptor.
Binding Affinity
They quantify the strength of the interaction between a drug candidate and its target receptor.
Receptor binding assays
Some receptor binding assays also measure the ability of a drug candidate to activate or inhibit the receptor’s downstream signaling pathways.
Functional Activation
Biochemical/Enzyme Activity Assays
• Enzyme Inhibition
• Enzyme Kinetics
• High-Throughput Screening
These assays determine the ability of a drug candidate to inhibit the activity of a target enzyme, which can be a potential
mechanism of action.
Enzyme Inhibition
Enzyme activity assays can provide information on the kinetic parameters of an enzyme-drug interaction, such as the rate of catalysis or inhibition.
Enzyme Kinetics
Enzyme activity assays are well-suited for high-throughput screening of
large chemical libraries to identify potential drug candidates.
High-Throughput Screening
Permeability Studies
• Passive Diffusion
• Active Transport
• Blood-Brain Barrier
Measures the ability of a drug candidate to passively diffuse across cell membranes, which is crucial for oral bioavailability.
Passive Diffusion
Evaluates the potential for a drug candidate to be a substrate or
inhibitor of membrane transporter proteins.
Active Transprt
Assesses the likelihood of a drug candidate to cross the blood-
brain barrier and reach the central nervous system
Blood-Brain Barrier
Metabolic Stability Studies
• Metabolism Pathways
• Metabolite Profiling
These studies identify the major
metabolic pathways and enzymes
involved in the biotransformation of a
drug candidate.
Metabolism Pathways
Metabolic stability assays can provide a
comprehensive understanding of the
drug’s metabolic fate and potential
metabolite-mediated effects.
Metabolite Profiling
