Lecture 17: Pre-Clinical Saftey Testing

Block 8

Why Are Animals Used in Research

• To advance scientific understanding – understand physiology

• As models to study disease – models of pathology

• Preclinical models used to develop and test potential forms of treatment

• To protect the safety of people, animals and the environment – used to determine hazards

Requirements for Drug Approval

• Must demonstrate effectiveness and safety for the intended condition, while complying with regulations

• Multiple regulatory agencies assess risks and benefits to ensure patient safety

  • Must understand the magnitude of the risks and act to minimise them

• Toxicologists help determine safe and effective dose ranges

• Animal and in-vivo models used to assess risks and inform clinical use

  • Help determine safe dose range

Making A Medicine, From Pharmacokinetics to Clinical Testing

• Pharmacokinetics and safety testing continue throughout Phases 1-3 of clinical trials

• Toxicology studies are ongoing, even during the drug's trial phase

Pre Clinical Saftey Testing in the UK

• The Home Office reports and publishes the number of procedures annually

• In 2019, 437,000 procedures were carried out for regulatory purposes, making up 25% of all experimental procedures for regulatory purposes

Regulatory Purpose/Procedure

• Involves routine production, efficacy, tolerance, toxicity, and safety testing

  • 33% of these procedures are for toxicity and safety testing

• Most toxicology procedures are carried out in the commercial sector

• 94% of procedures meet UK/EU legislative requirements

• Mice are the most commonly used species (30% of procedures), followed by chickens

Requirements For Saftey Testing & Use of Rodent and Non-Rodent Species

• Pharmaceuticals are assessed for potential human and environmental risks

• Animal studies generate hazard information

• Safety testing is required in rodents and non-rodents (except in exceptional cases e.g. terminal conditions) to minimise risk to human health

  • Conducted before and throughout the clinical phases of drug development programmes

• Two species are used to account for differences in drug processing, providing a more complete picture of risks involved

• Safety testing complies with safety legislation in the UK, EU, and globally, following the 3R ethical framework to reduce animal use and duplication

Regulatory Organisations

• MHRA – Medicines and Healthcare products Regulatory Agency (UK)

• EMA - European Medicines Agency (EU)

• FDA - Food and Drug Administration (USA)

• ICH - International Conference on Harmonization

• OECD -Organisation for Economic Co-operation and Development

• They produce guidelines and bring together regulatory agencies to respond to global developments and changes in the pharmaceutical sector → aim to prevent duplication

Hazard Vs Risk

• Hazard is the potential to cause harm

• Risk is the likelihood of harm under specific conditions

• If a drug is hazardous but only requires one dose to be effective or for a short period, risks may be considered suitable, but if a drug is hazardous and is required for a longer period, the risk of exposure to toxicity increases

Underlying Principle of Dose Administration

• A large dose is administered to a small number of animals to extrapolate results to humans receiving smaller doses

• Greater risk is acceptable for life-threatening conditions (e.g., chemotherapy), where high risks are tolerable

• Lower risks are prioritised for non-life-threatening conditions (e.g., contraceptive pill), as it is taken daily, and long-term safety is more critical

European Legislation For Acute Dose Toxicity Testing

• Demands that acute toxicity tests must be carried out in two or more mammalian species covering at least two different routes of administration

LD50

• Test created in 1927

• An index of acute toxicity (typically expressed as mg/kg

• Various doses of drug were given to groups of animals % mortality in a set time period (e.g. 2 days) and used to calculate the lethal dose for 50% of the group – calculate n.o of live and dead animals

Problems With LD50

• Only measures mortality – doesn't account for sub-lethal toxicity, altered physiology, or pathology

• Focuses on acute toxicity, not long-term effects

• Varies between species – results are not universally applicable

• Experimental conditions can drastically change results

• Doesn't measure idiosyncratic reactions – can't account for unexpected toxicity

• Requires many animals, causing suffering disproportionate to information gained

  • Toxicity takes many forms and cannot be measured purely in terms of increased mortality

• Removed from OECD guidelines due to ethical and scientific concerns

Fixed Dose Procedure (FDP, OECD TG 420)

• Proposed in 1984, as an alternative to the LD50 test

• The drug is given at one of the four fixed-dose levels (5, 50, 500, and 2000 mg/kg, i.v. and intended route) to 5 male and 5 female animals (allows sex differences to be identified):

  • Once administered, observation of animals over 14 days 

  • Humanely culled Autopsy (macroscopic and microscopic examination )

  • Identify drugs target organ(s) and their effects

Evident Toxicity

• Generated using Fixed Dose Procedure

  • Clear signs of toxicity seen at a given dose

  • Moderate toxicity

    • (not impending death or moribund condition)

• A sliding scale for toxicity – no signs of toxicity and death; want effects to be somewhere in the middle

Fixed Dose Procedure: Next Stages

• If no signs of toxicity occur at the initial dose - retest at a higher dose

  • Test another five male and five female mice

• If a dose is used that produces clear signs of toxicity but no mortality – no further testing needed

• If mortality occurs - retest at a lower dose to assess for evident toxicity

Advantages of FDP

• Fewer animals required

• Males and females are used

• The results relate to animal survival and evident toxicity

• Test through 2 routes of administration – IV and the intended route – results are more related to toxicity events seen at the target organs

• Once dose is identified from the acute toxicity test, it is taken forward to chronic repeat dose toxicity testing

Pre-Clinical Repeat Dose Toxicity Testing

• A compound surviving early tests can move to repeat-dose studies in animals.

• Estimates safe starting dose for human clinical trials.

• Involves testing on two mammalian species, including one non-rodent (e.g. birds, rabbits).

• Chronic toxicity testing lasts up to 2 years (typically 6-9 months).

• Studies the relationship between dose, animal response, and target organ in detail

Focus of Repeat Dose (Chronic) Toxicity Testing

• Investigates the effects of a test substance on vital systems/functions:

  • Central Nervous System: Motor activity, behavioural changes, coordination, reflex responses, body temperature.

  • Cardiovascular System: Blood pressure, heart rate, ECG, repolarisation/conductance abnormalities.

  • Respiratory System: Respiratory rate, tidal volume, haemoglobin oxygen saturation.

  • GI Tract, Liver, and Kidney Function: Function tests to assess organ impacts.

Types of Drug Toxicity

• Toxic Effects Related to Pharmacological Action:

  • Example: Proarrhythmic effects with antiarrhythmic agents, or bleeding with anticoagulants.

• Side Effects Unrelated to Pharmacological Action:

  • Often due to reactive metabolites or immunological reactions.

  • Example: Paracetamol’s metabolites damage the liver.

• Unpredictable and Uncommon Adverse Effects:

  • Detected after widespread use.

  • Example: Vioxx and the increased risk of cardiovascular disease.

Therapeutic Index

• A quantitative measure of the relative safety of the drugs effect → defines the range between non-toxic doses and the maximum effective dose

  • Therapeutic Index = Maximum Non-toxic Dose / Minimum Effective Dose.

• A high value indicates a safer drug with a favourable safety and efficacy profile

• Limitations:

  • doesn’t always guide clinical use effectively.

  • Example: Digoxin has a low TI but is still used, while Thalidomide had a high TI but was withdrawn due to severe side effects.

Mutations Tested in Mutagenicity and Carcinogenicity Studies

• In vitro tests are conducted to identify potential carcinogens and mutagens.

• Drugs may act to cause:

  • Gene (Point) Mutations: Changes in 1 or a few nucleotides.

  • Chromosomal Mutations: Structural aberrations and alterations in chromosomes.

  • Genomic Mutations: Changes in chromosome number, such as aneuploidy.

AMES Test

• Assesses the mutagenic potential of a drug.

• Process:

  • A mutant form of S. typhimurium is used, which needs exogenous histidine to survive.

  • Bacteria are plated on minimal media and exposed to the test drug, with or without a metabolic activation system.

  • Depletion of histidine in the media occurs.

  • After a set time, only bacteria that mutate back to the wild type (WT) can survive once histidine is used up.

    • Can measure drug-induced mutations back to WT

  • Mutagenicity of a drug is proportional to the number of colonies observed after a set time

• Used to identify which drugs cause gene mutations.

Key Features of In Vitro Tests for Mutagenicity and Carcinogenicity

• Tumourogenesis: Tests for the formation of neoplasms and tumours.

• Carcinogenicity: In vitro tests designed to assess the potential of a substance to cause cancer.

In Vivo Carciniogenicity Tests

• Aim: To detect tumorigenic properties, i.e., formation of neoplasms or cancerous tumours (via genotoxic or non-genotoxic mechanisms).

• Tests for Genotoxic Carcinogens: Drugs that directly interact with DNA, causing DNA damage or chromosomal aberrations - can be detected by genotoxicity tests.

• Nongenotoxic Carcinogens: Agents that may not directly damage DNA but alter gene transcription or signal transduction, affecting gene expression and cell signaling.

Conventional Long Term Carcinogenicity Bioassay Test

• Use rats and mice (both sexes).

• Use at least 3 dose levels of the drug and a concurrent control group (age-matched, untreated).

• Duration of study: Mice (18 months), Rats (24 months).

• Animals are dosed daily (Oral, dermal, or inhalation)

• Animal health features are monitored throughout the study.

• Key Assessment: Full pathological analysis of tissues and organs when the study is terminated.

• Substances that induce tumours in animals are considered potential human carcinogens unless proven otherwise.

Reproductive Toxicology

• Assess the toxicity of drugs on mammalian reproduction at all stages (embryo, fetus, offspring).

  • Critical for human risk assessment of drugs.

• Aims to identify toxic effects of drugs on reproductive systems through all stages of development.

• Testing involves one rodent and one non-rodent species.

• Observations conducted throughout the entire lifecycle to detect both immediate and long-term adverse effects.

Stages of Reproductive Toxicology Testing

• Premating to Conception:

  • Assess male & female reproductive function, gametogenesis, mating behaviour, and fertilisation.

• Conception to Implantation:

  • Assess female reproductive function, preimplantation development, and implantation.

• Implantation to Hard Palate Closure:

  • Assess female reproductive function, embryonic development, and major organ formation.

• Hard Palate Closure to End of Pregnancy:

  • Assess female reproductive function, fetal development, and organ development.

• Birth to Weaning:

  • Assess female reproductive function, neonate adaptation to extrauterine life, preweaning development and growth.

• Weaning to Sexual Maturity:

  • Assess post-weaning development, growth, sexual maturity, and offspring development

Types of In Vivo Study Types

• 1^st: fertility and early embryonic development

• 2^nd: embryo and foetal development in 2 different species

• 3^rd: pre- and post-natal development

First stage of reproductive toxicology: Fertility and Early Embryonic Development

• Assess the adverse effects of a drug, prior to mating in males and females and continues throughout mating and implantation in rodents

• Focus on fertility and early embryonic development.

• Female Rodents:

  • Assess effects on oestrus cycle, tubular transport, implantation, and pre-implantation development.

  • Treatment over 2-3 oestral cycles (~14 days) before mating, continuing through implantation.

• Male Rodents:

  • Assess effects on spermatogenesis and epididymis transport following 4 weeks of treatment before mating.

First stage of reproductive toxicology: Fertility and Early Embryonic Development - Key Parameters

• Sexual function

• Sperm analysis

• Pregnancy rate

• Implantation sites

• Litter size at term

• Gross fetal examination to identify deleterious effects of the drug

Reproductive Toxicology: Second stage: Embryo-foetal development

• Asses for any adverse effects on the female during the critical period of organogenesis

• Administer the drug to the pregnant animal during organogenesis, where the impact on the foetus is then examined – assess foetal survival, malformations present or any changes in histology

• Typically evaluated in 2 species, e.g.  rat and rabbit

Thalidomide

• Synthesised in 1953

• Appeared nontoxic in rodent models that a LD50 could not be established- large therapeutic effects/windows – seen as very safe

• Initially marketed as a sedative, but also found to be an anti-emetic (nausea)

• Prescribed to alleviated morning sickness in pregnant women

  • Morning sickness typically occurs in the first trimester – time when organogenesis occurs in humans

Teratogenicity of Thalidomide

• ~12,000 children born with disabilities due to thalidomide, leading to its withdrawal in 1961.

• Thalidomide is a racemic mixture with (R) and (S) enantiomers.

  • The (S) isoform is responsible for teratogenicity.

• The (S) thalidomide binds to the major groove of DNA at purines in promoter regions of genes (inc. those controlling angiogenesis in developing limbs).

• Subsequent testing found it to be a potent teratogen in zebrafish, chickens, rabbits, and monkeys, but not in rodents.

Reproductive Toxicology: Third Stage – Pre- and Postnatal Development

• Aim to assess any adverse effects following exposure of the maternal animal from implantation right the way through to lactation and weaning

• Evaluates the sustained effect of the drug on the pregnant and lactating female animal

• Also, asses the maturation and development of the offspring

• Effects induced in this period can be delayed – important that the development of the offspring is monitored up to sexual maturity

Reproductive Toxicology: Third Stage – Pre- and Postnatal Development: Key Parameters

• Survival

• Postnatal developments

• Non-lethal abnormalities e.g. changes in hearing or BP

• Behaviour

• locomotive

• cognitive impairments

Drugs Withdrawn Following Clnical Testing

• Even after licensing, medicines are continually monitored, which can lead to withdrawal from the market due to side effects.

• Some drugs make it through safety testing and are only withdrawn during clinical trials due to unexpected severe side effects.

  • TGN 1412: An immunomodulatory drug that caused a cytokine storm, leading to severe inflammatory reactions and chronic organ failure.

  • Terfenadine: Caused cardiac arrhythmias when interacted with erythromycin and grapefruit.

  • Cerivastatin: Led to skeletal muscle damage (rhabdomyolysis).

  • Rofecoxib (Vioxx): Increased risk of heart attacks and strokes.