Pre-clinical Toxicology

PRECLINICAL TOXICOLOGY

Dr. Roger Preston
School of Pharmacy & Biomolecular Sciences
Royal College of Surgeons in Ireland
Coláiste Ríoga na Máinleá in Éirinn

LEARNING OUTCOMES

• In Silico Toxicity Testing of Chemicals:

  • Describe the methodologies and applications of in silico modeling in predicting the toxicological profiles of chemical substances.

• In Vitro Toxicology Testing of Drugs:

  • Explain the procedures and significance of in vitro testing in assessing drug toxicity using cell cultures or cellular assays.

• In Vivo Toxicity Testing of Drugs:

  • Illustrate the procedures and importance of in vivo studies in investigating the toxic effects of drugs in living organisms.

• Major Toxicology Endpoints:

  • Discuss critical toxicological endpoints such as lethality, organ damage, and other measurable adverse effects resulting from exposure to toxic substances.

UNDERSTANDING THE DRUG LIFE CYCLE

• Identification of New Drug Candidates:

  • New drug candidates are identified through various methods, including high-throughput screening and rational design.

• Laboratory Studies:

  • Laboratory studies evaluate the safety and efficacy of drug candidates before they enter clinical trials involving human participants.

• Clinical Trials:

  • Clinical trials are designed to evaluate the effectiveness of drugs and monitor their side effects in human participants.

• Regulatory Approval:

  • Once clinical trials demonstrate safety and efficacy, drugs undergo regulatory approval for public marketing, followed by ongoing safety monitoring post-approval.

DRUG DISCOVERY PIPELINE

• Overall Process Length:

  • The complete process of drug discovery, from target identification to clinical trials, typically spans several years, categorized into distinct phases.

Phases:

1. Target Identification and Validation

   • Link disease targets to biomarkers.

   • Duration: 1-2 years

2. Lead Generation and Optimization

   • In silico screening aids in hit generation and confirmation.

   • Duration: 1-2 years

3. Preclinical Animal Studies

   • Safety studies on 20-100 subjects

   • Duration: 1-2 years

4. Phase 1: Safety Trials

   • Involve 100-300 participants; focus on evaluating drug safety.

   • Duration: 1-2 years

5. Phase 2: Efficacy and Safety Trials

   • Involve 1,000-3,000 subjects.

   • Duration: 2-3 years

6. Phase 3: Full-scale efficacy trials

   • Further safety evaluations.

   • Duration: 1-2 years

7. FDA Review and Approval

   • Regulatory review process.

IN SILICO TOXICOLOGY

• Definition:

  • In silico toxicology refers to the computer-based modeling used to predict chemical toxicity, aimed at reducing the reliance on animal testing.

• Benefits:

  • Helps identify potential chemical hazards early in drug development.

  • Reduces costs and improves overall safety during the drug development process.

CHEMICAL TOXICITY ASSESSMENT

• Computational Toxicology Methods:

  • Use diverse data sources to predict the safety profile of chemicals.

• Key Measurement:

  • LD50: Lethal Dose 50%, a commonly used metric in toxicology, is defined as the dose of a substance that causes death in 50% of a population.

  • Example: $LD50 = 354 ext{ mg/kg}$.

• Read-across Methodology:

  • A technique where data from similar substances is used to predict toxicity for the new chemical entity.

• Data Sources:

  • Toxicity Reference Database (ToxRefDB), Exposure Forecaster Database (ExpoCastDB).

IN VITRO TOXICITY TESTING

• Definition & Purpose:

  • In vitro toxicity testing involves cellular assays to assess the potential toxic effects of chemicals on cells, notably human cell-derived models.

• Types of Assays:

  • Cell Death Assays:

  • Evaluate the potential of chemicals to induce cell death via cytotoxicity or apoptosis.

  • Organ-specific toxicity assessments can be performed using cells from various organs.

Types of Cell Death:

1. Controlled Cell Death (Apoptosis)

   • A regulated process designed to eliminate damaged or unnecessary cells without harming nearby tissues.

2. Uncontrolled Cell Death (Necrosis)

   • Occurs due to injury or damage, leading to cell swelling and bursting, which can cause inflammation in surrounding tissues.

COMMON APOPTOSIS ASSAYS

• TUNEL Assay:

  • Detects DNA fragmentation, a key marker of apoptosis.

• Annexin V Staining:

  • Identifies early apoptotic cells by binding to phosphatidylserine on the cell membrane.

• Caspase Activity Assays:

  • Quantifies activation of caspases, enzymes crucial for apoptosis.

COMMON CYTOTOXICITY ASSAYS

• MTT Assay:

  • Measures cell viability based on metabolic activity, indicated by a color change.

• Trypan Blue Exclusion Assay:

  • Differentiates between live and dead cells by dye uptake observed under a microscope.

• LDH Release Assay:

  • Assesses cell membrane damage by measuring the release of LDH enzyme levels into culture medium.

ORGAN-ON-A-CHIP TOXICITY TESTING

• Overview:

  • A cutting-edge method utilizing 3-D models of organs where cells are grown on scaffolds and perfused with media, providing a more realistic testing environment.

• Advantages:

  • Enables the interaction of multiple cell types and better mimics in vivo conditions.

• Disadvantages:

  • Still lacks full in vivo representation.

IN VIVO TOXICITY STUDIES

• Use of Animal Models:

  • Commonly utilize rodents (mice and rats) due to their biological similarities to humans.

  • Larger animals like rabbits, dogs, and non-human primates are used to assess specific toxicological effects.

TYPES OF IN VIVO TOXICITY STUDIES

1. Acute Toxicity Studies:

   • Assess impacts after brief exposure; critical for determining immediate health risks.

   • Establish lethal dose (LD50), guiding risk assessments.

2. Sub-Acute Toxicity Studies:

   • Focus on effects following repeated exposure over a short period (usually 28 days).

   • Identify affected organs and adverse effects regarding dose-response.

3. Sub-Chronic Toxicity Studies:

   • Monitor long-term effects (up to 90 days) from continuous exposure to substances.

   • Evaluate body weight, organ health, and blood chemistry.

4. Chronic Toxicity Studies:

   • Conducted over extensive periods (6 months to 2 years) to assess prolonged exposure effects.

   • Key for establishing safe exposure limits and identifying vulnerable organs.

MULTIPLE TYPES OF TOXICITY STUDIES

• Progressive Design:

  • Each type of toxicity study has increasing duration and cost, with acute studies addressing immediate effects and chronic studies examining long-term implications.

• Genotoxicity and Reproductive Toxicity Studies:

  • Assess effects on genetic material and reproductive health, critical for evaluating comprehensive safety profiles.

TERMINOLOGY OF TOXICOLOGY END POINTS

• Defining Toxic Effects:

  • Essential to establish what constitutes a toxic effect, which can include:

  • Death

  • Organ damage

  • Illness

• Important Metrics: NOAEL, LOAEL, BMD:

  • NOAEL: No Observed Adverse Effect Level.

  • LOAEL: Lowest Observed Adverse Effect Level.

  • BMD: Benchmark Dose.

LETHAL DOSE 50 (LD50)

• Definition and Importance:

  • Identifies the dose causing death in 50% of subjects; serves as a straightforward toxicological endpoint.

• Challenges in Determination:

  • Difficulties arise when precise dosing leads to needing large quantities of test substances.

• OECD Guidelines:

  • Countries under the OECD have agreed to cease LD50 testing since 2000.

LD50 EXAMPLES

• Sucrose: 29.7 g/kg (rat, oral)

• Sodium Chloride: 3 g/kg (rat, oral)

• VX Nerve Gas: 0.14 mg/kg (human, skin estimate)

• Paracetamol: 2.4g/kg (rat), 340 mg/kg (mouse)

• Nicotine: 50 mg/kg (rat), 3 mg/kg (mouse)

• Botulinum Toxin: 1 ng/kg (human, iv estimate)

NO OBSERVED ADVERSE EFFECT LEVEL (NOAEL)

• Definition:

  • NOAEL is the maximum dose where no adverse effects are observed; crucial in toxicological risk assessments for determining safe exposure limits.

• Applications:

  • NOAEL values guide regulatory decisions and the design of subsequent safety testing in humans and animals.

LOWEST OBSERVED ADVERSE EFFECT LEVEL (LOAEL)

• Definition:

  • LOAEL represents the minimal dose causing observable harmful effects in subjects; essential for risk evaluations.

• Role in Regulatory Framework:

  • Helps set exposure limits for chemicals and drugs, protecting both human and environmental health.

NOAEL/LOAEL RESPONSE MODELING

• Graph Representation:

  • Depicts dose-response relationships to visualize toxic effects based on NOAEL and LOAEL thresholds.

REFERENCE DOSE (RfD)

• Definition:

  • A Reference Dose estimates a lifetime daily safe exposure to a chemical without adverse effects, significant in environmental risk assessments.

• Regulatory Use:

  • RfD is a critical factor for agencies to set safe exposure limits to protect public health from chemical pollutants.

MAXIMUM TOLERABLE DOSE (MTD)

• Definition:

  • The MTD indicates the highest dose that does not prompt unacceptable side effects during clinical trials.

• Importance in Pharmacology:

  • Balances drug efficacy with patient safety, guiding safe dosing practices for new therapies.

HUMAN EQUIVALENT DOSE (HED)

• Dose Translation:

  • Direct comparisons of doses across species (e.g., mg/kg in mice vs. humans) may be inaccurate; use of body surface area for ratios provides a more precise estimation.

• Species Considerations:

  • When employing multiple species in studies, utilize the most sensitive or relevant species for safety assessments.

MAXIMUM RECOMMENDED STARTING DOSE (MRSD)

• Definition:

  • MRSD is the highest initial dose regarded as safe for first-in-human clinical trials, crucial for safeguarding participant well-being.

• Calculation Method:

  • MRSD is derived from preclinical toxicity data, typically calculated as $MRSD = rac{NOAEL}{10}$ (with potential for higher numerator values depending on the context).

DO ANIMAL STUDIES EVEN WORK?

• Challenges and Limitations:

  • Many drugs toxic to animals often do not progress to clinical studies, leading to questions about predictive value.

  • Notable instances like the Thalidomide case highlight discrepancies between animal and human toxicological responses.

REVIEW OF LEARNING OUTCOMES

• Objective Recap:

  • Covered in silico toxicity testing, in vitro assessment methods, in vivo toxicity studies, and vital toxicological endpoints.