PRECLINICAL TESTING STRATEGY, PRECLINICAL TESTING

PRECLINICAL TEST

1. Pre-Clinical Testing Strategy, Vis-à-vis envisaged clinical Studies, Flow chart for development of preclinical testing Presented by: Abhishek Mondal iPhD /2023-2029/PT/02 Pharmacology & Toxicology Guided by: Dr. Venkateswara Rao Amara Assistant Professor Department of Regulatory Toxicology Dept: Pharmacology & Toxicology 1

2. Table of contents In silico testing In vitro testing In vivo testing PK-PD studies Relevance to Human Disease Flow chart for preclinical testing Reference Dept: Pharmacology & Toxicology 2

3. In Silico In silico studies are integral to drug discovery, offering computational methods to enhance the efficiency and accuracy of identifying and developing new therapeutic compounds. Applications in Drug Discovery Virtual Screening: Ligand-Based Virtual Screening: Identifies potential drug candidates by comparing the chemical features of known active compounds. Structure-Based Virtual Screening: Utilizes the 3D structure of a target protein to screen large libraries of compounds for potential binding affinity. Molecular Docking: Predicts the preferred orientation of a drug candidate when bound to a target protein, providing insights into the binding affinity and potential efficacy of the compound. De Novo Drug Design: Involves designing new drug molecules from scratch based on the structure of the target protein, optimizing their binding properties and biological activity. Pharmacophore Modeling: Identifies the essential features of a molecule required for its biological activity, helping to design and optimize new compounds. Dept: Pharmacology & Toxicology 3

4. Cont … QSAR (Quantitative Structure-Activity Relationship): Establishes relationships between the chemical structure of compounds and their biological activities, aiding in the prediction of the activity of new compounds. ADMET Prediction: Assesses the Absorption, Distribution, Metabolism, Excretion, and Toxicity properties of compounds, helping to predict their pharmacokinetic and safety profiles early in the drug development process. Benefits Efficiency: Speeds up the identification and optimization of drug candidates. Cost-Effectiveness: Reduces the need for extensive laboratory testing. Early Prediction: Identifies potential issues with toxicity and efficacy early in the development process. Dept: Pharmacology & Toxicology 4

5. In Vitro Testing In vitro testing refers to investigations and examinations outside a living creature, usually in a controlled laboratory setting, with biological components such as cells, tissues, or organs. "in vitro" is Latin for "in glass," referring to using glass containers such as Petri dishes and test tubes in these investigations . In vitro testing is mainly used for determining i ) Mechanism of Action studies ii) Cytotoxicity assay iii) Metabolism studies Mechanism of Action: In medicine, the word “mechanism of action” refers to how a medication or any other chemical brings about a certain effect inside the body. It helps to understand how a drug specifically imparts a particular target inside a cell, such as an enzyme, or cellular process. Dept: Pharmacology & Toxicology 5

6. Cont … Steps to determine the mechanism of action of drugs are listed below: Definite objective Selection of appropriate model Establishing Experimental conditions Preliminary Screening Target identification & validation Signal Transduction Pathways Gene expression analysis Proteomics & Metabolomics Functional Assay Imaging & microscopy Computational & Bioinformatics Analysis Data interpretation & validation Reporting & Documentation Dept: Pharmacology & Toxicology 6

7. Cont … Let’s consider the MOA of an anticancer to be determined by the steps to be followed Objective- Determine the MOA of a new anticancer compound Model selection- Use of human breast cancer cell line Preliminary screening-MTT assay to establish cytotoxicity Target identification- Conduct pull-down assay followed by mass spectrometry to identify potential target proteins Pathway analysis- Use western blot to analyze the change in MAPK & PI3k/Akt pathways Gene Expression- Perform RNA-Seq to identify differentially expressed gene Validation- Use CRISPR/ Cas9 to knock out the identified target gene & confirm the loss of compound efficacy Functional Assay- Measure apoptosis using Annexin V/ PI staining & flow cytometry Imaging- Use confocal microscopy to observe the localization of the compound within the cell Data interpretation- Analyze data, perform pathway enrichment analysis, and manuscript for publication Dept: Pharmacology & Toxicology 7

8. Cont.… Cytotoxicity studies- Cytotoxicity refers to the quality of being toxic to cells. It describes the capacity of certain agents, such as drugs, chemicals, or natural substances, to cause damage to cells, leading to cell dysfunction or death. The following steps are to be followed to perform cytotoxicity studies successfully Define the objective Selection of appropriate cell lines Culture conditions Preparation of test compound Experimental design Assay for cytotoxicity MTT assay MTS assay XTT assay Protocol of Assay Data Analysis Interpretation & Validation Dept: Pharmacology & Toxicology 8

9. Contd … An example of carrying out a cytotoxicity study is given below Objective- Assess the cytotoxicity of a novel anticancer compound in human breast cancer cells Cell line selection- Use MCF-7 cells and culture them in RPMI-1640 medium with 10% FBS Experimental design- Treat cells with the compound at a concentration ranging from 0.1 to 100 μ M for 48 hours. Assay- perform an MTT assay to measure cell viability Data Analysis- Normalize absorbance values, plot dose-response curve, and calculate IC50 Validation-Confirm results with additional assay( eg : LDH release, Annexin V/PI staining) Reporting-Prepare a comprehensive report including methodology, data analysis, and conclusion Dept: Pharmacology & Toxicology 9

10. Cont.… Metabolism studies: In vitro metabolism studies are essential for understanding how a compound is metabolized by biological systems, which helps predict its pharmacokinetics, potential drug-drug interactions, and possible toxic metabolites. Different steps to be followed to perform metabolism studies are as follows: Objective Selection of appropriate models Preparation & incubation Experimental design Metabolism stability Assay Identification of metabolites Enzyme kinetics & inhibition studies Phase II metabolism studies Data Analysis Interpretation & reporting Dept: Pharmacology & Toxicology 10

11. Cont … An example of an in vivo metabolism study is given below as follows Objective- Assess the metabolic stability and identify metabolites of a new drug candidate Model Selection- Use ham liver microsome and hepatocytes Experimental Design- Microsomal stability Assay: incubate the compound with microsomes and NADPH, sample at multiple time points Metabolite Identification: use LC-MS/MS to analyze samples Kinetic studies- Determine Km and Vmax using various substrate concentrations Phase II Metabolism: Incubate the compound with hepatocytes and cofactors(UDPGA, PAPS) to assess conjugation Data Analysis- Calculate metabolic half-life, intrinsic clearance, identify metabolites Reporting- Document all findings, discuss metabolic pathways and potential drug-drug interactions. Dept: Pharmacology & Toxicology 11

12. In Vivo Studies In vivo studies refer to experiments conducted within a living organism, such as animals or humans, to observe biological processes in their natural context. These studies are crucial for understanding the comprehensive effects of drugs, treatments, or interventions in a whole, living system. They provide valuable insights into the efficacy, safety, pharmacokinetics, and pharmacodynamics of therapeutic agents, and are essential for translating preclinical findings into clinical applications Dept: Pharmacology & Toxicology 12

13. Cont … List of steps to be followed for in vivo studies for animal models Objective Selection of appropriate animals Ethical consideration Study design Dosage & Administration PK studies Efficacy studies Safety & toxicology studies Data collection & analysis Interpretation & Reporting Dept: Pharmacology & Toxicology 13

14. Cont … Example of a workflow of in vivo animal model studies Objective- Assess the efficacy and safety of a new anticancer drug in a mouse xenograft model. Animal Model selection- Use immunodeficient mice(e.g. nude or SCID) implanted with human tumor cells. Study design- Groups: Divide mice into control and treatment groups Randomization: Randomly assign animals to groups Blinding: blind the personnel conducting the assessments to the treatment groups Dosing- Route & Frequency: Administer the drug orally once daily for 28 days Dose Range: Determine doses based on preliminary maximum tolerated dose studies Efficacy Assessment- Tumor Measurement: Measure tumor size using calipers twice weekly Endpoints: Primary endpoint is tumor size reductions, secondary endpoints include survival and body weight Safety Assessment- Clinical Monitoring: observe animals daily for clinical signs of toxicity Blood Sampling: Collect blood samples at baseline, mid- study,and end-study, and end-study for biochemical analysis. Necropsy and Histopathology: Perform necropsy and examine major organs histologically at the wnd of study Dept: Pharmacology & Toxicology 14

15. Cont … Data Analysis- Tumor Growth inhibition: Calculate the percentage of tumor growth inhibition compared to controls. Statical Test: Use appropriate statistical tests(e.g.- ANOVA,T-test) to analyze data Reporting- Efficacy Results: present tumor growth curves and statistical analysis Safety Profile: Summarize clinical observations, biochemical data, and histopathological findings Conclusion: Discuss the implication of the findings for the drug’s development Dept: Pharmacology & Toxicology 15

16. Cont … In vivo dose-response studies are crucial for determining the relationship between the dose of a compound and its pharmacological or toxicological effects in an animal model. These studies help identify the effective dose range, the optimal therapeutic dose, and the potential for adverse effects. In vivo toxicology studies are essential for evaluating the safety profile of new compounds, assessing potential adverse effects, and determining safe dosage ranges. These studies help identify toxic effects on various organ systems and provide data for risk assessment and regulatory submissions Dept: Pharmacology & Toxicology 16

17. PK-PD Studies Pharmacokinetics (PK) and Pharmacodynamics (PD) studies are essential for understanding the absorption, distribution, metabolism, and excretion (ADME) of a compound, as well as its pharmacological effects and mechanisms of action. These studies provide critical data for optimizing dosing regimens, assessing drug efficacy and safety, and supporting regulatory submissions Here’s a detailed guide on conducting PK/PD studies for ADME Objective Animal model Ethical consideration Study Design Dosing & Administration PK studies PD studies Endpoint measurement Data Analysis Interpretation & Reporting Dept: Pharmacology & Toxicology 17

18. Cont … Example Workflow for PK/PD Study: Objective: Characterize the PK and PD properties of a new antihypertensive drug in rats animal Model Selection: Use male Sprague-Dawley rats. Study Design: Groups: Divide rats into five groups: one vehicle control, one positive control, and three dose groups (e.g., low, mid, high). Randomization: Randomly assign rats to groups. Blinding: Blind the personnel conducting the assessments to the treatment groups. Dosing: Route and Frequency: Administer the drug orally once daily for 14 days. Dose Range: Determine doses based on preliminary studies. Endpoints: PK Endpoints: Collect blood samples at multiple times (e.g., 0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) to determine plasma drug concentrations. PD Endpoints: Measure blood pressure using a tail-cuff method at baseline, 1, 2, 4, 6, and 12 hours post-dose. Biochemical Analysis: Collect blood samples for serum biochemistry. Organ Weights and Histopathology: Collect and weigh major organs at necropsy and perform histopathological examination. Dept: Pharmacology & Toxicology 18

19. Cont … Data Analysis: Noncompartmental Analysis: Calculate PK parameters (Cmax, Tmax, AUC, half-life) Concentration-Effect Relationship: Plot drug concentration vs. blood pressure reduction. Statistical Tests: Use ANOVA to compare groups. PK/PD Modeling: Develop a PK/PD model to describe the relationship between plasma concentration and blood pressure reduction. Reporting: Results: Present PK and PD data, including dose-response curves and statistical analysis. Interpretation: Discuss the implications of the PK/PD findings for the drug’s therapeutic use. Conclusions: Provide recommendations for further development and clinical trials. Dept: Pharmacology & Toxicology 19

20. Cont … Pharmacokinetic/Pharmacodynamic (PK/PD) studies play a crucial role in determining the therapeutic window of a drug, which is the range of doses that elicit a therapeutic effect without causing significant adverse effects. These studies help in identifying the optimal dosing regimen, ensuring efficacy while minimizing toxicity guide on conducting PK/PD studies to establish the therapeutic window: Objective Selection of Appropriate Animal Models Ethical Considerations Study Design Dosing and Administration Endpoints and Measurements PK Studies PD Studies Data Collection & Data Analysis Interpretation and Reporting Dept: Pharmacology & Toxicology 20

21. Cont … Example Workflow for PK/PD Therapeutic Window Study: Objective: Determine the therapeutic window of a new anti-inflammatory drug in rats. Animal Model Selection: Use male and female Sprague-Dawley rats. Study Design: Groups: Divide rats into six groups: one vehicle control, one positive control (known anti-inflammatory drug), and four dose groups (e.g., low, mid-low, mid-high, high). Randomization: Randomly assign rats to groups. Blinding: Blind the personnel conducting the assessments to the treatment groups. Dosing: Route and Frequency: Administer the drug orally once daily for 28 days Dose Range: Determine doses based on preliminary studies. Endpoints: Efficacy Endpoints: Measure inflammation markers (e.g., cytokine levels, histopathological scoring of inflammation) at baseline, mid-study, and end-study. Toxicity Endpoints: Monitor clinical signs, body weight, food and water intake, biochemical and hematological parameters, organ weights, and histopathology. PK Endpoints: Collect blood samples at multiple time points (e.g., 0, 0.5, 1, 2, 4, 6, 8, 12, 24 hours) on day 1 and day 28 to determine plasma drug concentrations. Dept: Pharmacology & Toxicology 21

22. Cont … Data Analysis: Noncompartmental Analysis: Calculate PK parameters (Cmax, Tmax, AUC, half-life). Concentration-Effect Relationship: Plot drug concentration vs. inflammation markers Therapeutic Index Calculation: Determine the TI by dividing the toxic dose (TD50 or LD50) by the effective dose (ED50). Statistical Tests: Use ANOVA to compare groups. PK/PD Modeling: Develop a PK/PD model to describe the relationship between plasma concentration and anti-inflammatory effect. Reporting: Results: Present PK and PD data, including dose-response curves and statistical analysis. Therapeutic Window: Identify the MEC and MTC and summarize the therapeutic window Conclusions: Discuss the implications of the findings for the drug’s therapeutic use and safety profile. Dept: Pharmacology & Toxicology 22

23. Relevance to Human Disease In the context of preclinical studies, "relevance to human studies" refers to the extent to which the findings from these studies can be expected to predict or apply to human health and disease. This concept is crucial for determining the potential effectiveness and safety of new treatments, interventions, or drugs when they are eventually tested in humans. Disease Model: In vitro and in vivo disease models are critical for understanding human diseases and developing new therapies. These models aim to replicate aspects of human diseases to study their mechanisms, progression, and potential treatments. Types of Disease Models: In Vitro Models: Cell Lines: Human or animal cells cultured to study disease mechanisms at the cellular level (e.g., cancer cell lines, neuroblastoma cell lines) Primary Cells: Cells taken directly from human or animal tissues, maintaining more of their original characteristics (e.g., primary neurons, hepatocytes). Organoids: 3D structures derived from stem cells that mimic the architecture and function of organs (e.g., brain organoids, liver organoids). Tissue Cultures: Cultured slices or explants of tissues to study disease in a more complex environment (e.g., brain slices for epilepsy research). Dept: Pharmacology & Toxicology 23

24. Cont … In Vivo Models: Genetically Engineered Models: Animals genetically modified to mimic human diseases (e.g., transgenic mice with human genes). Spontaneous Models: Animals that naturally develop diseases similar to human conditions (e.g., certain strains of rats with hypertension). Induced Models: Animals in which disease is induced through physical, chemical, or biological means (e.g., chemically induced diabetes in rats). Xenograft Models: Human tissues or cells transplanted into immunodeficient animals (e.g., human tumor xenografts in mice). Relevance to Human Disease: Pathophysiological Similarity: Molecular Pathways: Models should replicate key molecular pathways involved in human disease (e.g., amyloid-beta production in Alzheimer’s disease models). Cellular Mechanisms: Models should exhibit cellular mechanisms relevant to the disease (e.g., insulin resistance in diabetes models). Tissue Architecture: For organ-specific diseases, models should mimic the tissue architecture and microenvironment (e.g., liver fibrosis models) Genetic Similarity: Human Genes: Use of human genes in animal models to replicate genetic aspects of human diseases (e.g., Huntington’s disease models with the human HTT gene). Genetic Modifications: Use of CRISPR/Cas9 and other gene-editing tools to create precise genetic mutations observed in human diseases (e.g., cystic fibrosis models). Dept: Pharmacology & Toxicology 24

25. Cont … Phenotypic Similarity: Clinical Symptoms: Models should exhibit clinical symptoms similar to human disease (e.g., tremors and rigidity in Parkinson’s disease models). Disease Progression: Models should replicate the disease progression seen in humans (e.g., stages of cancer metastasis). Applications in Human Disease Research: Mechanism of Disease: Pathogenesis Studies: Understanding the mechanisms of disease development and progression (e.g., studying the role of T-cells in autoimmune diseases). Biomarker Identification: Identifying biomarkers for disease diagnosis and progression (e.g., identifying blood biomarkers in Alzheimer’s models) Drug Discovery and Development: Target Identification: Identifying and validating therapeutic targets (e.g., identifying kinase inhibitors for cancer). Efficacy Testing: Testing the efficacy of new drugs in disease models before clinical trials (e.g., testing antiviral drugs in viral infection models). Safety Assessment: Assessing the safety and toxicity of new drugs (e.g., evaluating cardiotoxicity in heart disease models). Dept: Pharmacology & Toxicology 25

26. Cont … Therapeutic Strategies: Gene Therapy: Testing gene therapies and delivery methods (e.g., testing CRISPR-based therapies in genetic disease models). Immunotherapy: Evaluating new immunotherapeutic approaches (e.g., testing CAR-T cells in cancer models). Regenerative Medicine: Studying stem cell therapies and tissue regeneration (e.g., using stem cell-derived cardiomyocytes in heart disease models). Challenges and Limitations: Species Differences: Physiological Differences: Differences between animal and human physiology can limit the relevance of findings (e.g., differences in drug metabolism). Immune System Differences: Variations in immune system responses between species (e.g., immune responses in mice vs. humans). Complexity of Human Diseases: Multifactorial Nature: Many human diseases are multifactorial, involving genetics, environment, and lifestyle, which are challenging to replicate fully in models (e.g., cardiovascular diseases) Heterogeneity: Genetic and phenotypic heterogeneity in human populations (e.g., cancer heterogeneity). Ethical Considerations: Animal Welfare: Ethical concerns regarding the use of animals in research and the need for ethical approvals and humane practices. Alternatives: The development and validation of alternative methods to reduce animal use (e.g., in vitro models, computer simulations). Dept: Pharmacology & Toxicology 26

27. Cont … Biomarkers are critical in the context of human disease as they serve as measurable indicators of physiological states, pathological conditions, or responses to therapeutic interventions. They play a significant role in diagnosis, prognosis, disease progression monitoring, and treatment efficacy evaluation. Types of Biomarkers: Diagnostic Biomarkers: Used to detect or confirm the presence of a disease or condition (e.g., prostate-specific antigen (PSA) for prostate cancer). Prognostic Biomarkers: Used to predict the course or outcome of a disease (e.g., HER2/neu in breast cancer). Predictive Biomarkers: Used to predict the response to a particular therapeutic intervention (e.g., KRAS mutations in colorectal cancer). Pharmacodynamic Biomarkers: Used to indicate the biological response to a therapeutic intervention (e.g., blood pressure as a marker of antihypertensive therapy effectiveness). Surrogate Biomarkers: Used as substitutes for clinical endpoints to predict clinical benefit or harm (e.g., LDL cholesterol levels for cardiovascular risk). Relevance to Human Disease: Early Detection and Diagnosis: Disease Screening: Biomarkers can be used for screening populations to identify individuals at high risk or in the early stages of a disease (e.g., mammography combined with CA 15-3 for breast cancer) Early Diagnosis: Early detection through biomarkers allows for timely intervention, which can improve patient outcomes (e.g., hemoglobin A1c (HbA1c) for diabetes). Dept: Pharmacology & Toxicology 27

28. Cont … Prognosis and Disease Progression: Risk Stratification: Biomarkers help in stratifying patients based on their risk of disease progression or recurrence (e.g., B-type natriuretic peptide (BNP) for heart failure). Monitoring Progression: Regular monitoring of biomarkers can provide insights into disease progression or remission (e.g., viral load in HIV patients). Guiding Treatment Decisions: Therapeutic Targeting: Biomarkers can identify patients who are likely to benefit from specific treatments (e.g., PD-L1 expression in cancer immunotherapy). Personalized Medicine: Biomarker-driven approaches allow for tailoring treatments to individual patients, enhancing efficacy and reducing adverse effects (e.g., pharmacogenomic markers). Evaluating Treatment Efficacy: Response Monitoring: Biomarkers can be used to monitor the effectiveness of treatments, enabling adjustments in therapy as needed (e.g., tumor markers like CA-125 in ovarian cancer). Treatment Optimization: They help in optimizing treatment regimens by indicating therapeutic responses or resistance (e.g., C-reactive protein (CRP) levels in inflammatory diseases). Dept: Pharmacology & Toxicology 28

29. Cont … Applications in Human Disease Research: Cancer: Diagnosis: Tumor markers like CA-125 (ovarian cancer), CA 19-9 (pancreatic cancer), and PSA (prostate cancer). Prognosis: HER2/neu overexpression in breast cancer, indicating aggressive disease and poor prognosis. Therapeutic Monitoring: Circulating tumor DNA ( ctDNA ) for tracking tumor dynamics and response to treatment. Cardiovascular Diseases: Risk Assessment: Biomarkers like LDL cholesterol, high-sensitivity CRP ( hs -CRP), and troponins. Prognosis: BNP and NT- proBNP levels for heart failure prognosis. Monitoring Treatment: Troponin levels to assess the effectiveness of treatments following myocardial infarction. Neurological Disorders: Diagnosis: Amyloid-beta and tau proteins in cerebrospinal fluid for Alzheimer’s disease. Disease Progression: Neurofilament light chain ( NfL ) as a marker of neurodegeneration. Response to Therapy: Monitoring levels of biomarkers to assess response to disease-modifying therapies in multiple sclerosis Dept: Pharmacology & Toxicology 29

30. Dept: Pharmacology & Toxicology 30 Flow Chart for Pre-Clinical Testing Start Initial drug discovery & screening In Vitro Studies

31. Dept: Pharmacology & Toxicology 31 Lead Compound identification In Vivo efficacy Studies In vivo pharmacokinetic & pharmacodynamic

32. Dept: Pharmacology & Toxicology 32 In vivo toxicology studies(Acute, Sub chronic, chronic) Safety pharmacology studies Reproductive & Development toxicity studies

33. Dept: Pharmacology & Toxicology 33 Genotoxicity Studies Immunotoxicity Studies Carcinogenicity Study

34. Dept: Pharmacology & Toxicology 34 Risk Assessment & Mitigation Planning Data Analysis & Interpretation Regulatory Compliance

35. Dept: Pharmacology & Toxicology 35 Clinical Trial & Planning IND Submission

36. Reference Freyer , M. W., & Lewis, E. A. (2008). Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods in Cell Biology, 84, 79-113. Altelaar , A. F., Munoz, J., & Heck, A. J. (2013). Next-generation proteomics: towards an integrative view of proteome dynamics. Nature Reviews Genetics, 14(1), 35-48. Kurien , B. T., & Scofield, R. H. (2006). Western blotting. Methods, 38(4), 283-293. Freshney , R. I. (2015). Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. John Wiley & Sons. Guidance for Industry: S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals, U.S. Food and Drug Administration (FDA) Drug Metabolism and Pharmacokinetics in Drug Discovery: A Primer for Bioanalytical Chemists, Part I, Journal of Bioanalysis and Biomedicine Guidance for Industry: Content and Format of Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, Biotechnology-Derived Products, U.S. Food and Drug Administration (FDA) Dept: Pharmacology & Toxicology 36

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