APT Advance Pharmacological techniques.pptx

Advances in molecular pharmacology and an improved understanding of the mechanism of most diseases have created the need to specifically target the cells involved in the initiation and progression of diseases. This is especially true for most life-threatening diseases requiring therapeutic agents which have numerous side effects, thus requiring accurate tissue targeting to minimize systemic exposure. Recent drug delivery systems (DDS) are formulated using advanced technology to accelerate systemic drug delivery to the specific target site, maximizing therapeutic efficacy and minimizing off-target accumulation in the body. As a result, they play an important role in disease management and treatment. Recent DDS offer greater advantages when compared to conventional drug delivery systems due to their enhanced performance, automation, precision, and efficacy. They are made of nanomaterials or miniaturized devices with multifunctional components that are biocompatible, biodegradable, and have high viscoelasticity with an extended circulating half-life.

1. Advanced Experimental Techniques in Pharmacology Course Code-230IPSD06 Presented By- Miss. Dhanashri S. Punde Roll No- PH_04 Department - Pharmaceutical Science Guide - Dr . Aishwaryadevi B. Deshmukh Associate professor School of Pharmaceutical sciences, JSPM University, Pune 1

2. 2 Content: Introduction What is cell culture Types of cell culture Cell-based assays Molecular Biology Techniques for Drug Target Validation Protein Purification and Characterization high Throughput Screening Methods Cell-based assays advancement

3. 3 Introduction In modern pharmacology research, advanced techniques are indispensable for exploring biological mechanisms, identifying potential drug targets, and facilitating drug discovery. These techniques enable researchers to look deeper into cellular and molecular processes , leading to the development of safer and more effective therapeutic interventions. Importance of Advanced Techniques: Precision and Specificity Efficiency and Speed Insights into Molecular Mechanisms Personalized Medicine Advanced Techniques: Cell Culture Techniques and Cell-Based Assays Molecular Biology Techniques for Drug Target Validation Protein Purification and Characterization High-Throughput Screening Methods

4. Cell culture Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been already established. Types of cell culture Classified into three- Primary cell culture Adherent cell culture Suspension cell culture Secondary cell culture Cell line Finite cell line Continuous cell line 4

5. Cell-based assays Viability Assays Proliferation Assays Cytotoxicity Assays. Apoptosis Assays Migration and Invasion Assays Adhesion Assays Reporter Gene Assays Functional Assays High-Content Screening (HCS) 3D Cell Culture Assays 5

6. 6 Gene knockouts Gene Overexpression Protein Expression High-Throughput Screening (HTS) Genetic Association Studies Molecular Biology Techniques for Drug Target Validation Fig No:01 Molecular Biology Techniques for Drug Target Validation

7. 7 Aspect Protein Purification Protein Characterization Purpose To isolate a specific protein from a complex mixture To determine the properties and functions of the purified protein Techniques Affinity chromatography Ion exchange chromatography SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide) Gel Electrophoresis Ultracentrifugation Mass spectrometry X-ray crystallography Nuclear magnetic resonance (NMR) spectroscopy Western blotting Enzyme assays Circular dichroism (CD) spectroscopy Process Isolation: Extracting protein from cells or tissues Purification: Using various chromatography methods to separate the protein based on its properties (e.g., charge, size, affinity) Structural Analysis: Determining the 3D structure and molecular weight Functional Analysis: Assessing the protein's activity, stability, and interactions Examples of Techniques Affinity Chromatography: Uses specific interactions (e.g., antigen-antibody) to capture the protein Ion Exchange Chromatography: Separates proteins based on their charge Gel Filtration Chromatography: Separates proteins based on size Mass Spectrometry: Identifies protein composition and modifications X-ray Crystallography: Provides high-resolution 3D structure NMR Spectroscopy: Provides information on protein structure and dynamics in solution Application Producing pure protein samples for research or therapeutic use Understanding protein function, mechanism of action, and interactions with other molecules Outcome High-purity protein suitable for experimental use Detailed knowledge about the protein's structure, function, and biochemical properties Protein Purification and Characterization Table No:01 Protein Purification and Characterization

8. High Throughput Screening Methods High-throughput screening (HTS) methods are used in drug discovery and other fields to rapidly test large numbers of compounds or genetic materials for activity against a biological target. These methods enable the identification of potential drug candidates or lead compounds with desired properties. 8 A screening robot at the Conrad Prebys Center for Chemical Genomics. (Rebecca Bernot , St. Baldrick's Foundation)

9. 9 Aspect Description Purpose To rapidly test and evaluate large numbers of compounds for activity against a target. Techniques Biochemical Assays : Test isolated proteins or enzymes Cell-Based Assays : Test compounds in living cells Screening Scale High-throughput: Can screen thousands to millions of compounds Common Assay Types Enzyme Activity Assays: Measure changes in enzyme activity Binding Assays: Assays: binding affinity to target proteins Reporter Gene Assays: Measure gene expression changes in cells Equipment Used Automated Liquid Handlers : Dispense small volumes of reagents Microplate Readers : Detect assay signals in 96, 384 or 1536-well plates Robotic Systems: Handle and process large numbers of samples automatically Data Analysis Software Tools: Analyze large datasets, identify hits, and evaluate statistical Significance Hit Identification: Determine which compounds show desired activity Validation: Confirm hits through secondary assays High Throughput Screening Methods Details

10. 10 Applications Drug Discovery: Identify potential lead compounds Target Validation: Confirm biological relevance of targets Mechanistic Studies: Understand how compounds affect biological pathways Advantages Speed: Rapidly screens large libraries of compounds Efficiency: Automates processes to reduce human error and labor Scalability : Can easily scale up to screen more compounds or targets Challenges False Positives/Negatives: Requires careful assay design and validation Data Management: Handling and analyzing large datasets can be complex Cost: Initial setup for HTS equipment and compound libraries can be expensive Table No:02 High Throughput Screening Method details

11. Cell-based assays advancement : Organ-on-a-Chip Technology 3D Cell Culture CRISPR/Cas9 Gene Editing High-Content Screening (HCS) Induced Pluripotent Stem Cells (iPSCs) Optogenetics Microfluidics Single-Cell Analysis 11

12. 12 Technology Example Author Summary Organ-on-a-Chip Lung-on-a-Chip Sisodia, Y., Shah et al., 2023 Respiratory diseases result in significant mortality due to inadequate personalized and effective therapeutic interventions. Addressing this issue requires innovative approaches that replicate the complexities of the human lung. In vitro miniaturized bionic simulations, such as lung-on-chip devices, offer promising potential for developing successful treatments. This review explores the role of lung-on-chip microfluidic simulators in advancing pulmonary drug discovery and personalized medicine. It highlights how these devices mimic lung functions, including breathing patterns, elasticity, and vascularization, to create a realistic 3D pulmonary microenvironment. 3D Cell Culture Tumor Spheroid Model Riedl , A. et al.,(2023) Inhibition Sensitivity: 3D cultures displayed enhanced sensitivity to inhibitors of the AKT–mTOR–S6K and MAPK pathways compared to 2D cultures. For example, the MEK1 inhibitor (AZD6244) was effective in reducing cell proliferation in 3D but not in 2D cultures.Rewiring of Signaling: Upon inhibition of AKT–mTOR–S6K, there was increased ERK phosphorylation in 2D culture but reduced ERK signaling in 3D spheroids. Similarly, MEK1 inhibition decreased AKT–mTOR–S6K signaling in 3D but not in 2D cultures. CRISPR/Cas9 Gene Editing CRISPR/Cas9 for ALS Therapy Liu, Z., Guan et al.,(2023) CRISPR/Cas9 gene-editing technology holds promise for treating diseases by correcting harmful mutations or disrupting disease-causing genes with precision. Various Cas9 variants have been developed to manage complex genomic changes. However, effective in vivo delivery strategies are lacking, and future research may focus on nonviral vectors with target recognition. Disease-induced changes could guide targeted delivery or gene editing. This paper reviews developments in gene-editing types, delivery vectors, and disease characteristics, summarizes successful clinical trials, and discusses challenges in CRISPR/Cas9 applications for disease treatment. Table No:03 literatures for Cell-based assays Literatures survey for Cell-based assays

13. 13 Technology Example Author Summary High-Content Screening (HCS) HCS for Neurodegenerative Disease Jones, L. H. et al.,(2023). Combines imaging and data analysis Identifies new therapeutic targets Focus on neurodegenerative diseases Induced Pluripotent Stem Cells (iPSCs) iPSCs for Cardiac Disease Modeling Kim, H. D. et al., (2023) Model’s cardiac diseases Screens cardiotoxic drugs Provides patient-specific insights Optogenetics Optogenetic Control of Immune Cells Wang, X et al., (2023) Controls T cell activity with light High spatiotemporal precision Applications in immunotherapy Microfluidics Microfluidic Organ Chip for Liver Disease Park, S. E. et al., (2023) Mimics liver functions Studies disease progression Drug testing applications Single-Cell Analysis Single-Cell RNA Sequencing in Cancer Patel, A. et al., (2023) Highlights intratumoral heterogeneity Identifies rare cell types Provides insights into cancer biology

14. 14 References- Freshney RI. Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications. John Wiley & Sons; 2016. Masters JR. Animal cell culture: the basics. In: Doyle A, Griffiths JB, editors. Animal Cell Culture. Springer, Humana Press; 2000. p. 1-19. Baserga R. Cell Growth and Division: A Practical Approach. IRL Press; 1989. Mather JP, Roberts PE. Introduction to Cell and Tissue Culture: Theory and Technique. Springer Science & Business Media; 1998. Liu Z, Guan W, Wang M, Shen Y, Chen Y, Liao S, et al. CRISPR/Cas9-mediated TDP-43 knockout in ALS model iPSCs and its application in therapeutic screening. J Neurochem . 2023;160(4):745-58. Jones LH, O’Brien PJ, Gough AE, Mader MM, Turetsky AB. High-content screening for neurodegenerative diseases: Uncovering novel therapeutic targets. J Biomol Screen. 2023;28(6):920-33. Kim HD, Cho EJ, Jung YJ, Park JS, Lee SK, Han JW, et al. Induced pluripotent stem cell-derived cardiomyocytes for disease modeling and drug screening in cardiac diseases. Stem Cells Transl Med. 2023;12(3):432-45. Wang X, Yu C, Yang H, Lu Y, Shi C, Liang J, et al. Optogenetic control of T cell receptor signaling with high spatiotemporal precision. Nat Immunol. 2023;24(7):915-24. Park SE, Georgescu A, Huh D. Microfluidic organ chip models of human liver for studies of tissue dynamics and disease progression. Lab Chip. 2023;23(1):50-67. Patel AP, Tirosh I, Trombetta JJ, Shalek AK, Gillespie SM, Wakimoto H, et al. Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma. Science. 2023;379(3):408-14.

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