Drug Development Review PDF

These slides have been reduced down to the minimum needed to help you understand the basics of drug discovery. Pay special attention to slides with stars. New Drug Development Dr. Chris Cartmell Research Assistant Professor Depar...

These slides have been reduced down to the minimum needed to help you understand the basics of drug discovery. Pay special attention to slides with stars. New Drug Development Dr. Chris Cartmell Research Assistant Professor Department of Pharmacology University of Arizona [email protected] December 9, 2024 Slides curtesy of Dr. Qin Chen IND: Investigational New Drug NDA: New Drug Application Number of Compounds Pass Through Phases of Drug Development Phases of Clinical Trials  Phocomelia by Thalidomide (1950-1960) Thalidomide: Sedative, Antiemetic Used as antiemetic in pregnant women in Europe, Canada, Australia and parts of Asia, mid 1950 – mid 1960. Dr. Frances Kelsey, the reviewing medical officer for FDA in 1960, believed the drug lacked proof of safety and blocked thalidomide being sold in the US. Report from the World Health Organization on causes of current and projected future deaths Deaths due to infections from Anti-Microbial Resistant (AMR) strains  Types of Bioassays 1. Cell-based Assays  Non-mammalian cells  Fungal and Yeast  Parasites Anti-infective agents  Bacterial  Nematode  Plants Herbicide effects  Mammalian or and human cells  Primary  Immortalized  Stem 2. Cell-free Assays  Cell extract-based  Purified enzyme based (enzymatic assays: e. g.ELISA)  Types of screening in cells 1. Phenotypic approach: The phenotypic approach allows for the identification of active compounds that effect cells by unidentified interactions  Toxicity screening Positive result is visualized as an end-point of a multifaceted cellular process (e.g.) Advantage: No major previous knowledge of molecular targets Activity can be modulated by multiple mechanisms via interaction with a variety of targets Limited information about the mechanist of action  Mechanism informed screening: Reported gene assays Positive result is visualized as an end-point of a specific cellular process (e.g. ROS) Engineered cells Does not target an individual macromolecule Focus on specific signalling pathways Unlimited information about the mechanist of action 2. Molecular approach: affect specific molecular target inside the cell (Protein, DNA or RNA)  Hit/Lead Discovery: Biological Assay Screening Qualitative bioassays are used for assessing the physical effects of a substance that may not be quantified, such as abnormal development or deformity. Healthy Cell Line Dead Cell Line Cell viability assays evaluated using microscopy are qualitative Disc Diffusion Assay Well Diffusion Assay Microorganism based diffusion assays are qualitative  Hit/Lead Discovery: Biological Assay Screening Quantitative bioassays involve estimation of potency of a substance by measurement of the biological response that it produces. Liquid Microorganism Assay Liquid Assays are amendable to Media high-throughput Control (HTS) screening Positive Inhibition can be Control Dilution quantified by Series comparison to positive Organism and negative controls Control Comparison to a dilution series of a known antibiotic can Extracts and/or Fractions be used to give an estimation of potency Traditional Operation of Drug Discovery  Library Generation Plate Extraction Data Analysis Management Natural Source SPE HTS/LCMS Hit/Lead Discovery: Bioassay Guided Fractionation Unknown Factors: Number of compounds related to the observed activity of the crude extract? Are the compounds major, minor, or micro constituents of the original mixture? Difficulties: Sometimes the initially activity of the crude mixture diminishes as the mixture is fractionated Bioactivity can be a result of synergistic effects of two or more compounds. The active compound is present in so small an amount in the original mixture that it is only present in vanishingly small amounts as the fractionation process proceeds Loss of the active compound during some chromatographic step Compounds may have previously been described  Hit/Lead Discovery: Compound Dereplication Done to prevent unnecessary isolation of known compounds associated with known biological activity Most effective when multiple spectrometric data is present: Mass spectra, UV absorbance spectra, H-NMR spectra Spectra by RT Sample UV-vis Sample ESI+ spectrum Standard ESI+ spectrum Standard UV-vis Sample ESI- spectrum Standard ESI- spectrum Involves the comparison of spectrometric data to compound databases (commercial and in-house) Antibase, MarineLit, Dictionary of Natural Products (Chapman and Hall)  Normal Phase Fractionation: Elution (hexane:EtOAc:MeOH) 9:1:0 7:3:0 1:1:0 0:1:0 0:7:3 Determine Assay Bioactive Fractions X X X Analytical HPLC-MS  Crude Extract 1: Fraction 1:1:0 Two groupings of active fractions indicate there are at least 2 active components to Fraction 1:1:0 HPLC profile comparison carried out to determine which component(s) was ??? present in active fraction but not in adjacent non-active fraction Dereplication of first active, compound ID xanthomegnin (known antibiotic compound) Dereplication of second active did not ID compound, potential novel compound, decision made to continue with structure elucidation Example: Bioassay Guided Fractionation Original Crude Extract: Tested Positive against S. aureus in liquid assay Extract contains multiple components with wide range in polarity Initial Fractionation Done Active Fraction Using Normal Phase Flash Fractionated Using Chromatography RP-HPLC Purified Active Compounds Dereplicated Using LC-MS/DAD Hit/Lead Discovery: Hit Confirmation Compounds that were found active are re-tested using the same assay conditions to obtain an IC50 value Confirmed hits are assayed using a different assay which is usually closer to the target physiological condition or using a different technology (Orthogonal testing) The ideal is a molecule that interferes with only the chosen target, but not other, related targets Secondary Screening: Confirmed hits are tested in a functional assay or in a cellular environment Membrane permeability is usually a critical parameter Assess effectiveness of compound binding to the target Hit is screened against a panel of unrelated targets: the more unrelated targets a compound hits, the more likely that off-target toxicity will occur Confirmed hit compounds are then ranked according to the various hit confirmation experiments Hit structures are quickly checked in specialized databases to define patentability