Pre-clinical development

Drug discovery

i) What is drug discovery? Creating new compounds from scratch or isolating them from natural sources...  ...testing them in vitro - then on animal models...  ...determine their efficacy & safety...  ...before advancing them on to clinical trials for human testing!  Drug discovery combines the fields of synthetic & natural product chemistry

ii) Why do we need new drugs? Unmet medical needs  Tackle new diseases - i.e. AIDS, obesity, CJD Improve on low efficacy rates - dementia & cancer drugs Filter out unwanted/harmful side-effects - antidepressants, antipsychotics Overcome drug resistance - antibiotics  Treat diseases earlier so downstream costs for treatment/disease management are lower - Alzheimer's, spinal injuries  Cost of therapy - interleukins used in HIV therapy Social costs to individuals/countries - Depression  Create employment 

Steps in drug discovery

Therapeutic concept

Firstly, we define a particular disease with a therapeutic need to be met Examples might include: Alzheimer's disease - improve cognition  Schizophrenia - reduce frequency or severity of psychotic episodes  HIV - prevent virus from inflitrating cells 

Therapeutic concept = Identification of a biochemical, cellular or pathophysiological mechanism in a disease  Look at the various levels & stages of our chosen disease to see where the problem/s are occuring  Examples: Relationship of a gene mutation with pathology of disease How bacteria/virus/fungus infiltrates cell & destroys it   Identifying why the immune system is attacking certain cells/organs   

Target selection

Particular molecular target is selected & validated  Hone in on specific action we want drug to have  Example: Enzyme inhibitors  Target DNA  Receptor agonists & antagonists 

Target validation

i) High throughput screening  Used to filter millions of potential compounds into a few hundred  'Hits' are identified through their activity with a particular assay - show specific effects  'Artefacts' are the remaining few hundred drugs - show no specific effects  Use 2 different mechanisms: Biochemical assays: Assess protein-protein interactions  Provide info on nature of molecular interactions (binding affinity, regions of protein interactions & kinetics)  Cannot determine cellular impact - only structure & target (region & site)  Cell-based assays: Based on iPS culture  ​​​​​​​​​​​​​​Measure features such as: Growth Cell metabolism changes Morphology Changes in 2nd messenger activity  CBAs can distinguish agonists & antagonists - biochem cannot 

ii) Structure activity relationships (SAR) Relationship between chemical structure of a molecule & its biological activity  Useful substructures within the lead series are defined & maintained....  ...whilst other structural features that have no defined purpose are varied/changed  Medicinal chemists use chemical synthesis - insert new chemical groups into the compound & test their biological effects 

iii) Rational drug design  Uses natural endogenous mediator as lead compound & makes modifications (the biological macromolecule implicated in disease pathology acts as a starting point)  Based on the idea that modulation of a specific biological target may have therapeutic value  Alternative to blindly testing hundreds of molecules to see if one or more will bind to cellular/molecular targets  Difficult start point - they're usually too polar & have v high Mr  Chemists must: Reduce Mr Reduce number of polar OH groups  Eliminate chiral centres  'Me-too' drugs = use pre-existing drugs as a chemical start point  Need to make sig. improvements on 'parent' drug (due to patency issues) -  needs to have better absorption, pharmacokinetics or receptor affinity 

iv) Structure based design  Historically used to make enzyme inhibitors  Carried out in 5 steps: X-ray crystal structure of target in its biologically active form is found Ligand is co-crystallised with target protein  Ligand analysed to find its binding domain  Biological relevance of interactions confirmed by site-directed mutagenesis (changing DNA sequence of gene)  Ligand is continually redesigned to optimise interactions 

SARS, Rational drug design & Structure-based design eliminate futher artefacts  Reduce potential drug list from hundreds down to approx 10 - 'Lead series'

Lead finding

Drug metabolism/pharmacokinetic & toxicological properties of remaining potential drugs examined - DMPK & toxcitiy ipS assays  ~3 drugs remain in 'lead series' Liver activity assays Liver microsome/hepatocyte survival after drug exposure recorded - drug conc. & effects also noted  Lots of SAR info collected  Activity CYP450 enzymes assayed to predict drug interactions (allow us predict how drug will interact with P450 enzymes - multiple drug interactions!!)  Intestinal absorption assays Drug permeability through Caco-2 cells (Intestinal epithelial cells) Drug applied apical side cell - conc. measured on basolateral side  Determines absorption & bioavailability  BBB absorption assays in-vitro assays using brain endothelial cells  Drug applied blood-facing side of cells - measured on brain side  Compound profiling = Drug properties (pharmacoK, toxicity, metab) discovered in target validation & lead finding phase are compiled & catalogued - Other researchers can know properties of specific compounds without needing to re-use assays  

Lead optimisation

Drug optimisation = key!  Want to improve affinity, selectivity & DMPK parameters (plasma 1/2 life, oral bioavailability etc) of leads  Want to maintain physiocochemical properties - esp. aqueous solubility  Want to make the drug pharmaceutically 'easy-to-make' - intrinsic solubility (solubility @ pH where drug is un-ionised), dissolution rate & stability (want to have long shelf-life, no demand for V specific storage environment)  Pharmacists may carry out more tests in: Permability  Receptor-specificity, CYP450 inhibition/activation Genotoxicity 

Animal testing

i) Animal models  Why do we need to use animal models in pre-clinical drug development? Many human diseases involve interactions between multiple organs & tissues  These interactions cannot be fully understood through sole use of cell & molecular biological assays  Animal models allow researchers to view the physiological impact of a drug - not just its cellular/biochemical    Animal screening = expensive  Occurs after extensive in-vitro studies  Main aim of animal testing = Evaluation therapeutic index Ideal animal model: Produces valid results  Displays selectivity  Predictable (Oncology & CNS diseases are most unpredictable in animal models - researchers prefer use of iPs (induced pluripotent stem cells) derived from patients with onco/CNS genetic disease)  Reproducible results 

ii) Pharmacokinetics  Rat, dog, pig or primate models  Establishing ADME characteristics (See Pharmacokinetics lectures :) Bioavailabilty Tissue distribution Active metabolite formation Elimination test compound  Tests typically last less than 48hrs  Also used to establish appropriate administration methods (oral, bolus, intraperitoneal) 

iii) Toxicity  Acute vs subacute toxicity Chronic vs subchronic toxicity Reproductive toxicity

Reproductive toxicity Determine potential adverse effects of drugs on: Gametogenesis Foetal organogenesis Parturition Lactation Neonatal survival  Vitality of newborn  Carried out in rodents & rabbits  Divided into 3 segments: Segment I (pre-conception):  Treatment during gametogenesis Females = 60 days Males = 15 days  Segment II (pre-implantation): ​​​​​​​Treatment during gestation Rodents = day 6-15  Rabbits = day 6-18  Segment III (perinatal to postnatal): ​​​​​​​​​​​​​​Treatment through at least 15 days of gestation  Plus 21 days lactation