Post on 21-Feb-2022
PHA 3042 Modern Drug Development
Drug Discovery
- 10/10,000 compounds reach clinical trials - 21% of drugs fail due to safety - 66% due to lack of efficacy - 1/10 make it through the clinical trials - 12-15 years to market
Costs - $2 billion for research and development - 25% of medicines make a profit, 75% fail to recoup costs of R&D - Blockbuster revenue has decreased due to patents expiring and generics making some - Blockbuster drug produced >$1 billion per year - Worldwide Market: $1.3 trillion/year - US: $430 billion (33% of the market) - Australia: $10 billion (1% of the market)
Sources of Drugs Types of Discovery Serendipity: Accidental discoveries Rational Design: Understanding the target and structure (beta blockers) Chemical Modification: Chlorothiazide from antimicrobial → diuretic
Natural Sources - Accounts for 50% of drugs in clinical use - ½ of these come from plants used in traditional medicine - Even if now is synthetically made, it is from a natural source
Secondary Metabolites - Possess no obvious primary metabolic function - Not for growth of plants, but rather their survival (defence against predators) - Plants can create 3D structures that we cannot replicate in the lab
Bioactive Molecules Alkaloids
- Basic nitrogen-containing compound - White crystalline substance - Bitter → morphine, atropine
Glycosides - Contains sugar and non-sugar component - Cardiac glycosides (digoxin)
Plants as Sources of Drug - <10% systematically studied for bioactive compounds - Derived from roots, leaves, bark, seeds and flowers - 1/10,000 tested considered promising - Of these, 1 in 4 approved as a new drug
Need - 500mg pure compound may come from 50kg raw material - Up to 2kg of pure compound needed for clinical trials - Therefore, 200 tones raw material
Selecting a Plant Ethnopharmacology/Ethnobotany
1. Culture should be located in floristically diverse location (rainforest) 2. Culture remained n region for many generations 3. Tradition of passing plant knowledge from generation to generation
Consensus: Whole Village Specialist: Healer Chemotaxonomic Sampling
- Specific compounds are often only found in groups of related plants
Isolation of Active Compounds - Botanist must identify the plant - Stabilisation process – drying or preserved in alcohol - At the lab, pulverising it into powder and made into solvent for extraction - Obtain pure compounds - Chromatography, bioassay, toxicity testing, determine structure
Paclitaxel - Pacific Yew has anti-tumour activity – screening program - Supply problem with a low yield and grows slowly - Could use baccatin from related species as a precursor (semi-synthetic)
Animal Based Drugs - Insulin derived from pancreas of pigs and cows - Oestrogen from urine of pregnant mares - Heparin: Anticoagulant derived from bovine lungs and pig intestine
o Insulin and Heparin are now synthetic
Snake Venoms - Neurotoxic - Hemotoxic - Myotoxic, Cardiotoxic, Nephrotoxic
Captopril - Brazilian viper was causing intestinal contractions - Sergio Ferreria joined Vane’s lab, as he was interested in bradykinin - John Vane believed ACE was important in regulating blood pressure - Ang 1 and bradykinin increased in blood levels, therefore ACE was being inhibited - Peptide was not absorbed orally, and Squibb (Pharma) made it orally available and became their
first billion-dollar drug
Microorganism Based Drugs - Penicillin and statins are derived from fungi - Bacteria produced tetracycline
Identifying Drug Targets Protein Targets
- The gene has the inherited characteristic of the protein - There are 626 drugs with 125 targets - Ion channels, GPCR, ionotropic, kinase linked, enzymes, transporters - Most new drugs on the market are monoclonal antibodies
Finding Drug Targets 1. Analysis of pathophysiology - Understanding pathways involved in disease determines novel target 2. Analysis of mechanism of action of existing therapeutic drugs - Work “backwards” from known action to mechanism 3. Genomic Approaches - Most drugs targets are proteins, so encoded in genome
James Black and Beta Blockers - Went from the disease and went backwards to the target - Knew hypertension was due to vasoconstriction, and therefore worked to that
Genome - Not a “One gene → One protein → One target - A protein has accessory proteins, receptor dimers, etc.
Genome to Phenotype Drugs can affect:
- Expression of gene - Regulatory factors of gene products - Compensatory mechanisms of their functional activity or
physiological effects
Genomic Strategies Disease Genes Mutated genes which cause or predispose to the development of human disease
- Often not single-gene disorders - Most gene products found to be untargetable
Disease-Modifying Genes Altered expression or activity believed to induce the disease state Druggable Genes
Genes encoding proteins that are likely to possess binding domains that recognise drug-like molecules Disease Modifying Genes A better approach
- Targets reinforcing or compensatory genes to alter gene expression of non-mutated genes
- These are believed to induce disease or altered activity of the gene product
- This causes functional changes and effects the disease phenotype
- This is done by the following techniques
Gene Expression Profiling - DNA microarrays → extract mRNA - Convert back to cDNA - Label the healthy and diseased after RT PCR - Mix them in equal amounts - Green indicates that it is higher in control than
the disease - Red indicates it Is upregulated in the diseased
state - Yellow means it is unchanged
Gene Knockout/Transgenic Screening - Used for target validation, rather than discovery - Ensures that the gene is causing the disease or phenotype
Druggable Genes - Drugs that can bind to a gene
Orphan Receptor - An apparent receptor, usually identified by DNA cloning - Endogenous ligand is not known
Drug Screening Techniques High Throughput Screening (HTS) Target is incorporated into biochemical or cell-based assays and exposed to large number of compounds Requirements Validated, Tractable Targets
- Biochemical vs. cell based - Response often cell type-dependent
Chemical Diversity - Natural sources - Commercial libraries
Industrialised process - Miniaturisation (many well plates) - Cost-effective
Application of HTS - Screening library into the primary hit - Single concentration against the target - Primary screen is around 25-250K per assay - Secondary screen confirms affinity/potency - Uses multiple concentrations
Selecting HTS Assay Biochemical: Enzyme/Substrate reactions, binding affinity Cell Based (More common): signal transduction (cAMP), membrane transport, division and cytotoxicity Heterogeneous Format: Requires many steps Homogeneous Format: Mix and measure – more amenable to fully automated HTS - preffered
Advantages and Disadvantages
Cell Based Primary or Stem Cells
Radioactive Assays - Sensitive, robust (ligand binding) but is a one step
process - Ability of test compound to inhibit binding of radio
ligand to target
Scintillation Proximity Assay – SPA - Excitation of scintillate (bead with receptor/antibody) - Radioligand binds to it and the bead emits light - However, risks such as radioactivity (headaches), long read times
Fluorometric Assays 1. Fluorescent dye complexes with ions 2. Membrane Bound Dyes – signal changes with membrane potential 3. Membrane impermeable dyes – bind to intracellular targets, only leaky
cells stained
Fluorescent Dye Complexes with Ions - Monitor changes in intracellular ions - cAMP, Calcium, pERK, Na+ - Emission changes with activation or block of receptors or ion channels - Alpha 1 Agonist (Phenylephrine) → GPCR → IP3 and GAD → Calcium - This is detected when Fluo-3 is taken up into the cell to bind to calcium - Increased calcium = Increased fluorescence measured via FLIPR - Beta 1 Agonist (Isoprenaline) → A.C → cAMP
ALPHA Screen: cAMP - Needs to be in the dark - A donor bead and an acceptor bead - Artificial cAMP brings beads together - Excitation of bead and donor bead donates oxygen
to the acceptor bead - Light is then generated when they are in close
proximity Biological cAMP
- If produced by the cell it competes and displaced artificial cAMP
- This causes the beads not to bound, and decreases fluorescence
Increased cAMP = Decreased Fluorescence
Receptor Gene Assays - Gene expression is transfected cells quantified by linking promoter to reporter gene whose
expression can be monitored - Compounds activating or inhibiting promoter
detected - If gene transcription is occurring, it becomes visible
how second messengers modulate genes
Drug Screening Techniques: 2 Animal Research Pure (Exploratory) = Physiology Applied = Disease Toxicology
History - FDA disaster in 1938 started animal testing - 50’s and 60’s has thalidomide for pregnant women that caused malformations - This now needed pregnant women to be tested if marketed to them
Preclinical Trials - Must test in rodent and non-rodent (dog or non-human primate) - Efficacy in animal models - Drug metabolism and safety pharmacology
In Vitro Approaches
- Vascular bed and blood vessels (Nitric Oxide discovery) - Cell culture of single cell - Membranes and molecules
Advantages - Versatile and relatively simple - Many preparations: smooth, cardiac skeletal, brain etc - Measures a wide range of physiological responses
o Contraction, membrane excitability, synaptic function, vascular resistance
Disadvantages - Tissues normally from small laboratory animals vs. humans or other primates - Short term experiments - Preparations don’t normally survive
In Vivo - More complex using the whole animal
Advantages - Pharmacodynamics of drug - Long term drug administration effects - Off target actions and toxicity potential
Disadvantages - Ethical and legal constraints - Need to keep animal experiments to a bare
minimum
Translation from in Vitro to in Vivo to in Human - Acute in vivo may not translate to chronic in vivo effectiveness - Anti-depressants are different acutely compared to chronically
Predictive Value Very Good: Organ functions Less Good: Psychological/behavioural functions Not Good: Side effects, toxicity
3 Measures for Blood Pressure Tail Cuff Systolic Blood Pressure
- Non-invasive and chronic - Variable and less accurate, and stress’ animal
Intra-Arterial Cauterisation
- Semi-chronic, accurate with multiple parameters - Invasive and not suitable for chronic studies
Radiotelemetry
- Implant of a radiotelemetry device in peritoneal cavity - Tip of pressure transducer inserted in abdominal aorta - Radiofrequency implant continuously and remotely recorded - Chronic, accurate and allows further data processing - Invasive, expensive and less parameters measured