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