Pre-clinical development

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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

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Steps in drug discovery

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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

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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'

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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

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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

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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