DRUG DISCOVERY AND DEVELOPMENT LECTURE NOTES

OVERVIEW OF DRUG DISCOVERY AND DEVELOPMENT

Is there a need for a new drug?

Yes:

1. Medical needs
2. Commercial attractiveness
3. Competitive situation
4. Ease of development – clinical trials (However if the number of patients is low,
then the number of clinical trials volunteers will also be low. This is one
indication that your target market is low, which means that profits will be
extremely hard to obtain).
5. Cost of disease
6. Fits with the portfolio of various companies, e.g. pharmaceutical companies.

Is there a medical need for developing drugs?

 Diseases that are not adequately treated can be treated by drugs

 Other drugs could be improved:
o In terms of efficacy (effectiveness), e.g. the best drugs for chronic drugs don’t
treat all patients.
o Fewer side effects, e.g. NSAIDs have gastric side effects including discomfort
and ulceration in the long term.
 An ever-increasing medical burden:
o Ageing population
o Diseases of affluence (prevalence of type 2 diabetes is increasing due to
changing life-styles and obesity).

Commercial attractiveness (Investments must return profits to increase dividends per share).
This is dependent on:

 Market size
 Competition
 Limited patent life of around 20 years (12 of them will be when the drug is in
development).
o There is a need to replace off-patent drugs to prevent the competition
producing your drug legally.
o The life-cycle of a drug has to be legally maintained and kept exclusive
 New formulations require new patents, which can increase the life-
cycle of a drug.
 E.g. a new route of administration would require a new
formulation.
 Maintenance of a pipeline of drugs (2-3 launches per year to balance profits)
 Pharmaceutical companies aim for blockbuster drugs that generate billions of £/$ per
year.
o This makes it cost effective to discover, develop, test and market one drug.
o The sustainability of blockbuster drugs is questionable, as many failures come
about for every one blockbuster.
o Blockbuster drugs, once the patent finishes, will see their sales depleted as the
competition releases your formulated compound under their name.

Stages in Drug Discovery:

Before even applied research is carried out, various academic organisations have carried out
basic research, and these concepts are from what the pharmaceutical industry feeds off.
Applied research, clinical research and product registration and launch are the following
stages.

In the drug discovery stages specifically:

Target Identification and Validation  HIT finding  Lead Optimization  Early Clinical
Safety and Efficacy  Proof of Concept/Phase I  Phase II  Phase III  Registration 
Post-launch activities.

Target identification and validation encompasses a wide variety of scientific activities

focused on identifying new targets and confirming their role in disease. As our knowledge
grows about human biology, we are moving towards targeting cellular pathways of proteins
rather than individual proteins.

Hit finding entails development of robust assays to test small-molecule compounds in high
throughput screens. In the case of Biologics, this stage entails development of antibodies with
affinities to chosen targets.
In lead optimization, small molecule hits are chemically altered to improve their drug-like
properties. In the case of Biologics, at this stage, antibodies are modified to increase their
affinity for their target.

To establish an initial safety profile of the drug, extensive toxicological and safety
pharmacological profiles are done using in silico (computer), in vitro and appropriate animal
models. This occurs in early clinical safety and efficacy processes.

In Proof of Concept trials, the drug is for the first time given to humans – a small group of
patients or healthy volunteers – in order to verify the mechanism of action and to get an early
readout of the efficacy of the compound in human disease. In phase I trials, the drug is tested
in a small group of patients or healthy volunteers (20-80) to evaluate its safety, determine a
safe dosage range and identify side effects. Proof of concept and phase I trials are often
combined.

In phase II trials, the drug is given to a larger group of people (100-300) to test its
effectiveness, determine the effective dose range and to further evaluate its safety.

In phase III trials, the drug is given to large groups of people (1,000 to 3,000) to confirm its
effectiveness, monitor side effects, compare it to commonly used treatments and collect
information that will allow the drug or treatment to be used safely.

For the registration of a new drug, the results of all preclinical and clinical studies, the quality
data and description of the manufacturing process are compiled and submitted for review to
the regulatory authorities. If the regulators agree that the data proves the quality, efficacy and
safety of the drug, a marketing authorization is granted. From then on, a new drug can be
made commercially available to patients.

Post-launch activities – Once a drug is on the market, adverse effects need to be constantly
monitored and reported to the regulatory authorities. In addition, life-cycle programs –
including phase IV clinical trials – are often undertaken to add new indications or improve
existing formulations of the drug.

Target Identification and Validation:

Identify possible target using an underlying disease:

E.g. Gene mutations in humans

Sodium channel NaV17 is found in sensory nerves

A gain of function mutation causes ongoing pain

A loss of function mutation causes complete

insensitivity to pain but not to other sensations, e.g. hot
and cold sensation.
A NaV17 gain of function mutation also leads to red feet in erythmelalgia. Also familial rectal
pain was observed. The red feet were caused by spontaneous firing of this receptor that
caused the perception of pain. The return action potential released substance P in the feet,
which is a vasodilator and is responsible for the redness.

Altered gene/protein expression is seen in disease (gene microarrays for changed mRNA
levels).

Altered gene/protein expression can be induced into animal models of disease.

The defect is genetically modified in animals through knock-outs or over-expression.

Target validation

This is to confirm the role of a molecule in disease.

We use a “tool” compound that interferes with the molecular pathway. Also, antibody work is
becoming progressively more useful due to the specificity of antibodies.

We can use an antibody to neutralise a protein.

We can use RNA interference to knock-down specific mRNA and consequent expression of
encoded proteins, and through this and various other methods, we can induce a knock-out or
an over-expression in genetically modified animals. However, this takes a long time (around
18 months).

For some companies, there is a move to target cellular pathways of proteins rather than
individual proteins for many diseases. Cellular activities are based on interacting biochemical
pathways, and measurements are taken based on the amount of interference with the pathway.
We still have to compensate for altered activity in other regions of the pathway.

How are new drugs discovered?

1. Serendipity/luck, e.g. penicillin.

2. Folk remedies, e.g. aspirin from willow bark, opiates from the opium poppy and taxol
(microtubule stabiliser) from ewe tree.
3. Replacement therapies, e.g. insulin and thyroxine.
4. Rational drug design, e.g. cimetidine and captopril
5. High throughput screening
Rational ligand design:

Ligand based design requires:

1. Knowledge of other molecules that bind to biological targets

2. The modification of chemical structures systematically:
a. To measure the biological activities quantitatively
b. To derive characteristics required to bind to the target (pharmacophore)
c. To discover effective compounds
3. We can discover potential drugs without knowing the structure of the target molecule
a. E.g. receptor ligands have been used to discover antagonists, which is how H2
receptor agonists and antagonists were discovered.
b. Enzyme substrates can be used to discover potential drugs, e.g. captopril (ACE
inhibitor).

Structure based drug design requires:

1. Knowledge of the 3D structure of the target (often done in silica)

2. X-ray crystallography
a. Perhaps with a ligand “docked” in the active site
3. NMR spectroscopy
4. Computer modelling
5. The structure of related proteins could be used – homology model
a. The design of an inhibitor for the enzyme cathepsin S was based on the
structure of the active site for other members of the capsaicin family
(capsaicin C and K).
6. The use of information to design compounds that bind with high affinity and
selectivity to the target.

The above summarise the target identification and validation stages.

Hit finding is achieved by high throughput screening:

Large pharmaceutical companies screen around 1 million compounds in each discovery

program. This is based on compounds that were made in old discovery programmes and new
libraries of compounds.

Hit finding requires the development of:

- Assays that give reliable and robust results with a good signal to noise ratio
- A human target molecule assay
- Equipment that can handle large numbers of compounds. Anything from 96
to 3456 well plates may be used and the light intensity is measured from each
plate, and normally this stage should take one week to complete.
Miniaturization is needed as well as very small volumes of compound in
order to carry out this stage.
- Automation and robotics carry out the assays.

Smaller biotechnology companies use smaller libraries, e.g. kinase or GPCR specific
libraries. These libraries are designed to contain molecules that are more likely to act on the
chosen target, e.g. ion channels, GPCRs and enzymes (e.g. cysteinal proteases).
High throughput screening begins with 1 million compounds being screened against the
target in one concentration.

20,000 active compounds are re-tested to confirm the activity.

Hundreds of active compounds are then tested to determine their activity quantitatively, e.g.
how dilute you can make the solution while it still produces maximal response (potencies of
different compounds).

The hits are then identified.

How to get from the hit to the lead candidate:

From the initial hit, identify any structurally similar molecules and group them into their
chemical classes.

Omit any classes with obvious liabilities, e.g. toxicity (possibly sue to highly reactive groups,
which could lead to the alkylation of DNA).

Choose compounds that are suitable for chemical modification.

Selectivity for the target over a related family member can help to narrow down the search
field.

Start medicinal chemistry programme to chemically modify the hits to determine if the yield
structure-activity relationship is changed, and to whether the changes in structure leads to
clear increases or loss of activity. Affinity and selectivity are the 2 main categories to worry
about.

Find out the activities in another assay, e.g. cellular assay.

Determine the in vitro activity using a target from an animal species that may be used for in
vivo studies, e.g. rat or mouse.

Physico-chemical properties (solubility and stability) should be determined.

Permeability/efflux studies in cultured cell lines should also be carried out.

Metabolic stability in liver microsomes (human and rat) should be studied, as to whether the
compound is a substrate or inhibitor of P450 enzymes (major metabolic enzymes) to
determine the effectiveness of first pass metabolism.

It’s 4:29AM....
Lead optimization:

A lead provides a potential template for a drug but may not have:

- Sufficient potency and selectivity

i. Broad screen against many other hundreds of receptors,
enzymes and channels.
- Acceptable drug metabolism and pharmacokinetic properties (oral
bioavailability in rodents, life time in circulation etc)
- Safety date, e.g. does it affect the hERG ion channels found in the heart –
these are potassium channels that, if affected, can lead to arrhythmias.
- Any data comparing it to any marketed gold standard compound would be
useful
- Information about species variation
- Functional data – detailed in vivo profile in disease model.

Lead optimisation aims to turn an early lead into a molecule that is suitable to administer to
humans.

The iterative process includes all the assays/tests used for lead identification.

Provisional patent filing covers lead compounds. However if you file a patent too early then
you will get reduced protected sales time, but if you file it too late then another company may
patent the same drug.

Use of animal models:

Most early studies use in vitro assays using molecules or cells.

Some secondary assays may use isolated tissues, including blood vessels, urogenital/airways
smooth muscle and/or brain slices.

Whole animals are used to determine possible effects on general physiology (early safety
tests) and possible therapeutic actions in suitable animal models of the disease.

We can develop a translational model that can be used in animals and humans (Proof of
concept/phase I stage). This would show that the compound is attaining concentrations that
engage the target molecule.

The translational model could include a PET ligand. This is a very short-lived isotope
that also binds to the target, and if you candidate demonstrates a high affinity for the
drug then it will act as an antagonist to PET binding.

Functional effects could also be demonstrate in other translational models.

Early pre-clinical safety and efficacy testing:

This is to establish a safety profile for the lead compounds. In silico methods (databases of
reactivities and toxicological properties) and in vitro toxicological tests using bacteria and
mammalian cells are used.

This is also to demonstrate pharmacokinetic profiles of the compound in animals, e.g.

rodents. We could need to find the plasma concentrations of compounds that are active in
animal models.

We would need to demonstrate physiological readouts, end organ histopathology, mechanism

tests and/or related safety/toxicological tests and find the toxic dose (to see if it is far away
from therapeutic dose to set limits for trials) to pass the in vivo safety/toxicology tests.

In later development, longer term toxicological studies are used to study reproductive
toxicology in fertility and reproductive terms of the drug. Normally, trials are carried out on
healthy, young male volunteers.

Other activities required for the programme:

1. Synthesis of large quantities of compound for safety/toxicology and clinical studies.

a. Often new synthetic routes are devised for bulk synthesis
b. Undertaken by specialist groups of chemists
c. Highest approved standards
d. High purity of compound
All of the above are important for safety/toxicology and clinical studies.

2. Formulation of compound developed

a. Drugs are given with excipients and solubility can be critical.
b. To improve the delivery of compound is essential. For example, a capsule that
breaks open in pH2 of the stomach isn’t useful if the drug isn’t targeted there.

3. Stability of compound is tested under various conditions, including temperature and

pH and it needs to be stable in formulation for the same time taken in clinical trials.
Biological Reagents:

There is an increasing search for biological agents for therapies, including vaccines (however
as more diseases are cured, less money can be made here), antibodies, RNA interference
(emerging market) and gene therapy/stem cells.

For antibodies, biotech companies and large pharmaceutical companies have predicted 35%
of their new development candidates in the early future will be antibodies. This is due to their
high specificity is a single antibody is developed.

Phase I/Proof of Concept Clinical Trials:

Phase 1:

The compound is tested in a small group of patients or healthy volunteers (20-80 people).

Single and multiple dose studies are carried out over different time periods

Safety is evaluated

A safe dosage range is evaluated

Initial side effects are identified

Proof of Concept trials:

The compound is given for the first time to humans

To a small group of patients or healthy volunteers

To verify the mechanism of action

Does it have the expected pharmacological action in humans?

Obtain early readouts of efficacy of compounds in human disease

Use translational model, possibly with biomarker enzymes as well

Often, Proof of Concept and phase I trials are combined and the elapsed time period is around
1 year.

4:51AM...head going empty and eyes drooping. Lots still left to do.
Phase 2 and 3 Clinical Trials:

Phase 2 trials:

The drug is given to a larger group of patients with the disease (100-300)

Effectiveness is tested

Effective dose range is established

Comparison to placebo treatment and possibly positive control drug is done

(in the analgesic market – most placebos have a large effect)

Further evaluation of studies is carried out

Time elapsed is around 2-5 years

Phase 3 trials:

The drug is given to a larger group of patients (1000-3000)

Effectiveness is confirmed

Side effects are monitored

Comparison to commonly used treatments is carried out

Collection of other information that will allow the drug to be used safely is carried out

Time elapsed is 2-4 years

Registration:

Required results of all pre-clinical and clinical studies, including chemistry, pharmacology,
toxicology, safety, pharmacokinetics, clinical data, case reports and labelling data. The
description of manufacturing process and quality data is also needed.

This is all compiled and submitted electronically to regulatory authorities, e.g. MHRA (UK),
FDA (USA) and/or EMEA (Europe).

Elapsed time is between 1-3 years, but a lack of cross-country harmonization of processes
may draw this process longer.
Approval by providers:

UK: National Institute for Health and Clinical Excellence (NICE) decides whether the drug
will be available on the NHS or not. Obviously it boosts sales if the NHS buys the drug.

NICE is an independent organisation responsible for providing national guidance on the

promotion of good health and the prevention and treatment of ill health.

It guides the NHS on the use of existing and new medicines, treatments and procedures.

They ask whether new drugs provide good value for money while weighting up the costs and
benefits for treatments.

USAL Private medical insurers/Medicare decides which drugs they will pay for.

Post-launch activities:

Once the drug is on the market, adverse effects are constantly monitored and reported to
regulatory authorities, including long-term safety data, side effect information and drug
interactions.

Life cycle programmes include phase 4 clinical trials to add new indications and to improve
existing formulations.

See lectures notes for some stats about numbers and costs.

The main reason for drug failure is efficiency in humans.

The most successful areas for drugs are in cardiovascular and arthritis treatment.

The highest costing stage of the drug development stage is phase 3.

Drugs are life saving and enhance the quality of life.

Drugs reduce the cost of hospitalization.

Drugs reduce the number of work-days lose due to illness and visits to healthcare
professionals.

Only 10% of annual health care budget is required to meet the drug costs to the NHS.

Is there any philanthropy? Some large companies invest resources in diseases of the
developing world. Malaria kills 1M a year and GSK released data on 13k compounds and
provided facilities for 60 scientists to work in GSK Labs.

Novartis Tropical Disease Research Centre in Singapore is used for the treatment of malaria,
TB and dengue fever. There are initiatives with medicines for malaria ventures.
Example of drug discovery programme – IMPORTANT FOR ESSAY IN EXAM ON
ANALGESICS – Novel treatment for chronic pain.

Pain is the largest CNS market and is valued at $20BN

There is a high unmet medical need

Current therapies are associated with poor efficacy, high incidence of adverse events (e.g.
drowsiness and other CNS effects) and there is a high off-label drug use that demonstrates the
dissatisfaction with current approved therapies.

300M people suffer from chronic pain. The biggest areas are post-op pain (70M), neuropathic
pain (26.8M and includes radiculopathy, diabetic neuropathy, post-hepatic neuralgia
(excruciating pain of permanent shingles), HIV, stroke, reflex sympathetic dystrophy and
spinal cord injury), inflammatory/nociceptive (52.9M) that include osteoarthritis, rheumatoid
arthritis and fibromyalgia, and visceral pain (68.3M) that includes IBS, IBD, pancreatitis and
interstitial cystitis. Migraine and tension also affect 69M.

NSAIDs sold the most in 2006 with £8.18BN, second to narcotic analgesics at $5.52BN.

Acute and chronic pain:

Acute – protective pain. This is pain that is experienced when one senses a noxious/damaging
stimulus. Thermal models of pain exist that time how long your reflexes take to kick in and
include hot plate, tail flick and cold plate tests. Also noxious pressure can be experienced in
mechanical acute pain.

Chronic pain no longer serves a protective function and can be very painful in the absence of
noxious stimuli. Chronic pain is hypersensitivity to various stimuli including mechanical,
thermal and chemical stimuli as well as spontaneous pain.

The pain pathway is from the peripheryyyyyyy...... to the brain!

Possible sites of intervention are peripheral nerves, spinal cord
locations and ascending pathways. Sensitization in periphery and
centrally are faucets of pain. There are many potential molecular
targets and different types of peripheral nerve fibres (touch, cold
etc). One type response to noxious stimuli that include heat,
noxious chemicals like acid and inflammatory mediators.

Don’t remember the time here....

TRPV1 as a target for pain treatment:

TRPV1 is transient protein potential protein V1 and it is found in nociceptors. It creates an

ion channel that is permeable to calcium and sodium ions, but is located only in sensory
nerve fibres that respond to various painful stimuli.

TRPV1 was cloned and expressed in cells:

It was activated by heat (generally over 42 degrees  average pain threshold)

It was also activated by capsaicin (hot ingredient in chilli, and endogenous,

structurally related proteins also activate TRPV1)

Activated by acid solutions

Activated/sensitised by inflammatory mediators acting via GPCRs, e.g. bradykinin

and prostaglandins

Activation of TRPV1 causes pain fibres to fire action potentials.

Potential molecular mediators of pain are evoked by various stimuli

This discovery of this protein stimulated the pharmaceutical and biotechnology companies to
invest in drug discovery programmes for TRPV1 antagonists.

In-Vitro Assays for TRPV1:

The primary in vitro assay used TRPV1 that was expressed in mammalian cells, including rat
and human cells.

TRPV1 activation caused calcium ions to enter the cells, and this was proven by using
calcium-sensitive dyes as measurements. This was an easy assay that was suitable for high
throughput screening.

Different stimuli can be used to activate TRPV1, e.g. capsaicin and acidic solutions.

Large chemical libraries were screened for this and potential chemical classes of TRPV1
inhibitors were identified.

A secondary in vitro assay was carried out where sensory neurones were isolated from a rat
and calcium sensitive dyes were injected into the neurones.
Optimization of TRPV1 antagonists:

The medicinal chemistry programmes optimized the in vitro activity in a quest for increased
solubility, stability, retention of activity, orally active compounds that penetrate the blood-
brain barrier to access terminals in the spinal cord (this is a major problem for drugs acting in
the central nervous system).

2 approached were used in animal models of chronic pain.

Freund’s adjuvant in the paw stimulated inflammation that lasts around one day. This was the
inflammatory model, and paw swelling was seen due to increased paw sensitivity to mild
pressure, touch and heat.

A neuropathic pain model was also carried out, and this was induced by partial nerve injury,
also leading to an increased sensitivity to mild pressure and touch.

Genetically modified mice lacking TRPV1 showed reduced heat hypersensitivity after
inflammation, and this result is consistent with the role for TRPV1 in pain sensation.

Sensitivity was measured by graded increasing pressures to stimulate pain for both
neuropathic and inflammation models.

The percentage reversal of hypersensitivity was shown to be as good as NSAIDs with the
TRPV1 antagonist.

Compounds were tested in animal models of chronic pain and the inhibition was active in
both inflammatory and neuropathic pain models. Further studies showed the effects in other
animal models of chronic pain, e.g. cancer and osteoarthritis. This suggests a broad spectrum
analgesic that could treat a wide variety of pain conditions = $$$$$$$$$$$$$$$$$$$$$$$$$.

An acceptable profile in safety/toxicology studies was also proven.

Into Clinical Trials:

Compounds from several companies were taken into Phase I and Proof of Concept trials.

A low dose of one compound was shown in the proof of concept trials to inhibit the action of
capsaicin applied to the skin of human volunteers, with nerve-evoked vasodilation and
reddening of skin as effects.

Another compound displayed the effect of an unexpected large increase in body temperature
in some human volunteers, so trials were stopped.

The worst case of increased body temperature was a one day increase in body temperature
from 37 degrees to 41 degrees, and it would have continually risen had it not been for more
drug intervention. This shows that the compound also had poor pharmacokinetics as it
remained in the body (fighting the induced decrease in temperature) for 3 more days.
Do all TRPV1 antagonists cause increases in body temperature?

Not all in animals – but reasons are unclear.

Continued concerns about the safety liabilities of TRPV1 antagonists.

In another trial, volunteers had reduced sensation to noxious heat, e.g. stating that a 42 degree
bath was too cold.
LECTURES 1 AND 2 – RECEPTORS AS DRUG TARGETS – MEASURING AGONIST
ACTIVITY.......5:25AM...Don’t ever forget student life!!!!

Homeopathy is bullshit.

Relies on crap.

Diluting makes it more potent??

A drug is a chemical that affects physiological function in a specific way.

It can be an element, e.g. platinum as an anticancer drug, small molecule, e.g. majority of
drugs, peptide (RMM in the thousands), large protein or oligonucleotide.

Drugs often show stereo-specificity with one isomer being more active than the other. This is
because the receptor will be complementary to one isomer, as the other one won’t fit into the
3D binding site.

A receptor is a target protein for a drug. Receptors can be enzymes, e.g. COX for aspirin,
transporters, e.g. 5Ht reuptake transporter for fluoxetine, ion channels, e.g. lidocaine and
transmitter/hormone receptors (salbutamol).

Note some drugs do not act on proteins. Alternate targets include DNA (some anticancer
drugs work straight here) and buffers (to reduce stomach acid).

Drug effects are concentration dependent.

A subthreshold level leads to no effect, as the concentration is too low to have a noticeable
efficacy, and the chance of a drug finding its receptor is also extremely low.

The therapeutic concentration is when the benefits exceed the risk.

A high concentration (overdose) bears a lot of side effects, and increases the risk over the
benefits.

Dose is not necessarily a concentration.

A potent agonist produces a strong response at a low concentration. Also, a potent antagonist
prevents a response at a low concentration.

Law of mass action: Substrate + enzyme --- (binding)  Substrate Enzyme complex ---
(catalytic activity) enzyme + product. This isn’t usually reversible.
Drug + receptor --- (binding)  Drug Receptor Complex --- (receptor activation) DR*
(activated drug receptor). The reaction is usually reversible as the drug is unchanged.
Agonists help to promote the formation of DR*, how however this is all dependent on
probabilities.

Guy’s Bar is awesome!! Gonna miss it sooo much!

Non-specific binding is the binding that occurs between a drug and anything apart from its
receptor. If we subtract this from the total binding that occurs, we will get a measured answer
for the total amount of drug that is bound to receptors. We can then see how many receptors
are also present.
The drug concentration can be called Xd, and the number of receptors is equal to the total
amount of bound drug minus the amount of non-specific binding. The number of receptors
bound by a drug is the number in the DR complex. K+1 and K-1 are forward and backward
rate constants, and the elements on either side multiplied by their rate constants are equal.

Remember these derived formulas!!

A Scatchard plot displays the total amount of bound drug against the proportion of bound to
free drug.

Sorry about copy and pasting all the graphs but they’d look exactly the same in paint anyway.

Also, Dear Future Self....Stay interesting and don’t ever be afraid to try out something new.
Better to try something and then never regret any missed opportunities than never try at all.
See where life takes you.

Kd is the equilibrium dissociation constant = k-1/k+1. K MINUS ONE OVER K PLUS ONE

Kd is a physiochemical constant like Avogadro’s number. The Kd is the same for a given
receptor and drug combination in any tissue and species anywhere in the universe.

Kd can be used to identify an unknown receptor.

A drug’s Kd can identify possible receptors.

A receptor’s Kd (same thing) can identify potential agonists.

The Hill-Langmuir equation gives the proportion of receptors bound by an agonist (Pa) in an
easy-to-live-with equation.

The Ka is a measure of agonist affinity for its receptor. It is the concentration of agonist that
is required to occupy 50% of the available receptors and is a function of the agonist and
receptor combination. Its units are 1/M as K-1(s^-1) and K+1 (M s^-1).

Ka can tell give us selectivity and affinity comparisons.

From this table, we can say that carbachol has a lower

affinity than Oxo-M for M1-M4 receptors. The higher
the Kd, the more drug required to elicit a 50%
response. Carbachol however shows M1 and M3
selectivity, and Oxo-M’s Kd values don’t indicate a
particular subset selectivity.

Generally, odd numbered receptors are structurally

similar and same for even. E.g. M1 and M3 are quite
similar in structure, and M2 and M4 are as well.
An agonist binds to a receptor and elicits a response.

A + R affinityAR efficacy receptor activation AR*

These responses can anything from the opening of an ion channel, G-protein activation or
activation of an enzyme. This all depends on the receptor type.

The EC50 is the effective concentration that produces 50% of the maximal effect. It is a
functional measure and is a product of both affinity and efficacy.

The IC50 is the concentration required to produce 50% inhibition.

The LD50 is the dose to produce death in 50% of the subjects, but this is unethical and isn’t
used anymore.

According to Dose-Response (strictly speaking we’re not measuring dose, just concentration)
curves, we can determine relative potencies of various drugs according to their EC50 values.
The EC50 represents the concentration for 50% response. The Ka represents the
concentration for 50% occupancy.

Most biological agonists need less than 1% occupancy to observe their EC50 values – high
efficacy!

Partial agonists have affinity for the receptor but low efficacy and will not produce a
maximum response.

A partial agonist could have a competitive inhibition (antagonist) effect on a full one.

Inverse agonists represent a recent concept. In the past, most experiments assumed basal
activity of a receptor was zero.

However, receptors may activate without the presence of an agonist OMG OMG! Then, some
drugs might reduce the number of activated receptors at rest. These are termed inverse
agonists.

If observed for infinity, a receptor that is forever without a ligand will at some point activate
spontaneously.
Summary:

 Homeopathy: giving patients medicines that contain no medicine whatsoever

 Herbal medicine: giving patients unknown doses of an ill-defined drug of unknown
effectiveness and unknown safety
 Pharmacology is based on physiochemical constants that describe the binding of
drugs to receptors
 An agonist is a drug with affinity (Ka) and efficacy, both of which can be quantified
 A full agonist has high efficacy, i.e. produces a maximal effect before all available
receptors are occupied
 A partial agonist has low efficacy, i.e. occupies all of the available receptors without
producing a maximal response
 An inverse agonist reduces constitutive receptor activation
LECTURES 3 AND 4 – RECEPTORS AS DRUG TARGETS – MEASURING
ANTAGONIST AFFINITY: WHY ANTAGONISTS ARE MORE USEFUL THAN
AGONISTS IN DEFINING RECEPTORS

Not going to discuss:

 Chemical antagonists, e.g:

o Buffers
 Pharmacokinetic antagonism, e.g:
o Alter uptake
o Alter distribution metabolism of another drug
 Physiological antagonism, e.g:
o Act on opposing physiological systems
o Muscarinic receptor agonists slow the heart while β-adrenoreceptor agonists
speed up the heart. These are 2 agonists that cause antagonist effects to each
other.
o One is a physiological antagonist of the other.

Antagonists covered here:

1. Competitive reversible antagonist (B)

2. Competitive irreversible antagonist (Bi)
3. Non-competitive antagonist, e.g. channel blockers and G-protein uncouplers
4. Other

Competitive Reversible Antagonists (B):

Receptors can bind to agonists and antagonists.

Competitive reversible antagonists have affinity but zero efficacy. They bind to receptors but
induce no response. However, it binds in such a way to prevent agonist binding (competitive
inhibition).

Most competitive reversible antagonists have a higher affinity than agonists (agonists’
affinities are often in the mM range whereas antagonists have an affinity in the nM range).

B + R  BR
Types of agonist graph (with competitive antagonist position noted):

Antagonist binding is described by the same formula as for agonists, but here we’re
describing the antagonist. Where PB is the proportion of receptors bound by the antagonist:

PB = (XB/KB) / (XB/KB) + 1

KB is the dissociation rate constant for the antagonist – it is the concentration required to elicit
50% receptor occupancy. KA is exactly the same reading, but for agonist concentration.

Some antagonists, e.g. atropine, have a high affinity for M1, 2 and 3 but also have a non-
selective KB.

Pirenzepine has a low affinity for M2 and M3 but a higher affinity (still low overall) for M1
receptors. This is a selective antagonist that is used clinically as the M1 receptor subtype
induces gastric acid secretion.

The best antagonists are not synthetic ones – they are the ones that are products of evolution.

Binding of an agonist and a competitive agonist:

A + R   AR. From this, we can derive:

XA + (Ntot – NA – NB) k-1 k+1 NA

Where Ntot is the total number of receptors, NA is the number bound by the agonist and NB is
the number bound by the antagonist.
No receptor can be bound by both an agonist and a competitive reversible antagonist at the
same time.

We can combine the above derivation with the same derivation from the B + R  BR
equation to give overall the Displacement-Binding Equation:

PA = (XA/KA) / (XA/KA) + (XB/KB) + 1

And

KB = (PAXB) / XA/KA(1-PA) - PA

This is the dissociation-binding equation.

If we radiolabel the agonist and incubate it with a tissue and then remove the excess agonist
and record the radioactivity, we have the amount of radioactivity at 100% receptor
occupancy.

Then we can add an antagonist and observe the drop in radioactivity as the antagonist
displaces the agonist. A graph can then be constructed:

As the antagonist affinity increases, the graph shortens and vice versa.

With a major assumption, we can also derive that DR – 1 = XB/KB. DR = dose ratio and this
formula is the Gaddum equation.

This can then be linearised to the following Schild equation:

Log10 (DR – 1) = 1xLog10 (XB) – 1xLog10 (KB)

Increasing the concentration of the agonist can overcome the effect of a competitive
antagonist.

The big assumption is that the PA in both cases is the same. The agonist concentration axis
above should read Log10 [Agonist].

For a pure competitive antagonist, the graph moves to the right in a parallel shift.

A Schild plot is a plot of the log10 reading of the antagonist concentration against the log10
reading of the dose ratio – 1. It has to have a gradient of 1.

Log10 (DR – 1) = 1xlog10 (XB) – 1xlog10 (KB)

Or

Log10 (DR – 1) = 1 x log10 (XB) + pA2.

The pA2 is the negative log10 of the concentration of antagonist required to shift he agonist
concentration response curve two times to the right. The pA2 is when 50% of the receptors
are occupied by the antagonist, therefore pA2 = KB. This means that we do not have to do
binding experiments to measure the equivalent of the KB, so if pA2 = 9, then the
concentration is 1 x 10-9M = 1nM.

The pA2 value is independent of the agonist used.

pA2 readings are consistent as long as the same receptor (often hardly any species variation
as well between receptors) and same antagonist are used.

pA2 does not change as the agonist changes. It also doesn’t change with the tissue location
for the same receptor. It does however change with different receptors and also does change
with different antagonists.

Why the KB (pA2), i.e. antagonist, is better at defining receptor affinity than the KA, i.e.
agonist:

We can compare binding data (KB) with functional data (pA2).

Competitive antagonists have zero efficacy. The KA can change when the receptor is bound
by its effector. Uncoupled receptors have high affinities for agonists, and a receptor when
coupled to a G-protein has lower affinity for the agonist. Also the type of G-protein can
change the affinity.

Competitive irreversible antagonists (Bi):

Bi + R  BiR

There is no reverse reaction.

Competitive irreversible antagonists have affinity but zero efficacy, and they bind to the
receptor but induce no response. However, they bind in such a way to prevent agonist binding
(competitive) and it is permanently bound through covalent bonds to the receptor, until the
receptor is broken down.

Irreversible competitive antagonists are time dependent:

The lines will descend further as time continues. There is no parallel shift, and the maximal
response decreases as the receptors that are bound by Bi are effectively inactivated.

Partial agonists can act as competitive antagonists as they will compete with the full agonists
for the receptors, but only promote a minuscule response.

Non-competitive antagonists don’t bind onto the binding site but still prevent any efficacy.
Most non-competitive antagonists are channel blockers.

E.g. PicTx acts on GABAA receptors as a channel blocker.

E.g. Pertussis toxin blocks G-protein coupling in Go/i receptor. It is a G-protein uncoupler.

Non-competitive antagonists share the same graph as on the previous page but aren’t time
dependent. There is a fall in maximum response but there may or may not have some
rightward shift.

Allosteric modulators are neither agonists nor antagonists. They enhance the action of other
ligands.

Benzodiazepines act on GABAA receptors to increase the affinity for GABA and to increase
the efficacy of it as well.
Think I missed a lecture, so lecture numbers are going out of the window. 3:08AM.

INTRODUCTION TO BIOLOGICS

What is a biologic?

It is a product that is derived from living sources, e.g. humans, animals, bacteria and viruses.

Examples include blood proteins, recombinant proteins (e.g. erythropoietin), fusion proteins
and antibodies.

An Fc domain helps to improve the half life of two Fab molecules, which when put together,
all constitute the antibody.

Antibodies or immunoglobulins are produced by B cells. They comprise of heavy and light
chains, and each antibody molecule has a unique structure. Antibodies can bind to and
neutralise pathogens or prepare them for uptake and destruction by phagocytes.

The Fab is the antigen-binding fragment.

The Fc is the complement binding domain.

Antibodies can work by neutralisation, opsonisation and/or complement activation.

Neutralisation involves the antibody preventing bacterial adherence by sticking to the surface
of the bacteria.

Opsonisation involves the antibody promoting phagocytosis.

Complement activation involves the antibody activating the complement, which enhances
opsonisation and also the lyase of some bacteria.

Antibodies exist as different isotypes, each with different functions and properties.

IgM is a secretory molecule that isn’t clinically relevant due to its low affinity to its target
molecules.

IgG has a half life of around 20 days, so it is very clinically useful.

Also, the classical potency of complement activation is high for IgG1, IgG3 and IgM and is
low for IgG2 while being non-existent for IgG4.

The chance of placental transfer occurring is highest in IgG1, then IgG3 then IgG2.

Nomenclature of biologic therapies:

The suffix indicates the class of biologic therapy:

-Cept = human receptor fusion protein, e.g. etanercept (binds to

TNF) and abatacept.

-Ximab = chimaeric monoclonal antibody, e.g. infliximab (normally

given with immunosuppressant, e.g. methotrexate) and rituximab
(targets B cells). The problem with chimaeric monoclonal antibodies
is that since they are derived from rodents, an immune response is
initiated against them in humans.

-Zumab = humanised monoclonal antibody, e.g. efalizumab (targets

VLA4 to stop integrins or T-cells entering the brain – it is important
to look for ways to try and eliminate the PNC side effects.

-Umab = fully humanised monoclonal antibody, e.g. adalimumab

and ustekinumab.

Since there are many biologics that can be used to treat the same locations, e.g. TNF
molecule, before resistance and tolerance has been developed, we can swap the treatment of
the patient.
Therapeutic antibodies in the clinic:

The use of infliximab in the treatment of moderate to severe plaque psoriases proved to be a
huge success compared to taking corticosteroids, which had many side effects. Topical
creams were also very hard to apply.

How to minimise immunogenicity:

Strategies for generation of fully human antibodies:

E, f, g and h talk about phage display techniques, which include expressing the antibody on a
bacteriophage, then purifying the solution of the bacteriophage and isolating the antibody.
Monoclonal antibody discovery automation: B cells on plate  Isolate B cells producing
required antibody  Freeze required B cells for later use  Isolate variable region

Isolation of individual B cells: cluster fluorescent molecules around the B cell and pick out
the cell of interest.
Fab fragments increase the half life, as well as applying to PEG. Certolizumab is used in the
treatment of rheumatoid arthritis.
Abatacept is used in the treatment of rheumatoid arthritis.

Etanercept is used in the treatment of rheumatoid arthritis and Crohn’s disease

Drug target selection can be achieved in many ways, e.g. genetic screening and non-
prejudicial screening.

Sclerostin protein is used for bone growth and in some patients it is overexpressed, leading to
huge bone growth.

Candidate optimisation: Antibody Structure and Activity:

1. Modify structural properties of antibody

a. Decrease immunogenicity through in silico/vitro/vivo techniques, normally by
making single base pair mutations, therefore the antibody loses some affinity
for its antigen
b. Isotype-dependent upon therapeutic effect required, e.g. target neutralisation,
cell depletion or immune tolerance.
i. Complement fixation properties (e.g. could remove or mutate residues
in Fc region)
ii. Antibody-dependent cell cytotoxicity properties
c. Pharmacokinetics
i. Isotype
ii. Post-translational modifications
1. Glycosylation
 2. PEGylation (if it doesn’t have a high half life – normally
caused by a lack of an Fc region).

Considerations of biologic therapy in humans:

1. Determine optimal biologic dose

a. Pharmacodynamics and pharmacokinetics. You could mutate the biomarker,
e.g. in antibody-modulated CRP, there is no need for a phase II study as proof
of concept has already been carried out.

2. Determine toxicological threshold

a. Maximum tolerated dose is not usually included in clinical development plan
of investigation Mab

3. Route of administration
a. FIH - IV and SC routes of administration (SC preferred – easy for patient to
do)

4. Evaluation of proarrhythmic effects

a. For NBEs, monitor as part of safety studies, not early on in clinical
development (as for NCEs)

5. Immunogenicity studies (FIH)

a. No predictive animal models
b. Alteration of pharmacokinetics and possibly pharmacodynamics

6. Comparability studies (to find consistency in comparability)

a. Determine the final manufacturing process as early in clinical development as
possible

Then scale up the antibody!!!!

1 to 250mg is used for in vitro experiments.

50-100 litres can isolate enough products for first-in-man studies.

Large scale capacities are needed for preclinical and clinical use. Examples of large scale
machinery include manufacturing plants and bioreactors.

Cells Produce antibodies  Get rid of cells  Concentrate the isolated antibodies
(approximately to 100mg/ml)

SELECTIVE DRUG DELIVERY

A drug is an active pharmaceutical ingredient (API) that most likely has a small molecular
weight. It is an organic species that is synthesised by a medicinal chemist. A drug can also
possibly be a peptide or oligonucleotide (prepared by biotechnological means).

A medicine is an active pharmaceutical ingredient plus excipients. Excipients are materials

that should be inert and safe. Their sole function is to act as a carrier or platform for the
active pharmaceutical ingredient.

Fewer and fewer molecules are getting approval each year as they don’t pass phase 1-4 trials.

Consequence of New Discovery Practices:

 Maximised pharmacological activity, in terms of:

o Receptor affinity
And
o Receptor selectivity
However, the development of molecules to have a high affinity for a particular receptor led to
many hydrophobic molecules being produced and developed. These molecules are very hard
to turn into medicines.

 Increased hydrophobicity (see above)

Trends resulting from New Discovery Practices:

 Decreased water solubility

 Decreased permeability.

The decreased water solubility and permeability of the drugs themselves lead to poor
interactions with membranes.

Why should we design selective drug delivery products?

 To transfer the therapeutic at the right site and minimise delivery to healthy cells.
 To minimise the dose
 To minimise unwanted effects by exposure of the therapeutic to healthy tissues

The benefits of selective drug discovery include:

1. The optimisation of interaction of the drug with its site of action at the right frequency
and rate. I.e. right dose, right site and right duration.
2. The reduction of the side effects of the drug by restricting distribution to the target
sites.

Only specific drugs actually need targeting.

Paul Ehrlich coined the term the magic bullet in biology. He worked on the selective staining
of tissues for histology, and in particular the selective staining of bacteria. He suggested that
is a compound could be made that selectively targeted a disease-causing organism, then a
toxin for that organism administered with the targeted molecule would kill only harmful
microorganisms/cells that it is targeted to.

There are pharmacokinetic considerations related to drug targeting that include:

1. Drugs with a high total clearance (a lot are excreted) are good candidates, i.e.
hydrophilic drugs.
 2. The carrier-mediated transport is suitable for response sites with a relatively small
blood flow. This is because the lower the drug supply, the lower the dosage reached.
3. The higher the rate for elimination of the free drug from either the central or response
compartments the greater the need for targeted drug delivery.
4. To maximise the targeting effect, the release of the drug from the carrier should be
restricted to the response compartment (target site). The biochemistry of the target
tissue needs to be considered.

Requirements:

1. Requirements include the understanding of the biology involved in the disease

process, e.g. in cancer cells, there is an overexpression of GFR and folate receptors.
Cancer cells also secrete a lot of metalloproteinases.

2. The utilisation of these processes to obtain selective drug delivery, taking into account
the patho-physiology, biochemistry and chronicity (how the disease evolve?) of the
disease.

If we understand the disease better, then we can understand how to target it better.

The ideal targeted drug should be targeted to the disease site, it should be able to access the
site, it should be able to stay in the site for long (retention) and it should also know when to
deliver the actual drug from its protein capsules.

Changes can take place in disease to physiology and pathological processes. For example, the
expression of different receptors occurs in cancer states, and also changes in capillary
permeability occur. Remember angiogenesis also occurs.

Improving accessibility is required for:

 Disease of the CNS

This is because the main problem of the brain is the blood brain barrier. There are
receptors in the blood brain barrier and if we carry a drug attached to a ligand of these
receptors, the drug can easily access the brain.

 Diseases of the immune system

We could target macrophages for example to alter behaviour and activity

 Cancerous states
  Some cardiovascular diseases
 Arthritic disease

If a drug is delivered intracellularly but has high cellular permeability, it can diffuse rapidly
away from the site of location. We need to consider cellular kinetics, e.g. rate of uptake, rate
of excretion and rate of retention.

Timing affects the chronicity of the disease. We could deliver the drug before the precursor
cells become active disease cells, or afterwards. We could even silence gene expression
through siRNA. Chronopharmacology refers to the responsiveness of the target cell and the
influence of polymediator cascades.

Drug delivery and targeting systems: compound / purpose.

The active delivery system / therapeutic effect.

The carrier system (soluble or particle) / to effect a favourable distribution of the drug, to
protect the drug from metabolism, to protect the drug from early clearance.

A homing mechanism or a homing device (passive and/or active targeting) / To specifically

target the drug to the target cells or target tissue.

Active targeting utilises molecular mechanisms. Passive targeting doesn’t use any molecular
mechanisms for targeting.

Soluble macromolecular carriers include antibodies or ligands with polymers:

Poly(hydroxypropylmethacrylate), poly(lysine), polyaspartic acid, poly(styrene co-

maleic acid anhydride) and polyethylene glycol (PEG(.

PEG is the most widely used soluble carrier.

Particle carries include:

Liposomes, micelles, nanoparticles, PLGA microspheres (poly-lipo glutamic acid), proteins,

solid lipid nanoparticles and dendrimers (polymer is in the shape of 3 branches here).

Factors affecting drug delivery and targeting systems include the endothelial lining of the
blood circulation, the anatomical location of the target (decreased reached dose if the target is
far away) and the macrophages’ mononuclear phagocytic system (to identify foreign
particles) or the reticuloendothelial system (RES).

Soluble carriers don’t activate the immune system, but have a potential to.
Macrophages eventually contribute to the majority of drug clearing.

There are 3 main types of blood capillaries:

A continuous capillary is found in most places of the general circulation. It has a

subendothelial basement layer that is fully continuous.

Fenestrated capillaries are found in the exocrine glands and in the pancreas. They have
fenestrations of around 60nm width, which are sealed by a membranous diaphragm. The
subendothelial basement membrane is continuous.

Sinusoid capillaries are sometimes called discontinuous capillaries. They are found in the
liver, spleen and bone barrow. The endothelium contains various gaps of various size
(normally around 100nm) that are known as sinusoids. The subendothelial basement
membrane is either absent in the liver or fragmented and interrupted in the spleen and bone
marrow. It is very easy for a drug to enter these sinusoids, which is why sometimes drugs can
be collected in the liver rather than going to their target tissue.

If drugs are larger than most of the sinusoids, they end up in the lungs that then has with it a
risk of embolism. We hardly administer drugs greater than the 100nm sinusoids.

Kuppfer cells are present in the luminal membrane of the endothelium of capillaries. They act
ac macrophages and phagocytose harmful debris, foreign substances and dead cells that pass
through the blood.

Mononuclear Phagocytic system:

Fixed cells include macrophages in the liver (Kuppfer cells), spleen, lung, bone marrow and
lymph nodes.

Mobile cells include blood monocytes and tissue macrophages.

The mononuclear phagocytic system functions include:

1. The removal and destruction of bacteria

2. The removal and destruction of denatured proteins
3. Antigen processing and presentation
4. Storage of inert colloids (substance microscopically dispersed evenly in another
substance)
5. Assisting in cellular toxicity

Mononuclear Phagocytic system particle clearance:

Particles of sizes 0.1 to 7μm are cleared by the Kuppfer cells in the liver.
Negatively charged liposomes and positively charged vesicles are rapidly cleared. Neutral
vesicles remain longer.

Hydrophobicity particles are covered by blood proteins, opsonins, which help phagocytosis
and opsonisation.

Opsonisation explains why liposomes are cleared from the blood very rapidly.

Opsonisation:

 Macrophages secrete proteins

 Proteins interact with liposomes and/or other particles
 Macrophages are attracted to these proteins and recognise them.
 Macrophages phagocytose the proteins and their attached colloid.

Selective drug delivery to tumours: Cancer-targeted drug delivery

Enhanced permeation and retention effect:

In normal blood vessels, there are tight contacts between pericytes and endothelial cells. The
P/EC ratio is almost 1:1.

In tumour blood vessels, the contacts are much looser than in a normal vessel. The pericyte to
endothelial cell ratio is much below 1:1 and this is an indication of a tumour being present.

Targeted drug delivery to cancer increases circulation time, enhances uptake in cancer and
enhances specificity.

Nanoparticle kinetics:

When series gamma scintigrams of a KS patient after pegylated liposomes containing 111In-
DTPA were analysed, the circulating particles were trapped in the tumours after 2 days.
This is because pegylation (PEG) tricks the immune system and makes these liposomes
unrecognisable.

Particles and macromolecules can target tumours by size and functionalities:

Passive targeting:

 Enhanced permeation and retention (EPR)

 Targeting of RES cancers
 Size and surface chemistry

Active targeting:

 Surface chemistry allows functionalization with target molecules, e.g:

o Antibodies, e.g. Herceptin attached to nanoparticles
o Folic acid attached to dendrimers
o Carbohydrates attached to gold nanoparticles

Soluble macromolecular carriers: Monoclonal antibodies for the treatment of cancer:

We need targeting (i.e. recognition) of tumour-associated antigens and angiogenic vessel

antigens, as newly-formed vessels secrete different proteins to normal blood vessels.

Mechanism of action:

 Induction of apoptosis
 Induction of cytosis
 The carrying of a toxic payload of drug/toxin!!!!!!!!!!!!!!!!!!!!!!
 Inhibition of angiogenesis
 Coagulation of blood in the angiogenic vessels
 Enhancement of natural immune responses – cancer vaccine.

Most antibody-based drugs and drug carriers are IgGs, which have 2 heavy chains, 2 light
chains, antigen-binding sites (variable regions), a hinge region and disulphide bonds.

BR96 – Doxorubicin by Bristol Myers Squibb labs is an anti-CEA (carcinoembryonic antigen

– present on solid tumours). There is a hydrazone link to the drug present on the heavy chain
region. This link is pH sensitive and comes off in low pH, i.e. when taken from the late
endosome to the lysosome. Also, targeting for damaged cells is possible.

The conjugate is taken up by endocytosis and the drug is released intracellularly when the pH
drops below 6.5. The antigen may also be present in the bloodstream as well.

From a phase 1 clinical trial, doxorubicin loading was too low, the targeting system was poor
due to the dose-limiting toxicity of the effects in the gastrointestinal tract and the drug was
not released fast enough as the receptor recycles and undergoes exocytosis from tumour cells.
The whole conjugate was being recycled rather than the active drug being separated.

Soluble macromolecular carriers: Polymer-Drug Conjugates

Polymers are widely used as biomedical materials as the safety of implanted materials is
known and we can scale up the manufacturing process.

Polymers are widely used in pharmacy as pharmaceutical excipients (oral formulations),

components of devices and CR products and as solubilisers in parenteral formulations.

Polymer-Drug Conjugate: Lysosomotopic Drug Delivery

This phrase was coined by De Duve after he won the Nobel prize in 1975 for the discovery of
lysosomes. He thought of the concept of delivering drugs via the lysosomal compartment of
the cell. The use of polymer-drug carriers was proposed.

A polymer-drug carrier is where a polymer is connected to the drug by a biodegradable link.

Doxorubicin and cisplatin readily diffuse into cells but have a large side effect profile.

A polymer-drug conjugate has a targeting group in its polymer that binds to a receptor on the
cell surface, causing endocytosis of the polymer-drug conjugate. Then the linkage between
the polymer and the drug is broken and the drug is subsequently released into the cell while
the polymer is exocytosed.

Anthracycline Antibiotics: Doxorubicin

It is a natural product that can be used in the treatment of leukaemia, breast cancer and
combinations of other solid tumours. The usual doses are 60-80mg/m2 every 3 weeks.

However, the dose is limited due to the toxicology and side effect profile of the drug, which
includes neutropenia and cardiotoxicity (max cumulative dose 550mg/m2). Nausea, vomiting
and hair loss can be other side effects.
As a prodrug, doxorubicin joined onto a polymer is stable, but when cathepsin B (lysosomal
protease) comes along, it cleaves the joinage of the doxorubicin to the linker inside the cell,
and the drug is released. This drug operates by passive targeting.

Gamma camera imaging showed that the polymer drug has a longer blood circulation time
and doesn’t localise in the lung, liver or spleen.

Optimisation of tumour targeting by the Enhanced Permeability and Retention Effect begins
with selective uptake of the polymer conjugate by the EPR effect and the uptake of the
polymer conjugates by endocytosis-release of the drug intracellularly.

Active targeting: Liver selective delivery for the treatment of primary and secondary liver
cancer.

Galactose promotes targeting to asialoglycoprotein receptor in the liver.

Clinically viable particles for tumour targeting:

 The membranes (liposomes)

 The matrix (GRAS polymers, lipids and proteins)
 The solid core (metals, e.g. gold)

Large, multilamellar vesicles have poor stability but high uptake in the liver.

Small unilamellar vesicles have improved stability compared to large multilamellar vesicles.
However they have a mid-range level of liver uptake, and sonication is possible to make it
smaller.

Polymer-coated vesicles (PEGylated) are also known as stealth liposomes and are much more
stable and long circulating.

Various types of liposomes:

1. Liposomes with water-soluble drug entrapped into the aqueous interior or

incorporated into the liposomal membrane.

2. Antibody-targeted immunoliposome with antibody covalently coupled to

phospholipids or hydrophobically anchored
 3. Long-circulating liposomes grafted with a protective polymer, e.g. PEG that shields
the liposome from the interaction with opsonins.

4. Long-circulating immunoliposome or preferably to the distal end of the grafted

polymeric chain

5. New-generation liposome, the surface of which can be modified. Attachment of

protective polymer or label, the incorporation of possibly charged lipids allowing for
the complexation with DNA, the incorporation of stimuli-sensitive lipids, the
attachment of stimuli-sensitive polymer and the attachment of cell-penetrating
peptide, the incorporation of viral components are example of some of the things we
can do to the surface. In addition to a drug, the liposome can be loaded with magnetic
particles for magnetic targeting and/or with colloidal gold or silver particles for
electron microscopy.

When introducing PEG to a liposome, it makes it unrecognisable to macrophages. What if

we want it to be targeted to macrophages?

DaunoXome is a drug contained in a vesicle made of disteraolyphosphatidylcholine,

cholesterol and Daunorubicin in an 18:7:1 weight ratio. It is the only formulation approved as
a first line therapy of HIV-associated Kaposi’s sarcoma. The clinical dose is 40mg/m2 by
infusion for over 60 minutes every 2 weeks. It is diluted 1:1 with dextrose before use.

Doxil is presented in a stealth liposome of phosphatidylcholine, cholesterol, MPEG-

distearolylglycerol-phosphoethanolamine (MPEG-DSPE) in a weight ration of 3:1:1. The half
life of Doxil is 4.7 hours. The appropriate dosage guide is 10-20mg/m2 every 3 weeks by 30
minutes infusion. The maximum cumulative dose is limited to 550mg/m2. It is used as a 2nd
line treatment of Kaposi’s disease that has progressed, or prior combination, or for use in
patients intolerant to conventional therapy.

There are various ligands that can be used for active targeting, e.g. antibodies, parts of
antibodies – F(ab)2, Fab, ScFv and diabody. Also non-antibody agents can be used, e.g.
transferrin in siRNA treatment and folate in cancer treatment. Also nanocarriers are an
emerging platform for cancer therapy.

The various nanobased carriers for cancer detection and therapy include immuno-binding
fusion protein, carbon nanotube, micelles, dendrimers, polymeric carriers, liposomes,
nanoshells and polymer-conjugate drug/protein.
The therapeutic benefits of nanoparticles include:

 Solubility
 Carrier for hydrophobic entities
 Multifunctional capability
 Active and passive targeting
 Ligands; size exclusion
 Reduced toxicity

An increase in solubility, stability and specificity = a reduction in toxicity and increase in

efficacy.

Studies show that PEG inhibits/delays the release of drug from the liposome. Nanoparticles
remain for longer and don’t release their drugs in one go. How could we activate when these
nanoparticles release their material? Why not activate them by outside sources, e.g. heat by
laser. This gets round the PEG problem as the nanoparticles, even with PEG can still release
their contents whenever you want it.

A drug isn’t a medicine. Currently most medicines depend upon controlling plasma levels of
the drug within the therapeutic window and involve partitioning from the plasma. Selective
drug delivery depends upon either passive or active targeting. The pharmacokinetics of drug
delivery from such systems will depend upon that of the particle/macromolecule employed.

Nanoparticles are mostly used in rheumatoid arthritis, cardiovascular and cancer disease
states.

ARE PLANTS STILL A USEFUL SOURCE OF NEW DRUGS?

Are plants still a useful source of new drugs?

Drugs developed from plants in the past are still being developed into newer derivatives.

Drugs discovered more recently are still being used as therapeutic agents and are still being
developed.

Some new drugs are discovered from plants, and there is a hope for future drug discoveries
from plants.

However there are pertinent issues influencing new drug discovery from plants.

Ethnopharmacology is the scientific study of traditional medicinal uses of plants by human

communities.

Ethnomedicine is the use of plants as medicines by humans.

Out of all the flowering plants in the world, 90% have been unexploited. 25% of
pharmaceuticals are from plant sources and 40% are from fungi.

There is a concern with biodiversity and the disappearance of it along with habitats.

Ethnopharmacology data vs random selection (Random selection = random usage of plants to

find a therapeutical use):

Medical use of plants by humans has been occurring since the beginning of records. 65% of
the human population use plants as medicines in healthcare, and in Africa, 80% depend n
traditional medicine.

The usefulness of plants as medicinal agents can be developed by the isolation of active
compounds, the development of improved drugs from novel and unknown sources, the use of
more advanced pharmacological tools and the use of herbal remedies as starting points.

Here’s Lil Jon:

Advantages of ethnopharmacological data:

Human beings have used plants as medicines for thousands of years, and the effectiveness
and safety was known.

Many drugs in current use are developed from organised traditional medicine.

Fabricant & Farnsworth 2001 review of 122 globally drugs found that 94 of them came from
plants and 80% of the current used is related to traditional use.

For every 10,000 pure compounds that are tested, only 1 will reach FDA marketing and that
would be after 10 years and $231M in development.

Databases are now available that contain the details of thousands of plant ethnomedicines.

Folk-law traditional usage of plants isn’t protected through Intellectual Property Rights.

Other methods of plant screening:

1. Random selection
Followed by screening for specific phytochemicals, biological assays, e.g. anti-cancer, anti-
HIV. The rate of translation into human usage is very low.

2. Combinatorial chemistry

Lots of molecules are synthesised so there are many possible combinations. Millions of
related compounds are generated in a short time.

Drugs from plants so far: aspirin (Salix spp and meadowsweet), quinine (Cinchona), cocaine
(Erythroxylum), digoxin (Digitalis) and reserpine (Rauvolfia serpentine).

Some important medicines have been developed from poisons: atropine and hyoscine from
Atropa belladonna, curare from Strychnos guianensis and physostigmine from Physostigma
venosum.

Aspirin is from willow bark. The willow is native to Europe, Asia and parts of North
America. It has been used in China and Europe for centuries for pain and fever relief.

The first aspirin was made from meadowsweet, which is common in Europe, East America
and Canada. The salicin-active principle of meadowsweet and willow aspirin is that it is
converted to salicyclic acid in the body.

All aspirin today is synthetically produced, and aspirin is the most widely-used NSAID and is
still the standard for most NSAIDs. The structure-activity relationship is very important here.

Originally, aspirin was too irritating to be taken orally.

Drugs developed from salicyclic acid:

Salicyclic acid (from salicin) is used externally only as it is too irritating. Therefore,
derivatives were made for systemic use, including esters of salicyclic acid, salicyclate esters
of organic acids and salts of salicyclic acid.

Substitutions on the COOH and OH groups change the potency and toxicity. The ortho
position of the OH group is important for salicyclate action. Substitutions are also possible on
the benzene ring, e.g. in diflunisal (difluorophenyl derivative).

Therapeutic uses of salicyclates:

Treatment of low intensity pain, e.g. headache, arthritis, period pains and backache.

To lower elevated body temperature as an antipyretic.

Treatment of inflammatory conditions.

Treatment and prophylaxis in coronary heart disease, post-operative deep vein thrombosis
(inhibits platelet aggregation) and in heart attack and stroke cases.
Possible cancer protections are being reviewed.

This old drug has many new uses.

Quinine is the main alkaloid of the cinchona tree bark. It was used for fevers and tertians
(rhythm of getting ill at certain times) in 1633. In 1677, it was recognised and included in the
pharmacopoeia.

Therapeutic uses of quinine (very bitter):

Anti-malarial treatment (not prophylaxis), especially in chloroquine-resistant and multi-drug

resistant bacteria. It is more toxic and less effective than chloroquine, but there is increasing
use of chloroquine.

Quinine-resistant malaria is a possibility.

Malaria is a life-threatening parasite disease, transmitted by Anopheles mosquito

(plasmodium parasite) discovered in 1880.

It is the leading cause of disease, threatening social and economic development of around
half of the world’s population. There were 216 million cases of malaria and 655000 deaths in
2010, but a recent study suggests up to 1.2 million deaths per year occur due to unexpected
adult vulnerability. Women and children are the most susceptible.

Malaria is now eliminated from most temperate countries, but it used to be widespread in the
16th century. 30,000 travellers are affected annually worldwide with 6753 cases in the UK
between 2002 and 2006.

Antimalarial drugs face the problem of being ineffectual against various resistant strains of
the parasite. There is a need for affordable and effective new anti-malarial drugs. Anti-
malarials that are made not for profit are hard to come by, although WHO initiatives are in
place to develop these.

Roll Back Malaria by the WHO was made to develop the prevention, early
detection/diagnosis and ensure prompt treatment of malaria. A cost-effective and long-acting
vaccine needs to be developed. The Medicine for Malaria Venture is a non-profit making
partnership that is developing new, effective and affordable anti-malaria drugs.

Medicine for Malaria Venture is a private and public partnership launched in 1999 that
encourages research into innovative drugs with anti-malarial activity.

A combination therapy was suggested in 2004, and drugs for use in pregnancy are also being
tested.
The way forward for this program is to test other MMV strategies, e.g. increasing knowledge
of the parasite, implementing new and effective tools and intervention methods, e.g.
insecticide-treated mosquitoes.

One new drug developed is artemisin that is introduced to treat chloroquine-resistant malaria.

Artemisin is effective against deadly and drug-resistant malaria. It is derived from the
Artemisia herb that has been used in China for thousands of years. 40 countries are using this
drug as their 1st line treatment. There is a too great demand and insufficient supply.
Artemisin-based combination therapies are available, which offer an inexpensive alternative
to normal therapy.

Cocaine is found in the leaves of the coca shrub. The leaves were chewed socially by
throughout South America and the Andes. It is a stimulant that combats fatigue and the
effects of high altitude, with euphoric side effects.

The anaesthetic properties of cocaine were discovered by chance in the late 19th century. It
was firstly isolated in 1869 and introduced to medicine in 1884. Soon afterwards, it was used
in infiltration and conduction anaesthesia. It is the foundation for current knowledge of local
anaesthetics.

In 1892, there was a search for synthetic substitutes to the addictiveness and toxicity of
cocaine. Procaine was synthesised in 1905 and that as well as lidocaine, bupivicaine and
tetracaine are widely used as local anaesthetics today.

There are many different species of the Erythroxylum coca shrub.

Digoxin was firstly discovered by William Withering in 1875, who described the efficacy and
toxicity of foxgloves leaves (common plant). It treated dropsy by trial and error and there is a
variance in the relative potencies according to type of plant that the drug was isolated from.

The cardioactive glycoside in digitalis was extracted and separated in the mid-19th century. In
the mid-20th century, the compounds were clearly known, the dosages were worked out and
digitalis extracts were standardised.

Digoxin and digitalis both increase the force of contraction on the heart (positive inotropic
effect). The heart rate decreases (negative chronotropic effect), and this is used in the
treatment of heart failure.

Reserpine is an alkaloid extract of the Indian shrub Rauvolfia serpentina. The medicinal use
of the root of the shrub is described in many ancient writings. Later descriptions of its
usefulness in blood pressure and as an anti-psychotic are also noted. It was introduced into
Western medicine in the mid 1950s.
Reserpine was isolated and identified in 1949 synthetically from the natural product. It was
the 1st drug known to interfere with the sympathetic nervous system and was heralded in the
modern era of effective pharmacological control of blood pressure. It is cheap and effective,
but was overtaken by newer drugs due to the side effect profile, especially as the CNS effects
included sedation, an inability to concentrate and depression.

Some important medicines, e.g. curare, atropine and hyoscine and physostigmine were
developed from poisons.

Curare causes death from skeletal muscle poisoning. It is made from a variety of Strychnos
species, from which samples were brought to Europe in the late 16th century.

Purified fractions of curare were originally used in patients with tetanus and spastic disorders.
The structure of tubocurarine was then established and in 1942, curare was used for the 1st
time as a muscle relaxant in general anaesthetic. The nicotinic cholinoceptor is the 1 st
receptor-ion-channel complex that was isolated, sequenced and reconstructed in 3D.
Derivatives have been developed.

Curare derivatives include metocurine (dimethyl tubocurarine), which is synthetically made

and is 3 times more potent than curare.

Alcuronium is semisynthetic and is from S totoxifera.

Gallamine is the synthetic substitute for curare.

Decamethonium was developed by exploring the structure/activity relationships of curare

plants.

Succinylcholine has a curare-like action and is used for short-duration relaxation.

Atropine is an inhibitor of the autonomic nervous system. Atropine and hyoscine are
alkaloids of Atropa belladonna. Belladonna plants have been used by ancient Hindu
physicians for centuries. Deadly nightshade was used as poison in the Middle Ages and by
the Romans.

It used to be used as a beauty aid for women by dilating their pupils. Hyoscine is an
antispasmodic that is used for treatment of travel sickness.
Physostigmine, aka eserine, was obtained from the Calabar/ordeal bean, brought to England
in 1840 and the pharmacological properties were investigated. The pure alkaloid
(physostigmine) was isolated in 1864 and is still used in the treatment of glaucoma.

Recent discoveries from plants include taxol and galantamine.

Taxol was isolated from the bark of the Yew tree. It inhibits mitosis and is therefore useful to
treat the uncontrolled cell division in aggressive cancer. It interferes with normal microtubule
function and clinical trials in 1983 showed promise against breast, lung, oesophagus, head
and neck cancers.

Taxol was approved for the treatment of ovarian cancer in 1992 and is currently used for
ovarian, breast and lung cancer. It is also used for Kaposi’s sarcoma (tumour from herpes).
Ongoing trials to determine its effectiveness against other cancers continue.

1,200kg of bark only gives us 10g of taxol. Taxol is now semisynthetic due to the inadequate
supply of the natural source:

To begin with we start with the precursor (10-acetylbaccatin) from the Taxus species.

Plant fermentation and propagation of the taxus cell line is implemented

A possible alternate source for taxol production is the fungi that live off the Yew tree.

Synthetic approaches to taxol production are available, but the need for other derivatives is
present, e.g. need for docetaxel.

Taxol (paclitaxel) has a taxane ring. The side chain at C13 is essential for anti-tumour
activity. Docetaxel (taxotere) is developed by modifications at C-13. Docetaxel is more
potent that taxol and most other anticancer drugs. It is useful against ovarian and breast
cancer.

Currently used anticancer agents from plants include vinblastine, irinotecan, topotecan and
etoposide.

Galantamine was shown to reverse tubocurarine-induced muscle paralysis (inhibition of

acetylcholinesterase). It is now available in the USA for treatment of mild to moderate
Alzheimer disease. It is unproven that it improves impotence or sexual dysfunction due to
psychological issues.

Available drugs for the treatment of mild to moderate Alzheimer’s disease include
galantamine, donepezil and rivastigmine.

One major issue for future drugs is the Intellectual Property Rights pertaining to drug
development, as the knowledge of these drugs is traditional knowledge that technically
belongs to the indigenous population.

There is a need for pharmaceutical agents from plants. Other plants of the living world will
also contribute to the search for more novel drugs. More plants are still unexplored and may
contribute to advancements in biotechnology (e.g. genetic transfer and manipulation), plant
tissue culture and to plant constituents as substrates for combinatorial chemistry synthesis.

Traditional medicine will help in the fight against HIV/AIDS. There is a 75% correlation
between currently used medicines and their traditional use.
The Convention on Biological Diversity is a legally binding treaty that commits its 180
parties to ensuring the:

Conservation of biological diversity (the variability among living things and the
ecosystems they inhabit)

The sustainable use of its components

The equitable sharing of benefits arising out of the utilisation of genetic resources

Hoodia gordonii is a bitter-tasting succulent that grows wild in the Kalahari desert. It has
been used by the indigenous San people for thousands of years to stave off hunger pangs
when hunting. Its amazing appetite-suppressing quality has attracted many pharmaceutical
organisations. This offers promise for 300 million obese people worldwide but there was
intellectual property rights controversy as the San people didn’t have an equitable share in the
benefits. This culminated in a legal battle for benefit sharing.

India is leading the way in IPR protection of traditional knowledge and traditional medicine.
See: The African Model Law for the Protection of the Rights of Local Communities, Farmers
and Breeders, and for the regulation of access to biological resources in relation to
international law and institutions.

SAFETY PHARMACOLOGY

Primary pharmacodynamic studies investigate a mode of action and/or the effects of a

substance in relation to its desired therapeutic target.

Secondary pharmacodynamic studies investigate the mode of action and/or effects of a

substance that aren’t related to the desired therapeutic target.

Safety pharmacology studies investigate the potential undesirable pharmacodynamic effects

of a substance on the physiological functions in relation to exposure in the therapeutic range
and above.

The question of whether drugs are poisons can be answered in terms of the risk received vs
benefit gained.

Every drug is a poison at high enough doses/exposures. The therapeutic window is a window
of opportunity to treat a given disease effectively while staying in the safe dosage range.
We must take into account pharmacokinetics and the exposure profile as this can dramatically
alter the safety window. Ultimately, this is what pharmacology practices are trying to predict.

A robust definition of the therapeutic window is as a cornerstone to all pharmaceutical safety

assessments. We can predict the efficacious plasma concentration (Ceff) by:

 Estimations from preclinical efficacy models, e.g. blood pressure reductions in

hypertensive rat models.
 The efficacious plasma concentration is only confirmed in phase II efficacy clinical
trials several years down the line of development.

The No Observable Effect Level (NOEL) is the highest dose or exposure level at which no
noticeable effects are seen in safety assessments.

The No Observable Adverse Effect Level (NOAEL) is the highest dose or expose level at
which no adverse effects are seen in safety assessments.

The appropriate therapeutic window defines the entire early clinical development of a project,
e.g. the starting dose, highest dose etc.

Therapeutic indication is an important consideration when considering the impact of the

therapeutic window:

 Severe, life-threatening diseases can tolerate medications with smaller therapeutic

windows, e.g. oncology agents can progress despite the incidences of cardiotoxicity
that is due to the risk vs benefit argument.
 Non-life threatening indications have little toleration for safety effects, e.g. urinary
incontinence

The incidence of drug attrition, withdrawal and adverse drug reactions (ADRs) are greatest
with cardiovascular, central nervous system and hepatic side effects.

For marketed drugs, the main side effects are noticed in the CNS and GI tract. In clinical and
non-clinical drugs the main side effects are in the cardiovascular and hepatic systems.

The majority of withdrawn drugs have had cardiovascular and hepatic side effects.

Adverse drug reactions in humans fall into 5 categories:

Type A: Dose-dependent, predictable from primary, secondary and safety pharmacology.
This is the main cause of ADRs (> 75%) but these are rarely lethal.

Type B: Idiosyncratic response that isn’t predictable and may not always be dose related.
These are responsible for 25% of ADRs but the majority of lethal ADRs.

Type C: Long term adaptive changes that commonly occur with only a few classes of drugs.

Type D: Delayed effects, e.g. carcinogenicity and teratogenicity, but this type is of a very low
incidence.

Type E: Rebound effects following discontinuation of therapy. This commonly occurs with
some classes of drug.

Causes of acute adverse drug effects:

1. Augmented, super-therapeutic, effect of interaction with the primary molecular target,

e.g. bradycardia with a beta-blocker.
2. Interactions with the primary molecular target present in non-target tissues, e.g.
sedation by antihistamines.
3. Interactions with secondary molecular targets, e.g. cardiac ion channels and other
receptors.
4. Non-specific effects.
5. Pharmacologically-active metabolites.

Safety pharmacology began developing in 1975 and is still developing. See the historical
review of the “Origins, practices and future of safety pharmacology,” by Bass et al in the
Journal of Pharmacological and Toxicological Methods.

Regulatory requirements – ICH guidelines:

The ICH is the International Conference on the Harmonisation of Technical Requirements for
the Registration of Pharmaceuticals for Human Use. This is a tripartite organisation that
consists of regulatory authorities of Europe, Japan and the United States.

They make recommendations on ways to achieve greater harmonisation int eh interpretation

and application of technical guidelines and requirements for product registration in order to
reduce or obviate the need to duplicate the testing carried out during the research and
development of new medicines.

See ICH safety guidelines forms S7A and S7B.

ICH S7A objectives of safety pharmacology:

1. To identify undesirable pharmacodynamic properties of a substance that may have

relevance to its human safety, i.e. hazard identification
 2. To evaluate adverse pharmacodynamic and/or pathophysiological effects of a
substance observed in toxicology and/or clinical studies, i.e. risk assessment.
3. To investigate the mechanism of the adverse pharmacodynamic effects observed
and/or suspected, i.e. risk management/mitigation.

Studies that pharmaceutical companies have to complete include lead identification, lead
optimisation, candidate nomination, preclinical trials, phase I trials, phase II trials, phase III
trials and phase IV trials.

Regulatory safety studies include acute in vivo general toxicology studies, in vivo
genotoxicity and mutagenicity studies, chronic in vivo general toxicology studies,
developmental and reproductive toxicology studies, carcinogenicity studies and safety
pharmacology studies.

What Makes You Beautiful – Harry’s solo at Walkabout, remember that future Jameel!

There are changing reasons for drug attrition, so there is an increasing importance placed in
drug safety.

Historically, exposure and bioavailability has limited the success of drug discovery. If the
drug never sees the target, what are its chances of working? Industry has worked hard to fix
this, e.g. early evaluation of pharmacokinetics and drug metabolism through high throughput
LCMS with good success. No company wants to waste money on development of a drug that
will go wrong. They’d rather spend money analysing which compounds can go wrong and
discontinuing those projects, saving money in the long term.

As well as regulatory safety studies, there are also non-regulatory ones that include
high/intermediate throughput in in-vitro assays and in vivo toxicology and safety
pharmacology studies.

Undesired effects can come from non-specific interactions and secondary intracellular
interactions (can also be beneficial or neutral).

Strategies for early safety assessments:

1. Target safety
 a. Augmented, super-therapeutic, effect of interaction with the primary
molecular target, e.g. bradycardia with a beta-blocker.
b. Interactions with the primary molecular target present in non-target tissues,
e.g. sedation by antihistamines.

2. Compound safety
a. Interactions with secondary molecular targets, e.g. cardiac ion channels and
other receptors.
b. Non-specific effects due to physiochemical nature of the compound, e.g.
phospholipids, chelation of DNA.

General strategies for Target Safety Assessment include finding out more about a target,
including an in-depth review about it. This can be accessed from literature and internal
knowledge. For example, these sources of information can be utilised to identify a gene that
if knocked out can cause a particular phenotype in transgenic animals.

Another strategy for target safety assessment includes observing whether the proposed
mechanism of action affects the CNS or PNS. Tool compound identification also asks the
question of whether the drug is available to test safety.

Target tissue characterisation asks where the target is expressed, and there are tools available
to target transcription (mRNA).

Qualitative tools include the examination of cellular resolution through RT-PCR, ISH,
emulsion and/or by autoradiography.

Quantitative tools measure changes in transcripts and these tools include qPCR (Taqman),
LCMD and densitometric ISH.

Analyses can be directed towards both whole tissue and specific areas, e.g. the entire heart of
a rat, a dog’s SAN and/or a human’s AVN.

General Considerations for Compound Safety Assessment – wide ligand profile:

The wide ligand profile gives a broad spectrum view of a compound’s all round
pharmacology, e.g. what the compound hits other than its target. Binding or functional assays
of receptors/enzymes/kinases that represent a cross section of the “druggable proteasome”
can also be carried out.

Wide ligand profile – In Vitro Biological Fingerprints:

This is a tiered approach to testing. It is a primary screening mechanism involving a 10μmd

duplicate. The percentage of inhibition data is converted to a heat map with an IC50 follow-
up if inhibition occurs in more than 30% of the assays carried out. There is no surprise that
most successful drugs have a limited secondary pharmacology.

General Considerations for Compound Safety: Chemical toxicity

Some chemical structures are known to be associated with increases in toxicity. How the drug
is metabolised and drug to drug interactions (DDIs) are also important:

DDIs occur when one drug alters the rate or extent of absorption, distribution,
metabolism or excretion of another drug.

The result may increase toxicity of a given drug due to higher than expected exposure,
e.g. cimetidine reduces the clearance of theophylline, causing an increase in adverse
effects as theophylline is metabolised by cytochrome P450 that is inhibited by
cimetidine.

A comprehensive analysis of a drug clearance mechanisms are undertaken to understand

potential drug-drug interactions.

Adverse drug reactions are the 3rd leading clinical cause of death in the USA. We are failing
to protect patient populations.

Some drugs in clinical use have cardiovascular or respiratory side effects:

There is a difference in emphasis between safety pharmacology and general toxicology

studies:

Basic Objective of Safety Pharmacology:

 To build confidence in safety (CIS) and bring attrition into the preclinical phase so we
can catch unsuccessful compounds early.

Safety pharmacology and toxicology data is used to select the starting dose and range for
phase I studies.

ICH form S7A states that regulatory agencies require, as a minimum, non-clinical safety data
for the 3 systems deemed critical for life: cardiovascular system, respiratory system and
central nervous system.

Safety pharmacology also supports the understanding of adverse effects in clinical trials.

Key aspects of the ICH S7A and B documents:

 Follow-up studies and supplemental studies must be conducted prior to first human
exposure.
 Core battery should ordinarily be conducted to Good Laboratory Practice. Follow-up
and supplemental studies should be conducted to GLP to the greatest extent feasible.
 In vivo and in vitro test systems may be used.
 For in vivo studies, the clinical route of administration should be used where feasible.
  The dose-relationship and time course of the adverse effects should be investigated.
 Doses and concentrations should include and exceed the primary pharmacodynamic
or therapeutic range.
 Human-specific metabolites should also be evaluated.
 The same general principles also apply to the ICHS7B

Conditions under which supplementary studies may not be needed/reduced:

 For locally applied agents, e.g. dermal or ocular), where the pharmacology of the test
substance is well characterised and where systemic exposure or distribution to other
organs or tissues is demonstrated to be low.
 For cytotoxic agents for treatment of end-stage cancer patients. However, for
cytotoxic agents with novel mechanisms of action, there may be value in conducting
supplementary studies.
 For biotechnology-derived products that represent a novel therapeutic class and/or
those products that no not achieve highly specific receptor targeting, a more extensive
evaluation by supplementary studies should be considered.
 Additional exceptions include new salts having similar pharmacokinetics and
pharmacodynamics.

Cardiovascular assessment – ECG & Haemodynamic parameters:

 Blood pressure, heart rate and ECG-intervals as a minimum (some pharmaceutical

companies measure cardiac contractility).
 Drugs that increase blood pressure are not tolerated for many therapeutic indications
o Even with the increases are very small (e.g. 5mmHg), over time this can cause
an increased incidence of stroke, coronary heart disease and heart failure.
o Exception being chemotherapeutic agents, e.g. Sutent (Sunitnib)

Torsades de Pointes – QT interval assessment:

 This is a big issue for the pharmaceutical industry

 Inappropriate block of hERG channels can cause asynchrony and lead to QT
prolongation, which can lead to certain arrhythmias, e.g. Torsades de Pointes. This
includes a rapid heart rate, reduction in cardiac output and reduced brain perfusion.
 Biomarker for hERG blockade is ventricular repolarisation that is measured through
QT-interval prolongation.

Cell based tests, tissue based tests and in vivo tests are used together to make an integrated
risk assessment:
Cell based tests include work on cardiac ion channels, e.g. hERG that can also be achieved by
manual or automated patch clamp of the membrane of single cells. The block or opening of
individual ion channels can be measured.

Tissue based tests include work done on the whole heart, e.g. whole heart work and work on
Purkinje fibres. All cardiac ion channels would be measured here and the action potential can
be measured.

In vivo tests include whole animal tests, e.g. rat, dog or monkey, and these animals are either
anaesthetised or conscious. ECG, haemodynamics and other complex measures, e.g. organ
blood flow can be measured.

Cardiovascular assessment in conscious telemetered animals:

These animals are permanently implanted with a telemeter that produces high quality
dynamic data in free roaming dogs. The battery life is 2 years and the usage is continuous. A
reliable signal is produced in enclosures up to 4mm2 in size. There is a stable, consistent ECG
wave definition that can be observed, and similar devices are in place for small animals, e.g.
rat/mouse for early safety assessment.

There are many CNS adverse effects, e.g. lethargy, sedation, nausea, insomnia, suicidal
intentions, disorientation and anxiety.

Drug adverse effects on the nervous system:

Most of these impact the quality of life and mostly don’t have an effect on the risk to life.

Some are life-threatening as they decrease the patient’s respiratory drive, leading to
respiratory arrest. Others decrease the sympathetic outflow, leading to cardiovascular
collapse and others lead to a loss of consciousness, seizures and convulsions.

Some drug adverse effects on the nervous system are indirectly life-threatening, e.g.
drowsiness, cognitive impairment, motor inco-ordination, dizziness and visual disturbances.
These, for example, could affect driving performance.

Attrition during preclinical and clinical development leads to delays to registration, product
labelling and market withdrawal. There is a commercial issue attached if a drug has to be
tested many times for side effects.

Approaches to studying adverse effects on the nervous system:

 1. In vitro approaches – neuronal cultures, in vitro electrophysiology (ion channels,
neurones and slices).

2. In vivo approaches – behavioural/neurological studies, neurophysiological recordings

(e.g. EEG, ERG, EMG, BAER and nerve conduction velocity), neurochemical studies
(e.g. in vivo microdialysis and biomarkers) and neuroimaging processes (e.g. MRI,
MRS, PET and SPECT)

3. A post mortem approach is to use a neurohistopathology test

ICH S7A and CNS safety pharmacology:

Core battery studies include motor activity, behavioural changes, coordination, sensory/motor
reflex responses and body temperature should be evaluated. E.g. a Functional Observation
Battery (FOB), modified Irwin’s or any other appropriate test can be used.

Follow-up studies include behavioural pharmacology, learning and memory, ligand-specific

binding, neurochemistry, visual, auditory and/or electrophysiology examinations and more.

Autonomic elements of the FOB include salivation, lacrimation and excessive urination.
Neuromuscular elements of the FOB include posture, gait and tremor.
Sensorimotor elements of the FOB include touch response, startle reflex and the grasping
reflex. Behavioural elements of the FOB include arousal, ease of removal and time to
exit drawn circles and explore a new environment.
The reason for the lack of sedation with new generation antihistamines if their low brain
penetration. This is one of the impacts of the blood-brain barrier.
Tier 2 methods to assess motor coordination include beam walking (time how long it takes
the animal to fall off the beam. Also Gait analyses and an accelerated rotarod (automatically
spinning circle and time how long the rat takes to fall off) can be used.

Seizure liability:

This is a convulsion that is better defined as violent involuntary contraction or a series of

contractions of the voluntary muscles. A tonic seizure is a prolonged contraction and a clonic
seizure has alternate contraction and relaxation of the muscles.

A seizure is due to abnormal, excessive or synchronous neuronal activity in the brain

(detected on an EEG) that may or may not progress to an outwardly visible effect, e.g.
convulsion.

Rodent precipitant models of seizure:

Traditionally used models include chemically-induced seizures (usually pentylenetrazole) and

electroshock seizure models. However they are declining in use and may be superseded by
EEG measurements.

Electroencephalographies (EEGs) can be performed on rats, mice, guinea pigs, dogs and
primates. They include an EEG telemetry device implanted under anaesthesia. After several
days of recovery, recordings are made in conscious, freely-moving animals.

Spikes and waves on the EEG indicate seizure activity, which can also be stimulated
chemically, e.g. by 30mg/kg of ketamine.

Hippocampal slice preparation and the detection/investigation of seizure liability:

This happens in mice, rats and guinea pigs. The recording of population spikes is taken from
a hippocampal brain slice. Upon the addition of a convulsant drug to the slice, epileptiform
population spikes are noticed
on recordings.
Drug Dependency and Abuse Potential:

Regulatory guidance is given on this issue. A non-clinical assessment of abuse potential s

required for any compound that may have CNS activity, i.e. a large proportion of the
pharmaceutical industry’s portfolio.

EMEA/CHMP/SWP/94227/2004 Guidance Document of Dependence Liability:

This document proposes a tiered approach to detect and characterise abuse-dependence

liability of all new CNS-active substances.

We start with pharmacology in vitro and in vivo of the NAS (new something substance?!)
and its human metabolites, looking for early indicators of dependence, e.g. interaction with
receptors known to be involved in abuse-dependence. Timing: before human studies.

If there are flags, investigate withdrawal symptoms after repeat-dosing, self-administration

and drug discrimination studies. Timing: Before marketing authorisation application.

Assessment of Abuse Potential:

Regulatory authorities recommend the utilisation of three main animal models:

1. Drug discrimination
a. These models measure the subjective effects of a compound similar to those of
a known drug of abuse.
2. Self-administration model
a. Will a compound initiate and maintain drug seeking and taking?
3. Physical dependence and withdrawal model
a. Does cessation of chronic treatment (greater than 2 weeks) result in a
withdrawal syndrome?

The rat drug discrimination model involves pretreatment with a vehicle, pretreatment with a
training drug and then pretreatment with the test drug.

Over time, if the drug is giving pleasurable effects to the rat, when given a choice of two
buttons to press – one for vehicle administration and one for drug administration, then there is
a dependence associated with the drug.
Drug self-administration models go by the following process:

1. Training drug administration

2. Extinction
3. Test drug (full dose response in every animal)
4. Training drug
5. Test drug PR

Responses per minute are measured for the different drugs. If the rat responds more times
(i.e. presses the button that requests more of this drug) to a certain drug, then there might be a
dependence associated with it.

Physical dependence and withdrawal:

Physiological manifestations are related to the cessation of drug administration. Withdrawal

symptoms in humans include sweating, tremor, vomiting, anxiety, insomnia and muscle pain.

Respiratory Aspects:

Non-inhaled drugs can also have effects on respiratory function. A plethysmography in

conscious rats and also telemetry and measure respiratory effects, as well as effects on pump
function and gas exchange.

For inhaled drugs, adverse effects should also be considered, e.g. irritation, cough and
tolerance.

Very few clinical adverse effects are related to respiratory function. We also detect relatively
few drug effects on respiratory function in safety pharmacology studies.

Free moving plethysmograph:

A chamber is semi-sealed so that it develops its own unique pressure that subsides during a
respiratory cycle. Tidal volume and respiratory rate are derived from a change in volume
produced by the movement of air into and out of the lungs and the movement of the thorax
and abdominal wall produced by an animal in this chamber.

Bias the flow through the chamber to prevent CO2 accumulation and O2 depletion. A similar
technique is used in humans. However, with animals, a very small signal is picked up any
additional movements, e.g. sniffing, grooming etc., disrupts the respiratory signal. Therefore
the animals need to be acclimatised to the chamber.
GI system:

The digestive tract is the system of organs that take in food and digest it to extract energy and
nutrients while expelling the remaining waste. The major functions of the GI tract include
ingestion, digestion, absorption and defecation.

GI safety is important in gastric emptying (e.g. measured by phenol red emptying), acid
secretion (e.g. shay rat – ligated stomach at the start of the duodenum to prevent gastric
emptying) and transit (e.g. charcoal meal that can be detected).

An integrated risk assessment provides your finding with an implication. Anything to the
right of the X’s should not be worthwhile investigating.

How good are the models? All DC molecules are tested in SP models before clinical
assessments. The predictivity of the models is questionable.

Valentin et al derived an analytical tool for determining the sensitivity, specificity and
predictive capability of a safety pharmacology model in relation to the clinical outcome.
The sensitivity of a model is defined as the ratio of true positives divided by the sum of true
positives plus false negatives. A high sensitivity reflects a low rate of false negatives.

The specificity of a model defines the proportion for incidence of drugs without an effect in
humans that is correctly identified by the model. Therefore, it is a measure of the number (or
incidence) of the true negative outcomes. The specificity of a model is defined as the ratio of
true negatives divided by the sum of true negatives plus false positives. A high specificity
reflects a low rate of false positives.

The predictive capacity or accuracy of a model defines how often a model will predict true
positives or negative outcomes in humans. The predictive capacity of a model is the ratio of
true positives and true negatives divided by the total number of drugs evaluated. Thus, a high
predictive capacity reflects a greater confidence to detect the presence or the absence of an
effect in both non-clinical and clinical situations when using a particular model.

The 3Rs are applied in S7A by avoiding the unnecessary use of animals, by using new
technologies and methodologies in accordance with good scientific principle and by avoiding
the discomfort of pain in un-anaesthetised animals.

Combination studies are also performed, which can also lead to the application of the 3Rs in
terms of reduction of number of animals used. Irwin et al derived a device to be used in
combination studies. A cage has a metabolic waste collection system, an infusion and
sampling pump and sample storage area. This upholds the reduction and refinement principle
as more than one set of data can be received here from only one animal.
The future of safety pharmacology is to bring more safety aspects into preclinical levels.

Summary:

 Safety Pharmacology is a rapidly emerging and evolving discipline that aims to

predict functional side effects to human volunteers and ultimately to patients.
 The ICH guidelines (S7A/B) on safety pharmacology enable us to adopt a science-
drive approach.
 Good predictivity is achieved by good science – a stepwise, logical progression
through the preclinical and clinical phases by using validated and optimised tests.
 Very few of the agents that give notable Type A adverse effects in the clinic would
not be detected using a science-driven approach.
 Above all else, the quality of the data is the key to everything.

Biggest…..lecture…..ever.