Pharmacology L1, L3 and L4

Question 1

Which of these is the chemical, generic and trade name?

N-acetyl-para-aminophenol, paracetamol/acetaminophen, panadol

Answer 1

N-acetyl-para-aminophenol = chemical

Paracetamol/acetaminophen = generic (mix of chemicals)

Panadol = trade

Question 2

What are main targets of drugs?

Answer 2

Receptors (GPCRs in particular), enzymes, transporters, channels

Question 3

What is the most common receptor involved in drug signalling pathways?

Answer 3

GPCR receptors

Question 4

What are the 8 steps of the drug discovery process?

When does discovery end and development start?

When do we obtain our lead and candidate?

Answer 4

Target selection –> target validation –> lead discovery –> lead optimisation –> pre-clinical development –> clinical development –> regulatory approval –> release to market

Discovery ends after lead optimisation yields candidate drug(s).

Lead is obtained after “lead discovery” phase; candidate obtained after “lead optimisation”

Question 5

What are the 3 methods of identifying drug targets?

Answer 5

Analysis of pathophysiology (e.g. we want to find a drug for depression) (forward approach: disease to target to drug)

Analysis of mechanism of action of existing drugs (e.g. a drug we know works, but we don’t know how) (backwards approach: drug to target)

Genomic approaches (e.g. identify errors in transcription and translation; search for druggable genes)

Question 6

What are “druggable genes”?

Answer 6

Genes which code for proteins that are involved in disease and can be targeted by drugs

Question 7

How was fluoxetine discovered?

Answer 7

Analysis of pathophysiology (forwards approach) –> disease to target to drug –> researchers noticed that there existed a serotonin transporter on pre-synaptic neurons which decreased serotonin in synaptic cleft –> based on physiology of neuron, researchers created transporter inhibitor, Fluoxetine

Similar story with ACE inhibitors –> studies of renin-angiotensin system in hypertension made researchers select angiotensin converting enzyme as the target for drug action

Question 8

How were benzodiazepines discovered?

Answer 8

Benzodiazepines just worked for anxiety and sleep disorders, so physicians prescribed them without question their mechanism of action –> later discovered GAMA receptors were their targets –> similar drugs developed upon discovery of GAMA receptors

Question 9

How can we validate a target once we have found it?

Answer 9

KO the gene coding for the target protein in animal models; express the gene ex-vivo in a cellular system with siRNA (e.g. ASOs)

Question 10

What are the 3 consecutive steps we can take for finding lead compounds during the “lead discovery” phase when starting from nothing but a target?

Answer 10

1. Screen a bunch of compounds in chemical library
2. Identify potential leads, called hit candidates (primary hit candidates, validated hit candidates)
3. validate those hit candidates, obtain around 3 lead compounds

Question 11

What is the difference between a hit candidate and drug candidate/candidate drug?

Answer 11

Hit candidate is a potential chemical that could turn out to be a lead. Drug candidates are leads which have been optimised to enter pre-clinical trials.

Hit candidates themselves are NOT leads.

Question 12

What are the 4 sources of new drugs?

Answer 12

1. screening chemical libraries (e.g. high throughput screening)
2. rational drug designing
3. serendipity
4. screening natural products

Question 13

Explain the 4 types of rational drug design?

Answer 13

RDD is about determining the structure and function of a target receptor/molecule and using this information to develop drug treatments instead of relying on serendipity.

• Designing brand-new drug based on pathophysiology of disease (e.g. identification of ACE enzyme involved in renin-angiotensin; generating drug derived from viper venom, modifying it with medicinal chemists, and generating ACE inhibitors)
• exploiting side effects (e.g. sulphanilomide; extracting the part of a chemical that elicits a side effect, modifying it and marketing it as a drug for some other condition)
• Quantitative structure activity relationships, QSAR (using a virtual “screening” tool to assess the structures and activities of chemicals in library and matching them to your receptor)
• structure-based design (virtual assessment of receptor structure + identification of binding pockets/surface features that can be targeted by drug)

Question 14

What are primary hit candidates?

Answer 14

Chemicals that have affinity for receptor, but no efficacy.

They are what you get after that first round of primary screening of chemical libraries.

Question 15

What is high throughput screening?

Answer 15

Main method of screening chemical libraries for possible lead compounds for our target; 1000-5000 compounds screened against target simultaneously in cell-based assay

Question 16

How many compounds/chemicals from our chemical library can be screened against our target simultaneously?

Answer 16

1000-5000

Question 17

Lead discovery: How many compounds do we screen? How many primary hit candidates do we get? How many validated hit candidates do we get? How many lead candidates do we end up with?

Answer 17

• 100,000 - 1,000,000 compounds/chemicals from chemical library are screened
• 100 - 1,000 primary hit candidates
• 10 validated hit candidates
• 3 lead compounds

Question 18

How do we go from 100-1,000 primary hit candidates to our 10 validated hit candidates? I.e. how do we validate our primary hit candidates. What are the key features of a validated hit candidate

Answer 18

Look for affinity, efficacy (whether it activates/inhibits receptor) and selectivity (chose compounds that have minimal off-target effects)

Validated hit candidates (x10) have the following features:

• Activity at receptor as inhibition or activation
• drug-like properties (e.g. low MW <500, clogp <5, aromatic ring in structure)
• defined, novel structure

VHC are not “drugs” because aren’t optimised for selectivity, toxicity and ADME properties in humans yet

To get from VHC to lead, we send the VHC to medicinal chemists for SAR modifications to improve pharmacokinetic properties and reduce toxicity

Question 19

What are the requirements for a compound to be classified as a “drug”?

Answer 19

Drugs (i.e. candidate drugs) are leads which have been optimised and are in pre-clinical development –> have had selectivity, efficacy, toxicity, ADME tests done

Question 20

What is clogP?

Answer 20

A measure of lipophilicity. Optimal is <= 5 but not too low –> high clogP means lipophilic… if too low then drug is hydrophilic, and will not be able to enter into bloodstream and be distributed

Question 21

What is QSAR?

Answer 21

Quantitative structure activity relationships –> technique which involves predicting the activity and properties of an unknown group of molecules based on analysis of an equation connecting structures and molecules to their respective measured activity and property.

Question 22

Simply state the 4 types of rational drug design techniques?

Answer 22

1. design a structure from scratch based on pathophysiology (e.g. design a serotonin transporter blocker)
2. exploit side effects of existing drugs
3. QSAR
4. (receptor) structure-based design

Question 23

When do medicinal chemists with their cyclical structure-activity relationships modifications get involved?

Answer 23

When deriving lead from validated hit candidates, and when optimizing the lead into a candidate drug

Question 24

What is involved in lead optimization?

Answer 24

We are attempting to perfect the leads as much as we can into candidates - we are constantly communicating with medicinal chemists (MCs) as they modify the leads with structure-activity analysis (SAR), and enhance pharmacokinetic and pharmacodynamic/efficacy properties, making new compounds in the process (of course, since they tweak and modify the existing structure)

Question 25

At what stage are animals first involved?

Answer 25

All stages, starting from receptor identification!

Question 26

What are the 4 main reasons why drugs fail in clinical trials?

Answer 26

No desired efficacy in humans —> adverse side effects —> financial/commercial reasons —> undisclosed

Question 27

What are the 4 stages of pharmacological profiling/assessment?

Answer 27

1. radioligand binding assays to assess affinity
2. in-vitro profiling in animals
3. in-vivo profiling (acute and chronic) in animals
4. humans

Question 28

Give an example of why in-vivo testing is so important and why we cannot just rely on in-vitro testing?

Answer 28

Clonidine, an alpha2-agonist induced vasoconstriction in isolated blood vessels in-vitro, but when tested in-vivo in rodents, it actually lowered BP because of an unknown action at the CNS

Question 29

How well do organ function studies, psychological/behavioural function studies, and side effect/toxicity studies translate from animals to humans?

Answer 29

Organ function = well
psychological/behavioural = satisfactory, but limitatious
side effect = bad/poor predictive value (e.g. human monoclonal antibody treatment cytokine storm)

Question 30

Can animal models of human disease be acute and chronic? Give examples of how we can do this

Answer 30

Yes. Acute = inducing seizures, histamine-induced bronchoconstriction, LPS injection to induce septic shock etc.

Chronic = drug-induced diabetes, Parkinsons, hypertension; diet induced hypercholesterolemia, diabetes; surgically induced stroke, aortic banding; inbred/transgenic disease lines

Question 31

How many candidate drugs enter pre-clinical studies?

Answer 31

1-3

Question 32

Summarise what happens in pre-clinical testing?

Answer 32

Investigating toxicity/safety in animals (and maybe re-testing efficacy), but the main idea is toxicity.

Question 33

What are the 3 types of toxicity?

Answer 33

1. systemic (acute or chronic)
2. carcinogenic
3. teratogenic

Question 34

Summarise how we test for acute systemic toxicity

Answer 34

3 dose levels —> one per animal —> 14 days
2 mammalian species of different sexes
Assess clinical signs (urinalysis and hematology tests; mortality, lethargy, body weight)
Monitor drug targets as a measure of selectivity via autopsy
Doses are not cumulative

Question 35

What is the NOAEL?

Answer 35

No observed adverse effects level (NOAEL) –> the highest dose possible which does not cause any undesirable adverse toxic effects at all. This dose could still be within the therapeutic range, just not within the toxic range

Question 36

What is the MTD?

Answer 36

Maximum tolerated dose (MTD) –> the maximum dose that will produce a desired effect without unacceptable toxicity (some toxicity is acceptable)

Question 37

Summarise how we test for chronic systemic toxicity

Answer 37

3 levels: sub-acute (14-day) + sub-chronic (28 OR 90 days) + chronic (9-12 months study)
Doses are cumulative doses, oral administration
2 relevant species with different sex

Question 38

What is the formula for the TI?

Answer 38

median toxic dose/median effective dose

Question 39

Do we want a narrow or wide TI?

Answer 39

Wide, so that the difference between median effective and median toxic dose is the greatest and there is room for dosing errors

Question 40

Summarise the Ames test

Answer 40

Carcinogenic toxicity test done in-vitro (acute testing) —> culture his- salmonella with drug and liver extract —> if drug is not carcinogenic, no bacteria will grow (all will stay his-) —> if drug is carcinogenic, it should induce mutations in his- bacteria, turning them into his+ bacteria and bacteria will grow in the culture

Question 41

What are the 2 reasons why the Ames test is conducted with liver extract?

Answer 41

1. pro-drug needs to be activated

2. we need to check that metabolites aren’t toxic/carcinogenic

Question 42

Briefly summarise acute and chronic carcinogenic toxicity testing?

Answer 42

Acute = done in-vitro with bacteria (Ames test), no animals required
Chronic = done in-vivo in animals over time but can be done after approval for clinical phase 1 trials

Question 43

What are the 3 time points/opportunities to test for teratogenicity?

Answer 43

1. before mating
2. at implantation through to birth
3. during last trimester through to lactation/breast-feeding

Question 44

In summary, what were the respective roles of the overall discovery phase, pre-clinical development phase and clinical development phase?

Answer 44

Discovery phase = assessing efficacy in animals alone

Pre-clinical = assessing safety in animals/toxicology (and also re-testing efficacy)

Clinical = assessing safety/toxicology and efficacy in humans

Question 45

Describe phase 1, phase 2, phase 3 and phase 4 of clinical trials

Answer 45

Phase 1 = assessing toxicity/safety with healthy participant
Phase 2 = assessing initial efficacy in small group of patients (can be a control/experimental group)
Phase 3 = more thorough efficacy testing in larger group of patients with control and experimental group
Phase 4 = post-marketing surveillance in the whole population

Question 46

What are the sub-phases in phase 1 and phase 2 of clinical testing

Answer 46

1a = single dose to healthy participants
1b = repeated dose to healthy participants 

2a = initial efficacy testing
2b = dose selection

Question 47

In which phase of clinical trials are there experimental and control groups?

Answer 47

Phase 2

Question 48

Terminology to know: open label, single-blind, double-blind, randomization, cross-over design, parallel design, placebo, comparator

Answer 48

Ok

Question 49

What is the main positive and main negative of a cross-over study design?

Answer 49

\+ve = within-patient controls
-ve = 1st component might interfere with the second, ethical dilemma emerges and takes longer (2 x 12 weeks)

Question 50

What are the 2 schemes (not groups/organizations) that researchers can obtain in order to get approval for clinical trials?

Answer 50

Clinical trial exemption (CTX) –> obtain this scheme by applying directly to TGA
Clinical trial notification (CTN) –> obtain this by applying to the HREC (human research ethics committee)

Question 51

What is the “number needed to treat” (NNT) reported in clinical trial results?

Answer 51

NNT = 1/absolute risk reduction (decimal) = 1/(success in treatment group-success in control group)

–> success in treatment group = (# of treated people with success / total # of treated people)

It is the number of people you need to treat, in order for 1 to get a beneficial response (e.g. if you give the drug to 100 people, and only 1 gets a good response)