L5 - CF and Small Molecules II

Question 1

What mutation does Ivacaftor target?

Answer 1

G551D mutation

Question 2

What mutation does Lumacaftor target?

Answer 2

F508 mutation

Question 3

What does a western blot of CFTR show?

Answer 3

2 bands
Mature – glycosylated – higher molecular weight
Immature – not yet glycosylated

Question 4

Why does F508 CFTR remain immature?

Answer 4

Gets sent for degradation due to mis-folding

Question 5

Why did they look at the ratio of mature CFTR to total band density?

Answer 5

F508 mutation – protein is not fully glycosylation

Wanted to find a drug that promoted glycosylation and trafficking –> would therefore see more CFTRT Band C (mature)

Question 6

How did they test if Lumacaftor allowed the production of more mature CFTR? - rat

Answer 6

Tested on rat thyroid cells homozygous for F508 CFTR
Ran western blots after treating cells with different concentrations of Lumacaftor
Increased concentration of Lumacaftor –> increases amount of mature CTFR

Question 7

How did they test if Lumacaftor allowed the production of more mature CFTR? - human

Answer 7

Tested on HBE cells homozygous for F508 CFTR
Added amiloride - so only focus on Cl channel function
Added cAMP agonist - stimulated CFTR
Added different concentrations of Lumacaftor
- Saw a concentration dependent increase in Isc
Added CFTR inhibitor
- Decrease in Isc
- Increase in currents is therefore down to activation of mutant CTFR
– More CFTR in membrane as well due to Lumacaftor

Question 8

What did the randomised, double-blind, placebo controlled trial with Lumacaftor show?

Answer 8

Monitored FEV1 - expressed data as change in % of predicted FEV1
No obvious impact of the drug - didn’t reach clinical setting

Question 9

What results did they see when looking at sweat chloride when treating with Ivacaftor or Lumacaftor?

Answer 9

With Ivacaftor - shift of -55mM
With Lumacaftor - shift of -8mM
- Much poorer than Ivacaftor with G551D mutations
- Sweat chloride isn’t deadly it’s just a clinical marker

Question 10

What is the average sweat chloride of a patient with the F508 mutation?

Answer 10

Sweat chloride around 100mmol

Question 11

Why did they decide to test a combination treatment of Ivacaftor and Lumacaftor?

Answer 11

Lumacaftor- corrector – drives CFTR to the surface

Ivacaftor - potentiator – helps open the channels (more than normal)

Question 12

What experiments did they do in the phase 3 clinical trial of the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 12

1108 patients homozygous for F508
Over 12 years
1 - Ivacaftor and 600mg of Lumacaftor every 24 hours
2 - Ivacaftor and 400mg of Lumacaftor every 12 hours

Question 13

What results did they get from the phase 3 clinical trial of the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 13

Test - after 2 week lung function improves – 3% improvement
- The 10% improvement continually seen with Ivacaftor and G551D is rarely seen with this treatment
Placebo
- Lung function slightly decreased
- Even in placebo 22 patients had 5% improvement in lung function

Question 14

What is a Forest plot?

Answer 14

Whole range of parameters you are interested in during a clinical trial
Look at whether placebo or treatment patients have a better profile

Question 15

What results did they get from forest plot of the phase 3 clinical trial of the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 15

For all the groups – treatmnet patients did better than placebo patients
- Combination therapy has a posiitve impact - just not big enough impact

Question 16

What results did they get for time to first pulmonary exacerbation for the phase 3 clinical trial of the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 16

Placebo – 60% didn’t have a problem

Treatment – 75% didn’t have a problem

Question 17

What results did they get for events leading to hospitalisation or events leading to IV antibiotic treatment for the phase 3 clinical trial of the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 17

Treatment – reduced compared to placebo

- Showed importance of dosing regimes

Question 18

Overall what does the data suggest for the combination treatment of Ivacaftor and Lumacaftor on the F508 mutation?

Answer 18

Lumacaftor can traffic F508 CFTR to the membrane and it is functional
In F508 patients Lumacaftor alone not sufficient to improve symptoms
However if Lumacaftor traffics F508 CFTR to the membrane the addition of the potentiator Ivacaftor can enhance function and relieve symptoms

Question 19

What is another potential combined treatment to treat the F508 mutation?

Answer 19

Elexacaftor, tezacaftor and ivacaftor

• Tezacaftor and Elexacaftor – corrector
• Ivacaftor – potentiator

Question 20

Results for elexacaftor, tezacaftor and ivacaftor combined treatment on patients containing F508 and MF - CTFR Band C relative intensity

Answer 20

Not a lot of mature CFTR with F508
Elexacaftor and Tezacaftor – small increase
Elexacaftor, Tezacaftor and Ivacaftor – small increase
In patients with one F508 mutation – combinations
are not having as big an impact as when patients are
homozygous for F508

Question 21

Results for elexacaftor, tezacaftor and ivacaftor combined treatment on patients homozygous for F508 - CTFR Band C relative intensity

Answer 21

Not a lot of mature CFTR with F508
Tezacaftor and ivacaftor – marked increase
Elexacaftor – marked increase
Elexacaftor and Tezacaftor – large increase
Elexacaftor, Tezacaftor and Ivacaftor – large increase

Question 22

Results for elexacaftor, tezacaftor and ivacaftor combined treatment on patients containing F508 and MF - Isc - Cl transport

Answer 22

Tezacaftor and ivacaftor – small increase

Elexacaftor, Tezacaftor and Ivacaftor – large 4 fold increase

Question 23

Results for elexacaftor, tezacaftor and ivacaftor combined treatment on patients homozygous for F508 - Isc - Cl transport

Answer 23

Tezacaftor and ivacaftor – small increase

Elexacaftor, Tezacaftor and Ivacaftor – large 5-6 fold increase

Question 24

Overall what do all the results of the triple therapy with elexacaftor, tezacaftor and ivacaftor show?

Answer 24

Elexacaftor and Tezacaftor in combination are having the biggest effect at driving CFTR to the membrane
Ivacaftor is then increasing its open probability

Question 25

Results for elexacaftor, tezacaftor and ivacaftor combined treatment on patients homozygous for F508 - FEV1 data

Answer 25

Tezacaftor and Ivacaftor
- 3% increase in lung function 
- Same as Lumacaftor and Ivacaftor 
- Drop in Cl concentration by 10mmol 
Elexacaftor, Tezacaftor and Ivacaftor
- 14% increase in lung function 
- Even better than Ivacaftor on G511D patients 
- Drop in Cl concentration by 45mmol 
Placebo 
- Worsening of patients 
- Increase in Cl concentration

Question 26

What is the cost of Ivacaftor a year per G551D patient?

Answer 26

£189,000

Question 27

Why is the cost of Ivacaftor worth it?

Answer 27

Patients then need less of other medication

They are hospitalised less often

Question 28

What is the cost of Ivacaftor and Tezacaftor a year per patient?

Answer 28

£104,000
Not as effective so cheaper than Ivacaftor alone
Patients still need other therapies and hospital treatments

Question 29

You are a researcher trying to identify alternative pharmacological targets to treat CF (F508 mutation). Can you identify an alternative membrane target, name a drug that would target this protein and explain why it could work?

Answer 29

ENaC
- CFTR is known to interact and inhibit ENaC
- When CFTR is mutated, this inhibition is lost –> overactivation of ENaC –> enhanced Na absorption –> enhanced water absorption –> reduced height of liquid layer even more than just due to loss of Cl secretion
ENaC activity is enhanced in CF
- Use an ENaC antagonist - e.g. amiloride
- Blocking ENaC would prevent the absorption of Na into the cells across the apical membrane
- This would prevent the paracellular transport of water between the cells
- Reduce water loss from the ASL –> increases ASL height
- Normal mucous clearance achieved
Other Cl channels in the membrane also able to excrete Cl –> can’t replace CFTR though
Previous work not successful - 2 big limitations
- Systemic effects
– E.g. renal – diuresis and hyperkalaemia
- Short term effects on the lung