Therapeutic Potential of Small Molecules in CF

Question 1

Why are recurrent infections in CF patients a problem?

Answer 1

These infections lead to inflammation = tissue damage

Eventually this tissue damage is so great that life can not be supported

Question 2

List current treatments for CF patients which treat symptoms of the disease and not the cause

Answer 2

Nebulised antibiotics 
Inhaled bronchodilators 
Mycolytics 
Nebeulised hypertonic saline 
Oral antibiotics
Pancreatic enzymes 
Fat soluble vitamins 
Steroids 
Exercise/ physiotherapy 
High energy supplements

Question 3

Which drug company did Aurora buy in 2001?

Answer 3

Vertex pharmaceuticals

Question 4

Why were Vertex initially going to close the CF drug screening programme?

Answer 4

It did not have a good cost benefit analysis

However, the CF foundation injected millions of dollars so that the programme was kept open

Question 5

Name two drugs (with a brief description) that were found as a result of the screening programme

Answer 5

1) VTX-770 (IVAKAFTOR)
Is a potentiator i.e. keeps channels open for longer
Licensed for use in the UK for G551D patients
2) VTX-809
Corrector, not licensed in the UK but is when used in combination with VTX-770 (combination = orkambi)

Question 6

Why is it desirable to make a drug which targets delta F508?

Answer 6

It is the most common CF allele in the world with some contries having the allelic frequency as high as 90% in their CF patients

Question 7

Describe the G551D mutation

Answer 7

Gating mutation
So in the drug screen they looked for drugs which could open the channel
Mutation in NBD1
Type 3 mutation
13% of CF patients have this mutation and have severe symptoms

Question 8

How did they screen for compounds which increased the Po of CFTR in the G551D mutation?

Answer 8

They used a cell based fluoresence assay where the compound changed colour with a change in mV
E.g. if CFTR opens the mV changes due to Cl- secretion
They tested 228,000 chemicals and found VTX-770

Question 9

Describe the delta F508 mutation

Answer 9

Phenylalanine deletion
Type II trafficking defect (also slightly lower Po than WT)
Patients have severe symptoms
Screen looked for a compound to drive CFTR to the membrane

Question 10

How did they screen for compounds which increased the trafficking of CFTR in the delta F508 mutation?

Answer 10

CFTR is glycosylated at the membrane = higher MW band
So they looked for compounds which gave this higher MW band
164,000 chemicals were tested
They found VTX-809

Question 11

Describe the first experiment which suggested that VX-770 could be used potentially in G551D patients

Answer 11

Fisher rat thyroid cells overexpressed WT or G551D CFTR
In WT forskolin increased cAMP so caused an increase in short circuit current due to Cl- secretion
However, in the G551D cells there was no increased Cl- secretion by forskolin due to reduced gating function
However adding Fsk and VX-770 increased Cl- secretion
A CFTR inhibitor blocked the response to VX-770

Question 12

Why is Forskolin needed?

Answer 12

For cAMP priming so the channels are open

Question 13

Is the increase in function by VX-770 enough?

Answer 13

Yes - it is not as big as the WT but we only need 15-20% functioning CFTR for health

Question 14

Why is VX-770 known as a potentiator?

Answer 14

Single channel recording show single channel currents
When the drug is used with Pka the channel spends more time in its open configuration
Therefore it is a potentiator because it allows the channel to open

Question 15

How did they show VX-770 was effective in native tissue?

Answer 15

Tissue was taken from a CF patient (primary bronchial epithelial cells)
They then looked as SSC
1) When FSK was added alone there was no increase in SSC
2) They then increased concentrations of VX-770 which increased the SCC
3) A CFTR inhibitor was added which blocked the SCC

Question 16

Describe the results seen when patient tissue was used to test the effect of VX-770

Answer 16

In the WT tissue FSK increased current by 56uA/cm2
However, in the CF patients it only reduced it to 2.9uA/cm2 which is only 5% of the WT
However, VX-770 = increase to 27uA/cm2 which is 50% of the WT which is enough for normal function
Clinical suggestions show a 15% increase is sufficient to alleviate symptoms in CF patients

Question 17

Describe how VX-770 was shown to effect cilia beat frequency in vitro

Answer 17

WT cells beat at a frequency of 16Hz
G551D mutations = cilia are beant so the frequency is only 2.5Hz
VIP and Vx-770 shows a restored ciliary beat frequency

Question 18

How did they perform the clinical trial for VX-770?

Answer 18

Randomised, double blind placebo controlled trial used
Monitored FEV1 as a percentage of their predicted FEV1
The trial lasted 48 weeks where patients even received a placebo or 150mg every 12 hours of VX-770

Question 19

What was the patients average FEV1 % of predicted and sweat chloride before the trial started?

Answer 19

FEV1 % of predicted was approx 63%

Sweat chloride was 100.1 mM

Question 20

Describe the results in the clinical trial for FEV1, pulmonary events and sweat Cl

Answer 20

Placebo patients had no improvement in FEV1
Patients on ivakaftor did have an increase in FEV1
Patients with the drug also were more event free - 67% compared to 41% on the placebo
Sweat chloride reduced below clinical threshold around 50 mmol/l

Question 21

Why did they measure nasal potential difference and why?

Answer 21

Vte was measured across the nasal epithelium
A cl- free solution was squirted in to the patients nose to promote Cl- secretion
Isoprotanerol was also added because it binds to B receptors to activate CFTR via cAMP
Patients with VX-770 had a negative shift in Vte as more Cl- secretion was occuring
This tells them that VX-770 enhances the function of CFTR

Question 22

How did they screen for a molecule which could help CF patients with the delta F508 mutation?

Answer 22

They looked at the ratio between mature (glycosylated) and imature CFTR
This was shown on western blots

Question 23

What did the initial experiment suggest about VX-809 in rat fisher thyroid cells

Answer 23

Increasing the concentration of VX-809 increased the ratio of glycosylated to unglycosylated CFTR
SCC also increased with VX-809

Question 24

What type of drug is VX-809

Answer 24

It is a corrector as it thought that misfolding of CFTR has been corrected so the drug now can get in to the membrane

Question 25

Describe the pulse chase experiment

Answer 25

HEK cells were overexpressing CFTR and were exposed to radioactive met and cyst
These are incorporated in to new CFTR so it is now radioactive
The compound was then removed and cells were left for different durations
In the chase they measured the ratio of mature and immature CFTR using radioactivity
This ratio depends on how long the cells have been left

Question 26

What was the effect of VX-809 in the pulse chase experiment?

Answer 26

WT CFTR over time shifted towards being mature
Delta f508 mutant did not and overtime the immature CFTR is reducing but there is no increase in mature CFTR
However, with VX-809 there is more CFTR being made which is mature

Question 27

Why was the combination of XV-809 and VX-770 suggested?

Answer 27

The delta F508 mutant did not work as it should do once it was in the membrane
Po of WT CFTR us around 0.4 but delta F508 has a low Po
This could be increase with VX-770 which is a potentiator = increase to around 0.6

Question 28

How was the clinical trial for VX-809 carried out?

Answer 28

Randomised, double blind, placebo controlled study
FEV1 % of predicted normal was measured - positive value means it has moved closer to normal
The trial lasted 28 days and patients were homozygous for delta F508

Question 29

What were the results of the VX-809 clinical trial?

Answer 29

There was no obvious improvement in patients despite the in vitro data being strong
There was a moderate improvement in sweat Cl- but only by 6mmol = still above clinical threshold

Question 30

What is the combination of VX-770 and VX-809 called?

Answer 30

Orkambi

Question 31

Describe results from the orkambi clinical trial

Answer 31

Orkambi has a high cosy but caused a bigger decrease in sweat chloride than VX-770 alone
However, this was still only by 14mmol/l so is still above clinical threshold
FEV1 also improved but only moderately when 600mg of VX-809 and 250mg VX-770 was used

Question 32

Describe the phase 3 trial for orkambi

Answer 32

All patients were homozygous for delta F508 and were over 12 years of age (approx 1100 people)
Patients had different mg of the combination over 24 weeks
There was a moderate increase in lung function - usually of more than 5%, very few had more than 10%

Question 33

Why do patients respond differently in clinical trials?

Answer 33

Patients may respond differently due to their genetic background and their compliance

Question 34

What is a forest plot?

Answer 34

Where patients are split in to groups according to age, gender etc and the effect of the drug is plotted in terms of if it is better than the placebo

Question 35

How did orkambi affect pulmonary exacerbation?

Answer 35

60% of patients on placebo had not had one but 70-80% on orkambi had not had an event
This is therefore expected to reduce costs associated with hospitalisation

Question 36

How much does Ivakaftor and orkambi cost each year per patient?

Answer 36

Ivakaftor = £189,000
Orkambi = £104,000

Question 37

Which of these drugs are licensed in the UK?

Answer 37

Ivakftor is as the improvement is large and no other treatments are needed
However, orkambi is not as effective so is not licensed as the cost benefit analysis is poor
However, there is a push to get it licensed
There is also the potential for a new drug to be developed called tersakaftor

Question 38

Why was the initial proposal of gene therapy dropped in the 1990s?

Answer 38

There were many problems as airway cells are readily replaced and new cells will not have the WT CFTR
There was also concerns on using adenoviruses

Question 39

Describe the non-viral CFTR gene therapy trial in 2015

Answer 39

Phase 2b clinical trial used liposome complexes rather than viruses
140 patients took part where they either had nebulised 0.9% saline or pGM169/GL67A
Trial lasted 28 days and patients recieved 9 doses

Question 40

Why is the control for this trial not ideal?

Answer 40

The best control would be to use scrambled mRNA for the CFTR gene however this would not be safe for patients

Question 41

What were the results from the non-viral CFTR gene therapy trial?

Answer 41

Patients stabilised - they did not improve but they did not get worse like patients on the placebo
However, if given early enough then they could maintain a better lung function throughout life
Most patients had a positive response but some did not
FEV1 and FVC was better with the treatment and gas trapping was also reduced

Question 42

What is the issue when we begin to succesfully treat CF patients lung problems?

Answer 42

They start to die from other problems where gene therapy will not work