Therapeutic potential of small molecules in cystic fibrosis

Question 1

Characteristics of cystic fibrosis

Answer 1

Failure to clear mucus and bacteria 
increased risk of infection 
inflammation 
tissue damage 
decreased lung function

Question 2

Examples of current treatments

Answer 2

Nebulised antibiotics
inhaled bronchodilators 
mucolytics
pancreatic enzymes 
steroids
physiotherapy

Question 3

What are the 2 new drug developments?

Answer 3

VTX-770 (ivakaftor)- potentiator- licensed for the use foe the G551D mutation
VTX-809- corrector- licensed in the US but not the UK

Question 4

Whats the most common mutation in CF?

Answer 4

deltaF508- 90% allelic frequency worldwide

Question 5

What are the characteristics of the G551D mutation and how did they screen for drugs?

Answer 5

Gating mutation, type 3 mutation, mutation found in NBD1, glycine to aspartate, 1-3% have it, severe symptoms
Screening- carried out cell-based fluorescence vm assays- cell changes its fluorescence when the Vm changes- screened 228,000 chemicals- looked for a change in fluorescence- VTX-770

Question 6

What are the characteristics of the deltaF508 mutation and how did they screen for drugs?

Answer 6

Trafficking mutation, class 2 mutation, phenylalanine deletion, severe symptoms
Screening- cell-based immunoblot assay- looking at mature post golgi CFTR levels
looked for drugs associated with mature CFTR- screened 164,000 chemicals- VTX-809

Question 7

What experiments did they do with fisher rat thyroid cells? Whats the drawback?

Answer 7

1. Exposed G551D mutant cells to forskolin, and forskolin + VTX-770
2. Exposed mutant cells to PKA and ATP, then added VTX-770
   over expression studies- not in native cells- not as accurate

Question 8

What experiment did they do other than the overexpression study?

Answer 8

Took primary human upper airway cells from CF patients
added amiloride- because not interested in ENac
add forskolin
then added VX-770 in increasing concentrations
measured the short-circuit current
did the same in WT cells- got the WT Fsk response
mutant fsk response= 5% of WT
mutant fsk+ drug response= 48% of WT
Suggested that only need 15% of functioning channels to alleviate CF symptoms

Question 9

How is the ASL affected in CF patients? What effect does VTX-770 have?

Answer 9

ASL is decreased

double mutant + drug= increase in ASL, still below WT

Question 10

What was the main clinical trial for VX-770? What were the main results?

Answer 10

Monitored FEV1 as a % of predicted
expressed data as a change in % of predicted FEV1
Ivacaftor improved function immensely- FEV stays improved
proportion of event-free subjects was higher for those on ivacaftor
drop in sweat chloride below threshold for CF for patients on ivacaftor

Question 11

How did VTX-770 affect the Vte of the nasal epithelium?

Answer 11

Should see a negative shift in in the Vte if you have functional CFTR channels- Cl secretion= negative shift
ivacaftor enhanced the function of CFTR- seen by the positive relationship between the increased conc of the drug and the increasingly negative Vre

Question 12

How is deltaF508 CFTR seen on a western blot?

Answer 12

See a lower band on the WB- immature CFTR

Question 13

What 2 things does VTX-809 do to mutant channels?- seen in fisher rat thyroid cells

Answer 13

Increases the function of the mutant channels

increases amount of mature F508 CFTR

Question 14

What was the pulse-chase experiment for?

Answer 14

Expose the cells to radioactive methionine- immature CFTR incorporates the radioactive meth- labelled compounds removed and and cells left for different periods of time- look in each group at the ratio of mature vs immature CFTR
mature CFTR= glycosylated
immature= not glycosylated

Question 15

What was the result of the pulse chase experiment?

Answer 15

When the mutant cells were exposed to VTX-809- amount of mature CFTR increased, and immature CFTR decreases
in comparison to mutant cells + vehicle control

Question 16

How does VX-809 affect the deltaF508 mutant CFTR?

Answer 16

In NIH cells- expressing mutant CFTR
with the drug- see that when the CFTR is trafficked to the membrane- Po is close to the WT
if you use 770 + 809= Po even higher

Question 17

How does VX-809 affect the SCC? How do we know it effects CFTR channels?

Answer 17

When VX-809 added in increasing concentrations
see an increase in response
when a CFTR channel inhibitor is used- response drops to 0

Question 18

What people were used in the VX-809 clinical study? what happened

Answer 18

18-19 CF patients
homozygous for deltaF508
same experi as VX-770
results:
no pattern of activity at all 
nothing obvious
moderate improvement in sweat chloride- but chloride conc still above threshold for CF
small effect mediated by VX-809

Question 19

What is orkambi, how effective is it?

Answer 19

Combination therapy- VX-770 and 809
minimal benefit to patients
positive values of change in FEV2
moderate improvement when highest conc of 809 is used

Question 20

What happened in the phase 3 clinical trial?

Answer 20

1108 patients
all homozygous for deltaF508
3 GROUPS: placebo, lumacaftor 400mg and ivacaftor 250mg, lumacaftor 600mg and ivacaftor 250mg
positive values
small improvement in lung function
nothing compared to VX-770 and G551D mutant

Question 21

What factors affect the severity of mutations/ response to a drug?

Answer 21

non-coding regions- severity of symptoms

compliance

Question 22

What does a forest plot show, how was it for the combo therapy?

Answer 22

Shows us if the treatment is better than the placebo- 0= no difference between the two
across lots of groups- see small improvement in FEV1
but only a few show improvement in line with ivacaftor and G551D

Question 23

Why is Orkambi not licensed yet?

Answer 23

It’s not that effective- doesn’t have an impact like ivacaftor has
thus patients still have to take majority/ all of their current treatments
and there isnt a large drop in hospitalisations

Question 24

What therapy has re-merged?

Answer 24

Gene therapy- using liposome complexes instead of adenoviruses

Question 25

What are the downfalls of gene therapy?

Answer 25

Cells in the airways are replaced every few days- new cells wont retain engineered DNA
foreign particles can trigger an immune response

Question 26

What was the downfall to the first non-viral CFTR gene therapy trial?

Answer 26

Their controls werent what they wanted
a good control- scramble the CFTR DNA and place it in a liposome complex
this isnt ethically appropriate- scrambled DNA= mutant proteins, make the patients worse
they had to use saline instead
Therefore the positive result- could be due to the liposome formulation, and not the actual DNA

Question 27

What were the results of the trial?

Answer 27

The complex stabilised airway function- patients didnt get worse, whereas patients with the placebo did
Saw an increase in chloride secretion- wasnt significant though