Therapeutic potential of small molecules in cystic fibrosis Flashcards
Characteristics of cystic fibrosis
Failure to clear mucus and bacteria increased risk of infection inflammation tissue damage decreased lung function
Examples of current treatments
Nebulised antibiotics inhaled bronchodilators mucolytics pancreatic enzymes steroids physiotherapy
What are the 2 new drug developments?
VTX-770 (ivakaftor)- potentiator- licensed for the use foe the G551D mutation
VTX-809- corrector- licensed in the US but not the UK
Whats the most common mutation in CF?
deltaF508- 90% allelic frequency worldwide
What are the characteristics of the G551D mutation and how did they screen for drugs?
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
What are the characteristics of the deltaF508 mutation and how did they screen for drugs?
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
What experiments did they do with fisher rat thyroid cells? Whats the drawback?
- Exposed G551D mutant cells to forskolin, and forskolin + VTX-770
- Exposed mutant cells to PKA and ATP, then added VTX-770
over expression studies- not in native cells- not as accurate
What experiment did they do other than the overexpression study?
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
How is the ASL affected in CF patients? What effect does VTX-770 have?
ASL is decreased
double mutant + drug= increase in ASL, still below WT
What was the main clinical trial for VX-770? What were the main results?
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
How did VTX-770 affect the Vte of the nasal epithelium?
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
How is deltaF508 CFTR seen on a western blot?
See a lower band on the WB- immature CFTR
What 2 things does VTX-809 do to mutant channels?- seen in fisher rat thyroid cells
Increases the function of the mutant channels
increases amount of mature F508 CFTR
What was the pulse-chase experiment for?
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
What was the result of the pulse chase experiment?
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
How does VX-809 affect the deltaF508 mutant CFTR?
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
How does VX-809 affect the SCC? How do we know it effects CFTR channels?
When VX-809 added in increasing concentrations
see an increase in response
when a CFTR channel inhibitor is used- response drops to 0
What people were used in the VX-809 clinical study? what happened
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
What is orkambi, how effective is it?
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
What happened in the phase 3 clinical trial?
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
What factors affect the severity of mutations/ response to a drug?
non-coding regions- severity of symptoms
compliance
What does a forest plot show, how was it for the combo therapy?
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
Why is Orkambi not licensed yet?
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
What therapy has re-merged?
Gene therapy- using liposome complexes instead of adenoviruses
What are the downfalls of gene therapy?
Cells in the airways are replaced every few days- new cells wont retain engineered DNA
foreign particles can trigger an immune response
What was the downfall to the first non-viral CFTR gene therapy trial?
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
What were the results of the trial?
The complex stabilised airway function- patients didnt get worse, whereas patients with the placebo did
Saw an increase in chloride secretion- wasnt significant though
