what are the current treatments available for CF?
- treatments only aleviate symtoms and dont treat the cause
- oral and nebulised antibiotics (e.g. tobramycin)
- nebulised hypertonic secretion (stimulate H2O secretion)
- steroids
- exercise
- bronchodilators
- mycolytics
- pancreatic enzymes
- fat soluble vitamins
- physiotherapy
what are the 2 small molecules that have been developed to help in the treatment of CF?
VX-770 - ivacaftor
VX-809 - lumacaftor
what is ivacaftor?
VX-770, a potentiator - increase channel Po (can be used on the G551D mutation)
what is lumacaftor?
VX-809, a corrector - ensure correct trafficking of CFTR to the membrane
what is the drug name for the combinaiton of Vx-770 and VX-809?
orkambe
where abouts on the CFTR protein are deltaF508 and G551D found?
In NBD1
how was VX-770 found to be a contendor in the treatment of G551D mutations?
- drug screening
- cell based floursescnce assay (Vm)
- When Vm changes - cell changes flourescence - looked for cells which changed flourescence as changed Vm - looked for which one effected CFTR
how was VX-809 found as a contender to treat delta F508 CF mutation patients?
- drug screening
- cell based immunoblot assay - looked for mature/post golgi levels - in the membrane?
what experiment using fisher rat thyroid cells showed the effects VX770 has on cells?
- WT and G551D mutation cells used
- showed VX77- can work to open up mutant channels in high levels of cAMP
- channels have to be primed by cAMP (forskolin) first in order to VX770 to have an effect
- VX770 and fsk work together to increase Isc
- when CFTR inhibitor added - reverse effects of fsk and 770 - shows effects are due to CFTR change
- VX770 can enhance mutant CFTR channels
what did the single channel CFTR current show when VX770 was added?
- in fisher rat thyroid cells
- Po of mutant CFTR increases - causes mutant CFTR channels to open
What did experiments using human bronchial epithelial (HBE) cells show about forskolin responses?
- WT fsk response - 56uA/cm2
- G551D/F508 fsk response - 2.9 uA/cm2
- G551D/F508 Vx770 fsk response - 27uA/cm2 (48% of WT - sufficient as only 15-20% of channel function required)
- shows VX770 fsk enhances response of mutant CFTR channels
Using HBE cells how was ASL effected by CFTR mutations and vx770?
- When CF cells treated with VX770 and VIP, ASL increases and moves closer to WT levels than before
- shows could have an actual beneficial effect in patients due to this increased channel function - could increase ASL
How was cilia beat frequency affected with treatment of VX770 (HBE cells) ?
When CF cells were treated with VIP and VX770 - cilia beat frequency increased to WT levels - more functional - could clear mucous more effectively in humans in vivo?
how were the clinical trials for VX770 in G551D CFTR mutations carried out?
- randomised double blind trials
- placebo used
- 50mg of drug (or placebo) given every 12 hours
- 48 weeks
- at least 1 G551D mutation in patients
- start with similar parameters
what were the results from the VX770 clinical trial in treatment of G551D mutation %FEV1?
- %FEV1 increased 10% with treatment of VX770 - maintained for 48 weeks
- placebo FEV1% no improvement - even a slight deterioration in FEV1%
- has potential to improve quality of life
what were the results from the VX770 clinical trial in treatment of G551D mutation - events?
- with treatment with VX770- 67% of patients were event free - no pulmonary exaccerbations
- with placebo - less patients were event free - only 41 event free
what were the results from the VX770 clinical trial in treatment of G551D mutation - sweat [Cl-]?
- in patients treated with VX770 - sweat chloride concentration is reduced below the clinical threshold - maintained for 48 weeks
- In placebo - no change in sweat chloride
what does VIP do?
stimulate CFTR
is there a cost benefit for the use of VX770?
Yes, patients on VX770 cost less in hospital admissions and other treatments and there is a significant improvement to the quality of life - is a cost benefit
What was shown in fisher rat thyroid cells about VX-809 treatment with F508 CFTR?
With increasing concentrations of VX809, there was an increase in both the maturation and function of F508 CFTR
what usually happens to mutant f508 CFTR?
It remains in an immature (unglycosylated form), as it does not reach the golgi - gets to ER and then undergoes multiple rounds of the CNX cycle - as it is misfolded - gets degraded via ERAD
what were the steps in the pulse chase exp looking at F508 CFTR in HEK cells?
- RA label amino acids with 35S methionine and cysteine
- Then any new immature made CFTR is 35S labelled
- 35S compounds are removed and cells are left for different durations
- some 35s is mature (glyc) and some stays immature (unglycosylated)
- ratio of these depends on how long cells left and what pharmacological manipulation cells are exposed to (e.g. vehicle or vx770)
what were the results from the pulse chase exp in HEK cells?
- the longer WT cells were left - the more mature protein formed , and there was less immature protein
- in F508 cells there was a decrease in the immature form (lots of it to start with) and no/ little mature protien - degradation was going on - not maturation
- F508 exposed to VX809 - decrease in immautre form - forced to become mature - more mature form than with no VX809`
What was shown in Po when NIH cells expressing f508 CFTR were treated with VX809
The Po of f508 cftr when cells were treated with vx809 increased (became closer to WT)- shows that CFTR has been trafficked to membrane and is functional when it bcomes trafficked
- with both VX809 and VX770 the response is enhanced
