L6 - Alternative Treatments Flashcards
What was originally thought was going to be the magic bullet for CF treatment
Using gene therapy to introduce WT anf fucntional CFTR back into cells
Describe the progression of gene therapy
Started with adenoviruses but progressed to using liposome complexes
Describe the conditions of the non viral CFTR trial
140 patients tested, 9 doses every 28 days
62 placebo, 78 with p/GM169/GL67A
(ombination of cationic liposome (GL67A) and plasmid DNA expressing CFTR (pGM169)
Placebo was a 0.9% saline
Describe the change in airway function for the non viral CFTR gene therapy
In placebo see a steady decline in lung function
In the gene therapy group - there is no improvement in lung function but lung function DOES NOT GET ANY WORSE
What determined how effective the gene therapy was
Determined by individuals - some responded well - others didnt
Could be due to differences in the genetic background of these patients
Was is gas trapping
Describe what effect would be seen on gas trapping if this treatment was working
Secondary obstruction of the small airways - likely to occur in infants with CF
If treatment is working would expect to see a reduced incidence of gas trapping
What was used a direct measure of how well the gene therapy was being taken up by cells
Looking at the change in bronchial DNA
describe the change in bronchial DNA for the placebo group
No change - this is expected they have not been exposed to the foreign DNA
Describe the change in bronchial DNA for the gene therapy group
Had a range of success
In two of the patients it was completely uneffective
Describe how the chrloride response was measured
Injection of a chloride free ISOPROTENEROL solution
This low EC Cl gives a huge driving force for Cl secretion across the apical membrane can mesaure thisby measure the nasal potential
Describe the chloride response in the treatment group
Those who had had the gene therapy see the most chloride secretion
What does all of the data presented in the non viral CFTR gene therapy suggest
That there is the provision lung function stabilisation
However the population was heterogeneous - some responded well others not so well
What is one critisism of the non viral gene therapy study
Saline is not the best control
Should (if in vitro) have used scrambelled CFTr and liposomes - this is for ethical reasons
What is the function of ENaC in CFTR patients
Massively enhanced
In the upper airway what it the relationship/interaction between CFTR and ENaC
direct inhibition
If CFTR is mutant what is the knockon effect on ENaC
There is no longer any inhibition of ENaC
Lose Cl secretion and enhance Na absorption
Thus height of the ASL is EVEN LOWER than expected
If we know CFTR function is enhanced then what would one potential alternative treatment be?
Block ENaC - could this lead to an increase in the height of the ASl
Most obvious first experiment to block ENaC
What were the reuslts of this?
Using amiloride
This was not effective at all
if amiloride was ineffective what could be tried
SPLUNC1
What is SPLUNC1
A naturally occuring ENaC antagonist
What does SPLUNC1 stand for
Secreted protein short plalate lung and epithelial clone 1
Describe what normally happens to ENaC in the absence of SPLUNC1
Normally enzymes such as trypsin etc. catalyse a proteolytic cleavage of ENaC which increases the Po and thus Na absorption is increased
What then is the function of SPLUNC1
Binds to ENaC preventing cleavage
Suppresses Na absorption across the apical membrane
Describe what was seen adding trypsin in the absence of splunc1
Currents enhanced - SPLUNC1 cleaves ENaC leading to an increase in Po
What was the effect of adding SPLUNC1 wihtout any trypsin
Currents recorded were significantly lower
Why were currents decreased when SPLUNC1 added on its own WITHOUT ANY TRYPSIN
Blocks the endogenous cleavage by other enzymes in the cell
Describe what was seen when adding SPLUNC1 and trypsin
Currents were smaller than when without any SPLUNC1
What was used to take forward the SPLUNC1 experiments …
S18
What is S18
What was the first question that needed to be adressed when looking at its function
It is the ENaC inhibitory domain of SPLUNC1
Need to check that it still inhibits ENaC when it is without the rest of the protein
Describe the premise of the experiments used to ensure that S18 binds to and inhibits ENaC
Using competitive binding experiments with an alexa fluor 488 SPLUNC1
When SPLUNC1 binds ENaC fluroescence can be detected -
Describe the results of the competitive binding experiment at pH 6
Not alot is happening here …
Shows ACIDIFICATION INHIBITS THE BINDING
What were the results of the SPLUNC1/S18 competitive binding experiment
Where s18 and SPLUNC1 are both present there is less relative fluorescence than when just SPLUNC1 alone
This is because s18 competes for ENaC binding and flourecne is only seen if SPLUNC1 binds … so with compeitive inhibition there is less SPLUNC1/ENaC binding
Conclusions that can be drawn from the SPLUNC1/S18 compeitive binding experiment?
What is the next question that needs to be addressed
That S18 binds to ENaC at the same place
Does this binding prevent cleavage in the same way as SPLUNC1 does
With drug experiments what should be used as a control
the vehicle only control
E.g. the substance the drug was dissolved in (for insoluble compounds this is usually DMSO)
Describe the affect on the ASL of the exposing HBE cells to following
Vehicle only
Amiloride
S18 (why)
Height low
No change - dont really know why
Height of the ASL has increased - Na absorption blocked les driving force for the movement of the water out of the ASL between cells
Describe the effect on the ASL height when started above optimum height ~30um of the following
Vehicle
Amiloride
S18
Height starts too high and ends up too low for both vehicle control and the amiloride
For s18 the height of the ASL stabilises at a higher level
Bringing the height of the ASL down is mediated through the absorption of Na through ENaC - with this inhibited ENaC isnt able to bring the level down as much
Looking at amiloride sensitive transepithelial potential and short circuit currents what does a big gap mean
Big gap = big function of ENaC
How much ENaC is contributing to the potential (the more this value the more ENaC is providing to the transepithelial potential)
what was the effect of S18 on both the transepithelial potential and the short circuit current
With S18 ENaC was only contributing 1mV to the Vte (75% inhibition)
with s18 also reduced Isc
Sum up the effect of S18 in the HBE cells
S18 inhibits the proteolytic cleavage of ENaC
This decrease the Po of the channel and thus the currents are reduced
How does the cell model for the alveolar cell differ from the upper airway cells
Here both CFTR and ENaC absorb
CFTR is involved in Cl absoprtion and NOT Cl secretion
What will the effect of an ENaC blocker in the alveolar cells mean for the height of the ASL
Increase the height of the ASL since Na reabsorption is blocked here
Describe the effect of the following on alveolar surface liquid height
Vehicle
Amiloride
S18
Low
No change
Height of the layer increases
What is one of the benifits of using S18
There are no systemic effects
What is a systemic effect
An off target effect at another organ
Where would it be expected that S18 would have an off target effect? why is this
At the kidney
Amiloride (another ENaC blocker has effects at the kidney
Effect of S18 and amiloride on URINE FLOW RATE
S18 has no impact on urine flow rate
Amiloride increase since less Na reabsorbed
Effect of S18 and amiloride on fractional extretion of Na
s18 has a minimal impac
Amiloride has an impact here since ENaC is blocked less na can be reabsorbed and thus fractional excretion will increase
Effect of S18 and amiloride on fractional excretion of K
S18 - no significant difference
amiloride - decreased fractional excretion of K since Na absorption drives K secretion by the principal cells of the collecting duct
Effect of S18 and amiloride on the plasma [K]
S18 -no significant difference
Amiloride - over time increases the levels of plasma K, chages the excitability of nerves etc. people would be at risk of cardiac arrhytmias and sudden cardiac death
Why don’t mice make the best models for cystic fibrosis
Since mice also have Ca activated Cl channels which can take over from CFTr when this is mutated
What is the effect of S18 on the height of the ASL in WT mice
Increases the height of the ASL
What is the effect of S18 in mice models of cystic fibrosis
Incrased height of the ASl
Since mice arent ideal models of CF was model organism was used to tes S18
How was this model made
What was measured
sheep
Pharmacological model - CFTR was blocked
Tracheal mucous velocity was measured
Results from the Sheep CF model
Tracheal mucous velocity initially decreases as a CFTR inhibitor is added
When treated w/ S18 shows a sustained recover in increase of TMvelocity
Same effect is seen when amiloride is added but these effects are TRANSIENT AND SHORT LIVED
