Lecture 5-Small Molecules Flashcards
Describe current treatments in CF
Learning Objective
Discuss drug screening approaches in CF
Learning Objective
Discuss evidence that VX-770 acts as a potentiator and has therapeutic benefit
Learning Objective
Discuss evidence that VX-809 acts as a corrector but does not have therapeutic potential when used in monotherapy approaches
Learning Objective
Discuss how VX-770 & VX-809 can have therapeutic benefit in combination
Learning Objective
Discuss the current gene therapy position
Learning Objective
Cystic fibrosis & lungs
• Depletion PCL • Failure to clear mucous / bacteria • Infection • Inflammation • Tissue damage Decrease lung function
Aim of treatment
Treat symptoms and not the cause
-specific to mutation
Current treatments
- Nebulised antibiotics - tobramycin
- Inhaled bronchodilators
- Mycolytics – pulmozyme
- Nebulised hypertonic saline
- Oral antibiotics
- Pancreatic enzymes
- Fat soluble vitamins
- Steroids
- Exercise
- Physiotherapy
- High energy supplements
Vertex pharmaceuticals - current 2 drugs
VTX-770 – ivakaftor / kalydeco * - potentiator
VTX-809 - corrector
Licensed in US not Uk
VTX-770 (Ivakaftor)
Potentiator
VTX-809
Corrector
Class IV
Conduction
Class III
Regulation
Class VI
High turnover CFTR
Class II
Trafficking
Class I
Null production
CFTR variation
Regional variation in mutation frequency exists
F508 90% allelic worldwide
G551D
- Look at drugs that opened the channel
- Glycine to aspartate
- Type III
- severe symptoms
- Both mutations in NBD1
- 13% of uk Pop
- Look for a change in fluorescence – possible change due to CFTR
- Identified VX-770
Delta F508
Phenylalanine deletion
- Type II
- up to 90%
- severe symptoms
- Trafficking defect
SCREENING
- cell - base immunoblot assay mature CFTR levels
- VX-809
Mature CFTR
- post Golgi
- Glycosylated version – high MW – makes to mem mature
CFTR- lower ban immature MW Lower
Fisher rat thyroid expressing WT or G551`D CFTR
- addition of Forskolin little increase in Cl secretion because CFTR gating means they’re not opening
- G551D FSK and VX-770 increase in secretion but not as big as Wt
- CFTR inihibtor - site of current goes down
- mutant poor stimulation - gating defect
- mutant doesnt open but if it is exposed to VX770 it opens channel function enhanced
Channels need priming by
PKA and cAMP
SCC in Fisher rat thyroid cells - over-expression system
WT vs G551D
G551D- CFTR PKA/ATP
- downward deflection show the channels are opening
- VX770 - channel mostly in open configuration - therefore classed as a potentiator as it potentiates opening of the channels
Isolation of upper airway cells - tissue from patient- native primary tissue
G551d mutation and delta F508
- SCC
- added amiloride to block ENac
- overtime added forskolin
- dose response curve 770
- see graph
- G551D/F508 Vx-770 FSk response- 50% of wt function sufficient to rescue secretory function to the upper airway cell
- CL secretion at 50% is sufficient for normal function
VIP
stimulate cAMP in cells
Volume of ASL
G551D/F508 + VIP+ 770
increases layer or ASL not to wt but still some increase
Cilia beat frequency
Vip/770 - Increase in freq just below wt
- better than 770 0n its own
- cilia bent when height of layer down
Vx770 & G551D Clinical Trial
- Randomised double blind trial 48 Weeks
- monitored FEV as a % of predicted
- Expressed data as change in % of predicted FEV
- +ve value closer to predicted
- -ve further from predicted
- promising result/huge improvement
- increase in event free in VX-770 67% patients
- drop in sweat chloride below the threshold
VX-770 other name
Ivacaftor
Sweat Chloride threshold
60 mmol
Measurement of transepithelial potential in nasal epithelium
- Squirt cl free solution (to promote the driving force for chloride secretion) on apical side to promote Cl secretion
- also add isoprotenerol
- if functional chlorde channels show a negative shift in the transepithelial potential in the nasal passage
- increase in chloride- increase in negative shift
- NO EFFECT OF CHLORIDE FREE SOLUTION WITH ISOPROTENEROL IN G551D MUTANTS
Isoprotenerol
Beta receptor agonist stimulates cAMp therefore activates CFTR
Second clinical trial - Transepithelial potential in the nasal epithelium
Oral dose
VX-770 at 25mg/75mg/150mg
- not a lot at 25mg
- change at higher conc - more negative shift in the potential
- VX-770 enhances function of the chloride channels
- direct measurement of chloride secretion
- improved function of CFTR
VX-809
in vitro data very positive the clinical data less so
- aim to get CFTR to be mature
Difference between immature and mature CFTR
Mature - Glycosylated
Immature- Not yet glycosylated
- Delta F508 remains immature and sent for degradation due to mis-folding
VX-809 Data
little mutant protein trafficked to the membrane
- increase of VX-809 improvement in traffcking
- 30% of delta F508 in mature form and making it to the membrane
- As Vx-809 increase – increase in SCC increase in cl secretion
VX-809 Mode of action
Expected that it corrects misfolding and then you get mature cftr getting to membrane
Pulse chase experiments HEK+ CFTR
- strengthened evidence for effectiveness
- HEK - overexpresssing CFTR
- expose cells to radioactive methionine and cysteine
- compounds taken up and a.a incorporated
into CFTR produced
-removed outside radiation and left the cells in wells for different durations
-looked at ratio of mature vs immature CFTR - map overtime how the Vx-809 was forcing mutant channels to the membrane
- amount of immature CFTR goes down over time (only radioactive)
- mutant little being matured as it breaks down
- immature changing to mature with VX-809
**Not the same as wt but sufficient to push mature CFTR to membrane ***
