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BMS 346- Epithelial physiology in health and disease > Lecture 5-Small Molecules > Flashcards

Lecture 5-Small Molecules Flashcards

1
Q

Describe current treatments in CF

A

Learning Objective

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2
Q

Discuss drug screening approaches in CF

A

Learning Objective

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3
Q

Discuss evidence that VX-770 acts as a potentiator and has therapeutic benefit

A

Learning Objective

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4
Q

Discuss evidence that VX-809 acts as a corrector but does not have therapeutic potential when used in monotherapy approaches

A

Learning Objective

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5
Q

Discuss how VX-770 & VX-809 can have therapeutic benefit in combination

A

Learning Objective

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6
Q

Discuss the current gene therapy position

A

Learning Objective

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7
Q

Cystic fibrosis & lungs

A 
•	Depletion PCL
•	 Failure to clear mucous / bacteria
•	 Infection
•	 Inflammation
• Tissue damage
Decrease lung function
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8
Q

Aim of treatment

A

Treat symptoms and not the cause

-specific to mutation

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9
Q

Current treatments

A
  • Nebulised antibiotics - tobramycin
  • Inhaled bronchodilators
  • Mycolytics – pulmozyme
  • Nebulised hypertonic saline
  • Oral antibiotics
  • Pancreatic enzymes
  • Fat soluble vitamins
  • Steroids
  • Exercise
  • Physiotherapy
  • High energy supplements
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10
Q

Vertex pharmaceuticals - current 2 drugs

A

VTX-770 – ivakaftor / kalydeco * - potentiator

VTX-809 - corrector

Licensed in US not Uk

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11
Q

VTX-770 (Ivakaftor)

A

Potentiator

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12
Q

VTX-809

A

Corrector

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13
Q

Class IV

A

Conduction

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14
Q

Class III

A

Regulation

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15
Q

Class VI

A

High turnover CFTR

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16
Q

Class II

A

Trafficking

17
Q

Class I

A

Null production

18
Q

CFTR variation

A

Regional variation in mutation frequency exists

F508 90% allelic worldwide

19
Q

G551D

A
  • 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
20
Q

Delta F508

A

Phenylalanine deletion

  • Type II
  • up to 90%
  • severe symptoms
  • Trafficking defect

SCREENING

  • cell - base immunoblot assay mature CFTR levels
  • VX-809
21
Q

Mature CFTR

A
  • post Golgi
  • Glycosylated version – high MW – makes to mem mature
    CFTR- lower ban immature MW Lower
22
Q

Fisher rat thyroid expressing WT or G551`D CFTR

A
  • 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
23
Q

Channels need priming by

A

PKA and cAMP

24
Q

SCC in Fisher rat thyroid cells - over-expression system

A

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

25
Q

Isolation of upper airway cells - tissue from patient- native primary tissue

A

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
26
Q

VIP

A

stimulate cAMP in cells

27
Q

Volume of ASL

A

G551D/F508 + VIP+ 770

increases layer or ASL not to wt but still some increase

28
Q

Cilia beat frequency

A

Vip/770 - Increase in freq just below wt

  • better than 770 0n its own
  • cilia bent when height of layer down
29
Q

Vx770 & G551D Clinical Trial

A
  • 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
30
Q

VX-770 other name

A

Ivacaftor

31
Q

Sweat Chloride threshold

A

60 mmol

32
Q

Measurement of transepithelial potential in nasal epithelium

A
  • 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
33
Q

Isoprotenerol

A

Beta receptor agonist stimulates cAMp therefore activates CFTR

34
Q

Second clinical trial - Transepithelial potential in the nasal epithelium

A

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

35
Q

VX-809

A

in vitro data very positive the clinical data less so

- aim to get CFTR to be mature

36
Q

Difference between immature and mature CFTR

A

Mature - Glycosylated
Immature- Not yet glycosylated
- Delta F508 remains immature and sent for degradation due to mis-folding

37
Q

VX-809 Data

A

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
38
Q

VX-809 Mode of action

A

Expected that it corrects misfolding and then you get mature cftr getting to membrane

39
Q

Pulse chase experiments HEK+ CFTR

A
  • 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 ***

BMS 346- Epithelial physiology in health and disease (13 decks)

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