Lecture 5 Small Molecules and CF

Question 1

Which pharmaceutical company has been leading the way in the development of novel small molecules in the treatment of CF

Answer 1

Vertex pharmaceuticals

Question 2

Which class of small molecule drug can be used to treat the G551D mutation

Answer 2

Potentiators

Question 3

Which class of small molecule drug can be used to treat the ΔF508 mutation

Answer 3

Correctors

Question 4

What is the name of the drug licenced in the treatment of patients with the G551D mutation

Answer 4

VTX-770/Ivacaftor

Question 5

Which small molecule has been shown to be beneficial in the treatment of patients with the ΔF508 mutation in CFTR

Answer 5

VTX-809/Lumacaftor

Question 6

Which Vertex small molecule combination treatment has been investigated in the treatment of CF

Answer 6

Orkambi – combination of VTX809 and VTX770 (Ivacaftor and Lumacaftor)

Question 7

What happens in regard to the PCL in the lungs of patients with CF

Answer 7

There is a depletion of the PCL on top of the apical membrane of the cell which means that the cilia are bent over and cannot beat as effectively

Question 8

Recall some of the symptoms of CF

Answer 8

Failure to clear mucus increased risk of infection and inflammation subsequently resulting in tissue damage

Question 9

What is often the cause of death in CF patients

Answer 9

A decrease in lung function associated with insufficient lung tissue remaining to support life (occurs in 70% of cases)

Question 10

Outline the airway epithelium cell model

Answer 10

On the basolateral membrane there is a Na+/K+ATPase and a K+ channel that act to set a negative membrane potential. The basolateral Na+/K+ATPase also acts to keep [Na+]I low and set up a basolateral driving force for Na+ influx with K+ and 2Cl- through NKCC1. Na+ brought in by NKCC1 recycles over basolateral membrane via the Na+/K+ATPase with K+ also recycling over a basolateral K+ channel. The action of NKCC1 means that Cl- accumulates inside the cell creating a high [Cl-]I. On the apical membrane CFTR results in the net secretion of Cl-. In addition Na+ moves back into the cell at the apical membrane through ENaC. The balance of Cl- secretion and Na+ absorption determines the height of the airway surface liquid layer.

Question 11

What is true of all the main CF treatments currently available

Answer 11

These only treat the symptoms and not the cause of CF

Question 12

How can we treat the inflammation caused by CF

Answer 12

Steroids

Question 13

How can we treat the thickened mucus in CF patients

Answer 13

Mucolytics such as dnase/pulmozyme which breaks down the connections between mucins and decrease its viscosity

Question 14

How can salbutamol and beclomethasone be used to help managed CF

Answer 14

These are bronchodilators which open up the airways. Beclomethasone is also a corticosteroid which means it will also have anti-inflammatory properties

Question 15

Describe how nebulised hypertonic saline can be used to treat CF

Answer 15

By inhaling hypertonic saline it creates a driving force for water movement into the PCL. This acts to help restore the PCL and clear mucus

Question 16

Why might CF patients need pancreatic enzymes fat soluble vitamins and high energy supplements

Answer 16

Pancreatic insufficiency is common in CF so the enzymes can replace this. Alongside that the fat-soluble vitamins and high energy supplements ensure that sufficient levels of nutrients are absorbed

Question 17

Briefly describe the difference between potentiators and correctors in how they work

Answer 17

Potentiators increase the open probability of the channels already inserted into the membrane whereas correctors increase trafficking of CFTR to the membrane.

Question 18

Which classes of CFTR mutations do small molecule drugs target

Answer 18

Classes I II III and VI

Question 19

Discuss the allelic frequency of the ΔF508 mutation

Answer 19

The ΔF508 mutation is the most common mutation in CF with a worldwide allelic frequency of 90%. However its incidence does differ between countries. In the UK it accounts for aroun 75% of CF patients in Spain and Italy 50% but then in Denmark it accounts for 98% of all CF patients

Question 20

CF drugs targeting which mutation in CFTR will be the most beneficial

Answer 20

ΔF508

Question 21

Patients with CF need to have mutations in both CFTR alleles T or F

Answer 21

T – its autosomal recessive

Question 22

Patients with CF have the same mutations in both CFTR alleles T or F

Answer 22

F – they can possess different mutations in each copy of the CFTR gene

Question 23

Where are the two most common CF mutations found within the protein

Answer 23

In NBD1

Question 24

What class of CFTR mutation is the G551D mutation

Answer 24

Class III – Gating defect

Question 25

What is the significance of G551D incidence in the UK

Answer 25

It’s a lot higher than worldwide average. 13% in the UK compared to 1-3% overall

Question 26

Describe the screening assay used to identify drugs that may be able to treat G551D CF

Answer 26

This is a cell-based fluorescence Vm assay. A fluorescent compound is injected into cell where its fluorescence changes with membrane potential. Chemicals that change Vm can be screened by looking for changes in the fluorescence of the molecule

Question 27

What class of mutation is the ΔF508 CFTR mutation

Answer 27

This is a class II trafficking defect mutation

Question 28

What does ΔF508 mean

Answer 28

There is a deletion of a phenylalanine residue at position 508 in the CFTR protein

Question 29

Describe the screening assay used to identify drugs that may be able to treat ΔF508 CF

Answer 29

This is a cell-based immunoblot assay that looks for the mature post-golgi CFTR levels. CFTR exists in two forms in the cell immature and mature with the mature CFTR being the form found in the membrane. A drug successful in trafficking CFTR to membrane will increase the levels of mature CFTR relative to immature CFTR. This can be observed by a change in band intensity on a western blot with chemicals that increases the intensity of the higher mature band relative to the lower immature band proving desirable

Question 30

How does mature and immature CFTR differ

Answer 30

The maturity is due to several glycosylation events with mature CFTR having a higher molecular weight (appears higher on the western blot)

Question 31

Below is a graph of current generated in cells expressing G551D CFTR against time with the addition of a number of different compounds. Describe what this data shows

Answer 31

This data shows that upon addition of forskolin there is an increase in current generated. In addition if VTX-770 is then added there is a further increase in the current generated in the cells. Addition of a CFTR inhibitor is able to alleviate the effects of VTX-770 suggesting that the action of VTX-770 on increasing current is entirely mediated by CFTR

Question 32

What happens when VTX-770 is added to cells expressing G551D CFTR before being treated with forskolin

Answer 32

Cells treated with VTX-770 before forskolin do not generated increased currents. This is because cAMP is required to prime the CFTR channels for opening

Question 33

Ivacaftor requires cAMP to be high in order to exert its effects T of F

Answer 33

T

Question 34

What are the effects of forskolin on the ISC measured for the wild type CFTR channel

Answer 34

Forskolin increases the short circuit current of CFTR from 8 to ~50μA cm-2

Question 35

Using the data below describe the effects of VTX-770 on the ISC of the G551D CFTR

Answer 35

There is a much smaller increase in the ISC for G551D CFTR treated with forskolin than in wild type (from 2μA cm-2 to 5μA cm-2). However upon the addition of FSK and VTX-770 there is a much bigger increase in ISC to around 20μA cm-2. This increase in ISC is entirely reversed when a CFTR inhibitor is added further suggesting that the action of VTX-770 on ISC is through acting solely on CFTR

Question 36

Describe the single channel recording experiments for wild type and G551D CFTR

Answer 36

In the presence of PKA/ATP there were some downward deflections indicating channel opening. However the downward deflections increased when VTX-770 added representing that it had increased the P0 of G551D CFTR when ivacaftor was added

Question 37

What is the limitation of using overexpression models in CFTR and which in vitro experiments would be better

Answer 37

Overexpression models don’t mimic what is seen in vivo as the CFTR channel if often expressed in cells that would normally express it and hence could lack interacting subunits and other essential regulatory proteins. A better model is to use patient cells who already possess the desired CFTR mutation

Question 38

Describe the difference in response to forskolin of patient bronchial epithelial cells compared to healthy controls. What was the effect of adding VTX-770

Answer 38

The patient cells showed a much smaller increase in current in response to forskolin which was 5% of wild type. However upon addition of increasing concentrations of VTX-770 there was a dose-dependent increase in currents generated by the cells to ~48% of wild type (27μA cm-2) at the maximum dose.

Question 39

What is the significance of vasoactive intestinal peptide (VIP) on CFTR activity

Answer 39

VIP raises cAMP levels and subsequently stimulates CFTR

Question 40

What does the data below suggest about the effects of VTX-770 on the ASL volume and the ciliary beat frequency of the CF patient bronchial epithelial cells

Answer 40

Wild type human bronchial epithelial cells have an ASL volume of ~40μl whereas those possessing CFTR mutations have an ASL volume of around 18μl. In the presence of VIP and VTX-770 there is an increase in the ASL volume to ~35μl almost consistent with wild type. Hence VTX-770 in the presence of high cAMP can increase the height of the PCL. In addition VTX-770 increases beat frequency which along with VIP restores beating to almost wild type levels

Question 41

How are the effects of drugs on the functioning of the lungs usually tested in clinical trials

Answer 41

Clinical trials investigating the effect of CF small molecule drugs usually monitor lung functions as FEV1 as a % of its predicted value. This data is expressed as a change in % of predicted FEV1 over time. A positive value represents and FEV1 closer to its predicted value as a result of the drug a subsequently a positive impact. Conversely a negative value means that the recorded FEV1 has got further away from predicted and there is a negative impact of drug

Question 42

Patients in the VTX-770 clinical trials all had similar baseline data such as height weight BMI FEV1 and sweat Cl-. Why was this important

Answer 42

So that group receiving the placebo and the treatment were the same and could therefore be compared to determine the sole impact of the drug alone

Question 43

What does this data show about the effects of VTX-770 on the absolute change in FEV1 5 of predicted

Answer 43

This shows that 2 weeks after starting VTX-770 there was a 10% improvement in lung function in patients receiving the drug compared to placebo. This improvement was sustained throughout the trial

Question 44

What were the effects of VTX-770 on the sweat Cl- concentrations of people with at least 1 G551D mutation in their CFTR

Answer 44

On average the sweat Cl- levels in patients receiving VTX-770 dropped by around 55mM to Sweat Cl- levels below clinical threshold for CF diagnosis

Question 45

What was the effect of ivacaftor on the nasal potential

Answer 45

There was a dose-dependent negative shift in nasal potential with increasing concentrations of VTX-770. This negative shift in nasal potential is consistent with Cl- secretion

Question 46

In order to VTX-770 to work it requires cAMP levels to be elevated. How was this achieved in the nasal potential experiments

Answer 46

Injection of the βAR agonist isoproterenol which increases cAMP and activates PKA which in turn leads to increased activity of CFTR

Question 47

How is VTX-770 now used in the clinic

Answer 47

Ivacaftor is licenced in the treatment of CF patients over the age of 6 that possess at least one G551D CFTR mutation

Question 48

What is the downside of ivacaftor in the treatment of CF

Answer 48

It is extremely expensive costing around £189000 per patient per year

Question 49

Immature CFTR is not glycosylated T or F

Answer 49

F – it is glycosylated but it requires additional glycosylation to become the mature CFTR protein

Question 50

The ΔF508 mutation means that less of the CFTR protein is glycosylated and maturated. What is the effect on the protein as a result of this

Answer 50

ΔF508 CFTR remains immature and is then sent for degradation due to its misfolding

Question 51

What do the results below show about the effects of VTX-809 on the relative proportion of mature CFTR and the currents generated by the cells as a result of treatment with the compound

Answer 51

The graph of the left shows that as the concentration of the VTX-809 drug is increased so too does the proportion of the CFTR protein that is mature as a fraction of the total CFTR protein. The maximum proportion of mature CFTR only reaches 30% however this is believed to be sufficient for normal physiological function. The graph on the right show that increasing the concentration of VTX-809 increases the currents generated in the cells with maximum currents generated of around 40μA cm-2

Question 52

Describe the pulse-chase experiments used to investigate the effects of lumacaftor on the proportion of mature CFTR in cells

Answer 52

HEK cells were made to overexpress wild type or ΔF508 CFTR. The pulse denotes the addition of radioactive methionine and cysteine residues into the culture media (35S-containing). These radioactive residues get incorporated into the proteins that the cell makes so that any new protein produced will be radioactive (so too will CFTR). The media is then washed off and the cells are left for different periods of time. Then the chase refers to a series of western blots at different times to track the manufacture and process of the CFTR protein. These time samples allow you to follow where radioactivity is. Some of the 35S-containing CFTR will be glycosylated and mature represented by the higher band on the western blot whereas other 35S-containing CFTR will remain non-glycosylated and stays immature. The ratio between mature and immature CFTR can be measured and used to determine the effect of VTX-809 compared to a vehicle control. This ratio these depends on how long cells left and what pharmacological manipulation

Question 53

Below are the results of the CFTR pulse chase experiments. Describe what this data shows

Answer 53

For the wild type CFTR protein there is initially lots of immature CFTR and no mature CFTR. Over time less immature protein is seen and more glycosylated mature CFTR is represented by the increasing intensity of the upper band. Hence more of the CFTR protein becomes glycosylated and matures. In comparison the ΔF508 CFTR cells initially show lots of immature CFTR. However over time the radioactive signal decreases and there is only a tiny increase in mature CFTR levels. This implies that the ΔF508 CFTR is being sent for degradation and is not being glycosylated and maturing. Interestingly for the ΔF508 cells treated with VTX-809 the western blots look much more like wild type. There is initially lots of immature CFTR and no mature CFTR. But over time less immature protein is seen and more glycosylated mature CFTR is represented by the increasing intensity of the upper band. This upper band is restored almost to the same intensity as is seen in the wild type CFTR cells indicating that the VTX-809 compound is promoting the glycosylation and maturation of the ΔF508 CFTR protein

Question 54

ΔF508 CFTR protein works relatively well once inserted into the membrane T or F

Answer 54

T

Question 55

What is the effect of VTX-809 on the open probability of cells overexpressing ΔF508 CFTR

Answer 55

The ΔF508 CFTR open probability is around 0.1 compared to the wild type channels where it is 0.4. However exposure of ΔF508 cells to VTX-809 restores open probability to wild type levels

Question 56

What is the result of combining treatment of VTX-809 with VTX-770 on the open probability of cells overexpressing ΔF508 CFTR

Answer 56

Exposure of cells to VTX-809 and VTX-770 enhances P0 above wild type levels hence VTX-770 potentiates CFTR open probability and activity

Question 57

Which patients were recruited for the VTX-809 clinical trial

Answer 57

19 patients homozygous for the ΔF508 CFTR mutation

Question 58

What was the result of the VTX-809 clinical trial in terms of the change in FEV1 and sweat Cl-

Answer 58

There was no improvement in FEV1 as a % of predicted in patients receiving VTX-809. In addition VTX-809 had a very minimal impact on sweat Cl- levels creating an 8mM drop in sweat Cl-. This is compared to the VTX-770 trial in G551D patients where treatment elicited at 55mM drop in sweat Cl-.

Question 59

Recall the evidence for the ivacaftor/lumacaftor combination therapy based on the data on the change in sweat Cl- levels

Answer 59

Patients treated with 150mg of lumacaftor alone saw a small decrease in sweat Cl- levels by around 5mM after 14 days. Then upon addition of 250mg of ivacaftor in combination saw a much bigger decrease in sweat Cl- down 12mM compared to baseline. This negative shift was reversed when patients came off the combination therapy. Hence there was a much greater impact of lumacaftor when given in combination with ivacaftor although this was not to the extent as was seen in the G551D patients with VTX-770 alone.

Question 60

What evidence suggested that ivacaftor/lumacaftor combination therapy could be beneficial in improving lung function

Answer 60

Patients treated with 600mg of lumacaftor along with 250mg of lumacaftor saw a 6% increase in lung function

Question 61

A larger okambi trial also measured the change in % of predicted FEV1 in patients receiving two different doses of the combination therapy. Outline the results of this

Answer 61

Both orkambi treatments resulted in an improvement however this was only on average a 3% improvement in FEV1 % of predicted. In fact there was a wide range of effectiveness of the combination therapy with 46% of combination patients seeing a 5% improvement but then 27% of combination patients seeing a 10% improvement.

Question 62

What did the forest plot data from the orkambi trial reveal about the effects of the treatment on the measured parameters

Answer 62

The combination therapy was better than placebo in every measured parameter

Question 63

In ΔF508 patients VX-809 alone is sufficient to improve symptoms T or F

Answer 63

F – it is not

Question 64

In G551D patients VX-770 alone is sufficient to improve symptoms T or F

Answer 64

T

Question 65

Evidence has shown that VX-809 can increase ΔF508 trafficking to the membrane however what else is required to relieve symptoms

Answer 65

A potentiator is required such as ivacaftor

Question 66

What is the downside to CF small molecule treatments such as ivacaftor and lumacaftor

Answer 66

They are extremely expensive; it costs £189000 for ivacaftor per year per patient and £104000 Orkambi per year per patient

Question 67

Orkambi is licenced for use in CF patients in the UK T or F

Answer 67

F

Question 68

Why is ivacaftor licenced for treating CF but lumacaftor/okambi not

Answer 68

Although ivacaftor costs more than Orkambi it has a much greater benefit to patients. It prevents more hospital admissions and other treatments compared to the combination therapy and hence has a greater saving in terms of healthcare cost

Question 69

Why weren’t early gene therapy approaches to treating CFTR largely unsuccessful

Answer 69

There were issues with side effects due to using viral vectors

Question 70

What new method of gene therapy is being investigated in the treatment of CF

Answer 70

The use of liposome complexes in order to introduce the wild type CFTR into patient airway cells

Question 71

Describe the setup of the recent CFTR gene therapy trial investigating the potential of liposome complexes in delivering CFTR

Answer 71

62 patients were given placebo of nebulised 0.9% saline as it was deemed unethical to introduce a scrambled DNA sequence. Another group of 78 patients received the pGM169/GL67A plasmid nebulised using liposome complexes. Patients were nebulised with the DNA at 28-day intervals consisting of 9 doses and the effects of this on the % of predicted FEV1 as well as a number of other indicators were measured

Question 72

What was seen in terms of the change in % of predicted FEV1 in the CFTR liposome complex gene therapy trial

Answer 72

The patients who received placebo had their % of predicted FEV1 decrease indicating a decline in lung function. The gene therapy did not lead to an overall increase in % FEV1 of predicted however these patients did see maintained lung function.

Question 73

What additional results were seen in the CFTR gene therapy trial

Answer 73

The forest plot data showed that the gene therapy was better than placebo for every variable. Gene therapy was notably better in FEV1 FVC and gas trapping parameters. There was an increase in bronchial DNA score in patients receiving gene therapy indicating the CFTR copy number increased. There was also a marginal increase in Cl- secretion in mean recordings for all patients

Question 74

What does the data below indicate about the overall effect of CFTR gene therapy compared to control on the percentage change in FEV1 compared to predicted

Answer 74

This data shows that overall placebo patients had a decline in lung function and a decrease in the change in FEV1 as a % of predicted. In comparison the patients receiving pGM169/GL67A on average saw no change in FEV1 as a % of predicted. Some of these patients improved and some got worse lung function

Question 75

Outline the current gene therapy position in the treatment of CF

Answer 75

Gene therapy can lead to a stabilisation of lung function. However due to the heterogeneous population used in current clinical trials it is difficult to determine its efficacy. Some patients have responded well others less well.

Question 76

Ivacaftor is what type of CF drug?

Answer 76

Potentiator

Question 77

Lumacaftor is what type of CF drug?

Answer 77

Corrector