cystic fibrosis, existing and emerging therapies

Cystic fibrosis, existing and emerging therapies Thomas Ferkol MD

Markey Pathway Conference

Cystic fibrosis: a historical timeline

Cystic fibrosis (CF) of the pancreas was described by Andersen.

The sweat defect was discovered by diSant'Agnese and colleagues when they noticed that many of the infants presenting with heat prostration during the “great summer heat wave” in New York City had CF.

Cystic fibrosis was identified as an autosomal recessive disease.

The fundamental physiologic defects were clearly established by Knowles and colleagues and Quinton as the failure of cAMP regulation of chloride transport.

The genetic defect for CF was located on chromosome 7.

The gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) was identified by positional cloning.

Cystic fibrosis transmembrane conductance regulator was established to be a cAMP-regulated chloride channel by complementation studies.

1938

1953

1983

1965

1985

19901989

Endocrine diabetes

Genitourinary• male infertility

Sweat Gland Dysfunction• hypochloremic, hyponatremic alkalosis

Respiratory• chronic cough • recurrent sinopulmonary infections• bronchiolitis/asthma• nasal polyposis• Staphylococcus aureus pneumonia• Pseudomonas aeruginosa endobronchitis

Cystic fibrosis: clinical presentations

Gastrointestinal• meconium ileus• meconium plug syndrome• distal intestinal obstruction syndrome• rectal prolapse• neonatal hyperbilirubinemia• failure to thrive• hypoproteinemic edema• hypovitaminosis• recurrent pancreatitis• biliary cirrhosis and portal hypertension

Cystic fibrosis: epidemiology

Population

Caucasian (US)

Caucasian (Great Britain)

Hispanic

African American

Native American

Asian (US, England)

Israel

Southern Europe

Epidemiologic

1 in 1,900-3,700

1 in 2,400-3,000

1 in 8,000-9,000

1 in 15,300

1 in 40,000

1 in 10,000

1 in 5,000

1 in 2,000-4,000

Newborn screening

1 in 3,400-3,800

1 in 2,200-3,200

--

--

--

--

--

--

Cystic fibrosis: median survival age, 1940-2007

0

5

10

15

20

25

30

1940 1950 1960 1970 1980 1990 2000

Med

ian

surv

ival

age

(yea

rs)

Year

3537.8

Cystic Fibrosis Foundation Registry, 2007.

2010









1938

1953

1983

1965

1985

19901989

K+

Na+

Normal Cystic fibrosis

Cl-

Na+ Cl- Cl- Na+ Cl-

H2OH2O

Na+

K+2Cl-

K+

Na+

Na+

K+2Cl-

ENaC

CFTR

Cl-a

Cystic fibrosis: airway inflammation

Cystic fibrosis: nasal transepithelial potential difference

0

-10

-20

-30

-40

-50

-60

0 4 6 8 10

PD (m

v)

Time (m)

amiloride

Cl- free

forskolin

ATP

CF

normal

PD









1938

1953

1983

1965

1985

19901989

ATP

ADP

Golgi

ER

Endosome

ATP ADP

PKAATP

ADP

Nucleus

Class 4: CFTR mutants that have altered channel properties, e.g., R117H

Class 1: premature termination of CFTR

mRNA translation, e.g., S489X

Class 3: regulatory mutants that fail to

respond normally to activation signals,

e.g., G551D

Class 2: CFTR degradation in the endoplasmic reticulum, e.g., F508

Class 5: decreased functional CFTR synthesis or transport, e.g., A455E

Classes of cystic fibrosis-causing mutations

Cystic fibrosis: clinical phenotype associated with CFTR mutations

Milder lung disease

Pancreatic sufficiency

Abnormal sweat chloride

Mild lung disease

Pancreatic sufficiency

Equivocal sweat chloride

R117H (5T)

3849 + 10kB C-to-T

2789 + 5 G-to-A

R334W

G85E

G91R

R347P

R347H

R347L

R117H (7T)

3849 + 10kB C-to-T

G551S

D1152H

A455E

Severe lung disease

Pancreatic insufficiency

Abnormal sweat chloride

F508

G542X

G551D

W1282X

N1303K

R553X

3120 + 1G-to-T

1078 del T

R75X

unaffected

Prospects for correcting cystic fibrosis: level of correction Chillon M, et al. N Engl J Med. 1996; 332:1475.

100% (wt, 9T/9T)

50% (wt, 9T, and mutant CFTR)

10% (wt protein, 5T/5T)

5% (wt protein, 5T, and severe mutant)

4% (R117H, 7T, and severe mutant)

1% (R117H, 7T, and severe mutant)

<1% (G551D, F508)

vas deferens

sweat ductairway

pancreas

Tissue affected CFTR activity

Pathogenesis of lung disease in cystic fibrosis

Defective CF gene

Defective/deficient CFTR

Bronchial obstruction

Infection

Bronchiectasis

Inflammation

Abnormal airway surface milieu

Davis PB, et al. J Respir Crit Care Med. 1996;154:1229.

Treatment of cystic fibrosis lung disease

Defective CF gene



Infection

Bronchiectasis

Inflammation


Decrease mucus viscosityAugment clearance

Chest physiotherapyMucolytics (rhDNase)

Hypertonic saline

Decrease bacterial load AntibioticsMacrolides

Reduce host response CorticosteroidsIbuprofen

Replace damaged lungs Transplantation

100

80

60

40

20

00-1 2-5 6-10 11-17 18-24 25-34 35-44 >45

P. aeruginosa

S. aureus

H. influenzae

B. cepacia

Age (y)

Perc

enta

ge p

ositi

ve

Cystic fibrosis: organisms isolated from the lower respiratory tract

Data compiled from Cystic Fibrosis Foundation Patient Registry, 2007.

Effect of Pseudomonas aeruginosa acquisition in cystic fibrosis

0.9

0.8

0.7

0.6

Cum

ulati

ve s

urvi

val

1.0 Late acquisition (>6y)

Early acquisition (<6y)

malesfemales

6 8 10 12 14 16Age (y)

Demko CA, et al. J Clin Epidemiol. 1995;48:1041

CFTR Antibacterial proteins

Impaired phagocytosisIncreased adherence

Impaired antimicrobial activity

asialoGM1

Impaired mucociliary clearance

Cystic fibrosis: bacterial colonization


Defective CF gene



Infection

Bronchiectasis

Inflammation



Chest physiotherapyMucolytics (rhDNase)

Hypertonic saline




Cystic fibrosis: pathology

Cystic fibrosis: radiological findings

respiratory epithelium

IL-8

pmn

Normal Cystic fibrosis

NE TNF-

mac

IL-1O2-

Cystic fibrosis: airway inflammation

Anti-inflammatory agents in cystic fibrosis: corticosteroids

A four year, randomized double-blind, placebo-controlled trial that compared the efficacy of two doses (1 mg/kg/d and 2 mg/kg/d) of alternate-day prednisone therapy with placebo in children with CF.

Eigen H, et al. J Pediatr. 1995;126:515.Lai HC, et al. N Engl J Med. 2000;342:851.

FVC

(% p

redi

cted

for a

ge

-6-5-4-3-2-101234

6 12 18 24 30 36 42 48 mos

1 mg/kg 2 mg/kg

placebo

p = 0.0001

high-dose arm stopped

An five-month, randomized, double-blind, placebo-controlled trial that examined the efficacy of azithromycin in patients with CF (age > 6 years, N = 251), chronically colonized with P. aeruginosa, and lung disease (FEV1 > 30% predicted for age).

Anti-inflammatory agents in cystic fibrosis: azithromycinSaiman L, et al. JAMA. 2003;290:1749.

FEV

1 (%

pre

dict

ed fo

r age

)

-6-5-4-3-2-101234

28 84 168 196 days

azithromycin

placebo

5 p = 0.009

Drug stopped

0

5

10

15

20

25

30

1940 1950 1960 1970 1980 1990 2000

Med

ian

surv

ival

age

(yea

rs)

Year

3537.8

Cystic Fibrosis Foundation Registry, 2007.

2010

anti-Staphylococcusantibiotics

anti-Pseudomonasantibiotics

airway clearance

inhaled antibiotics

inhaled mucolytics

Cystic fibrosis: median survival age, 1940-2007


Defective CF gene



Infection

Bronchiectasis

Inflammation



Mucolytics (rhDNase)Chest physiotherapy




Block Na+ uptakeIncrease Cl- efflux

AmilorideUTP/ATP

Hypertonic saline

Cystic fibrosis: alternative therapies to effect bioelectric properties of the respiratory epithelium

ClCa

CFTR

ENaC

Cl-

Cl-Na+ Na+Cl-

CF Altering other channels

AmilorideUTP/ATP

Hypertonic saline

Aerosolized hypertonic saline for the treatment of cystic fibrosisElkins MR, et al. N Engl J Med. 2006;354:229.

0

25

50

75

100

0 12 24 36 48

Period of observation (w)

Surv

ival

free

of s

ympt

om-

defin

ed e

xace

rbati

ons

(%)

hypertonic saline

control

9.2 w 36 w

An 48-week, randomized, double-blind, parallel-group trial that examined the efficacy of inhaled hypertonic saline in patients with CF over 6 years of age.

p = 0.001


Defective CF gene



Infection

Bronchiectasis

Inflammation








VX809VX770

PTC124

Increase CFTR proteinActivate mutant form

AmilorideUTP/ATP

Hypertonic saline

Golgi

ER

Endosome

Nucleus

Cell membrane

apical trafficking

G551D CFTR

Golgi

ER

Endosome

Nucleus

Cell membrane

Proteasome

degradation

F508 CFTR

Low temperature

Glycerol

Cystic fibrosis: correcting CFTR dysfunction Zeitlin P. N Engl J Med. 2004;351:606

VX809translation

transcription

post-translational folding

VX770

Cystic fibrosis: correcting G551D CFTR dysfunction

A four-week, randomized placebo-controlled trial that compared the effect of regular treatment with VX770 with placebo in CF patients with G551D mutation.

Accurso FJ, et al. N Engl J Med. 2010;363:1991.

20

40

60

80

100

120

3 14 21 28 days

[Sw

eat c

hlor

ide]

(mm

ol/L

)

0

VX770, 150 mg

placebo

VX770, 250 mg

25

[Sw

eat c

hlor

ide]

(mm

ol/L

)

-10

-8

-6

-4

-2

050 100 200

Cystic fibrosis: potentiating delF508 CFTR dysfunction

A two-week, randomized double-blind, crossover trial that compared the effect of regular treatment with VX809 with placebo in CF patients with delF508 mutation.

Gentamicin-induced correction of CFTR function in patients with cystic fibrosis and CFTR stop mutationsWilschanski M. N Engl J Med. 2003; 349:1433.

0

-2

-4

-6

-8

0 0.3 0.6 0.9 1.2

Gentamicin concentration (%)

Resp

onse

of n

asal

PD

to c

hlor

ide-

free

isop

rote

rinol

(mV)

p = 0.03pre-treatment

post-treatment


Defective CF gene



Infection

Bronchiectasis

Inflammation




Hypertonic saline





AmilorideUTP/ATP

VX809VX770

PTC124


Gene therapyProvide normal gene

Infectious diseases (40)Human immunodeficiency virus (37)Other viral diseases (3)

Monogenic diseases (58)Alpha1-antitrysin deficiency (2)Chronic granulomatous disease (3)Cystic fibrosis (23)Familial hypercholesterolemia (1)Fanconi anemia (4)Gaucher disease (3)Hunter syndrome (1)Ornithine transcarbamylase deficiency (1)Purine nucleoside phosphorylase deficiency (1)Severe combined immunodeficiency disease (6)Leukocyte adhesion deficiency (1)Canavan disease (3)Hemophilia (5)Muscular dystrophy (1)Amyotrophic lateral sclerosis (1)Junctional epidermolysis bullosa (1)Neuronal ceroid lipofuscinosis (1)

Cancer (405)

Other diseases(66)Peripheral artery disease (24)Arthritis (4)Arterial restenosis (3)Congestive heart failure (1)Coronary artery disease (21)Alzheimer disease (2)Ulcer (3)Bone fracture (1)Peripheral neuropathy (1)Parkinson disease (2)Eye disorders (4)Erectile dysfunction (1)Intractable pain (1)

Active or completed human gene therapy protocols

Pretreatment In vivo transfection In vitro transfectionPretreatment In vivo transfection In vitro transfection

Administration of an adenovirus containing the human CFTR cDNA to the respiratory tract of individuals with cystic fibrosis

Crystal RG, et al. Nat Genet. 1994;8:42.

A controlled study of adenovirus-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis

Patient No.

Cohort 1123

Cohort 2123

Cohort 3123

Cohort 4123

MOI

111

101010

100100100

100010001000

vehicle-treated

no no no

no no

no

no no no

no no no

vector-treated

nonono

nonoyes

yes no yes

yes no yes

CFTR mRNA

Knowles MR, et al. N Engl J Med. 1995;333:823.

PD

(mV)

10

0

-10

-20

-30

-40

-5 0 5 10

PD

(mV)

10

0

-10

-20

-30

-40

-5 0 5 10Days Days

A controlled study of adenovirus-vector-mediated gene transfer in the nasal epithelium of patients with cystic fibrosis

Knowles MR, et al. N Engl J Med. 1995;333:823.

Effectiveness of current gene transfer vehicles used in cystic fibrosis

Gene transfer vehiclesAdenovirusAdeno-associated virusRetrovirus Murine leukemia virus LentivirusCationic liposomesMolecular conjugates

DNA deliveryyesyes

NDNDyesyes

RNA expression yesno

NDND

maybe no

CFTR functionnono

NDNDno

maybe

Prospects for gene therapy of cystic fibrosis: submucosal gland

http://www.medicine.mcgill.ca/dynhist/histoimages

epithelium (+)epithelium (+)

submucosal glands (++)submucosal glands (++)

duct (++++)duct (++++)

SiSites of CFTR expression in the human airwaySiSites of CFTR expression in the human airway

Engelhardt JF, et al. J Clin Invest. 1994;93:737.

Prospects for gene therapy of cystic fibrosis: obstacles

Respiratory epithelial cells vs submucosal glands. Unavailable target receptors. Inability to bypass physical and functional barriers in the airway. Possible biologic unsuitability of the airway epithelium as a target tissue. Immunologic consequences. Relevant outcome measure.


Defective CF gene



Infection

Bronchiectasis

Inflammation








AmilorideUTP/ATP

Hypertonic saline


Gene therapyProvide normal gene

VX809VX770

PTC124

cystic fibrosis, existing and emerging therapies

Documents

sweat defect

genetic defect

heat prostration

great summer heat wave

new york city

positional cloning

autosomal recessive

complementation studies