Diagnosis of Cystic Fibrosis

Types of test

Description

Outcome

Prenatal

Amniocentesis

Choronic villus

Remove fluid from sac in uterus around the baby test Remove tissue sample from placenta test

New born screening

Guthrie blood spot test

Check for increased [immunoreactive trysinogen]

Carrier (1 normal chr & 1 abnormal chr)

Blood and saliva

Test for faulty gene. Success rate:9/10

Types of test

Description

Outcome

Pilocarpine ionotophoresis (sweat)

Localised sweating induced by pilocarpine( muscuranic Ach receptor agonist)

Determine [Cl- ] >60mmol CF confirmed 40-60mmol intermediate, more likely to be diagnostic in infants *proceed to nasal potential difference if uncertain

Others

Lung function

a)Amount of air breathe in and out b) Forced expiratory volume(FeV1) c) How well lungs deliver O2 delivered in blood
Sputum sample cultured to check for Pseudomonas Aeruginosa Aggressive stage

Sputum

Boeck,D.K. et al. (2006)

Boeck,D.K. et al. (2006)

Advantage/Disadvantage of Diagnosis
1) Nutritional benefit 2) Early access to specialised care 3) Decrease time of diagnostic uncertainty 4) Ability to counsel parents for prenatal testing 1)(sweat test) False positive occurs when the patient already has fluid retention 2)(Sweat test)False negative inadequate amount of sweat 3)Identify healthy heterozygote carriers as potential patients 4) Patients with classic cystic fibrosis will be unaccounted for

There is no cure for CF yet!

Maintaining lung function

Alleviating Symptoms

Managing complications

Therapeutic Strategy

Nutrional therapy

Correction of Malfunctions of gene

Gene therapy: 1) Viral 2) Non-viral

Clearing Airways Fight infections Loosening / removal of mucus Imbalance of ions causes mucus to become thick and sticky, blocking airways Therapeutics: Physical therapy: Chest physical therapy common technique is clapping with cupped hands on the front and back of the chest. Mechanical devices also can help loosen lung mucus. Chest clapper, a hand-held device that mimics the effect of cupped hands clapping over the ribs Inflatable vest, a device worn around the chest that vibrates at high frequency Breathing devices, usually a tube or mask through which you exhale while performing breathing exercises Mucolytics: Dornase Alfa a.k.a. Pulmozyme to thin mucus Aka Recombinant human Dnase Hypetonic saline solution Dry osmotic hydrator Mannitol Completed phase 3 , available in some countries

Thick mucus in lungs and sinus is ideal breeding ground for bacteria Medication: Azithromycin(antibiotic pill Tobramycin/TOBI by Novartis Pharmaceuticals, USA( antibiotic inhalant) Aztreonam Lysinate (AZLI from Gilead Sciences)( antibiotic inhalant) High concentration of drug can be used on site of infection with minimal side effects Ibuprofen(anti-inflammatory)

Managing complications Liver Fatty liver (non-symptomatic) Cirrhosis Treatment: Transplant (severe) Ursodeoxycholic acid(bile acid) dissolve obstructions Oesophagus (gastro-oesophageal reflux) Treatment: Prokinectic plus antacid Surgery(severe) Reproductive Bilateral absence of Vas deafrens Treatment: Assisted reproductive technology + sperm aspiration

Nutritional benefits Pancreas Insufficient exocrine secretion Pancreatic enzyme replacement therapy(PERT)increase absorption of fat soluble vitamins, co-enzymes

Feeding tube Insufficient nutrition absorption Tube from nose to stomach or surgically implant into abdomen

Mutations

Drug

Mechanism of Action

Class I

Gentamicin (aminoglycoside anitbiotics)

Suppresses premature termination codonallows amino acid to be incorporatedtranslation continues CFTR protein can be made

Class II

4-phenylbutyrate (4PBP): Histone deacetylase inhibitor( HDACi)

Induced increase of proinflammatory cytokine IL8decrease in proteosomes,Nf-kB etc increase F508 CFTR protein increase trafficking of ions

Class III

Genistein, Kalydeco*

assumed to function as potentiatorenhances Cltransport of mutant CFTR

Class IV

Genistein, Kalydeco* Same as for class III

Class V

Milrinone ( class III phosphodiesterase inhibitor)

Sustains elevated cAMP level. Theoretically improves Cl- conductance ( results seen in mouse model)

Class VI

No known research yet

Latest treatment
1st FDA (Food Drug Association) approved drug for genetic level (2012 Name of drug: Kalydeco Created by Vertx pharmaceuticals and cystic fibrosis foundation Other names: VX-770/ Ivacaftor CTFR- potentiator
Increases the ability of CFTR( sensitizes) to transport ions across the cell membrane Clinical trials showed patients had decline in chloride level in sweat (Cystic Fibrosis foundation, 2011)

Targets G551D mutation

Gene therapy
Gene therapy possible as CF is a single gene disorder Aims:
Treat the disease underlying cause Reduce the high therapeutic burden that patients currently have to endure To transfer functional CFTR gene to repiratory epithelial cells To correct Cl- transport

Gene therapy: non-viral

Gene is administered via nebulizer (inhaler)which consists of lipid globules which encase a circular DNA (plasmid)
PGM 169:
DNA plasmid expressing CFTR gene created to maximise efficiency (negatively charged) Produce CFTR coupled with production of ions( hCEF1 promoter) during CFTR activity Does not contain CpG island which triggers immune respons

(GL-67A)
Lipid ( positively charged, safe, stable, improve gene transfer)

Gene therapy: viral

Modified viruses, with virulence gene removed, designed to transfer gene into cells Viruses are
Good at targeting/ entering cells Modified no replication or destruction of cells However, not able to expand to fit entire genetic material larger than its own genome size Might trigger immune response to prevent repeated treatment or create illness to patients

Viral therapy
Lentiviral vector programme
Developed by UK consortium Pseudotyped Simian immunodeficiency virus (SV40) with Sendai vector (SeV) 2 surface envelope proteins (HF and N) increase effeciency of airway transduction (functional CFTR Cl- channel) Favourable results:
No need for preconditioning with agents to damage epithelium Effect lasted 15months in murine nose (in vivo)

Viral therapy
Parainfluenza virus (vector) Created In vitro model of human cystic fibrosis ciliated surface airway epithelium (CFHAE) Favourable results:
Sufficient CFTR produced (100 fold) Rectified Bioelectric defect (Na+ , Cl- ) Restored Airway Saline Levels(ASL) regulated Improved mucus transport

Future of CF therapy
Current clinical trials: 1) F508del: Lumacaftor (VX-809) +ivacaftor (VX-770) Phase II; phase III planned 2) F508 del: VX-661+ ivacaftor Phase II 3) Class I : PTC/other 10 Ataluren Phase III (primary endpoint negative) 4) Antibiotic against Pseudomonas gave favourable results: Liposomal preparation of Amikacin + Ciproflocaxin Levofloxacin (inhalant at 1st trial)

Future of CF therapy

Despite the number of trials, progress into actual clinical products has been slow However, increasingly more and more clinical trials are reaching phase IIIhave a good chance of becoming licensed products for a range of diseases.

In a nutshell

Boyle,M.P. ,and Boeck,K.D. 2013)

Reference (therapeutics)
Boeck, D.K., Wilschanski, M., Casteilani, C., Taylor, C., Cuppens, H., Dodge, J., and Sinaassapel, M. (2006). Cystic Fibrosis: Terminolgy and diagnostic alogorithms. Thorax 61(71), 627-635. Cohen, T.S., and Prince, A. (2011). Cystic Fibrosis: a mucosal immunodificeincy syndrome. Nature Medicine 18, 509-519. Davies, C.J., Alton, E.W., and Bush, A., (2007). Cystic Fibrosis. British Journal of Medicine 355, 1255-1259. Hurley, M.N., Camara, M., and Smyth, A.R. (2012). Novel Aprroaches to Treatment of Pseudomonas Aeruginosa infection in cystic fibrosis, European respiratory Journal , 1-19. Ledford, H. (2012). Drug bests: Cystic Fibrosis Mutations. Nature 482(7384), 145. Mitomo, K., Griesenbach, U., Inoue, M., Somerton, L., Meng, C., Akiba, E., Tabata, T., Ueda, Y., Frankel, G.M., Farley, R., Singh, C., Chan, M., Munkonge, F., Brum, A., Xenariou, S., Escudero-Garcia, S., Hasegawa, M., and Alton, E.W. (2010). Toward gene therapy for cystic fibrosis using a lentivirus pseudotyped with Sendai virus envelopes. Moleculara Therapy 18(6), 1173-1182. Moskowitz, S.M., Chimiel, J.F., Sterner, D.L., Cheng, E., Gibson, R.L., Marshall, S.G., and Culting, G.R. (2008). Clinical Practice and Genetic Counselling for Cystic Fibrosis and CFTR related Disorders. Genetics in Medicine 10, 851-868. Roque, T., Boncoeur, E., Saint-Criq, V., Bonvin, E., Clement, A., and Tabamyo, J.J. (2008). Proinflammatory effect of sodium 4-phenylbutyrate in deltaF508-cystic fibrosis transmembrane conductance regulator lung epithelial cells: involvement of extracellular signal-regulated protein kinase 1/2 and c-Jun-NH2-terminal kinase signaling. The Journal of Pharmacology and Experimental Therapeutics, 326)3), 949-956.

Reference(therapetics)
Rowe, S.M., Borowitz, D.S., Burns, J.L., Clancy, J.P., Donaldson, S.H., RetschBogart, G., Sagel, S.D., and Ramsey, B.W. (2012). Progress in Cystic Fibrosis and CF ttherapeutics Development Network. Thorax 67(10), 862-890 Zeitlin, P.L. (1999). Novel Pharmacological therapy for Cystic Fibrosis. Journal of Clinical Investigation 103(4), 447-452 Zhang, L., Button, B., Gabriel, S.E., Burkett, S., Yan, Yu., Skiadapoulos, M.H., Dang, Y.L., Vogel, L.N., McKay, T., Mengos, A., Boucher, R. C., Collins, P.L. and Pickles, R.J. (2009). PLoS Biology 7(7), e1000155. Sharma, G.D., Bye, R.B., McColley, S.A., Windle, M.L., Callahan, C., and Cataletto, M.E., 2013. Cystic Fibrosis Treatment and Management [online]. Available at: < emedicine.medscape.com/article/1001602-treatment > [Accessed on: 28th February 2013] University of Missouri, 2011. [online]. Genistein and Analogues for treatment of Cystic Fibrosis Available at: <www.ibridenetwork.org/mu/ Genistein_ and_Analogues _for treatment_ of _Cystic _Fibrosis [Acessed on: 1st March 2013] National Heart, Lung and Blood Institute, 2011. How is Cytic Fibrosis Diagnosed? National Institute of Health [online]. Available at: www.nhlbi.nih.gov/health/healthtopics/topics/cf/diagnosis.html [Accessed on : 1st February 2013]

Reference(therapeutics)
Cystic Fibrosis Foundation, 2012. Therapies for Cystic Fibrosis. [online]. Available at: www.cff.org/treatments/theraapies/#clearing_the_airwways [Accessed on: 3rd February 2013] Mayo Foundation for Medical Education and Research, 2012. Cystic Fibrosis:Complicatios. [online]. Available at: www.mayoclinic.com/health/cysticfibrosis/0500287/DSECTION=complications> [Accessed on: 1st March 2013]