Phenylbutyrate increases SMN gene expression in vitro and in vivo

1. Phenylbutyrate increases SMN gene expression in vitro and in vivo Christina Brahe Università Cattolica del S.Cuore, Roma

2. SMA is caused by homozygous absence of the SMN1 gene SMN1 SMN2 tel cen

3. SMA patients have one or more SMN2 genes which modulate the disease severity SMN2 severe form SMN2 SMN2 mild form These genes produce an insufficient level of SMN protein

4. Hypothesis Particular drugs may activate the SMN2 genes to produce more SMN protein SMN SMN improvement of muscle strength

5. There are drugs that open up the DNA to allow the expression of genes at a higher level inactive gene HDAC active gene HAT condensed chromatin accessible chromatin

6. Phenylbutyrate belongs to this class of drugs • FDA approved • for the treatment of another pediatric disease • is generally well tolerated • crosses the blood-brain barrier

7. Study of the effect of phenylbutyrate on SMN2 expression in cell cultures from SMA patients Results • Increase in SMN transcripts (precursor of protein) • in most but not all cultures • the increase in transcripts ranged from 50%-400% • increase in SMN protein

8. Maximum increase in SMN2-fl transcript levels 500 450 400 350 300 % increase in SMN2-fl transcripts 250 200 150 100 50 patient 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 SMA I SMA II SMA III

9. Increase in SMN protein 120 16 h 100 24 h 80 % increase in SMN protein 60 40 20 SMA I SMA II SMA III

10. Study of the effect of phenylbutyrate on SMN2 expression in leukocytes of SMA patients • Phenylbutyrate was administered to 6 patients • (4 type II and 2 type III) and 3 parents for 7 days • blood samples were taken before, during and after the trial • leukocyte SMN2 transcripts were measured

11. 900 800 T1 T2 500 T3 400 T4 T7 300 Mean 200 100 0 -100 PB increases SMN expression in leukocytes 900 800 500 400 SMN-fl transcripts (%) relative to T0 300 200 100 0 -100 1 2 3 4 5 6 M2 M3 F6 1 2 3 4 5 healthy untreated controls patients parents Brahe et al., EJHG 2005

12. Open pilot trial • 10 patients with SMA II (age: 30 months-12 years) • Treatment with phenylbutyrate for 13 weeks • (1 week on, 1 week off) • Efficacy was evaluated by using a functional motor scale

13. Functional motor scale Three point scoring system: 2 scores for unaided, 1 for assistance and 0 for inability 20 items Score 2 points Score 1 point Score 0 Score Touch one hand to head flexes head to hand unable ( )

14. Results on the functional scale before 35 3 weeks 30 25 9 weeks 20 score 15 10 5 0 2.6 3.5 3.6 3.8 4.1 6.1 6.3 7.7 9.7 12.7 Age (years) Mercuri et al., 2004

15. Mean scores in treated patients and control group 22 20 18 16 14 score 12 10 8 6 4 2 T0 T1 T2 T0 T3 (6 months) treated patients untreated controls Mercuri et al., 2004

16. Conclusions of the open trial • improvement of functional ability • children <5 years had a more obvious improvement • great variability of response (as previously observed • in vitro)

17. Large randomised, double-blind, placebo controlled trial • Enrolled :106 patients SMA II or non-ambulant type III • Age:30 months -12 years • Drop out:14 (13%) • Schedule:intermittent (1 week on, 1 week off) for 13 weeks • Assessments: • Hammersmith functional motor scale • myometry (>5 years) • FVC (>5 years)

18. Problems to be solved • More reliable outcome measures • (more sensitive functional motor scale and quantitative muscle • testing, etc) • protocol design to achieve the maximum response • (dosage, continuous or pulsedadministration, duration) • gain information on the mechanisms underlying the • difference in response

19. Istituto di Genetica Medica Università Cattolica, Roma Danilo Tiziano Carla Angelozzi Federica Borgo Anna Maria Pinto Daria Darelli Tiziana Vitali Giovanni Neri Istituto di Neurologia Università Cattolica, Roma Eugenio Mercuri Ospedale Bambino Gesù Rome Enrico Bertini Acknowledgements: Patients and parents FSMA USA FSMA Italy A.S.A.M.S.I. Telethon - Italy