Finding a Cure for Cerebral Palsy

1. Finding a Cure for Cerebral Palsy Sidhartha Tan Clinical Professor Department of Pediatrics

2. TIMING Sentinel event? ANATOMICAL Early event? Birth asphyxia? Maternal Placental Fetal

3. Causes of Cerebral Palsy Hypoxia Asphyxia Birth Trauma Ischemia Stroke Maternal Infections Infections of Fetus & Infant Prematurity Genetic Metabolic

4. Pathogenesis

5. Major Etiology:Fetal Hypoxia-Ischemia • Spectrum of adverse outcomes • stillbirths • post-natal death • cerebral palsy and motor disorders • mental retardation • neonatal seizures • learning disabilities

6. Basis of Repetitive Hypoxia Fetal Heart Rate Uterine contractions 40 min later

7. Early Treatment Hypothermia -only treatment for term hypoxic-ischemic encephalopathy Cooling Cap trial Intermediate EEG group

8. Why We Need Better Treatments Whole Body Cooling Trial Hypothermia

9. Placenta Umbilical cord Scenario of Sustained Hypoxia Acute Placental Insufficiency Abruptio placenta

10. Objectives • Determine the neurobehavioral deficits of newborn rabbit pups following preterm hypoxia-ischemia to the fetus.

11. Model of Uterine Ischemia

12. Preterm Hypoxia 22 25 31.5 0 Gestation (Days) 32=P1 Neuro- behavior tests Sustained Hypoxia-Ischemia

14. All Animal Models Are Calibrated Models Derrick et al. J Neuroscience 2004; 24:24-34

15. Hypertonia Similar to Dystonic Hypertonia High Flexion Low Muscle Activity Extension High Rest Slow Fast Slow Rest Fast Control Dystonia in H-I Derrick et al. J Neuroscience 2004; 24:24-34

16. Hypertonia persists P1 P5 P11 Tan et al. J Child Neurol 20:972–979, 2005

18. Summary of Neurobehavior Tests • Increased tone and postural deficits • Hypertonia resembles dystonia • Deficits in ability to smell • Intensity and Duration of motor movements of Head and Limbs • Locomotion: Circular, straight line. • Righting reflex • Suck and swallow and aversive head turn

19. Fetal ADC Predicts Hypertonia

20. Summary of Fetal MRI • Prenatally, MRI identifies all the rabbits to get the severe CP phenotype – ADC allows study of critical early events • The IMMEDIATE fetal response during HI and reperfusion determines EVERYTHING

21. Nitric Oxide NO• Possible Mechanisms Reactive Nitrogen Species • Energy Failure • Free radicals • Excitotoxicity • Inflammation

22. Injured Cell Population 120 ) 0 5 1 100 x ( s 80 l l e c 60 y * h t 40 l a e 20 H 0 Non-Hypertonic Hypertonic Risk ADC Risk ADC

23. nNOS is Critical No hypertonia Hypertonia 0.3 m a A d N 0.2 o R t d m e z S i l O a 0.1 N m n r o n 0.0 y y d d d d h h a e a e t t r r l l e e a a u u D D j j e e n n H H I I

24. Clinical Scenario • Mother walks into the office • Some non-reassuring event • Biophysical profile • Fetal HR • Doppler velocimetry • Non-invasive test to determine status of fetal brain • Starting point of treatment

25. Prevention • Problems with identifying the timing and onset of the insult • 75% of CP is antenatal • What combination of etiologic factors? • NO PREVENTIVE STRATEGY at present

26. New Inhibitors by Fragment Hopping 7-NI JI-8 Richard B. Silverman

27. Selectivity To nNOS 7-NI JI-8 Richard B. Silverman

28. New Treatments

29. Preterm Hypoxia 22 25 31.5 0 Gestation (Days) 32=P1 Therapy Neuro- behavior tests Sustained Hypoxia-Ischemia

30. Prevention of CP by 7-NI Saline 20 7-nitroindazole s t 16 i K t i b 12 b a R 1 8 P f o . 4 o N 0 Normal Moderate Severe Dead Lei et al Dev Neurosci in press

31. JI-8 decreased NOS activity in fetal brain 120 120 y t i 100 v 100 i * t c 80 80 a 60 60 S O 40 40 N 20 20 n=9 n=10 n=11 n=11 0 0 Saline JI-8 Saline JI-8 24 hr Acute Ji H, et al, Annals Neurol 2009; 65(2):209-17

32. Prevention of CP by New nNOS Inhibitors Saline 20 HJ619 s t i JI-8 K 16 t i b b a 12 R 1 P 8 f o . o 4 N 0 Normal Moderate Severe Dead Ji H, et al, Annals Neurol 2009; 65(2):209-17

33. Tetrahydrobiopterin – BH4 • Important cofactor for number of aromatic amino acid hydroxylases • phenylalanine to tyrosine (phenylketonuria) • tyrosine to L-dopa, • tryptophan to 5-hydroxytryptophan. • and nitric oxide synthase (NOS).

34. Motor Disorders due to Defects in THB4 synthesis Synthesizing enzyme mutations result in dopa-responsive dystonia. Metabolic enzyme mutations result in motor disabilities and mental disturbances

35. Low biopterin levels in premature fetal brain 1.2 1.0 0.8 Biopterin (nmol/g wet tissue) 0.6 0.4 0.2 0.0 22 29 32 Postconceptional Age (days)

36. Prevention of CP by BH4 Analog Vehicle Sepiapterin 20 s t i 16 K t i b b 12 a R 1 P 8 f o . o 4 N 0 Normal Mild Severe Dead

37. Xanthine Oxidase Xanthine Uric acid O2 H2O2

38. Xanthine Oxidase Inhibition 24 Saline s Allopurinol t i 20 K t i b 16 b a R 12 1 P f 8 o . o N 4 0 Normal Moderate Severe Dead

39. The Rabbit Model of CP Offers Unique Opportunities We can test key concepts of CP in an animal model for translation into clinical studies Antenatal global hypoxia-ischemia results in neurobehavioral deficits suggestive of cerebral palsy. The hypertonia manifested in rabbits is mostly suggestive of dystonic hypertonia, a small proportion being spastic hypertonia, using a human classification. Not all fetuses behave the same. Implications for design of animal experiments. CP phenotype can be prevented by administration of drugs – designer drugs nNOSi, allopurinol, tetrahydrobiopterin.

40. Late Treatment of CP Treatment is usually symptomatic and focuses on helping the person to develop as many motor skills as possible or to learn how to compensate for the lack of such abilities.

41. Late Treatment Allied health therapy Exercise Casting Constraint-induced therapy Oral medications Chemodenervation Intrathecal baclofen Selective dorsal rhizotomy Orthopedic surgery

42. Advantages of Human Umbilical Cord Blood Cells • Rich source of pluripotent and multipotent stem cells • Possibly provide growth factors for neurogenesis • Possibly promote angiogenesis • ALLOGENEIC • Lack of need for full HLA monitoring • Negative for blood borne pathogens • Rapid availability • Less acute and chronic GvHD (after chemotherapy ablation and HUCB) • Retained graft versus leukemia effect

43. Rabbit Model of CP E22 E31 P5 C-s P1 P11 40min H-I HUCB • Peripheral infusion of HUCB cells or media or saline after birth at E31. • 1.0 ml HUCB cells obtained from JK’s lab, 5.0x106/ml, or 1 ml media or saline • Injection via external jugular vein or abdominal wall vein; 4 hrs after C-section • Determination of Mild and Severe Groups made at E31.

44. HUCB cells Increases Mortality

45. HUCB Cells Improves Motor Function in Severe Group

46. HUCB Cells Improves Motor Function in Mild Group

47. HUCB Cells Labeled with Magnetic Particles 4.7T in vivo Gadofluorine Feridex

48. HUCB Cells Labeled with Feridex E29 fetuses subjected to HI, C-section at E30, HUCB cell injection and sacrificed at E31, perfusion fixed in 4% paraformaldehyde after saline wash.

49. Conclusions • This is the first study to test the effect of HUCB cell therapy in an animal model of global hypoxia-ischemia. • HUCB cells increases mortality. • In survivors with cerebral palsy phenotype, HUCB partially reverses motor deficits in both severely and mildly affected newborn rabbit kits.

50. Problems • Is it possible to get a cure in a decade? • Focus on things that make a difference. • Divide the problem. Process goes from lab studies to FDA approval to clinical trials. • Hypothermia is a fairly accepted therapy. Even with hypothermia, there is a baseline of injury. • Common mechanistic pathways from HIE to TBI. • Pressure from parent groups.