1. Research Progress in Fragile X Syndrome – Brain Function to New Ideas for Treatment Elizabeth Berry-Kravis MD PhD Rush University Medical Center, Chicago Disclosures: EBK has received funding from Neuropharm LTD and Seaside therapeutics to conduct clinical trials in FXS, and has served as a consultant for Novartis Pharmaceuticals and Roche Pharmaceuticals

2. Fragile X Syndrome Form of X-linked MR/ID discovered by Martin and Bell 1943 Lubs fragile site 1969 1991 – Caused by mutations that inactivate FMR1 gene Prevalence – 1:4000 males and females Carriers – 1:100-250 females, 1:250-800 males All ethnic groups worldwide MOST COMMON KNOWN INHERITED FORM OF COGNITIVE DISABILITY

3. Features of Fragile X Syndrome Physical: large prominent ears, long face, large head, prominent jaw and forehead, midfacial hypoplasia, hyperflexible joints, large testis Intellectual Disability or LD Behavior problems: anxiety, hyperactivity, distractibility, perseveration, aggression Seizures – 15% Strabismus – 30% Medical: otitis, sinus, MVP, reflux, sleep apnea, loose stools, allergies

4. FXS Intellectual Disability Males - average adult IQ about 40 and mental age 5-6y, range severe ID to normal (mosaics) IQ scores higher when young, decline with age Specific cognitive profile Achievement and Adaptive skills higher

5. Fragile X Syndrome Characteristic cognitive pattern with prominent executive function deficits Strengths – –Receptive vocabulary – –Syntax – –Imitation – –Grammatical structure – –Visual memory – –Simultaneous processing – –Experiential learning Weaknesses – –Auditory processing – –Sequencing – –Abstraction – –Short-term memory – –Topic maintenance/ "connectedness" – –Mathematics – –Working memory – –Coordination/praxis

6. Why is Math so hard in FXS?

7. FXS Developmental Problems Motor delays in some Hypotonia - orofacial when young Fine motor problems - poor writing ability Gross motor clumsiness Speech/language delays Delayed imitative and symbolic play

8. FXS Developmental Problems Parents most frequently become concerned first about speech delay Other concerns that may lead to child’s initial evaluation: – –Motor delay – –Extreme hypersenstivity/defensiveness – –Hyperactivity or anxiety – –Cognitive delays or LD at school age Therapists for child with delay often first encourage testing – if parents don’t recognize magnitude of problem or doctors are discouraging testing

9. FXS Developmental Problems Most FXS patients can be identified with delay on developmental screens by 9-18 months Average age of diagnosis still between 3-4 years – no change in past 10 years (Bailey)Average age of diagnosis still between 3-4 years – no change in past 10 years (Bailey) Miss early interventionMiss early intervention New NFXF/FXCRC goal: diagnosis before age 2 – working on PediatriciansNew NFXF/FXCRC goal: diagnosis before age 2 – working on Pediatricians

10. FXS - Pattern of Speech/Language Deficits Most abnormal – –Jargon/tangential language – –“Jocular litanic phraseology” – –Perseverative speech – –Lack of use of gestures – –Talking to self – –Cluttering Less abnormal – –Fluency – –Prosody Strengths – –Grammar – –Vocabulary

11. Language Characteristics in FXS Relative to Normal Mental-Age Matched Controls and Developmentally Delayed Subjects Decreased intelligibility – –Vowels sounds more variable than normal developmentally matched controls – –Poor oromotor control – –Faster rate of speech Decreased length of utterances Increased self-repetitious and perseverative language Single word vocabulary a strength

12. Language in FXS vs Autism More impaired in non-verbal communication than autistic group More impaired in expressive language Less impaired in receptive language which is a strength Tangential language more prevalent than in autism or general developmental delay (possibly due to anxiety, inhibitory control deficits)

13. FXS Socialization Deficits Good understanding of facial expression – different from typical autism Deficits in peer entry Defcits in interpreting social cues – correlate with anxiety, attention problems, social problems Discrimination deficit - body language, hidden curriculum Friendly but social anxiety

14. FXS - Female Affecteds More mildly involved Average IQ 80 NVLD, VIQ>PIQ, poor math, very impaired executive function, distractibiity Same cognitive pattern as males Physical features/medical problems variably present Social/psychiatric disability common – anxiety/shyness, oddness Decreased education, job stability, socioeconomic status

15. FXS Treatment in Clinic - Supportive Early intervention Intensive speech therapy OT with sensory integration Inclusion in school as much as possible Educational curriculum, environment, teaching style matched to FXS cognitive profile Socialization program Behavior plan Behavior medications for ADD/anxiety Aggressive tx of otitis – tubes/audiology Manage sleep apnea Yearly eye exams Control seizures Orthopedics if needed Monitor for MVP/heart Genetic counseling Discuss reproductive options Rush FXS Clinic since 1992 > 400 patients

16. Psychopharmacology in Fragile X Syndrome Targets behavior to improve functioning Supportive, does not target underlying cognitive problem Only one prior controlled trial in FXS (N=15) shows Ritalin effective in 2/3 of boys Therapeutic decisions based on target largest problem symptom complex(es) – trial and error May need to treat multiple behavioral domains

17. Decision to Use Behavioral Medication Individual engaging in dangerous behaviors Individual is dysfunctional from behavior Individual could accomplish more or be higher functioning if specific behavior is managed – –Increase ability to participate – –Able to be in more inclusive setting Not necessarily “when all else fails” ALWAYS an adjunct to behavioral, environmental measures

18. FXS - Types of Medications and Indications* Problem Behavior Cluster Medication Class Medications Distractibility/ Hyperactivity Stimulants Methylphenidate, Adderall, Provigil Overarousal/ Hypersensitivity Alpha-agonists Clonidine, Tenex Anxiety/OCD/ Perseverative SSRIs Prosac, Zoloft, Celexa, Lexapro Mood LabilityAntidepressants, AEDs, Lithium Tricyclics, Effexor, Li, SSRIs, VPA, TPX, CBZ Aggression/Self abusive Atypical Antipsychotics, Risperdal, Abilify, Seroquel, Geodon *meds may be targeting several clusters

19. Abilify (aripiprazole) – New Mechanism Partial agonist (activator) at dopamine D2 and serotonin 5HT1a receptors, antagonist at 5HT2a May be particularly good in FXS because may help with ADHD symptoms (dopamine effect) as well as anxiety/irritability Likewise can get aggravation of irritability, perseveration, agitation due to dopamine effect in subgroup Social behaviors better in animal models compared to other antipsychotics Thus far best antipsychotic response rate for FXS – can give dramatic social improvements

20. Abilify – High Response Rate in FXS (2004-2007)

21. Supportive Psychopharmacology is Helpful in FXS… Berry-Kravis and Sumis, 2006 …but treating the underlying disorder would be better Rush Fragile X Clinic: 208 trials 136 patients 231 trials 123 patients 100 trials 58 patients 52 trials 52 patients Attention Hyperactivity Anxiety Mood Aggression Irritability Hyperarousal Oversensitivity

22. FMR1: The Fragile X Gene Carrier state passed through many generations before FXS Simple DNA test to diagnose FXS – measures repeat size

23. FMRP Expression and Disability 100 0 Disability FMRP Social anxiety/shyness Distractibility/hyperactivity Executive deficits Spatial perceptual deficits NVLD Intellectual Disability

24. The Fragile X Mouse (Knockout; K/O) fmr1 gene inactivated No active FMRP Subtle cognitive problems Audiogenic seizures Good neurobiological model to answer question: WHAT DOES FMRP DO?

25. McKinney et al, AJMG -B: Neuropsychiatric Genetics, 2005 Both FXS Mouse and Human Brains: Dendritic Spines are Abnormal in FXS with Immature Long Spines at Brian Connections FMRP regulates maturation of brain synapses (connections)

26. FMRP in Dendrites (Brain Connections) FMRP in RED in tip of neural dendrites and where processes are forming FMRP regulates proteins made at brain connections (in dendrites) - proteins have to be made in right amount for connection to mature and work right. Antar et al, J. Neurosci 2004

27. AMPA ribosome mGluR glu FMRP Normal Mature connection LTD AMPA ribosome mGluR glu Fragile X Immature connection (too weak) Excessive LTD – due to mGluR system overactivity mGluR Theory of Fragile X Dendrite How loss of protein regulation causes weak connections in FXS

28. New Research is Leading to Future Treatments for Fragile X Specific glutamate (group I mGluR) pathway regulated by fragile X protein (FMRP) in neurons Regulates strength of neural connections needed for learning Overactive when FMRP not there – messes up connections Can block pathway with new drugs (mGluR blockers) being developed Drug Normal Fragile X

29. AMPA ribosome mGluR glu Fragile X Immature connection (too weak) Excessive LTD – due to mGluR system overactivity Dendrite 1 2 3 4 X X AMPA Other systems Potential Mechanisms for New Treatments of FXS

30. AMPA ribosome mGluR glu AMPA ribosome mGluR glu Fragile X Immature connection (too weak) Excessive LTD Ampakine Stronger connection Ampakine Mechanism 3 Activate AMPA receptors Raise BDNF – bring AMPA receptors to synapse Targeted Treatments we have tried

31. AMPA ribosome mGluR glu AMPA ribosome mGluR glu Fragile X Immature connection (too weak) Excessive LTD Stronger connection Li Lithium Mechanism 1 Lithium blocks PI turnover, reduce mGluR signalling through PL-C/PK-C cascade, directly block protein synthesis by GSK3B block

32. Lithium and FXS Models Lithium corrects courtship memory deficits in dfxr mutant fly – McBride 2005 Lithium corrects open field hyperactivity and decreases audiogenic seizures in fmr1 k/o mouse - Bauschwitz – –FX-specific effect - lithium is convulsant in normals Corrects several learning tests, LTD in mouse Data from R. Bauschwitz Audiogenic Seizures

33. FXS Open-Label Lithium Trial Test concept of mGluR pathway inhibition 13 better at 2 mo, minimal toxicity ABC Total (range 0-174) 13 improved, p=0.005 Placebo change in CX516 study (N=25): -4 RBANS List Learning (range 0-40) 8 of 10 improved, p= 0.028 CX516 placebo change: 0.0 Also significant improvement in CGI, VAS, and Vineland Personal Daily Living Skills and Maladaptive Behavior 1 yr

34. Lithium Study Biomarker - ERK Activation in Lymphocytes ERK phosphorylation slower in K/O and FXS Berry-Kravis et al, JDBP 2008 N=11 Baseline mean: 4.87 min 2 Month Treatment mean: 4.11 min (p=0.007) (about 1 min is difference FXS and control) 1 Year Treatment mean: 3.56 min (P=0.00006) 2 mo 1 yr baseline

35. New Targeted Treatments in Trials - GABA Activators Data from FXS animal models – –GABA receptors abnormal – –Glutamate toxic to fragile X fly, GABA activators rescue Baclofen (available, used for spasticity) - GABA-A agonist that blocks presynaptic release of glutamate – –Can help irritability in FXS and autism – –Reverses abnormal behaviors and seizures in FXS mouse – –R form is much more active

36. R-Baclofen and the mGluR Theory – Decrease Excessive Glutamatergic Transmis sion Pre-synapticPost -synaptic mGluR5 Proteins FMRP Proteins GABA-B STX209 Mechanism 4

37. Clinical Trial of R-Baclofen (STX209) in FXS Started in December Goal is to show improves tantrums/aggression/agitation/irritability Placebo-controlled crossover with 4 weeks of titrated treatment each arm Tests before and after treatment each arm Age 12 and up first 10 participants, now age 6 and up Extension – can get drug after trial if it works Good side effect profile Have seen some positives during trial thus far

38. New Targeted Treatments in Trials - mGluR5 Blockers mGluR5 negative modulators = potent anxiolytics, also anticonvulsant MPEP is prototype but not for humans Being developed for anxiety disorders, neuropathic pain, irritable bowel Like lithium, would correct mGluR overactivity in most areas of brain and would correct oversynthesis of all FMRP- regulated proteins Unlike lithium, specific for mGluR system

39. AMPA ribosome mGluR glu AMPA ribosome mGluR glu Fragile X Immature connection (too weak) Excessive LTD Stronger connection Li mGluR5 blocker Fenobam Ampakine Combination: Fragile X connection corrected mGluR5 Blocker Mechanism 1

40. AMPA Receptor Rescue by MPEP Control FMRP Internal AMPA Surface AMPA FMRP Absent FMRP Absent and MPEP GluR1 = AMPA receptors that drive strength of neural connections Nakamoto et al. 2007

41. ALL ABNORMAL Phenotypes Tested in Fragile X Fly Normalized by MPEP Courtship memory deficits Mushroom body beta lobe fusion Lack of survival on commercial food (glutamate-containing) Odor shock long-term memory deficits McBride et al. 2005 Warren 2006 Bolduc 2007

42. ALL ABNORMAL Phenotypes in FXS Mouse Normalized by MPEP (or Other mGluR5 Blockers) Audiogenic seizures Epileptiform bursts Open field behavior Prepulse inhibition Dendritic spine shape AMPA receptor internalization Excessive protein synthesis Bauschwitz, 2006 Wong et al. 2006 These and other phenotypes in the fmr1 K/O mouse also all reversed by crossing fmr1 K/O to mGluR5 heterozygous mutant

43. MPEP Controls Audiogenic Seizures in FXS Mouse Video courtesy of R. Bauschwitz PhD WE CAN CURE THE FXS MOUSE!

44. But Mouse is not Man…

45. human mouse Human brain a lot more wiring time

46. mGluR5 Negative Modulators in Development for FXS FenobamFenobam –Old molecule – No major toxicity –Reduced anxiety in some adult phase II trials –Dropped - short half life/erratic pharmacokinetics (PK) –Found to be mGluR5 blocker (Porter et al. 2005) –2008 – orphan drug status for FXS – Neuropharm –The first mGluR5 blocker used in FXS

47. FENOBAM Open Label Escalating Single Dose Trial of Fenobam in FXS RUSH and UC Davis (Neuropharm and FRAXA) safety trial of 1 dose (50-150 mg), 12 adult FXS (6M, 6F), age 18-38, IQ 36-85  PPI improved 20% in 6/12 subjects (control test-retest group 2/13, p=0.03)  Observation of positive behavioral changes in 9/12 subjects  No fenobam-related adverse events

48. Prepulse: 50 ms, 75 db 1000Hz tone Prepulse interval: 60, 120, or 240 ms Obicularis Oculi EMG Obicularis Oculi EMG Auditory stimulus Auditory stimuli Amplitude (volts) Startle Alone Trials Prepulse Trials Latency 50 ms 105 db white noise Prepulse Inhibition (PPI) Studies

49. Hessl et al. AJMG 2008

50. P=0.03 PPI improved on fenobam Berry-Kravis et al. JMG 2009

51. mGluR5 Negative Modulators in Development for FXS STX107 – Seaside (from Merck)STX107 – Seaside (from Merck) –Good safety profile, twice daily dosing –In Phase I - FXS trials projected in 2010 AFQ056 – Novartis – –Good safety, twice daily dosing – –Randomized double-blind 28 day clinical trial in Europe just finished – 30 males with FXS – –Test behavior and cognitive measures – –New trial planned in Europe Other companies developing for other conditions (Addex, AstraZeneca)

52. mGluR5 Negative Modulators in Development for FXS RO4917523 – Roche – –Once daily dosing, good safety – –Starts Phase II trial in USA November 2009 – –Placebo controlled 6 week treatment – –60 males or females with FXS age 18 up – –Cognitive, behavior measures, eye tracking, PPI – –Lots of blood monitoring for PK 2 Phases - dose finding and max dose If safe and appears successful will extend to 6-17 age group

53. Potential Other Future TRIALS Minocycline – Mechanism 2 Better ampakines – Mechanism 3 Multicenter Abilify trial for FXS approval PAK inhibitors Combinations Still need work on design and outcome measures for these, many steps, but benefit may be overlap with autism pathways – treatments for autism too

54. Minocycline Works on MMP9 – one of proteins regulated by FMRP Too much MMP9 in FXS Minocycline inhibits the activity of the excess MMP9 Normalizes spines and some behaviors in FXS mouse Anitibiotic used for zits – available now – –Causes teeth to turn yellow if used before age 10 – –Trial in Canada – –Lots of use in USA – variable positives and negatives – hard to interpret definitively – –Big trial planned through FXCRC Only affects one FMRP protein target so might need to use with other things

55. New Measures of Function “Outcome Measures” for Clinical Trials

56. KiTAP Test Alertness Flexibility Go-no Go - Many subtests reliable in test-retest – will use in Roche study - Correlates with attention and hyperactivity measures - Crosses all levels

57. Eye Tracker as New Outcome Measure Validated this summer with test-retest to be ready for Roche mGluR trial in Fall – measures eye contact

58. NIH Study Shows Increased Rates of Cerebral Protein Synthesis in Fragile X Knockout Mice WT KO Qin et al (2005) J Neurosci 25:5087.

59. Current NIH Study to Prove Increased Protein Synthesis in Humans with FXS PET scans with anesthesia FXS males over 18 years All expenses paid to Washington No medications (or if on stimulants can stop for day of study) Will be very helpful in proving the mGluR theory in humans and supporting the new medication development Can be used to show the new meds work

60. Testing for FXS Modifiers and Biomarkers Many proteins in this pathway may be implicated in autism

61. Making Stem Cells from Skin Cells (iPS) …and brain cells from the stem cells for personal brain cell cultures to study treatment targets in different people with FXS

62. Newborn Screening for Fragile X Comes to 7 th Grade

63. Pilot study on newborn screening for FMR1 mutations using blood spots: a) Identification of the frequencies and the allele size distribution of the FMR1 premutation and full mutation alleles a)Assessment of the severity of clinical involvement during early development for newborn probands with premutation and full mutation alleles b)To document the degree of clinical involvement of the wide variety of fragile X-related phenotypes in the extended family of the newborn proband identified by newborn screening. CGG repeat N 24 (GCC) n 5’5’ CGG repeat c f cf Lane 1 2 3 4 5 The CGG linker protocol allows the detection of expanded alleles in both males and females Consent forms and information sheets approved by IRBs and being presented to each mother delivering a baby at Rush (Chicago) and UC Davis (Sacramento) since October 1, 2008 Sacramento and Chicago: 2000 blood spots collected

64. Newborn Screening for FXS Need follow up for positives with early intervention and genetic counseling at FXS clinic Benefits – –in early intervention right away – –avoid long process of diagnosis, extra tests – –avoid second (or third) affected child – –get diagnosis for symptomatic premutation carriers through cascade analysis of family Problems – –what to do with asymptomatic premutation carriers – –no cure If new treatments give partial “cure” – newborn screening will be key (treatments may work better earlier) Pilot program (30,000 births) in California, Illinois

65. Pilot FXS Newborn Screening Will address epidemiology issues (eg. mutation frequencies) Will address developmental questions (natural history of development and early intervention in pre and full mutation carriers) Will address whether screening and diagnosis is helpful or not for families

66. FXCRC Registry and Database National Registry to find people who want to be involved in research and clinical trials – going up Fall 2009 Database on participants with FXS entered to answer important questions about the disorder – hopefully up Summer 2010 Enroll at any FXS Clinic – OHIO Clinic De-identified at national level – names of participants kept at Fragile X Clinics Can specify types of research can do Need to sign consent for each person entered For registry: one page form to fill out for each person – self and relative forms

67. Acknowledgements FRAXA Research Foundation NIH – NINDS Kiwanis Spastic Paralysis Foundation National Fragile X Foundation Collaborators – –Sue Ellen Krause PhD – –Sandy Block MS – –Steve Guter MS – –Ed Cook MD – –Randi Hagerman MD – –Maureen Leehey MD – –Paul Hagerman MD PhD – –Chris Goetz MD – –Don Bailey PhD – –Glenn Stebbins PhD – –Pete Van der Klish PhD Lab Research Associate – –Lili Zhou MD Study Co-ordinators – –Kristina Potanos – –Dahlia Weinberg – –Rebecca Lara – –Foster Lewin – –Allison Sumis – –Crystal Hervey Students – –Ok-Kyung Kim – –Chinton DeSai – –Ravi Iyengar – –Hazel Perry Statistics – –Sue Leurgans PhD – –Joanna Wuu MS Cortex Pharmaceuticals – –Steve Johnson PhD – –Roger Stoll PhD Seaside Pharmaceuticals – –Randy Carpenter PhD Study Co-ordinators off site – –Tristan Jardini – –Penelope Decle – –Jennifer Cogswell Steve Porges PhD Mina Johnson PhD Isabel Boutet PhD Steve Hooper PhD Ivan Jean Weiler PhD Bill Greenough PhD Ning Weng PhD Mark Bear PhD Emily Osterweil PhD FXS Families