Research Progress in Fragile X Syndrome – Brain Function to New Ideas for Treatment

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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
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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 14000 males and females
• Carriers 1100-250 females, 1250-800 males
• All ethnic groups worldwide

MOST COMMON KNOWN INHERITED FORM OF COGNITIVE
DISABILITY
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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

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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

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Fragile X Syndrome Characteristic cognitive
pattern with prominent executive function deficits

• Weaknesses
• Auditory processing
• Sequencing
• Abstraction
• Short-term memory
• Topic maintenance/ "connectedness"
• Mathematics
• Working memory
• Coordination/praxis

• Strengths
• Receptive vocabulary
• Syntax
• Imitation
• Grammaticalstructure
• Visual memory
• Simultaneous processing
• Experiential learning

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Why is Math so hard in FXS?
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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

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FXS Developmental Problems

• Parents most frequently become concerned first
  about speech delay
• Other concerns that may lead to childs 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 dont recognize
  magnitude of problem or doctors are discouraging
  testing

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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)
• Miss early intervention
• New NFXF/FXCRC goal diagnosis before age 2
  working on Pediatricians

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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

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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

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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)

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FXS Socialization Deficits
Friendly but social anxiety

• 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

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FXS - Female Affecteds

• More mildly involved
• Average IQ 80
• NVLD, VIQgtPIQ, 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

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FXS Treatment in Clinic - Supportive
Rush FXS Clinic since 1992 gt 400 patients

• 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

• 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

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Psychopharmacology in Fragile X Syndrome

• Targets behavior to improve functioning
• Supportive, does not target underlying cognitive
  problem
• Only one prior controlled trial in FXS (N15)
  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

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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

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FXS - Types of Medications and Indications
meds may be targeting several clusters
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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

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Abilify High Response Rate in FXS (2004-2007)
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Supportive Psychopharmacology is Helpful in FXS
Rush Fragile X Clinic

Berry-Kravis and Sumis, 2006
but treating the underlying disorder would be
better
208 trials 136 patients
231 trials 123 patients
52 trials 52 patients
100 trials 58 patients
Anxiety Mood
Attention Hyperactivity
Aggression Irritability
Hyperarousal Oversensitivity
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FMR1 The Fragile X Gene
Carrier state passed through many generations
before FXS
Simple DNA test to diagnose FXS measures repeat
size
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FMRP Expression and Disability
100
Social anxiety/shyness
Distractibility/hyperactivity Executive deficits
Spatial perceptual deficits
FMRP
NVLD
Intellectual Disability
0
Disability
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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?

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Both FXS Mouse and Human BrainsDendritic Spines
are Abnormal in FXS with Immature Long Spines at
Brian Connections
FMRP regulates maturation of brain synapses
(connections)
McKinney et al, AJMG -B Neuropsychiatric
Genetics, 2005
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FMRP in Dendrites (Brain Connections)
FMRP in RED in tip of neural dendrites and where
processes are forming

• Antar et al, J. Neurosci 2004

FMRP regulates proteins made at brain connections
(in dendrites) - proteins have to be made in
right amount for connection to mature and work
right.
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Excessive LTD due to mGluR system overactivity
How loss of protein regulation causes weak
connections in FXS
AMPA
LTD
AMPA
AMPA
AMPA
FMRP
AMPA
AMPA
FMRP
Dendrite
Normal Mature connection
Fragile X Immature connection (too weak)
mGluR Theory of Fragile X
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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

Normal
Fragile X
Drug
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4
3
AMPA
AMPA
Other systems
2
X
X
1
AMPA
AMPA
Excessive LTD due to mGluR system overactivity
Potential Mechanisms for New Treatments of FXS
Dendrite
Fragile X Immature connection (too weak)
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Targeted Treatments we have tried
Stronger connection
Excessive LTD
AMPA
AMPA
Activate AMPA receptors Raise BDNF bring AMPA
receptors to synapse
AMPA
AMPA
AMPA
AMPA
Ampakine Mechanism 3
Fragile X Immature connection (too weak)
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Excessive LTD
Stronger connection
AMPA
AMPA
Lithium blocks PI turnover, reduce mGluR
signalling through PL-C/PK-C cascade, directly
block protein synthesis by GSK3B block
Li
AMPA
AMPA
AMPA
AMPA
Lithium Mechanism 1
Fragile X Immature connection (too weak)
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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
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FXS Open-Label Lithium Trial

• Test concept of mGluR pathway inhibition
• 13 better at 2 mo, minimal toxicity

1 yr
RBANS List Learning (range 0-40) 8 of 10
improved, p 0.028 CX516 placebo change 0.0
ABC Total (range 0-174) 13 improved,
p0.005 Placebo change in CX516 study (N25) -4
Also significant improvement in CGI, VAS, and
Vineland Personal Daily Living Skills and
Maladaptive Behavior
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Lithium Study Biomarker - ERK Activation in
Lymphocytes
ERK phosphorylation slower in K/O and FXS
Berry-Kravis et al, JDBP 2008
N11 Baseline mean 4.87 min 2 Month Treatment
mean 4.11 min (p0.007) (about 1 min is
difference FXS and control) 1 Year Treatment
mean 3.56 min (P0.00006)
baseline
2 mo
1 yr
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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

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R-Baclofen and the mGluR Theory Decrease
Excessive Glutamatergic Transmission
mGluR5
GABA-B
Proteins
mGluR5
Pre-synaptic
Post -synaptic
Mechanism 4
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Clinical Trial of R-Baclofen (STX209) in FXS

• Started in December
• Goal is to show improves tantrums/aggression/agita
  tion/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

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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

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Stronger connection
Excessive LTD
AMPA
AMPA
mGluR5 blocker Fenobam
Li
AMPA
AMPA
AMPA
AMPA
mGluR5 Blocker Mechanism 1
Fragile X Immature connection (too weak)
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AMPA Receptor Rescue by MPEP
Internal AMPA
Surface AMPA
Nakamoto et al. 2007
FMRP
Control
GluR1 AMPA receptors that drive strength of
neural connections
FMRP Absent
FMRP Absent and MPEP
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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
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ALL ABNORMAL Phenotypes in FXS Mouse Normalized
by MPEP (or Other mGluR5 Blockers)
Bauschwitz, 2006

• Audiogenic seizures
• Epileptiform bursts
• Open field behavior
• Prepulse inhibition
• Dendritic spine shape
• AMPA receptor internalization
• Excessive protein synthesis

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
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MPEP Controls Audiogenic Seizures in FXS Mouse
WE CAN CURE THE FXS MOUSE!
Video courtesy of R. Bauschwitz PhD
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But Mouse is not Man
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human
Human brain a lot more wiring time
mouse
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mGluR5 Negative Modulators in Development for FXS

• Fenobam
• 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

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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, p0.03)
• Observation of positive behavioral changes in
  9/12 subjects
• No fenobam-related adverse events

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Prepulse Inhibition (PPI) Studies
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Hessl et al. AJMG 2008
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PPI improved on fenobam
Berry-Kravis et al. JMG 2009
P0.03
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mGluR5 Negative Modulators in Development for FXS

• 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)

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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

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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
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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

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New Measures of FunctionOutcome Measures for
Clinical Trials
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KiTAP Test
Alertness
- Many subtests reliable in test-retest will
use in Roche study - Correlates with attention
and hyperactivity measures - Crosses all levels
Go-no Go
Flexibility
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Eye Tracker as New Outcome Measure
Validated this summer with test-retest to be
ready for Roche mGluR trial in Fall measures
eye contact
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NIH Study Shows Increased Rates of Cerebral
Protein Synthesis in Fragile X Knockout Mice
Qin et al (2005) J Neurosci 255087.
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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

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Testing for FXS Modifiers and Biomarkers
Many proteins in this pathway may be implicated
in autism
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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
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Newborn Screening for Fragile X Comes to 7th Grade
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• 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
• Assessment of the severity of clinical
  involvement during early development for newborn
  probands with premutation and full mutation
  alleles
• 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.

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
N24
5
c
f
(GCC)n
CGG repeat
c
f
CGG repeat
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Newborn Screening for FXS
Pilot program (30,000 births) in California,
Illinois

• 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)

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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

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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

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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

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