Transcript RESEARCH UPDATE IN PHENYLKETONURIA

RESEARCH UPDATE IN
PHENYLKETONURIA
Dr. Maureen Cleary
Great Ormond Street Hospital NHS Trust
Blood-brain barrier studies in
PKU
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Blood-brain barrier studies in PKU
Large Neutral amino acids
Essential fatty acids supplementation
Biopterin treatment
Ammonia lyase
Gene therapy
Blood-brain barrier studies
Phenylketonuria
• Monitor metabolic control by blood phe
• Preferable to measure phe at site of action
(brain) rather than point of delivery (blood)
1H-Magnetic Resonance
Spectroscopy
• Nucleus is magnetic
– Magnetic field causes all the magnetic nuclei to align
themselves to the major axis of the field
• A second magnetic field is applied
– Nuclei tilted to a specific angle
• When field removed they re-align themselves to
the major axis of the magnetic field
Magnetic Resonance Spectroscopy
• capable of identifying different molecules
• Same nuclei eg protons experience different local
magnetic fields
• Give rise to different MR spectra
• Area under peak proportional to concentration
NAA
Cr
Cho
1H-Magnetic Resonance Spectroscopy
in PKU
• Non-invasive assessment of changes in brain
metabolism
• Initial reports measure N-Acetyl-Aspartate,
NA Choline, inositol, creatinine
1H-Magnetic Resonance
Spectroscopy in PKU
• Normal NAA, choline, creatinine
• Suggests no demyelination
PKU and Magnetic Resonance
Spectroscopy (MRS)
• Rabbit made hyperphe
• MRS detected ‘phe’ peak
• Intensity correlated with brain phe on
postmortem
• Correlated poorly with plasma phe
MRS and brain phe
• 1995
– Detection and quantitation methodology of
brain metabolites in patients with PKU
NAA
Cr
Cho
MRS: normal
NAA
phe
MRS: PKU
NAA
phe
Measurement of phe
• Present in relatively small quantities
– Cf NAA, choline
– Need to use ‘difference spectroscopy’
– i.e. subtract spectra from non PKU controls
MRS of brain phe studies in PKU
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17 PKU (mean age 25.8 yrs)
10 healthy controls (25.3)
Early treated
6 off diet, 3 protein restricted, 8 on aa supp
(stopped 2 weeks pre-scan)
‘steady state’
» (Rupp et al., 2001)
MRS results
• Control brain phe mean 0.05, sd 0.025
• Blood versus brain linear relationship
• Blood to brain phe: 4:1
• Measurement error 0.03mol/kg ww
Blood- brain relationship
Pietz et al.,(1999)
Magnetic Resonance Spectroscopy
Weglage et al., 1998
• two siblings aged 17 and 30 yrs early
treated
• R408W/R408W
• IQ’s 90 and 77
• oral load phe
• max brain phe 12-23 hrs post phe load
• sib I blood 2448: brain 642 (IQ 90)
• sib II blood 2316: brain 804 (IQ 77)
Magnetic Resonance Spectroscopy
Weglage et al., 1998
• 4 untreated adults
• two IQ unobtainable ages 34 and 28 yrs
• blood 1320,1211/ brain 650,670
• two IQ 100 and 105 ages 33 and 31 yrs
• blood 1200, 1210/ brain <200, <200
• Suggests
– Intervariability of brain phe
– Explains different outcomes
• Only really explains unusual patients
MRS blood:brain
• Pietz et al., (1999)
– linear blood: brain 4:1
• Moller et al.,(2000)
– saturated at higher phe levels
• Moats et al. (2000)
– ?? exponential
Blood-brain relationships
Pietz et al.,(1999)
Moller et al.,(2000)
Moats et al., (2000)
Large Neutral amino acids
LNAA and PKU
• Large neutral amino acids compete for entry
to brain with phenylalanine
Large neutral amino acids and
PKU
• Administer large quantities of LNAA and
reduce phe entry to the brain
Large Neutral AminoAcids
and PKU
• What is the evidence that it should work?
– Earlier studies (animals or functional
testing)
– Later studies (humans) using Magnetic
Resonance Spectroscopy
LNAA and PKU
• Cerebral protein synthesis reduced in
hyperphe state in rats
• Improves upon supplementing with LNAA
– Binek-Singer & Johnson, 1982
LNAA and PKU: effect of supplements
on brain amino acids in animals
• Rats phe hydroxylase inhibited
– Phe load
– Phe load + LNAA
– LNAA group had lower brain phe and similar
blood phe
• Andersen & Avins, 1976
LNAA and cerebral function in PKU
• Valine, isoleucine and leucine supplements
– Reduced brain and CSF phe in rats
• Six patients with pku improved neuropsych
performance whilst taking VIL
– Berry et al., 1985
LNAA, PKU and MRS
• Later studies using MRS in humans
• One study
• Pietz et al., (1999)
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Six adults
Loading with oral phe 100mg/kg
Loading with oral phe plus LNAA
EEG testing
LNAA, PKU and MRS: Pietz et
al. (1999)
• Rise in brain phe occurred after loading
• This rise blocked when LNAA taken with
phe load
• EEG spectra abnormalities not seen when
LNAA ingested
LNAA study
• Brain phe after oral phe load
– mean preload
– mean post load 6 hrs
– mean post load 12 hrs
252
344
377
• Brain phe after oral phe + LNAA
– mean preload
– mean post load 6 hrs
– mean post load 12 hrs
226
235
210
Further considerations
• Is MRS sufficiently robust tool for
intervention studies?
• What are the relationships between BB phe
entry and actual brain tissue phe levels?
LNAA, MRS and MOUSE
• PAHENU-2 mouse model
– 0.5g/kg or 1.0 g/kg PreKUnil
– Reduction in blood phe and brain phe
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– Spectroscopy on homogenized mouse brain
– BCAT activity increased on LNAA
(only two mice in each group)
• Matalon et al, (2003)
Conclusions
• MRS can define a peak which is markedly
elevated in individuals with PKU compared
to normal spectra
• MRS can show reduction in this peak when
interventions occur such as LNAA
application
• MRS can show some unusual individuals
who have low brain phe and are ‘protected’
Conclusions
• Blood:brain barrier relationship not clear
• Extent of inter-individual variability not
clear
• Safety of long term LNAA not proven
• To use the technique in dynamic studies
need clarity of these changes through the
day
Essential fatty acids in PKU
Essential fatty acids in PKU
• Diet low in animal protein
– low intake alpha-linolenic acid
– low docoshexanoic acid
– importance in brain cell membrane
• Infant aminoacid formulae can be
supplemented with PUFA’s
• Should children’s formulae also be
supplemented?
Essential fatty acids in PKU
• AA product supplemented with fatty acids
• Children had higher levels of DHA than
unsupplemented group
• Considered more palatable than
unsupplemented formula
PKU and biopterin
PKU
• Phenylalanine
Tyrosine
Biopterin metabolism
Biopterin responsive PKU
• Should we be treating some
patients with biopterin?
Role of biopterin in PKU
• Biopterin co-factor for phe hydroxylase
• Inborn errors of biopterin detected by PKU
screening programme
• On biopterin those patient usually no longer
need phe restriction
Biopterin in PKU
• Recent observation that biopterin may
benefit phe hydroxylase deficient patients
Biopterin in PKU
• Hyperphe rather than classical PKU
• Mutations with residual activity
• Frequently (but not exclusively) missense
mutations within the coding region for the
catalytic domain
Biopterin and PKU
• Suggest loading test in all patients
• However newborn failed loading test
patients have subsequently been found to be
responsive
Biopterin and pku
• Cost of diet v. cost of biopterin
• Who would benefit?
• Does it benefit those with severe PKU?
• Is it safe in pregnancy?
• Trial later this year 2004
Alternative therapies
• Ammonia lyase therapy
• Recombinant phenylalanine ammonia lyase
• converts phe to trans-cinnamic acid in the
gut
• reduces plasma phe by approx 50% in PKU
mouse
New therapies
• Ammonia lyase treatment
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may be useful
needs further studies to test safety
may still need some diet
may be many years before available
PKU and Gene therapy
Alternative therapies
• GENE THERAPY
– Adv/RSV-hPAH infused into portal vein of
PAHenu2 mice
– phe levels normalised with sufficient dose
– comparable to 10-20% enzyme activity
– successful only in short term
– could not be duplicated due to immune
response to vector
Conclusions
• Research is fairly active in PKU
• Biopterin trial will find some individuals
with milder PKU who may benefit form
Biopterin treatment
• Enzyme treatment is underway