RESEARCH UPDATE IN PHENYLKETONURIA

Dr. Maureen Cleary

Great Ormond Street Hospital NHS Trust

Blood-brain barrier studies in PKU

• 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

phe

NAA

MRS: PKU

NAAphe

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

• 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)– 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 252– mean post load 6 hrs 344– mean post load 12 hrs 377

• Brain phe after oral phe + LNAA– mean preload 226– mean post load 6 hrs 235– mean post load 12 hrs 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

– 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– 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