Transcript Diet

New therapies in PKU
Gábor Rácz, MD, PhD
University of Szeged
Department of Pediatrics
Budapest
2010
Classical treatment
• Golden standard in PKU treatment: low Phe diet
Introduced as early as possible and continued as long as possible, preferably life-long.
• Enhanced new amino acid formulas
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better taste
better forms, ready to use formulas
better composition with LCPUFA, minerals, vitamins, prebiotics
• Low protein containing foods
Problems
Dietary therapy for HPA/PKU is a successful but difficult treatment. The constant
adherence to a restricted diet often leads to reduced compliance in adolescence and
beyond.
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Despite the remarkable success of public health programs that have instituted newborn
screening and early introduction of dietary therapy for PKU, there is a growing body of
evidence that suggests that neurocognitive, psychosocial, quality of life, growth, nutrition,
brain and bone pathology and maternal PKU outcomes are suboptimal. [Enns et al. 2010]
Meta-analysis: the majority of publications (140/150) that contained primary outcome data
presented at least one suboptimal outcome compared to control groups or standardized
norms/reference values in at least one of the following areas:
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neurocognitive/psychosocial (58/60)
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quality of life (4/6)
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brain pathology (3/32)
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growth/nutrition (29/34)
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bone pathology (9/9)
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maternal PKU (19/19)
Why new therapies?
Development of alternate therapies that would
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permit liberalization of dietary restrictions and
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simplify disease management
would greatly improve treatment of HPA/PKU
and the quality of life for the patients.
Targets
Diet:
Phe-free special AA mixture
Low-protein medical food
Natural low Phe proteins
Brain (blood):
Enzymatic breakdown
Large neutral amino acids
Liver:
intestinal
vascular
lumen
PAL
Digestive tract:
Enzymatic breakdown
Large neutral amino acids
LAT
BBB
cell
Targets
Diet:
Phe-free special AA mixture
Low-protein medical food
Natural low Phe proteins
Brain (blood):
Enzymatic breakdown
Large neutral amino acids
Liver:
PAH gene therapy
PAH enzyme
BH4 cofactor
chaperone
Digestive tract:
Enzymatic breakdown
Large neutral amino acids
Diet: Glycomacropeptides
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Only known natural protein (cheese whey) that is almost free of Phe – and Tyr, Trp.
Commercially available contains approximately 4 mg Phe/g GMP.
Somewhat better odor, taste compared to non-GMP special dietary products.
Mol Genet Metab. 2007; 92(1-2): 176–178.
Diet: Designer milk
• Transgenic rabbits were created to express low-phenylalanine
κ-casein in their milk [Baranyi 2007]
• Remodelling bovine milk: knock-out β-lactoglobulin, mutate Phe
residues in α-lactalbumin, modify PUFA content,... [Sabikhi 2004]
• Hi-tech milk processing (GMP) may be more acceptable to consumers
than transgenesis for altering milk composition.
Large neutral acid (LNAA) supplementation
• Phe could interfere with the transport of other LNAAs to the brain [Christensen 1953]
• Therapeutic effects due to:
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Competition with Phe for transport across
• intestine (and kidney) through LAT2 [Matalon 2006]
• brain, BBB through LAT1 (especially high affinity to Phe) [Andersen 1976]
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21% reduction in Phe level in CSF [Berry 1982]
Significant reduction (30-50%) of plasma and brain Phe levels.
Reduction in the influx of Phe into the brain what promotes proteins synthesis
[Pietz 1999]
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With higher Tyr and Trp concentrations reduction in behavioral disturbances and
depression usually attributes to diminished dopamine and serotinine brain levels
[Güttler 1986]
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Better production of myeline by oligodendrocytes.
LNAA had a positive effext on specific psychometric test (frontal lobe function)
[Schindler 2007]
Large neutral acid (LNAA) supplementation
• LNAA supplementation as a method of PKU treatment for
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adults over the age of 18 years
adolescense with poor dietary adherence
patients with untreated/late diagnosed PKU
NOT recommended for children and pregnanty woman with PKU
25-30% of daily protein from LNAAs
70-75% of daily protein from natural low-Phe products (vs 20% w/o LNAA)
Phe levels up to 1.5 mmol/l is allowed
60 kg 10 tbls 3× daily
There is still need for more long-term studies evaluating efficacy and safety of
LNAAs therapy.
LNAA medical foods
L-Tyrosine, L-Leucine, L-Tryptophan,
L-Isoleucine, L-Valine, L-Lysine ,
Artificial Lemon Flavoring, LThreonine, L-Arginine, L-Histidine, LMethionine
Lanaflex
NeoPhe
NeoPhe powder
PreKUnil
PheBLOC
dose
0.8 g/kg
1 tbl/kg
0.5 g/kg
0.5 tbl/kg
20% protein
needs
vitamines /
minerals
complete
not complete
not complete
not complete
not complete
Diet relaxation with LNAA
unlimited
dairy
meat
fish
nuts
bread, pasta
rice
potatoes, corn, peas
green leafy vegetables
fruits, juices
potato chips
chocolate
cookies
butter
partially restricted
very restricted
totally restricted
Enzyme substitution: PAL
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Phenylalanine-ammonia-lyase: enzyme from plants, yeast or bacteria,
involved in Phe degradation
PAL
+ NH3
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PEGylated Anabaena variabilis PAL to increase the activity and mask PAL from host
immune system
Tyr remains essential
PAL: preclinical studies
Mouse studies [Sarkissian 2008]:
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Plasma Phe profile of ENU2 mice during short-term 12-day
study with 3 sc bolus injections (on days 1, 4, and 8)
Pahenu2/enu2 PKU mouse model
PAL reduced Phe levels in brain tissue
and vascular space.
Reduced clinical manifestation of PKU,
including hypopigmentation.
The most therapeutically effective PAL
(Av, Anabaena variabilis) species was
one without the highest specific activity,
but with the highest stability; indicating
the importance of protein stability in the
development of effective protein
therapeutics.
Proc Natl Acad Sci U S A. 2008 December 30; 105(52): 20894–20899.
PAL: clinical studies
Phase 1 clinical study (Biomarin)
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Substantial blood Phe level reductions in the range of 36% to 97% (mean of 62%)
were observed in all patients at 0.1 mg/kg dose with a mean baseline blood Phe level
of 1113 umol/L. No notable blood Phe level reductions were observed by lower
dosing (0.001 to 0.03 mg/kg).
No serious immune reactions, including hives or anaphylactoid reactions, were
observed.
Seven out of 25 patients developed late mild to moderate injection-site reactions, of
which two also developed skin rashes without other symptoms.
http://www.bmrn.com/pipeline/peg-pal-for-pku.php
Tetrahydrobiopterin (BH4)
• Catalytic cofactor essential for the conversion of Phe to Tyr by the
phenylalanine-hydroxylase.
• Essential for the treatment of the BH4-dependent PKU.
• BH4 can completely reverse or reduce HPA in BH4-responsive
patients with PAH deficiency.
• How?
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mutation prevents PAH from folding correctly, reducing its activity
→ increased BH4 may facilitate folding by making chemical
environment more favourable
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mutation changes the shape of the BH4 binding site
→ increased BH4 increases the probability of binding even though the
site is abnormal
BH4 responsiveness
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BH4 resonsiveness is determined by the
allelic combination at the PAH locus.
The intesity of the response to BH4
therapy correlates with the residual
enzyme activity of the mutations.
Reduction in plasma Phe levels ≥30%
in 20-56% patients with (mild) HPA
Even some classical PKU patients may
show some response.
Blau, N. and H. Erlandsen (2004) Mol Genet Metab. 2004 Jun;82(2):101-11
BH4 responsiveness
BH4 treatment indication
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European Union: older than 4 years of age who have been shown to be
responsive to such treatment
United States: to reduce blood Phe levels in patients with HPA due to
BH4-sensitive PKU in conjuction with a Phe-restricted diet
BH4 benefits
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Patients are able to lower blood Phe level without additional diet restrictions when
off diet or relaxed diet.
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Increase Phe tolerance and still maintain control.
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Maintain more consistent control when Phe levels fluctuate greatly.
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But most of the patients still need at least some restriction of natural protein and
continue to use of low-Phe food.
Still many unsolved questions BH4 with Phe-restricted diet in prevention of
maternal PKU.
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A 29-year-old woman with R408W/F39L mutation, the blood
phenylalanine control was easily accomplished during the pregnancy. The
lack of nausea and vomiting during the first trimester suggests that the
occurrence of CHD in babies born to women with PKU may be reduced
with BH4. [Koch 2005]
Chaperone therapy
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PKU as misfolding disease, caused by mutations the impair correct protein folding
leads to loss of function due to degradation of mutant protein.
Chemical chaperones: cellular osmolytes glycerol, DMSO, glycine betaine,
deuterated water.
Pharmacological chaperones: strucure-directed small molecules that
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stabilize the wild-type PAH and PKU mutants
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and/or facilitate the folding of nonnative intermediate states toward the
native conformation
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by thermal up-shift of half denaturing temperature
3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one stabilizes
PAH, TH, TPH [Calvo 2008]
Pharmacological chaperones
Representative thermal denaturation profiles of WT-PAH
Effect of the hit compounds III and IV on PAH activity (A)
and protein levels (B) after 12-day administration of 5
mg/kg/d to WT C57BL/6 mice
J Clin Invest. 2008 August 1; 118(8): 2858–2867.
Enzyme replacement
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Phenylalanine-hydroxylase
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very complicated structure
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expressed only in liver
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necessity of full set of enzymes
to maintain BH4
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only 10% of PAH activity is
necessary for normal Phe
metabolism in mice [Ledley 1985]
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Orthotopic liver transplantation [Vajro 1993]
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Therapeutic uses of microencapsulated genetically engineered cells [Chang 1998]
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Hepatocyte transplantation [Harding 2010]
Gene therapy
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Discovery of PAH gene [Kwok 1985]
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Chromosome 12, 13 exons, 452 amino acids (27 Phe)
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Experimental protocols emerged to treat PKU with gene therapy
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Fatal systemic inflammatory response in OTC deficient patient following adenoviral gene
transfer in safety study [Raper 2003]
Complete and persistent phenotypic correction of phenylketonuria in mice by site-specific
genome integration of murine phenylalanine hydroxylase cDNA [Chen 2005]
PAH-based fusion proteins with delivery moieties based on the HIV-transactivator of
transcription peptide, and fragments of human hepatocyte growth factor aiming to specifically
target PAH to the liver [Eavri 2007]
Portal injection of recombinant rAAV [Harding 2006]
Muscles as target for gene therapy
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large total mass, high degree of vascularization
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stable environment for protein expression by persistent postmitotic nuclei, easy
accessibility
Gene therapy
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Long term corrections of murine HPA with the administration of rAAV that encode
the complete enzyem system necessary for BH4 synthesis and recycle - no adverse
reactions, therapeutic effect lasted for 40 weeks [Ding 2008]