Classical treatment Golden standard in PKU treatment low

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 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. 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: • neurocognitive/psychosocial (58/60) • quality of life (4/6) • brain pathology (3/32) • growth/nutrition (29/34) • bone pathology (9/9) • maternal PKU (19/19)

Why new therapies? Development of alternate therapies that would permit liberalization of dietary restrictions and 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: Digestive tract: Enzymatic breakdown Large neutral amino acids intestinal vascular lumen PAL 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 BH 4 cofactor chaperone Digestive tract: Enzymatic breakdown Large neutral amino acids

Diet: Glycomacropeptides • • • 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: Competition with Phe for transport across • intestine (and kidney) through LAT 2 [Matalon 2006] • brain, BBB through LAT 1 (especially high affinity to Phe) [Andersen 1976] 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] With higher Tyr and Trp concentrations reduction in behavioral disturbances and depression usually attributes to diminished dopamine and serotinine brain levels [Güttler 1986] 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 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 Neo. Phe powder Pre. KUnil Phe. BLOC dose 0. 8 g/kg 1 tbl/kg 0. 5 g/kg 0. 5 tbl/kg 20% protein needs vitamines / minerals 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 Phenylalanine-ammonia-lyase: enzyme from plants, yeast or bacteria, involved in Phe degradation PAL + NH 3 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]: Plasma Phe profile of ENU 2 mice during short-term 12 -day study with 3 sc bolus injections (on days 1, 4, and 8) Pahenu 2/enu 2 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) 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 (BH 4) • Catalytic cofactor essential for the conversion of Phe to Tyr by the phenylalanine-hydroxylase. • Essential for the treatment of the BH 4 -dependent PKU. • BH 4 can completely reverse or reduce HPA in BH 4 -responsive patients with PAH deficiency. • How? mutation prevents PAH from folding correctly, reducing its activity → increased BH 4 may facilitate folding by making chemical environment more favourable mutation changes the shape of the BH 4 binding site → increased BH 4 increases the probability of binding even though the site is abnormal

BH 4 responsiveness BH 4 resonsiveness is determined by the allelic combination at the PAH locus. The intesity of the response to BH 4 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

BH 4 responsiveness

BH 4 treatment indication 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 BH 4 -sensitive PKU in conjuction with a Phe-restricted diet

BH 4 benefits Patients are able to lower blood Phe level without additional diet restrictions when off diet or relaxed diet. Increase Phe tolerance and still maintain control. Maintain more consistent control when Phe levels fluctuate greatly. 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 BH 4 with Phe-restricted diet in prevention of maternal PKU. – A 29 -year-old woman with R 408 W/F 39 L 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 BH 4. [Koch 2005]

Chaperone therapy 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 – stabilize the wild-type PAH and PKU mutants – and/or facilitate the folding of nonnative intermediate states toward the native conformation – by thermal up-shift of half denaturing temperature 3 -amino-2 -benzyl-7 -nitro-4 -(2 -quinolyl)-1, 2 -dihydroisoquinolin-1 -one stabilizes PAH, 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 C 57 BL/6 mice J Clin Invest. 2008 August 1; 118(8): 2858– 2867.

Enzyme replacement Phenylalanine-hydroxylase – very complicated structure – expressed only in liver – necessity of full set of enzymes to maintain BH 4 – only 10% of PAH activity is necessary for normal Phe metabolism in mice [Ledley 1985] Orthotopic liver transplantation [Vajro 1993] Therapeutic uses of microencapsulated genetically engineered cells [Chang 1998] Hepatocyte transplantation [Harding 2010]

Gene therapy Discovery of PAH gene [Kwok 1985] Chromosome 12, 13 exons, 452 amino acids (27 Phe) Experimental protocols emerged to treat PKU with gene therapy 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 c. DNA [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 r. AAV [Harding 2006] Muscles as target for gene therapy – large total mass, high degree of vascularization – stable environment for protein expression by persistent postmitotic nuclei, easy accessibility

Gene therapy Long term corrections of murine HPA with the administration of r. AAV that encode the complete enzyem system necessary for BH 4 synthesis and recycle - no adverse reactions, therapeutic effect lasted for 40 weeks [Ding 2008]