Phenylketonuria: Complex results from a Simple metabolic block

PKU: A Disorder of Amino Acid Catabolism PKU: A Disorder of Amino Acid Catabolism Degrade Phe by Phenylalanine Ammonia LyaseDegrade Phe by Phenylalanine Ammonia Lyase

Protein Synthesis

Protein Synthesis

Catabolism

FurtherMetabolism

Missing/Alteredin PKUPhenylalanine Tyrosine

L-dopa

PKU

Fumarate +Acetoacetate

PAH

PAL

t-Cinnamic Acid + NH3

Phenylketonuria – Points of EmphasisPhenylketonuria – Points of Emphasis

1. PKU is a common autosomal recessive disorder

a. ~1/16000 births in US (350 – 500 new cases / year). There are at least 16,000 patients (pediatric – under care); another 30-40,000 PKU adults have irregular medical care.

b. Almost all PKU mutations result from defects in PAH. Most of these (~2/3) are missense mutants; ~1/3 are null. This presents a unique problem for therapy. A few PKU patients (1-2%) have defects in BH4 metabolism rather than PAH.

2. Maternal PKU is an increasing problem, as adhering to a strict diet to maintain Phe levels at NORMAL physiological levels during pregnancy can be difficult for many women.

3. Other therapeutic modalities are needed to replace, or as an adjunct to diet.

Maternal PKU – Severe mental retardation, Maternal PKU – Severe mental retardation, microcephaly, cardiac abnormalitiesmicrocephaly, cardiac abnormalities

Plasma Phe in Pregnant PKU Mothers at the Plasma Phe in Pregnant PKU Mothers at the University of Florida College of MedicineUniversity of Florida College of Medicine

Group 1, excellent control, 4 normal infants (100%); Group 2, marginal control, 6 normal* infants (60%), 4 affected, 1 no data; Group 3, inadequate control, 3 normal* infants (30%), 7 affected, 4 no data.

Outcomes: Affected Infants of Pregnant PKU Outcomes: Affected Infants of Pregnant PKU Mothers at the UF College of MedicineMothers at the UF College of Medicine

Group 2 Pregnancies Group 3 Pregnancies

1 2 3 4 5 6 7 8 9 10 11Microcephalic X X X X X X X X XLow Birth Weight X X X X X XCardiac Defect X XDevelomental Delay X X X XPremature Birth X XOther 1 2

Diagnostic criteria: Microcephalic, head circumference less than 3rd percentile for length and weight; Low Birth Weight, less than 5th percentile for gestational age; Cardiac Defect, clinical report; Developmental Delay, parental report; Premature Birth, 36 weeks or earlier; 1, parental reported behavioral problems; 2, Lobar Holoprosencephaly, clinical report. Group 1, excellent control, 4 normal infants (100%); Group 2, marginal control, 6 normal* infants (60%), 1 no data; Group 3, inadequate control, 3 normal* infants (30%), 4 no data.

I.I. Chemical mutagenesis and blind screening to identify BTBR mice Chemical mutagenesis and blind screening to identify BTBR mice with elevated serum phenylalanine levels. The F263S missense with elevated serum phenylalanine levels. The F263S missense mutation resembles human mutations.mutation resembles human mutations.

II.II. Serum Phe levels in PahSerum Phe levels in Pahenu2enu2 mice are similar to human PKU patients. mice are similar to human PKU patients.

III.III. Phenotypic similarities include mental deficits, mild ataxia, defects Phenotypic similarities include mental deficits, mild ataxia, defects in melanin biosynthesis, and reduced fertility.in melanin biosynthesis, and reduced fertility.

IV.IV. Female PahFemale Pahenu2 -/- mice exhibit a maternal PKU syndrome that can be -/- mice exhibit a maternal PKU syndrome that can be corrected by a Phe-restricted diet. corrected by a Phe-restricted diet.

The The PahPahenu2enu2 Mouse Model Mouse Model

PahPahenu2enu2 PKU and Heterozygote Mice PKU and Heterozygote Mice

Phenylalanine Ammonia Lyase (rAvPAL-PEG) Phenylalanine Ammonia Lyase (rAvPAL-PEG) Enzyme Substitution TherapyEnzyme Substitution Therapy

1.9 Angstrom resolution structure of the Anabaena variabilis tetramer. The red and blue ball structures show the four MIO prosthetic groups (PDB: 2NYN).

Phenylalanine Ammonia Lyase Structure

Phenylalanine ammonia lyase (PAL) converts Phe to ammonia and trans-cinnamic acid, which is readily excreted. The reaction mechanism is not completely understood but uses 3,5- dihydro-5-methylidene-4H-imidazol-4-one (MIO), formed by cyclization and dehydration of amino acid residues Ala-Ser-Gly.

Long-term rAvPAL-PEG Treatment of Male BTBR Long-term rAvPAL-PEG Treatment of Male BTBR PahPahenu2enu2 Mice Mice

Weeks

Ph

e L

ev

el,

mM

Ph

e L

ev

el,

mM

We

igh

t, g

ram

s

Brain Histology

Long-term rAvPAL-PEG Treatment of Female Long-term rAvPAL-PEG Treatment of Female PahPahenu2enu2 micemice

Possible Explanations – Female Non-responsePossible Explanations – Female Non-response

Serum Phe (mM)

BTBR Pahenu2 -/- Males 1.22 + 0.16

BTBR Pahenu2 -/- Females 1.72 + 0.21

BTBR Pahenu2 +/- 0.13 + 0.03

BTBR +/+ 0.10 + 0.03

Female Pahenu2 mice are more severely affected than males, perhaps increasing the requirement for functional, vector-delivered PAH protein. We are examining human patients for similar differences (IRB approved survey). This difference is clearly hormone related, but mechanism obscure.

12-16 week old Pahenu2 mice, 12 each sex

Coat Color Changes in Female Coat Color Changes in Female PahPahenu2 enu2 MiceMice

Pre-doseDay 6

Day 18 Day 46

Images of female BB375381 at indicated intervals. Dose of rAvPAL-PEG was biweekly 40 mg/kg.

Genotype

Wild type Het PKU

iNO

S c

ell c

ount

0

100

200

300

400

500

Activated Microglial Cell Infiltrates in Activated Microglial Cell Infiltrates in Female Female PahPahenu2enu2 Mice Mice

+/+ (Wild-type) BTBR +/- (Heterozygote) Pahenu2 -/- (PKU) Pahenu2

Microglial cells. iNOS immunoperoxidase stain, 40xBrain section H&E stain,

4x

Time (days)

1 8 15 22 29 36 43 50 57 64 71

iNO

S c

ell c

ount

0

100

200

300

400

500

600

Days post administration week 21: 2 inj/wk

Microglial Cell Infiltrates in PAL-Treated, and Microglial Cell Infiltrates in PAL-Treated, and Discontinued PAL-Treated Discontinued PAL-Treated PahPahenu2enu2 Mice Mice

3 Days post PAL 31 Days post PAL 42 Days post PAL

72 Days post PAL

Plasma Phe Levels in Female Plasma Phe Levels in Female PahPahenu2enu2 BB375381 BB375381

Correction of Maternal PKU with Correction of Maternal PKU with rAvPAL-PEGrAvPAL-PEG

Day 101 – Date of Birth

Female (BB375381) mated with an untreated Pahenu2 male

Day 110 – 9 Days pp Weight gain equal to PKU pups from a -/- x +/- mating.

However:5 females => 8 pregnancies 2 litters => 3 and 5 pups 6 neonatal death

More Successful Pregnancies with Modified 3x/week More Successful Pregnancies with Modified 3x/week PAL Treatment SchedulePAL Treatment Schedule

Better: 5 females, 10 pregnancies but only 4 successful (29 pups). WHY?

8 am Mon 4 pm Wed 4 pm Fri 10 mg/kg PAL

Plasma Phe levels, 10 mg/kg, 3x / Week PAL and Plasma Phe levels, 10 mg/kg, 3x / Week PAL and ADDITIONAL Blood Sample TimesADDITIONAL Blood Sample Times

Better: 5 females, 10 pregnancies but only 4 successful (29 pups). Speculate that inter-day plasma Phe levels are too high and perhaps also too low?

8 am Mon 4 pm Wed 4 pm Fri 10 mg/kg PAL

Dietary Phe Intake Dramatically Affects Plasma Phe Dietary Phe Intake Dramatically Affects Plasma Phe levels! 3x/week PALlevels! 3x/week PAL

Actual DataPKU Females

Simulated Daily Variation Superimposed On Actual Data from PAL-treated PKU Female MIce

Not perfect, but much better: 12 females, 29 pregnancies, 15 successful with 83 pups).

rAV-PAL Treated (6 mg/kg Daily Dose) Female PKU Mice. rAV-PAL Treated (6 mg/kg Daily Dose) Female PKU Mice. Phe Levels Throughout Time of DayPhe Levels Throughout Time of Day

Pathophysiology of PKU and Effects of PALPathophysiology of PKU and Effects of PAL

• Why are high levels of phenylalanine (Phe) teratogenic to brain?– Small brain size (PKU 80% of normal size)– Abnormalities of white matter, hypomyelination– Severe mental retardation

• Possible causes– Depressed glutamatergic synaptic transmission (Martynyuk,

Laipis, et al., 2005)– Microglial infiltration and reductions in dopaminergic cell

body density (Embury et al., 2007)– Reduced neurotransmitter synthesis (Pascucci et al., 2008)– Reduced cerebral protein synthesis (Wall & Pardridge, 1990;

de Groot et al., 2010)

Protein and mRNA Levels in Wild-type and PKU Protein and mRNA Levels in Wild-type and PKU BrainBrain

+/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- +/+ -/- Ctx Hip Str MBr BrSt Cbl

Myelin BasicProtein

Proteo-LipidProtein

P75

GAPdH

Neuro-FilamentMedium +/+ -/-

+/+ -/-

Neuro-FilamentLight

Microtubules and Post-translational Modification of Microtubules and Post-translational Modification of TubulinTubulin

Tubulin subunits are post-translationally modified by cleavage and the addition of a tyrosine residue.

This process is dynamic, and the tyrosination and detyrosination steps involve specific enzymes, aTubulin tyrosine ligase (TTL) and a tyrosine-carboxypeptidase.

In general detyrosinated microtubules are stable and tyrosinated microtubules are dynamic.

In neurons the tyrosination/detyrosination cycle may function to localize motor proteins, MAPs, and plus-end tracking proteins to specific cell locations (Hammond et al., Current Opinion in Cell Biology, 2008).

The tyrosination/detyrosination cycle is essential for normal brain development. The TTL-KO mouse dies perinatally with abberant neuronal network organization (Erck et al., PNAS, 2005).

Purification of Tubulin from Brain TissuePurification of Tubulin from Brain Tissue

Frozen brain tissue

Weigh and homogenize

High speed

Supernatant (1) Pellet - discard

Assemble MTs, 37°C plus paclitaxel

10% sucrose cushion

Supernatant (2)- discard

Pellet (3) Crude MTs

Cold depolymerize

Pellet (5) - discard

Low speed

Low speed

Supernatant (4)

Assemble MTs, 37°C plus paclitaxel

Supernatant (7) - discard

Pellet (6) MTs

Low speed

Resuspend MTs and digest with Carboxypeptidase A

kDa

200

116

97.4

66.2

45

31

MW 1 2 3 4 5 6 7 8

HPLC C18 reverse-phase column

“Experimental Phenylketonuria: Replacement of Carboxyl Terminal Tyrosine by Phenylalanine in Infant Rat Brain Tubulin.” J.A. Rodriquez and G.G. Borisy, Science 206, 463-465, 1979

Deproteinize, derivatize amino acids,

HPLC Determination of Mouse Brain Tubulin C-HPLC Determination of Mouse Brain Tubulin C-Terminal Modification by Tyr and PheTerminal Modification by Tyr and Phe

Wild type.

Heterozygote

PKU

Tyrosine and phenylalanine in brain tubulinC-terminal amino acids cleaved by carboxypeptidase A.

Genotype Tyrosine Phenylalanine S.D.

Wild type 86.6 13.4 5.4Heterozygote 94.7 5.3 0.07PKU 51.0 49.0 4.4

Amino acids as % of total. Values are the means of three separate determinations, three mice per genotype per prep, error bars = standard deviation.

Standard Curve: Blue – 31.25 µM; Green – 62.5 µM; Red – 125 µM; Black – 250 µM

Phe and Tyr on the C-terminal end of HELA Tubulin Phe and Tyr on the C-terminal end of HELA Tubulin is Dynamicis Dynamic

Tyrosine and phenylalanine as the carboxyl-terminal amino acid of HeLa cell tubulin. Purified tubulin cleaved by carboxypeptidase A.

Treatment Tyrosine Phenylalanine S.D.No added Phe 84 16 11.51 mM Phe 60 40 0.982 mM Phe* 58.4 41.6 n/a1 mM Phe/washout* 83.3 16.7 n/a

(Amino acids as % of total). Values are the means of two separate determinations, except * not repeated)

No added Phe

1 mM added Phe

Actin Merge Tubulin

Simultaneous Determination of Brain Amino Acids Simultaneous Determination of Brain Amino Acids and Neurotransmitter Levelsand Neurotransmitter Levels

Deproteinze with TCA, vacuum dry, Solubilize residue in coupling reagent

Derivatize amino acids with Phenylisothiocyanate (PITC)

Reverse-phase C18 columnDAD at 254 nm

Blood plasma

Frozen brain tissue: 4 mice, ½ brain each

Weigh, homogenize in perchloric acid5 mg/mL, prepare standards

Filter, transfer to vial

Reverse-phase C18 columnElectrochemical detection

Derivatize with o-pthalaldehyde

Reverse-phase C18 columnDAD at 338 nm

Neurotransmitters

Amino Acids

PAL Treatment Does Not Completely Normalize PAL Treatment Does Not Completely Normalize Plasma Amino Acid LevelsPlasma Amino Acid Levels

Brain Amino Acid Levels Generally Reflect Plasma Brain Amino Acid Levels Generally Reflect Plasma LevelsLevels

Brain amino acidsPlasma amino acids

Wild type PKU 1d 41d 58d

Co

nc

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/mg

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ht)

0

20

80

100

120TyrosineTryptophanPhenylalanine

Catacholamine Neurotransmitter Levels Do Not Catacholamine Neurotransmitter Levels Do Not Completely Correspond to Amino AcidsCompletely Correspond to Amino Acids

C) NeurotransmittersB) Brain amino acids

Wild type PKU 1d 41d 58d

Co

nc

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/g w

et

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600

800

1000

1200

1400

1600 NorepinephrineDopamineSerotonin

Wild type PKU 1d 41d 58d

Co

nc

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tra

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ng

/mg

we

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ht)

0

20

80

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120TyrosineTryptophanPhenylalanine

Behavioral Differences in PAL-treated PKU MiceBehavioral Differences in PAL-treated PKU Mice

Analysis of Behavior: Video RecordingAnalysis of Behavior: Video Recording

There are statistically significant differences in rearing behavior and line-cross rates in PKU vs WT or HET mice.

PAL-treated mice show improvements over untreated PKU controls and this improvement is LOST when drug treatment is halted.

ConclusionsConclusions

• Long term treatment of Pahenu2 mice results in:

– Reduction and stabilization of plasma Phe to physiological levels in Pahenu2 mice

– a decrease in microglial cell infiltration in the substantia nigra and other dopaminergic brain regions

– Near normalization of brain amino acids and neurotransmitter levels; changes in behavior/activity

• Correction of Maternal PKU Syndrome in female Pahenu2 mice, with successful pregnancy and post-partum survival of pups

• Treatment is well-tolerated and dose adjustments are easily made

• Disadvantages:

– Immunogenicity potential due to administration of an exogenous protein

– Non-oral route of administration

Phase II Clinical TrialPhase II Clinical Trial

• Long term treatment of human patients results in:

– Reduction and stabilization of plasma Phe to therapeutic or near physiological levels in a fraction of treated patients, within 4 months

– Treatment is well-tolerated in these patients and dose adjustments are easily made

– Subjective psychological improvement in some patients with NO change in plasma Phe levels

• Disadvantages:

– Immunogenicity potential due to administration of an exogenous protein; many patients show an immune reaction

– A further fraction of PKU patients develop tolerance after 6-12 months of treatment

– Non-oral route of administration (daily SQ injection, but well tolerated by patients)

Long-term correction of PKU in the Pahenu2 mouse by a modified form of

Phenylalanine Ammonium Lyase.P. Laipis1, W. Zeile1, J. Embury1, S. Bell3, P. Fitzpatrick3, R. Zori2, H.

McCune2, D. Musson3, C. O’Neill3, L. Tsuruda3

Univ. of Florida, College of Medicine, Departments of Biochemistry and Molecular Biology1 and Pediatrics2, Gainesville, FL 32610

BioMarin Pharmaceutical Inc.3, 105 Digital Drive, Novato, CA, 94949