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Phenylketonuria:

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Title: Phenylketonuria:


1
Phenylketonuria Complex results from a Simple
metabolic block
2
PKU A Disorder of Amino Acid Catabolism
Degrade Phe by Phenylalanine Ammonia Lyase
Missing/Altered in PKU
Phenylalanine
Tyrosine
PAH
Protein Synthesis
Protein Synthesis
PAL
PKU
Further Metabolism
t-Cinnamic Acid NH3
Catabolism
L-dopa
Fumarate Acetoacetate
3
Phenylketonuria Points of Emphasis
  • PKU is a common autosomal recessive disorder
  • 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.
  • 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.
  • 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.
  • Other therapeutic modalities are needed to
    replace, or as an adjunct to diet.

4
Maternal PKU Severe mental retardation,
microcephaly, cardiac abnormalities
5
Plasma Phe in Pregnant PKU Mothers at the
University 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.
6
Outcomes Affected Infants of Pregnant PKU
Mothers at the UF College of Medicine
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.
7
The Pahenu2 Mouse Model
  1. Chemical mutagenesis and blind screening to
    identify BTBR mice with elevated serum
    phenylalanine levels. The F263S missense
    mutation resembles human mutations.
  2. Serum Phe levels in Pahenu2 mice are similar to
    human PKU patients.
  3. Phenotypic similarities include mental deficits,
    mild ataxia, defects in melanin biosynthesis, and
    reduced fertility.
  4. Female Pahenu2 -/- mice exhibit a maternal PKU
    syndrome that can be corrected by a
    Phe-restricted diet.

8
Pahenu2 PKU and Heterozygote Mice
9
Phenylalanine Ammonia Lyase (rAvPAL-PEG) Enzyme
Substitution Therapy
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.
1.9 Angstrom resolution structure of the
Anabaena variabilis tetramer. The red and blue
ball structures show the four MIO prosthetic
groups (PDB 2NYN).
10
Long-term rAvPAL-PEG Treatment of Male BTBR
Pahenu2 Mice
Brain Histology
11
Long-term rAvPAL-PEG Treatment of Female Pahenu2
mice
12
Possible Explanations Female Non-response
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
13
Coat Color Changes in Female Pahenu2 Mice
Day 6
Pre-dose
Day 46
Day 18
Images of female BB375381 at indicated intervals.
Dose of rAvPAL-PEG was biweekly 40 mg/kg.
14
Activated Microglial Cell Infiltrates in Female
Pahenu2 Mice
/ (Wild-type) BTBR
/- (Heterozygote) Pahenu2
-/- (PKU) Pahenu2
Microglial cells. iNOS immunoperoxidase stain, 40x
Brain section HE stain, 4x
15
Microglial Cell Infiltrates in PAL-Treated, and
Discontinued PAL-Treated Pahenu2 Mice
3 Days post PAL
31 Days post PAL
42 Days post PAL
72 Days post PAL
16
Plasma Phe Levels in Female Pahenu2 BB375381
17
Correction of Maternal PKU with rAvPAL-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
gt 8 pregnancies 2 litters gt 3 and 5 pups
6 neonatal death
18
More Successful Pregnancies with Modified 3x/week
PAL Treatment Schedule
8 am Mon 4 pm Wed
4 pm Fri
10 mg/kg PAL
Better 5 females, 10 pregnancies but only 4
successful (29 pups). WHY?
19
Plasma Phe levels, 10 mg/kg, 3x / Week PAL and
ADDITIONAL Blood Sample Times
8 am Mon 4 pm Wed
4 pm Fri
10 mg/kg PAL
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?
20
Dietary Phe Intake Dramatically Affects Plasma
Phe levels! 3x/week PAL
Actual Data PKU Females
Simulated Daily Variation Superimposed On
Actual Data from PAL-treated PKU Female MIce
21
rAV-PAL Treated (6 mg/kg Daily Dose) Female PKU
Mice. Phe Levels Throughout Time of Day
Not perfect, but much better 12 females, 29
pregnancies, 15 successful with 83 pups).
22
Pathophysiology 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)

23
Protein and mRNA Levels in Wild-type and PKU Brain
Myelin Basic Protein
Proteo-Lipid Protein
Neuro-Filament Medium
/ -/-
Neuro-Filament Light
P75
GAPdH
/ -/- / -/- / -/- / -/-
/ -/- / -/- Ctx Hip
Str MBr BrSt Cbl
/ -/-
24
Microtubules and Post-translational Modification
of Tubulin
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, a Tubulin 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/detyrosinat
ion cycle is essential for normal brain
development. The TTL-KO mouse dies perinatally
with abberant neuronal network organization (Erck
et al., PNAS, 2005).
25
Purification of Tubulin from Brain Tissue
Frozen brain tissue
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
Weigh and homogenize
High speed
Supernatant (1)
Pellet - discard
10 sucrose cushion
Assemble MTs, 37C plus paclitaxel
Low speed
MW 1 2 3 4 5 6 7
8
kDa 200 116 97.4 66.2 45 31
Supernatant (2) - discard
Pellet (3) Crude MTs
Cold depolymerize
Low speed
Pellet (5) - discard
Supernatant (4)
Assemble MTs, 37C plus paclitaxel
Low speed
Pellet (6) MTs
Supernatant (7) - discard
Resuspend MTs and digest with Carboxypeptidase A
Deproteinize, derivatize amino acids,
HPLC C18 reverse-phase column
26
HPLC Determination of Mouse Brain Tubulin
C-Terminal Modification by Tyr and Phe
Tyrosine and phenylalanine in brain
tubulin C-terminal amino acids cleaved by
carboxypeptidase A. Genotype
Tyrosine Phenylalanine S.D. Wild
type 86.6 13.4
5.4 Heterozygote 94.7
5.3 0.07 PKU 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.
Wild type.
Heterozygote
PKU
Standard Curve Blue 31.25 µM Green 62.5
µM Red 125 µM Black 250 µM
27
Phe and Tyr on the C-terminal end of HELA Tubulin
is 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 11.5 1 mM Phe 60 40 0.98 2 mM
Phe 58.4 41.6 n/a 1 mM Phe/washout 83.3 1
6.7 n/a (Amino acids as of total). Values
are the means of two separate determinations,
except not repeated)
28
Simultaneous Determination of Brain Amino Acids
and Neurotransmitter Levels
Blood plasma
Deproteinze with TCA, vacuum dry, Solubilize
residue in coupling reagent
Derivatize amino acids with Phenylisothiocyanate
(PITC)
Reverse-phase C18 column DAD at 254 nm
Frozen brain tissue 4 mice, ½ brain each
Weigh, homogenize in perchloric acid 5 mg/mL,
prepare standards
Filter, transfer to vial
Reverse-phase C18 column Electrochemical detection
Derivatize with o-pthalaldehyde
Neurotransmitters
Reverse-phase C18 column DAD at 338 nm
Amino Acids
29
PAL Treatment Does Not Completely Normalize
Plasma Amino Acid Levels
30
Brain Amino Acid Levels Generally Reflect Plasma
Levels
Brain amino acids
Plasma amino acids
31
Catacholamine Neurotransmitter Levels Do Not
Completely Correspond to Amino Acids
C) Neurotransmitters
B) Brain amino acids
32
Behavioral Differences in PAL-treated PKU Mice
33
Analysis 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.
34
Conclusions
  • 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

35
Phase 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)

36
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. ONeill3, 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
37
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