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ICIEM 2006
Double Blind Placebo
Control Trial in PKU with
NeoPhe
Reuben Matalon1, Kimberlee Michals-Matalon1, Alberto
Burlina2, Alesandro Burlina2, Marcello Giovannini3, Laura
Fiori3, Elena Grechanina4, Peter Novikov5, James Grady1,
Stephen Tyring6, Flemming Guttler7
1University of Texas Medical Branch, 2University of Padova, 3University of Milan,
4University of Kharkiv, 5University of Moscow, 6University of Texas, 7Kennedy
Institute
Large Neutral Amino Acids (LNAA)
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Phenylalanine (Phe)
Leucine
Tyrosine
Tryptophan
Methionine
Histidine
Isoleucine
Valine
Threonine
Transport of LNAA to the Brain
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Km mmol/L
Phenylalanine (Phe)
0.12
Leucine
0.15
Tyrosine
0.16
Tryptophan
0.19
Methionine
0.19
Histidine
0.28
Isoleucine
0.33
Valine
0.63
Threonine
0.73
Pardridge, Inborn Errors of Metabolism in Humans.
MTP Press, 1980.
Km app
0.45
0.53
0.58
0.71
0.77
1.10
1.30
2.50
3.00
Andersen AE, Avins L
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LNAA injected to rat pups
Phenylalanine hydroxylase was ihibited by
parachlorophenylalanine
Brain phenylalanine decreased
1976 Arch Neurology 33:684
Tyrosine in The Treatment of PKU
Lou et al used Tyr 160 mg/kg in treated
patients with PKU
 Increased attention span
 Increased dopamine synthesis
1987 Acta Paediatr Scand 76:560
Tyrosine in Treatment of PKU
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Pietz et al. used high dose tyrosine in adults with
PKU and high blood Phe
No difference in treated group vs placebo
1995 J Pediatr 127:936
Tryptophan in Treated PKU
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Nielsen et al used tryptophan 4.5 gm/day to
treated PKU for 3 weeks
Showed a 3 fold increase in 5-HIAA in CSF
despite high blood Phe
1988 Dietary Phenylalanine and Brain Function.
Birkhauser
LNAA Supplementation in PKU
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Dotremont et al. used LNAA and a low
protein diet 0.6 gm/kg on 4 patients with
PKU
After 1 month subjects found with negative
nitrogen balance
Lysine was limiting amino acid
1995 J Inherit Metab Dis 18:127
Km (app) – Km (1 + ∑[aa]/Km]
This predicts that, if the plasma level of an
LNAA is much less than its value of Km,
then that amino acid will not compete
effectively for the carrier protein
Absolute and apparent Km values of neutral amino acids for the
neutral amino acid transporter in the BBB (Partridge, 1980)0
Amino acid
Typical
Km
App Km
plasma level
(mM)
(mM)
(mM)
LNAA’s
Phe
0.05
0.12
0.45
Leu
0.10
0.15
0.53
Tyr
Trp
Met
0.09
0.10
0.04
0.16
0.16
0.19
0.58
0.71
0.77
Isoleu
Val
Thr
0.07
0.14
0.19
0.33
0.63
0.73
1.3
2.5
3.0
Absolute and apparent Km values of neutral
amino acids for the neutral amino acid
transporter in the BBB (Partridge, 1980)
Amino acid
Typical
plasma level
(mM)
Km
(mM)
App Km
(mM)
Basic aa’s
His
Arg
0.05
0.10
0.28
0.09
1.1
0.40
Lys
0.30
0.10
0.25
LNAA Transport in Intestinal Mucosa Km
mmol/L
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Phenylalanine
1.0
Leucine
2.0
Valine
3.0
Methionine
5.0
Histidine
6.0
Competition effect is not likely to occur in tissue
other than brain unless high concentration of
amino acids is used
Pardridge, Inborn Errors of Metabolism in Humans. MTP Press,
1980.
Amino acid inhibition of Phe transport in Caco-2cells – 10uM Phe in buffer applied to monolayers
in presence of 1 mM concentration of each amino
acid
Inhibitor
LNAA’s
% inhibition
Leu
Tyr
Trp
55%
45%
36%
Basis Aa’s
Lys
His
50%
33%
Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)
PKU Mice on NeoPhe
phe mg/dl
Mice
Control
NeoPhe
78-
80-
83-
86-
159-
162-
F 577
F 579
F 582
23.8
23.7
28.8
21.4 19.4
25.1 28.4
21.9 22.2
24.9
33.1
20.9
18
8.3
10.3
11
14.8
8.3
F 584
23.8
30.7
7.8
12.4
30
25.3
PKU Mice on NeoPhe
phe mg/dl
Mice
Control
NeoPhe
78-
80-
83-
86-
159- 162-
F 585
F 586
F 588
20.6
23.2
21.6
21.7
25.7
21.7
24.4
21.2
24.2
19.8
21.9
24.4
8.5
13.5
10.9
12.3
11.3
10.9
Avg each time pt
Avg all Pre-LNAA
Avg all Post-LNAA
23.6
24.1
11.3
23.9
23.6
25.1
11
11.6
Pre- and Post-LNAA Blood Phe Levels
35
Pre-LNAA
Post-LNAA
Blood Phe [mg/dl]
30
25
20
15
10
5
0
1
2
3
4
5
6
Russia LNAA STUDY
Time
Phe
Tyr
KA
0’
3 days
µmol/l
718.8
668.4
mg/dl
11.98
11.14
µmol/l
53.9
91.3
mg/dl
0.98
1.66
3 days
3 days
523.2
376.2
8.72
6.27
103.4
108.3
1.88
1.97
Russia LNAA STUDY
Time
Phe
Tyr
KN
µmol/l
mg/dl
µmol/l
mg/dl
0’
707.4
11.79
42.9
0.78
3 days
3 days
3 days
607.2
572.4
585.6
10.12
9.54
9.76
126.5
159.5
83.6
2.30
2.91
1.52
Russia LNAA Study
Time
Phe
Tyr
KH
µmol/l
mg/dl
µmol/l
mg/dl
0’
635.4
10.59
33.0
0.60
3 days
554.4
9.24
242.0
4.40
3 days
322.2
5.37
94.6
1.72
3 days
136.2
2.27
110.0
2.00
3 days
102.6
1.71
94.0
1.71
Response of Blood Phe to LNAA
Ukraine
25
20
15
10
5
0
21 yo girl
µmol/l
0
24h
21
14.5
1260
870
72hr 1 wk 2 wk 3 wk 4 wk
16
7.9
960
474
12.7
762
7.1
13
426
780
Response on Phe on LNAA
Ukraine
25
20
15
10
5
0
0
36hr
72h
1wk
12 yo boy
20
13.8
12.75
10.4
µmol/l
1200
828
765
624
USA LNAA STUDY
Time
Phe
Tyr
GDL
µmol/l
mg/dl
µmol/l
mg/dl
0’
2 days
4 days
1290.6
1198.2
115.8
21.51
19.97
1.93
69.8
73.7
140.25
1.27
1.34
2.55
KM
0’
1540.2
25.67
30.8
0.56
8 days
883.8
14.37
53.8
0.98
0’
2 days
1978.2
1608.6
32.97
26.81
68.7
207.35
1.25
3.77
USA LNAA STUDY
Time
Phe
Try
ES
µmol/l
mg/dl
µmol/l
mg/dl
0’
1375.8
22.93
31.9
0.58
4-7 days
767.4
12.79
121.5
2.12
0’
965.4
16.09
58.8
1.07
2 days
828.6
13.81
156.2
2.84
RC
US Blood Phe and Tyr
NeoPhe Patient K 1 Week
µmol/L (mg)
Control
phe
1978.1(32.97)
1139.6 (25.66)
1456.2 (24.27)
tyr
1.25
0.62
0.62
NeoPhe
phe
tyr
µmol/L (mg)
1356.0 (22.6)
1308 (21.8)
1146 (19.1)
24% reduction
5.0
4.1
3.82
US Blood Phe and Tyr
NeoPhe Patient G 1 Week
µmol/L (mg)
Control
phe
mg/dl
tyr
NeoPhe
phe
mg/dl
tyr
1560 (26.0)
0.92
953 (15.89)
4.35
1764 (29.4)
1.9
505 (8.43)
3.32
56% reduction
US Blood Phe and Tyr
NeoPhe Patient K 1 Week
µmol/L (mg)
Control
phe
1978.1(32.97)
1139.6 (25.66)
1456.2 (24.27)
tyr
1.25
0.62
0.62
NeoPhe
phe
tyr
µmol/L (mg)
1356.0
(22.6)
1308 (21.8)
1146 (19.1)
24% reduction
5.0
4.1
3.82
US Blood Phe and Tyr
NeoPhe Patient G 1 Week
µmol/L (mg)
Control
phe
mg/dl
tyr
NeoPhe
phe
mg/dl
tyr
1560 (26.0)
0.92
953 (15.89)
4.35
1764 (29.4)
1.9
505 (8.43)
3.32
56% reduction
Figure 1. Blood Phe Response to
0.5g/kg NeoPhe in Patients with PKU
2000
1800
1600
IVS12nt1g>a/R261Q
IVS12nt1g>a/Y356X
IVS12ntg>a/IVS10nt11g>a
E280K/R408W
IVS12nt1g>a/IVS12nt1g>a
R261Q/R408W
R408W/R408W
IVS4ntg>t/R408W
umol/L
1400
1200
1000
800
R408W/R408W
E280K/E280K
F299C/IVS12nt1g>a
I65T/R408W
F299C/unk
600
400
200
0
Zero Time
1 week
Blood Phe
Paired t-test: p=0.001
Figure 2. Blood Phe Response to 1.0
g/kg NeoPhe in Patients with PKU
1800
1600
1400
umol/L
1200
IVS12nt1g>a/R261Q
IVS12nt1g>a/Y356X
IVS12nt1g>a/IVS10nt11g>a
E280K/R408W
IVS12nt1g>a/IVS12nt1g>a
ND
R408W/R252W
1000
800
600
400
200
0
Zero Time
1 week
Blood Phe
Paired t-test: p=0.006
Double-Blind Placebo Control on
patients in the US
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Patients were genotyped
Baseline Phenylalanine was determined 3
times
Placebo or Neophe was administered for one
week, 1tablet/kg/day
Blood Phe was determined 3 times
mg/dl
mmol/L
zero 1
26.1
1566
zero 2
29.36
1761
zero 3
27.2
1632
24 hrs
16.04
962
72 hrs
16
960
96 hrs
8.4
504
1 week
8.8
528
24 hrs
72 hrs
96 hrs
1 week
21.2
24.1
23.6
22.5
1272
1446
1416
1350
E280K/E280K
Before Neophe
Neophe
Placebo
mg/dl
mmol/L
zero 1
25.6
1536
zero 2
32.9
1974
zero 3
26.8
1608
24 hrs
14.3
858
72 hrs
16.8
1008
96 hrs
18.1
1086
1 week
12.2
732
24 hrs
72 hrs
96 hrs
1 week
19.2
20.6
24.8
23.7
1152
1236
1488
1422
299C/IVS12 nt1 g>a
Before Neophe
Neophe
Placebo
mg/dl
mmol/L
zero 1
16.09
965.4
zero 2
18.1
1086
zero 3
17.2
1032
24 hrs
12
720
72 hrs
14.1
846
96 hrs
10.1
606
1 week
11.4
684
24 hrs
72 hrs
96 hrs
1 week
16.2
18.2
17.1
16.12
972
1092
1026
967.2
F299C/Before Neophe
NeoPhe
Placebo
mg/dl
mmol/L
zero 1
24.1
1446
zero 2
23.0
1380
zero 3
21.1
1266
24 hrs
12.8
768
72 hrs
11.2
672
96 hrs
12.9
774
1 week
13.5
810
24 hrs
72 hrs
96 hrs
1 week
18.2
22.2
19.1
22.3
1092
1332
1146
1339
I65T/R408W
Before Neophe
Neophe
Placebo
Summary of Average Blood
Phe
Zero
µmol/l
NeoPhe
µmol/l
Placebo
µmol/l
E280K/E280K
1653
738.5
1350
F299C/IVS12ntgl>a
1706
921
1422
1027.8
712
967.2
1364
752
1339.2
F299C/-
I65T/R408W
Summary of Double Blind Study
1800
1600
micromol/L
1400
1200
E280K/E280K
1000
F299C/IVS12ntg1>a
800
F299C/-
600
I65T/R408W
400
200
0
zero
1 week
On Neo Phe
2 week
Placebo
Conclusion
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LNAA can reduce blood Phe levels when
given with meals
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Longer term double-blind placebo control
studies are needed
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Establishing the efficacy and safety of LNAA
can improve treatment of PKU
Acknowledgement
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This study was supported in part by grants
from Mid-Atlantic Connection for PKU and
Allied Diseases (MACPAD) & South Texas
Association for PKU and Allied Disease
(STAPAD)
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Generous supply of NeoPhe was given by
PreKulab, Denmark