NeoPhe in the Treatment of Phenylketonuria New Formulation of LNAA
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NeoPhe in the Treatment of Phenylketonuria
New Formulation of LNAA
2nd Ukraine Congress
On Clinical GeneticsOctober 2005
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UkraineElena Grechanina
RussiaPeter Novikov
DenmarkJytte Bieber Nielsen
Leah BrammerKirstin Ahring
USAReuben Matalon, M.D., Ph.D.
Kim Matalon
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Large Neutral Amino Acids (LNAA)
• Phenylalanine (Phe) • Leucine• Tyrosine• Tryptophan• Methionine• Histidine• Isoleucine• Valine• Threonine
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Transport of LNAA to the Brain
• Phenylalanine (Phe) 0.12 0.45• Leucine 0.15 0.53• Tyrosine 0.16 0.58• Tryptophan 0.19 0.71• Methionine 0.19 0.77• Histidine 0.28 1.10• Isoleucine 0.33 1.30• Valine 0.63 2.50• Threonine 0.73 3.00
Km mmol/L Km app
Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980.
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Andersen AE, Avins L
• LNAA injected to rat pups
• Phenylalanine hydroxylase was ihibited by parachlorophenylalanine
• Brain phenylalanine decreased
1976 Arch Neurology 33:684
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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
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Tyrosine in Treatment of PKU
• 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
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Tryptophan in Treated PKU
• 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
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LNAA Supplementation in PKU
• 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
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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
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Absolute and apparent Km values of neutral amino acids for the neutral amino acid transporter in the BBB (Partridge, 1980)0
Amino acid Typical plasma level (mM)
Km
(mM)
App Km
(mM)
LNAA’s
Phe 0.05 0.12 0.45
Leu 0.10 0.15 0.53
Tyr 0.09 0.16 0.58
Trp 0.10 0.16 0.71
Met 0.04 0.19 0.77
Isoleu 0.07 0.33 1.3
Val 0.14 0.63 2.5
Thr 0.19 0.73 3.0
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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 0.05 0.28 1.1
Arg 0.10 0.09 0.40
Lys 0.30 0.10 0.25
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LNAA Transport in Intestinal Mucosa Km mmol/L
• 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.
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Amino acid inhibition of Phe transport in Caco-2-cells – 10uM Phe in buffer applied to monolayers in presence of 1 mM concentration of each amino acid
Inhibitor % inhibition
LNAA’s
Leu 55%
Tyr 45%
Trp 36%
Basis Aa’s
Lys 50%
His 33%Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)
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78- 80- 83- 86- 159- 162-
F 577 23.8 21.4 19.4 24.9 18 11
F 579 23.7 25.1 28.4 33.1 8.3 14.8
F 582 28.8 21.9 22.2 20.9 10.3 8.3
F 584 23.8 30 25.3 30.7 7.8 12.4
PKU Mice on NeoPhe phe mg/dl
Mice Control NeoPhe
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78- 80- 83- 86- 159- 162-
F 585 20.6 21.7 24.4 19.8 8.5 12.3
F 586 23.2 25.7 21.2 21.9 13.5 11.3
F 588 21.6 21.7 24.2 24.4 10.9 10.9
Avg each time pt 23.6 23.9 23.6 25.1 11 11.6
Avg all Pre-LNAA 24.1
Avg all Post-LNAA 11.3
PKU Mice on NeoPhe phe mg/dl
Mice Control NeoPhe
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Pre- and Post-LNAA Blood Phe Levels
0
5
10
15
20
25
30
35
1 2 3 4 5 6
Blo
od
Ph
e [
mg
/dl]
Pre-LNAA Post-LNAA
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Denmark LNAA STUDY1 tablet/kg/day
Blood Samples
PKU 20 PKU 39 PKU 93 PKU 105 PKU 128 Avg
1 1436 1681 1697 1597 1627 1608
02 1262 1691 1591 1480 1602 1525
04 1164 1643 1526 1414 1407 1431
1 week 184 -58 220 184 268 160
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Denmark LNAA STUDY 2 tablets/kg/day
Blood Samples
PKU 20
PKU 39 PKU 93 PKU 105
PKU 128
Avg
08 1252 1739 1477 1413 1359 1448
09 1146 1537 1370 1233 1373 1332
11 1119 1556 1389 1179 1313 1311
15 1199 1650 1349 1222 1335 1351
2 week 237 31 348 375 292 257
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Time Phe Tyr
KA µmol/l mg/dl µmol/l mg/dl
0’ 718.8 11.98 53.9 0.98
3 days 668.4 11.14 91.3 1.66
3 days 523.2 8.72 103.4 1.88
3 days 376.2 6.27 108.3 1.97
Russia LNAA STUDY
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Time Phe Tyr
KN µmol/l mg/dl µmol/l mg/dl
0’ 707.4 11.79 42.9 0.78
3 days 607.2 10.12 126.5 2.30
3 days 572.4 9.54 159.5 2.91
3 days 585.6 9.76 83.6 1.52
Russia LNAA STUDY
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Russia LNAA StudyTime 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
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USA LNAA STUDYTime Phe Tyr
GDL µmol/l mg/dl µmol/l mg/dl
0’ 1290.6 21.51 69.8 1.27
2 days 1198.2 19.97 73.7 1.34
4 days 115.8 1.93 140.25 2.55
KM
0’ 1540.2 25.67 30.8 0.56
8 days 883.8 14.37 53.8 0.98
0’ 1978.2 32.97 68.7 1.25
2 days 1608.6 26.81 207.35 3.77
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USA LNAA STUDYTime 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
RC
0’ 965.4 16.09 58.8 1.07
2 days 828.6 13.81 156.2 2.84
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Response of Blood Phe to LNAAUkraine
0
5
10
15
20
25
21 yo girl 21 14.5 16 7.9 12.7 7.1 13
0 24h 72hr 1 wk 2 wk 3 wk 4 wk
µmol/l 1260 870 960 474 762 426 780
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Response on Phe on LNAAUkraine
0
5
10
15
20
25
12 yo boy 20 13.8 12.75 10.4
0 36hr 72h 1wk
µmol/l 1200 828 765 624
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US Blood Phe and TyrNeoPhe Patient K 1 Week
µmol/L (mg)
Control NeoPhe
phe tyr phe tyr
µmol/L (mg)
1978.1(32.97) 1.251356.0 (22.6) 5.0
1139.6 (25.66) 0.62 1308 (21.8) 4.1
1456.2 (24.27) 0.62 1146 (19.1) 3.82
24% reduction
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US Blood Phe and TyrNeoPhe Patient G 1 Week
µmol/L (mg)Control NeoPhe
phe tyr phe tyr
mg/dl mg/dl
1560 (26.0) 0.92 953 (15.89) 4.35
1764 (29.4) 1.9 505 (8.43) 3.32
56% reduction
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NeoPhe 0.5 g/kg in PKU Subjects
• 13 subjects
• Mean age 26.6 years
• 7 males, 6 females
• Mean decrease in blood Phe after one week 243 µmol/L
• Average decrease in blood Phe 22 %.
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NeoPhe 1.0 g/kg in PKU Subjects
• 7 subjects
• Mean age 25.2 years
• 5 males, 2 females
• Mean decrease in blood Phe after one week 377 µmol/L
• Average decrease in blood Phe 25 %.
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Figure 1. Blood Phe Response to 0.5g/kg NeoPhe in Patients with PKU
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Zero Time 1 week
Blood Phe
umol
/L
IVS12nt1g>a/R261Q
IVS12nt1g>a/Y356X
IVS12ntg>a/IVS10nt11g>a
E280K/R408W
IVS12nt1g>a/IVS12nt1g>a
R261Q/R408W
R408W/R408W
IVS4ntg>t/R408W
R408W/R408W
E280K/E280K
F299C/IVS12nt1g>a
I65T/R408W
F299C/unk
Paired t-test: p=0.001
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Figure 2. Blood Phe Response to 1.0 g/kg NeoPhe in Patients with PKU
0
200
400
600
800
1000
1200
1400
1600
1800
Zero Time 1 week
Blood Phe
umol
/L
IVS12nt1g>a/R261Q
IVS12nt1g>a/Y356X
IVS12nt1g>a/IVS10nt11g>a
E280K/R408W
IVS12nt1g>a/IVS12nt1g>a
ND
R408W/R252W
Paired t-test: p=0.006
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CONCLUSIONS
• For the first time mixture of LNAA can lower blood phenylalanine
• Using NeoPhe avoids lysine deficiency and negative nitrogen balance
• NeoPhe offers new options to treat PKU
• Diet can be more liberal with NeoPhe
• Still blood phe levels need to be controlled
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Acknowledgement
Participants in the study
Professor Elena GrechaninaKharkiv, Ukraine
Professor Peter NovikovMoscow, Russia
Dr. Jytte Bieber NielsenGlostrup, Denmark