ICIEM 2006ICIEM 2006
Double Blind Placebo Double Blind Placebo Control Trial in PKU with Control Trial in PKU with
NeoPheNeoPhe
Reuben MatalonReuben Matalon11, Kimberlee Michals-Matalon, Kimberlee Michals-Matalon11, Alberto , Alberto BurlinaBurlina22, Alesandro Burlina, Alesandro Burlina22, Marcello Giovannini, Marcello Giovannini33, Laura , Laura FioriFiori33, Elena Grechanina, Elena Grechanina44, Peter Novikov, Peter Novikov55, James Grady, James Grady11, ,
Stephen TyringStephen Tyring66, Flemming Guttler, Flemming Guttler77
11University of Texas Medical Branch, University of Texas Medical Branch, 22University of Padova, University of Padova, 33University of Milan, University of Milan, 44University of Kharkiv, University of Kharkiv, 55University of Moscow, University of Moscow, 66University of Texas, University of Texas, 77Kennedy Kennedy
InstituteInstitute
Large Neutral Amino Acids (LNAA)Large Neutral Amino Acids (LNAA)
Phenylalanine (Phe) Phenylalanine (Phe) LeucineLeucine TyrosineTyrosine TryptophanTryptophan MethionineMethionine HistidineHistidine IsoleucineIsoleucine ValineValine ThreonineThreonine
Transport of LNAA to the BrainTransport of LNAA to the Brain
Phenylalanine (Phe) Phenylalanine (Phe) 0.12 0.12 0.450.45 LeucineLeucine 0.150.15 0.530.53 TyrosineTyrosine 0.160.16 0.580.58 TryptophanTryptophan 0.190.19 0.710.71 MethionineMethionine 0.190.19 0.770.77 HistidineHistidine 0.280.28 1.101.10 IsoleucineIsoleucine 0.330.33 1.301.30 ValineValine 0.630.63 2.502.50 Threonine Threonine 0.730.73 3.003.00
Km mmol/L Km app
Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980.
Andersen AE, Avins LAndersen AE, Avins L
LNAA injected to rat pupsLNAA injected to rat pups Phenylalanine hydroxylase was ihibited by Phenylalanine hydroxylase was ihibited by
parachlorophenylalanineparachlorophenylalanine Brain phenylalanine decreased Brain phenylalanine decreased
1976 Arch Neurology 33:6841976 Arch Neurology 33:684
Tyrosine in The Treatment of PKUTyrosine in The Treatment of PKU
Lou et al used Tyr 160 mg/kg in treated Lou et al used Tyr 160 mg/kg in treated patients with PKUpatients with PKU
Increased attention spanIncreased attention span Increased dopamine synthesisIncreased dopamine synthesis
1987 Acta Paediatr Scand 76:5601987 Acta Paediatr Scand 76:560
Tyrosine in Treatment of PKUTyrosine in Treatment of PKU
Pietz et al. used high dose tyrosine in adults with Pietz et al. used high dose tyrosine in adults with PKU and high blood PhePKU and high blood Phe
No difference in treated group vs placeboNo difference in treated group vs placebo
1995 J Pediatr 127:9361995 J Pediatr 127:936
Tryptophan in Treated PKUTryptophan in Treated PKU
Nielsen et al used tryptophan 4.5 gm/day to Nielsen et al used tryptophan 4.5 gm/day to treated PKU for 3 weeks treated PKU for 3 weeks
Showed a 3 fold increase in 5-HIAA in CSF Showed a 3 fold increase in 5-HIAA in CSF despite high blood Phedespite high blood Phe
1988 Dietary Phenylalanine and Brain Function. 1988 Dietary Phenylalanine and Brain Function. BirkhauserBirkhauser
LNAA Supplementation in PKULNAA Supplementation in PKU
Dotremont et al. used LNAA and a low Dotremont et al. used LNAA and a low protein diet 0.6 gm/kg on 4 patients with protein diet 0.6 gm/kg on 4 patients with PKUPKU
After 1 month subjects found with negative After 1 month subjects found with negative nitrogen balancenitrogen balance
Lysine was limiting amino acidLysine was limiting amino acid
1995 J Inherit Metab Dis 18:1271995 J Inherit Metab Dis 18:127
Km (app) – Km (1 + Km (app) – Km (1 + ∑[aa]/Km]∑[aa]/Km]
This predicts that, if the plasma level of an This predicts that, if the plasma level of an LNAA is much less than its value of Km, LNAA is much less than its value of Km, then that amino acid will not compete then that amino acid will not compete effectively for the carrier proteineffectively for the carrier protein
Absolute and apparent Km values of neutral amino acids for the Absolute and apparent Km values of neutral amino acids for the neutral amino acid transporter in the BBB (Partridge, 1980)0neutral amino acid transporter in the BBB (Partridge, 1980)0
Amino acidAmino acid Typical Typical plasma level plasma level (mM)(mM)
KmKm
(mM)(mM)
App KmApp Km
(mM)(mM)
LNAA’sLNAA’s
PhePhe 0.050.05 0.120.12 0.450.45
LeuLeu 0.100.10 0.150.15 0.530.53
TyrTyr 0.090.09 0.160.16 0.580.58
TrpTrp 0.100.10 0.160.16 0.710.71
MetMet 0.040.04 0.190.19 0.770.77
IsoleuIsoleu 0.070.07 0.330.33 1.31.3
ValVal 0.140.14 0.630.63 2.52.5
ThrThr 0.190.19 0.730.73 3.03.0
Absolute and apparent Km values of neutral Absolute and apparent Km values of neutral amino acids for the neutral amino acid amino acids for the neutral amino acid transporter in the BBB (Partridge, 1980)transporter in the BBB (Partridge, 1980)Amino acidAmino acid Typical Typical
plasma level plasma level (mM)(mM)
KmKm
(mM)(mM)
App KmApp Km
(mM)(mM)
Basic aa’sBasic aa’s
HisHis 0.050.05 0.280.28 1.11.1
ArgArg 0.100.10 0.090.09 0.400.40
LysLys 0.300.30 0.100.10 0.250.25
LNAA Transport in Intestinal Mucosa KLNAA Transport in Intestinal Mucosa Kmm
mmol/Lmmol/L PhenylalaninePhenylalanine 1.01.0 LeucineLeucine 2.02.0 ValineValine 3.03.0 MethionineMethionine 5.05.0 HistidineHistidine 6.06.0 Competition effect is not likely to occur in tissue Competition effect is not likely to occur in tissue
other than brain unless high concentration of other than brain unless high concentration of amino acids is usedamino acids is used
Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980.
Amino acid inhibition of Phe transport in Caco-2-Amino acid inhibition of Phe transport in Caco-2-cells – 10uM Phe in buffer applied to monolayers cells – 10uM Phe in buffer applied to monolayers in presence of 1 mM concentration of each amino in presence of 1 mM concentration of each amino acidacidInhibitorInhibitor % inhibition% inhibition
LNAA’sLNAA’s
LeuLeu 55%55%
TyrTyr 45%45%
TrpTrp 36%36%
Basis Aa’sBasis Aa’s
LysLys 50%50%
HisHis 33%33%
Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)
78-78- 80-80- 83-83- 86-86- 159-159- 162-162-
F 577F 577 23.823.8 21.421.4 19.419.4 24.924.9 1818 1111
F 579F 579 23.723.7 25.125.1 28.428.4 33.133.1 8.38.3 14.814.8
F 582F 582 28.828.8 21.921.9 22.222.2 20.920.9 10.310.3 8.38.3
F 584F 584 23.823.8 3030 25.325.3 30.730.7 7.87.8 12.412.4
PKU Mice on NeoPhePKU Mice on NeoPhe phe mg/dl phe mg/dl
MiceMice ControlControl NeoPheNeoPhe
78-78- 80-80- 83-83- 86-86- 159-159- 162-162-
F 585F 585 20.620.6 21.721.7 24.424.4 19.819.8 8.58.5 12.312.3
F 586F 586 23.223.2 25.725.7 21.221.2 21.921.9 13.513.5 11.311.3
F 588F 588 21.621.6 21.721.7 24.224.2 24.424.4 10.910.9 10.910.9
Avg each time ptAvg each time pt 23.623.6 23.923.9 23.623.6 25.125.1 1111 11.611.6
Avg all Pre-LNAAAvg all Pre-LNAA 24.124.1
Avg all Post-LNAAAvg all Post-LNAA 11.311.3
PKU Mice on NeoPhePKU Mice on NeoPhe phe mg/dl phe mg/dl
MiceMice Control Control NeoPheNeoPhe
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
TimeTime PhePhe TyrTyr
KAKA µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl
0’0’ 718.8718.8 11.9811.98 53.953.9 0.980.98
3 days3 days 668.4668.4 11.1411.14 91.391.3 1.661.66
3 days3 days 523.2523.2 8.728.72 103.4103.4 1.881.88
3 days3 days 376.2376.2 6.276.27 108.3108.3 1.971.97
Russia LNAA STUDYRussia LNAA STUDY
TimeTime PhePhe TyrTyr
KNKN µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl
0’0’ 707.4707.4 11.7911.79 42.942.9 0.780.78
3 days3 days 607.2607.2 10.1210.12 126.5126.5 2.302.30
3 days3 days 572.4572.4 9.549.54 159.5159.5 2.912.91
3 days3 days 585.6585.6 9.769.76 83.683.6 1.521.52
Russia LNAA STUDYRussia LNAA STUDY
Russia LNAA StudyRussia LNAA Study
TimeTime PhePhe TyrTyr
KHKH µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl
0’0’ 635.4635.4 10.5910.59 33.033.0 0.600.60
3 days3 days 554.4554.4 9.249.24 242.0242.0 4.404.40
3 days3 days 322.2322.2 5.375.37 94.694.6 1.721.72
3 days3 days 136.2136.2 2.272.27 110.0110.0 2.002.00
3 days3 days 102.6102.6 1.711.71 94.094.0 1.711.71
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/lµmol/l 1260 870 960 474 762 426 7801260 870 960 474 762 426 780
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/lµmol/l 1200 828 765 6241200 828 765 624
USA LNAA STUDYUSA LNAA STUDYTimeTime PhePhe TyrTyr
GDLGDL µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl
0’0’ 1290.61290.6 21.5121.51 69.869.8 1.271.27
2 days2 days 1198.21198.2 19.9719.97 73.773.7 1.341.34
4 days4 days 115.8115.8 1.931.93 140.25140.25 2.552.55
KMKM
0’0’ 1540.21540.2 25.6725.67 30.830.8 0.560.56
8 days8 days 883.8883.8 14.3714.37 53.853.8 0.980.98
0’0’ 1978.21978.2 32.9732.97 68.768.7 1.251.25
2 days2 days 1608.61608.6 26.8126.81 207.35207.35 3.773.77
USA LNAA STUDYUSA LNAA STUDYTimeTime PhePhe TryTry
ESES µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl
0’0’ 1375.81375.8 22.9322.93 31.931.9 0.580.58
4-7 days4-7 days 767.4767.4 12.7912.79 121.5121.5 2.122.12
RCRC
0’0’ 965.4965.4 16.0916.09 58.858.8 1.071.07
2 days2 days 828.6828.6 13.8113.81 156.2156.2 2.842.84
US Blood Phe and TyrUS Blood Phe and TyrNeoPhe Patient K 1 WeekNeoPhe Patient K 1 Week
µmol/L (mg)µmol/L (mg)ControlControl NeoPheNeoPhe
phephe tyrtyr phephe tyrtyr
µmol/L (mg)µmol/L (mg)
1978.1(32.97)1978.1(32.97) 1.251.25 1356.0 (22.6)1356.0 (22.6) 5.05.0
1139.6 (25.66)1139.6 (25.66) 0.620.62 1308 (21.8)1308 (21.8) 4.14.1
1456.2 (24.27)1456.2 (24.27) 0.620.62 1146 (19.1)1146 (19.1) 3.823.82
24% reduction24% reduction
US Blood Phe and TyrUS Blood Phe and TyrNeoPhe Patient G 1 WeekNeoPhe Patient G 1 Week
µmol/L (mg)µmol/L (mg)
ControlControl NeoPheNeoPhe
phephe tyrtyr phephe tyrtyr
mg/dlmg/dl mg/dlmg/dl
1560 (26.0)1560 (26.0) 0.920.92 953 (15.89)953 (15.89) 4.354.35
1764 (29.4)1764 (29.4) 1.91.9 505 (8.43)505 (8.43) 3.323.32
56% reduction56% reduction
US Blood Phe and TyrUS Blood Phe and TyrNeoPhe Patient K 1 WeekNeoPhe Patient K 1 Week
µmol/L (mg)µmol/L (mg)
ControlControl NeoPheNeoPhe
phephe tyrtyr phephe tyrtyr
µmol/L (mg)µmol/L (mg)
1978.1(32.97)1978.1(32.97) 1.251.251356.0 1356.0 (22.6)(22.6) 5.05.0
1139.6 (25.66)1139.6 (25.66) 0.620.62 1308 (21.8)1308 (21.8) 4.14.1
1456.2 (24.27)1456.2 (24.27) 0.620.62 1146 (19.1)1146 (19.1) 3.823.82
24% reduction24% reduction
US Blood Phe and TyrUS Blood Phe and TyrNeoPhe Patient G 1 WeekNeoPhe Patient G 1 Week
µmol/L (mg)µmol/L (mg)
ControlControl NeoPheNeoPhe
phephe tyrtyr phephe tyrtyr
mg/dlmg/dl mg/dlmg/dl
1560 (26.0)1560 (26.0) 0.920.92 953 (15.89)953 (15.89) 4.354.35
1764 (29.4)1764 (29.4) 1.91.9 505 (8.43)505 (8.43) 3.323.32
56% reduction56% reduction
Figure 1. Blood Phe Response to Figure 1. Blood Phe Response to 0.5g/kg NeoPhe in Patients with0.5g/kg NeoPhe in Patients with PKUPKU
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
Figure 2. Blood Phe Response to 1.0 Figure 2. Blood Phe Response to 1.0 g/kg NeoPhe in Patients withg/kg NeoPhe in Patients with PKUPKU
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
Double-Blind Placebo Control on Double-Blind Placebo Control on patients in the USpatients in the US
Patients were genotyped Patients were genotyped Baseline Phenylalanine was determined 3 Baseline Phenylalanine was determined 3
timestimes Placebo or Neophe was administered for Placebo or Neophe was administered for
one week, 1tablet/kg/dayone week, 1tablet/kg/day Blood Phe was determined 3 timesBlood Phe was determined 3 times
E280K/E280K mg/dl mol/L
Before Neophe zero 1 26.1 1566
zero 2 29.36 1761
zero 3 27.2 1632
Neophe 24 hrs 16.04 962
72 hrs 16 960
96 hrs 8.4 504
1 week 8.8 528
Placebo
24 hrs72 hrs96 hrs1 week
21.2 24.1 23.6 22.5
1272144614161350
299C/IVS12 nt1 g>a mg/dl mol/L
Before Neophe zero 1 25.6 1536
zero 2 32.9 1974
zero 3 26.8 1608
Neophe 24 hrs 14.3 858
72 hrs 16.8 1008
96 hrs 18.1 1086
1 week 12.2 732
Placebo 24 hrs72 hrs96 hrs1 week
19.220.624.823.7
1152123614881422
F299C/- mg/dl mol/L
Before Neophe zero 1 16.09 965.4
zero 2 18.1 1086
zero 3 17.2 1032
NeoPhe 24 hrs 12 720
72 hrs 14.1 846
96 hrs 10.1 606
1 week 11.4 684
Placebo
24 hrs 72 hrs96 hrs1 week
16.218.217.1
16.12
97210921026967.2
I65T/R408W mg/dl mol/L
Before Neophe zero 1 24.1 1446
zero 2 23.0 1380
zero 3 21.1 1266
Neophe 24 hrs 12.8 768
72 hrs 11.2 672
96 hrs 12.9 774
1 week 13.5 810
Placebo 24 hrs72 hrs96 hrs1 week
18.222.219.122.3
1092133211461339
Zeroµmol/l
NeoPheµmol/l
Placeboµmol/l
E280K/E280K 1653 738.5 1350
F299C/IVS12ntgl>a 1706 921 1422
F299C/- 1027.8 712 967.2
I65T/R408W 1364 752 1339.2
Summary of Average Blood Summary of Average Blood PhePhe
0
200
400
600
800
1000
1200
1400
1600
1800
zero 1 week 2 week
On Neo Phe Placebo
mic
rom
ol/L
E280K/E280K
F299C/IVS12ntg1>a
F299C/-
I65T/R408W
Summary of Double Blind StudySummary of Double Blind Study
ConclusionConclusion
LNAA can reduce blood Phe levels when LNAA can reduce blood Phe levels when given with mealsgiven with meals
Longer term double-blind placebo control Longer term double-blind placebo control studies are neededstudies are needed
Establishing the efficacy and safety of LNAA Establishing the efficacy and safety of LNAA can improve treatment of PKUcan improve treatment of PKU
AcknowledgementAcknowledgement
This study was supported in part by grants This study was supported in part by grants from Mid-Atlantic Connection for PKU and from Mid-Atlantic Connection for PKU and Allied Diseases (MACPAD) & South Texas Allied Diseases (MACPAD) & South Texas Association for PKU and Allied Disease Association for PKU and Allied Disease (STAPAD)(STAPAD)
Generous supply of NeoPhe was given by Generous supply of NeoPhe was given by PreKulab, DenmarkPreKulab, Denmark