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)0Amino acidAmino acid Typical Typical

plasma level plasma level (mM)(mM)

KmKm(mM)(mM)

App KmApp Km(mM)(mM)

LNAA’sLNAA’sPhePhe 0.050.05 0.120.12 0.450.45LeuLeu 0.100.10 0.150.15 0.530.53TyrTyr 0.090.09 0.160.16 0.580.58TrpTrp 0.100.10 0.160.16 0.710.71MetMet 0.040.04 0.190.19 0.770.77IsoleuIsoleu 0.070.07 0.330.33 1.31.3ValVal 0.140.14 0.630.63 2.52.5ThrThr 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’sHisHis 0.050.05 0.280.28 1.11.1ArgArg 0.100.10 0.090.09 0.400.40LysLys 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% inhibitionLNAA’sLNAA’sLeuLeu 55%55%TyrTyr 45%45%TrpTrp 36%36%

Basis Aa’sBasis Aa’sLysLys 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 1111F 579F 579 23.723.7 25.125.1 28.428.4 33.133.1 8.38.3 14.814.8F 582F 582 28.828.8 21.921.9 22.222.2 20.920.9 10.310.3 8.38.3F 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 12.312.3F 586F 586 23.223.2 25.725.7 21.221.2 21.921.9 13.513.5 11.311.3F 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 11.611.6Avg all Pre-LNAAAvg all Pre-LNAA 24.124.1Avg 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 P

he [m

g/dl

]

Pre-LNAA Post-LNAA

TimeTime PhePhe TyrTyr

KAKA µmol/lµmol/l mg/dlmg/dl µmol/lµmol/l mg/dlmg/dl0’0’ 718.8718.8 11.9811.98 53.953.9 0.980.983 days3 days 668.4668.4 11.1411.14 91.391.3 1.661.663 days3 days 523.2523.2 8.728.72 103.4103.4 1.881.883 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 0.780.783 days3 days 607.2607.2 10.1210.12 126.5126.5 2.302.303 days3 days 572.4572.4 9.549.54 159.5159.5 2.912.913 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 1.271.272 days2 days 1198.21198.2 19.9719.97 73.773.7 1.341.344 days4 days 115.8115.8 1.931.93 140.25140.25 2.552.55

KMKM0’0’ 1540.21540.2 25.6725.67 30.830.8 0.560.568 days8 days 883.8883.8 14.3714.37 53.853.8 0.980.980’0’ 1978.21978.2 32.9732.97 68.768.7 1.251.252 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

RCRC0’0’ 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) 5.05.01139.6 (25.66)1139.6 (25.66) 0.620.62 1308 (21.8)1308 (21.8) 4.14.11456.2 (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 NeoPheNeoPhephephe tyrtyr phephe tyrtyr

mg/dlmg/dl mg/dlmg/dl1560 (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) 4.14.11456.2 (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 NeoPheNeoPhephephe tyrtyr phephe tyrtyr

mg/dlmg/dl mg/dlmg/dl1560 (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

0200400600800

100012001400160018002000

Zero Time 1 week

Blood Phe

umol

/L

IVS12nt1g>a/R261QIVS12nt1g>a/Y356XIVS12ntg>a/IVS10nt11g>aE280K/R408WIVS12nt1g>a/IVS12nt1g>aR261Q/R408WR408W/R408WIVS4ntg>t/R408WR408W/R408WE280K/E280KF299C/IVS12nt1g>aI65T/R408WF299C/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/R261QIVS12nt1g>a/Y356XIVS12nt1g>a/IVS10nt11g>aE280K/R408WIVS12nt1g>a/IVS12nt1g>aNDR408W/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

0200400600800

10001200140016001800

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