Double Blind Placebo Control Double Blind Placebo Control Trial in PKU with NeoPheTrial in PKU with NeoPhe

Reuben MatalonReuben Matalon11, Kimberlee Michals-Matalon, Kimberlee Michals-Matalon11, , Alberto BurlinaAlberto Burlina22, Alesandro Burlina, Alesandro Burlina22, Marcello , Marcello

GiovanniniGiovannini33, Laura Fiori, Laura Fiori33, Elena Grechanina, Elena Grechanina44, Peter , Peter NovikovNovikov55, James Grady, James Grady11, Stephen Tyring, Stephen Tyring66, Flemming , Flemming

GuttlerGuttler77, , Cláudia Braga8

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

Institute, Institute, Diagnósticos Laboratoriais Especializados, Brazil8

Large Neutral Amino Acids Large Neutral Amino Acids (LNAA)(LNAA)

• Phenylalanine (Phe) Phenylalanine (Phe) • LeucineLeucine• TyrosineTyrosine• TryptophanTryptophan• MethionineMethionine• HistidineHistidine• IsoleucineIsoleucine• ValineValine• ThreonineThreonine

Transport of LNAA to the Transport of LNAA to the BrainBrain

• 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 Phenylalanine hydroxylase was ihibited by ihibited by parachlorophenylalanineparachlorophenylalanine

• Brain phenylalanine decreased Brain phenylalanine decreased

1976 Arch Neurology 33:6841976 Arch Neurology 33:684

Tyrosine in The Tyrosine in The Treatment of PKUTreatment 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 Tyrosine in Treatment of PKUPKU

• Pietz et al. used high dose tyrosine in Pietz et al. used high dose tyrosine in adults with PKU and high blood Pheadults with PKU 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 Tryptophan in Treated PKUPKU

• Nielsen et al used tryptophan 4.5 Nielsen et al used tryptophan 4.5 gm/day to treated PKU for 3 weeks gm/day to treated PKU for 3 weeks

• Showed a 3 fold increase in 5-HIAA in Showed a 3 fold increase in 5-HIAA in CSF despite high blood PheCSF despite high blood Phe

1988 Dietary Phenylalanine and Brain 1988 Dietary Phenylalanine and Brain Function. BirkhauserFunction. Birkhauser

VIL in Treatment of PKUVIL in Treatment of PKU

• 1990 Berry et al used VIL (valine 150 1990 Berry et al used VIL (valine 150 mg/kg, isoleucine 150 mg/kg and leucine mg/kg, isoleucine 150 mg/kg and leucine 200 mg/kg) to reduce phe entry to brain200 mg/kg) to reduce phe entry to brain

• Substantial lowering of CSF Phe foundSubstantial lowering of CSF Phe found

• Tyrosine also was also substantially Tyrosine also was also substantially reduced in CSFreduced in CSF

• Am J Dis Child 144:539Am J Dis Child 144:539

LNAA Supplementation LNAA Supplementation in PKUin 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 protein diet 0.6 gm/kg on 4 patients with PKUwith PKU

• After 1 month subjects found with After 1 month subjects found with negative nitrogen balancenegative nitrogen balance

• Lysine was limiting amino acidLysine was limiting amino acid

1995 J Inherit Metab Dis 18:1271995 J Inherit Metab Dis 18:127

LNAA Supplementation LNAA Supplementation in PKUin PKU• Pietz et al. 6 males with PKU given a load (100 Pietz et al. 6 males with PKU given a load (100

mg/kg Phe) with and with out LNAAmg/kg Phe) with and with out LNAA

• Limited brain Phe entry by MRSLimited brain Phe entry by MRS

• EEG normal with LNAAEEG normal with LNAA

• EEG slowing without LNAAEEG slowing without LNAA

• Radiology 1996, 201:413Radiology 1996, 201:413

Km (app) – Km (1 + Km (app) – Km (1 + ∑[aa]/Km]∑[aa]/Km]

This predicts that, if the plasma This predicts that, if the plasma level of an LNAA is much less level of an LNAA is much less than its value of Km, then that than its value of Km, then that amino acid will not compete amino acid will not compete effectively for the carrier proteineffectively for the carrier protein

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)0transporter in the BBB (Partridge, 1980)0

Amino acidAmino acid Typical Typical plasma plasma level (mM)level (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 Absolute and apparent Km values of neutral amino acids for the of neutral amino acids for the neutral amino acid transporter in neutral amino acid transporter in the BBB (Partridge, 1980)the BBB (Partridge, 1980)

Amino acidAmino acid Typical Typical plasma plasma level (mM)level (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 LNAA Transport in Intestinal Mucosa KMucosa Kmm mmol/L mmol/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 Competition effect is not likely to

occur in tissue other than brain unless occur in tissue other than brain unless high concentration of amino acids is high concentration of amino acids is usedused

Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980.

Amino acid inhibition of Phe Amino acid inhibition of Phe transport in Caco-2-cells – 10uM Phe transport in Caco-2-cells – 10uM Phe in buffer applied to monolayers in in buffer applied to monolayers in presence of 1 mM concentration of presence of 1 mM concentration of each amino acideach amino acidInhibitorInhibitor % 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 Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)(1990)

Genotype of ENU2 Mice

N HET HOMO

(F263S)

LNAA in Treatment of Mice LNAA in Treatment of Mice with PKUwith PKU

• ENU2/ENU2 mice with PKUENU2/ENU2 mice with PKU

• Before treatment blood phe and tyrBefore treatment blood phe and tyr

• LNAA given in form of PreKUnilLNAA given in form of PreKUnil

• Dose 0.5 g/kg or 1.0 g/kgDose 0.5 g/kg or 1.0 g/kg

• Blood phe and tyr up to 6 weeks post Blood phe and tyr up to 6 weeks post treatment treatment

78-78- 80-80- 83-83- 86-86- 159-159- 162-162-

F 577F 577 23.823.821.21.

44 19.419.4 24.924.9 1818 1111

F 579F 579 23.723.725.25.

11 28.428.4 33.133.1 8.38.3 14.814.8

F 582F 582 28.828.821.21.

99 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 NeoPhe NeoPhe

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 NeoPhe NeoPhe

Pre- and Post-LNAA Blood Phe Levels in mice

0

5

10

15

20

25

30

35

1 2 3 4 5 6

Blo

od

Ph

e [

mg

/dl]

Pre-LNAA Post-LNAA

Double Blind Placebo Double Blind Placebo Control Trial in PKU with Control Trial in PKU with

NeoPhe in USNeoPhe in US

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 Figure 2. Blood Phe Response to 1.0 g/kg NeoPhe in Patients with1.0 g/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 Double-Blind Placebo Control on patients in the USon patients in the US

• Patients were genotyped Patients were genotyped

• Baseline Phenylalanine was Baseline Phenylalanine was determined 3 timesdetermined 3 times

• Placebo or Neophe was Placebo or Neophe was administered for one week, administered for one week, 1tablet/kg/day1tablet/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 Summary of Double Blind Study in USStudy in US

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 Longer term double-blind placebo control studies are neededcontrol studies are needed

• Establishing the efficacy and safety of Establishing the efficacy and safety of LNAA can improve treatment of PKULNAA can improve treatment of PKU

Double-Blind Study in Other Double-Blind Study in Other CentersCenters

•BrazilBrazil

•RussiaRussia

•UkraineUkraine

•ItalyItaly

EXPERIENCE IN BRAZIL

WITH LARGE NEUTRAL AMINO ACIDS 

Cláudia Braga1,2, Armando Fonseca1,2, Maria de Fátima Santos de Oliveira1, Maria Helena Tossman1, Maria Célia Appel1, Eduardo Vieira Neto2

Associação de Pais e Amigos dos Excepcionais (APAE-Rio)1

Diagnósticos Laboratoriais Especializados (DLE)2

Rio de Janeiro, Brasil

Apae-Rio

MethodsWe studied three patients (1male, 2 females), aged 13-14y, in a double blind study, in four weeks as the protocol:

Week 1: specific diet (aminoacids formula) Week 2: specific diet and placebo or NeoPhe (1 pill/Kg) Week 3: specific diet Week 3: specific diet and placebo or NeoPhe

PKU concentrations were determined three times a week during all the weeks by an enzymatic colorimetric method (NeoLISA PKU Kit- Intercientífica) in duplicate. All the aminoacids were determined also by tandem mass spectrometry in day five of every week.

One patient (case 3) presented vomits in the fourth week (NeoPhe)

Case 1. Case 1. RSLRSL

0

2

4

6

8

10

12

14

week 1 week 2 placebo

week 3 interval

week 4 NeoPhe

day 1

day 3

day 5

PK

U m

g%

En

zim

atic

me

tho

d

11,7mg% 7,95mg% 8,48mg% 6,5mg%PKU

average

Case 1. Phenylalanine Case 1. Phenylalanine Tandem mass spectrometryTandem mass spectrometry

0100

200300400

500600

700800900

week 1 week 2placebo

week 3interval

week 4NeoPhe

PKU mcmolL

44,5 %

Case 1. Tyrosine Case 1. Tyrosine Tandem mass spectrometryTandem mass spectrometry

0

10

20

30

40

50

60

70

week 1 week 2placebo

week 3interval

week 4NeoPhe

Tyr mcmol/L

Case 2. Case 2. TSATSA

0123

456

789

week 1 week 2NeoPhe

week 3interval

week 4placebo

day 1

day 3

day 5

6,9 mg% 4,7 mg% 6,5 mg% 7,46mg%

PK

U m

g%

En

zim

atic

me

tho

d

PKUaverage

32%

Case 2. Phenylalanine Tandem mass espectrometry

0

100

200

300

400

500

600

700

week 1 week2NeoPhe

week 3interval

week 4placebo

PKU mcmolL

Case 2. TyrosineTandem mass spectrometry

0

20

40

60

80

100

120

140

160

week 1 week 2NeoPhe

week 3interval

week 4placebo

Tyr mcmol/L

““

““Experience in Russia with Experience in Russia with Large Neutral Amino Acids”Large Neutral Amino Acids”

Peter NovikovPeter NovikovMoscow Research Institute Moscow Research Institute

of Pediatrics and Child of Pediatrics and Child Surgery, Moscow, RussiaSurgery, Moscow, Russia

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

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

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

Response Phe levels in PKU patients with NeoPhe administration (Russia)

Patient

AgeGender

Genotype

Mean Phe levels

(µmol/l) pre NeoPhe

Phe levels (µmol/l)

post NeoPhe

Decrease of Phe blood

levels (%)

Phe levels (µmol/l)

post Placebo

1 22 F R408W/ R252W 1261,19 782,6 37,9 -

2 12 M Not detected 1264,2 626,08 50,5 -

3 11 FR408W / R408W

1023,4692,3 32,4 -

4 12 FR408W / R408W

1023,4662,2 35,3 903

5 19 F R261X/R408W 1143,8 933,1 * 18,4 -

Means

15,2 1143,19 739,29 35,3 903

* - The patient stopped to receipt NeoPhe because girl has complications

ConclusionsConclusions• LNAA can decrease blood Phe levels in LNAA can decrease blood Phe levels in patients with classical PKUpatients with classical PKU

• Decreasing of Blood Phe levels is Decreasing of Blood Phe levels is accompanied by increased of blood Tyr levelsaccompanied by increased of blood Tyr levels

• The grade of the decrease of Phe levels The grade of the decrease of Phe levels can be higher after the short period without can be higher after the short period without LNAALNAA

• It will be necessary to check the different It will be necessary to check the different hypotheses of LNAA effectshypotheses of LNAA effects

Double-Blind Placebo study Double-Blind Placebo study in Ukarinein Ukarine

Elena Grechanina and Irene Novikova Elena Grechanina and Irene Novikova

University of Kharkiv, UkarineUniversity of Kharkiv, Ukarine

0

200

400

600

800

1000

1200

1400

Pre NeoPhe Post NeoPhe

R408W/ R252W

Not detected

R408W / R408W

R408W / R408W

R261X/R408W

Blood Phe response to 0.5 g/kg NeoPhe in 5 PKU patients

mol/

L

blood phe concentration

Response to NeoPhe : Double-Blind Study, Ukraine

Large Neutral Amino Acids – a novel treatment for PKU

Marcello Giovannini, Laura Fiori

Department of Pediatrics, San Paolo Hospital, University of Milan, Italy

   Week 1, on caps

(phe mg% - mcM/L)

Week 2, (no caps)

Week 3, on caps (phe mg% - mcM/L)

Basal Basal 16.7 - 984 - 18.3 - 1098

    

16.4 – 984 - 14.9 - 894

14.4 – 864 

- 11.9 – 714

12.9 – 774 

- 13.4 - 804

End of week 

12.7 - 762  -

15 - 900

Pl. phe decrease %

22%   18%(max 34%)

F, age 29

BW: 49 kg.

LNAA: 1/kg BW

LNAA

R158Q / -

NeoPhe-Placebo study in Italy- Padova

Alessandra Burlina and Alberto Burlina

Response to NeoPhe: Double-Response to NeoPhe: Double-Blind StudyBlind Study

Patient Age Baseline NeoPhe Placebo%

decrease

MC 26 1004 787.3 937.5 21.58

NV 15 597 157 554 73.70

BL 21 926 1225

AL 20 1067 1375

AcknowledgementAcknowledgement

• This study was supported in part by grants from This study was supported in part by grants from Mid-Atlantic Connection for PKU and Allied Mid-Atlantic Connection for PKU and Allied Diseases (MACPAD) & South Texas Association Diseases (MACPAD) & South Texas Association for PKU and Allied Disease (STAPAD)for PKU and Allied Disease (STAPAD)

• Generous supply of NeoPhe was given by Generous supply of NeoPhe was given by PreKulab, DenmarkPreKulab, Denmark

• To all Centers in Brazil, Italy, Ukraine and Russia To all Centers in Brazil, Italy, Ukraine and Russia for their participation in the studyfor their participation in the study