double blind placebo control trial in pku with neophe
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Double Blind Placebo Control Trial in PKU with NeoPhe. Reuben Matalon 1 , Kimberlee Michals-Matalon 1 , Alberto Burlina 2 , Alesandro Burlina 2 , Marcello Giovannini 3 , Laura Fiori 3 , Elena Grechanina 4 , Peter Novikov 5 , James Grady 1 , Stephen Tyring 6 , Flemming Guttler 7 , Cláudia Braga 8 - PowerPoint PPT PresentationTRANSCRIPT
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