2. 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, 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 1 University of Texas Medical Branch, 2 University of Padova, 3 University of Milan, 4 University of Kharkiv, 5 University of Moscow, 6 University of Texas, 7 Kennedy Institute, 1 University of Texas Medical Branch, 2 University of Padova, 3 University of Milan, 4 University of Kharkiv, 5 University of Moscow, 6 University of Texas, 7 Kennedy Institute, Diagnósticos Laboratoriais Especializados, Brazil 8

3. Large Neutral Amino Acids (LNAA) Phenylalanine (Phe) Phenylalanine (Phe) Leucine Leucine Tyrosine Tyrosine Tryptophan Tryptophan Methionine Methionine Histidine Histidine Isoleucine Isoleucine Valine Valine Threonine Threonine

4. Transport of LNAA to the Brain Phenylalanine (Phe) 0.12 0.45 Phenylalanine (Phe) 0.12 0.45 Leucine0.150.53 Leucine0.150.53 Tyrosine0.160.58 Tyrosine0.160.58 Tryptophan0.190.71 Tryptophan0.190.71 Methionine0.190.77 Methionine0.190.77 Histidine0.281.10 Histidine0.281.10 Isoleucine0.331.30 Isoleucine0.331.30 Valine0.632.50 Valine0.632.50 Threonine 0.733.00 Threonine 0.733.00 K m mmol/L K m app Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980.

5. Andersen AE, Avins L LNAA injected to rat pups LNAA injected to rat pups Phenylalanine hydroxylase was ihibited by parachlorophenylalanine Phenylalanine hydroxylase was ihibited by parachlorophenylalanine Brain phenylalanine decreased Brain phenylalanine decreased 1976 Arch Neurology 33:684

6. Tyrosine in The Treatment of PKU Lou et al used Tyr 160 mg/kg in treated patients with PKU Increased attention span Increased attention span Increased dopamine synthesis Increased dopamine synthesis 1987 Acta Paediatr Scand 76:560

7. Tyrosine in Treatment of PKU Pietz et al. used high dose tyrosine in adults with PKU and high blood Phe Pietz et al. used high dose tyrosine in adults with PKU and high blood Phe No difference in treated group vs placebo No difference in treated group vs placebo 1995 J Pediatr 127:936

8. Tryptophan in Treated PKU Nielsen et al used tryptophan 4.5 gm/day to treated PKU for 3 weeks 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 Showed a 3 fold increase in 5-HIAA in CSF despite high blood Phe 1988 Dietary Phenylalanine and Brain Function. Birkhauser

9. VIL in Treatment of PKU 1990 Berry et al used VIL (valine 150 mg/kg, isoleucine 150 mg/kg and leucine 200 mg/kg) to reduce phe entry to brain 1990 Berry et al used VIL (valine 150 mg/kg, isoleucine 150 mg/kg and leucine 200 mg/kg) to reduce phe entry to brain Substantial lowering of CSF Phe found Substantial lowering of CSF Phe found Tyrosine also was also substantially reduced in CSF Tyrosine also was also substantially reduced in CSF Am J Dis Child 144:539 Am J Dis Child 144:539

10. LNAA Supplementation in PKU Dotremont et al. used LNAA and a low protein diet 0.6 gm/kg on 4 patients with 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 After 1 month subjects found with negative nitrogen balance Lysine was limiting amino acid Lysine was limiting amino acid 1995 J Inherit Metab Dis 18:127

11. LNAA Supplementation in PKU Pietz et al. 6 males with PKU given a load (100 mg/kg Phe) with and with out LNAA Pietz et al. 6 males with PKU given a load (100 mg/kg Phe) with and with out LNAA Limited brain Phe entry by MRS Limited brain Phe entry by MRS EEG normal with LNAA EEG normal with LNAA EEG slowing without LNAA EEG slowing without LNAA Radiology 1996, 201:413 Radiology 1996, 201:413

12. 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 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

13. 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 Phe0.050.120.45 Leu0.100.150.53 Tyr0.090.160.58 Trp0.100.160.71 Met0.040.190.77 Isoleu0.070.331.3 Val0.140.632.5 Thr0.190.733.0

14. 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 His0.050.281.1 Arg0.100.090.40 Lys0.300.100.25

15. LNAA Transport in Intestinal Mucosa K m mmol/L Phenylalanine1.0 Phenylalanine1.0 Leucine2.0 Leucine2.0 Valine3.0 Valine3.0 Methionine5.0 Methionine5.0 Histidine6.0 Histidine6.0 Competition effect is not likely to occur in tissue other than brain unless high concentration of amino acids is used 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.

16. 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 Leu55% Tyr45% Trp36% Basis aa’s Lys50% His33% Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990)

17. Genotype of ENU2 Mice N HETHOMO (F263S)

18. LNAA in Treatment of Mice with PKU ENU2/ENU2 mice with PKU ENU2/ENU2 mice with PKU Before treatment blood phe and tyr Before treatment blood phe and tyr LNAA given in form of PreKUnil LNAA given in form of PreKUnil Dose 0.5 g/kg or 1.0 g/kg Dose 0.5 g/kg or 1.0 g/kg Blood phe and tyr up to 6 weeks post treatment Blood phe and tyr up to 6 weeks post treatment

19. 78-80-83-86-159-162- F 577 23.821.419.424.91811 F 579 23.725.128.433.18.314.8 F 582 28.821.922.220.910.38.3 F 584 23.83025.330.77.812.4 PKU Mice on NeoPhe phe mg/dl Mice Control NeoPhe

24. 78-80-83-86-159-162- F 585 20.621.724.419.88.512.3 F 586 23.225.721.221.913.511.3 F 588 21.621.724.224.410.910.9 Avg each time pt 23.623.923.625.11111.6 Avg all Pre-LNAA 24.1 Avg all Post-LNAA 11.3 PKU Mice on NeoPhe phe mg/dl Mice Control NeoPhe

26. Double Blind Placebo Control Trial in PKU with NeoPhe in US

27. Figure 1. Blood Phe Response to 0.5g/kg NeoPhe in Patients with PKU Paired t-test: p=0.001

28. Figure 2. Blood Phe Response to 1.0 g/kg NeoPhe in Patients with PKU Paired t-test: p=0.006

29. Double-Blind Placebo Control on patients in the US Patients were genotyped Patients were genotyped Baseline Phenylalanine was determined 3 times Baseline Phenylalanine was determined 3 times Placebo or Neophe was administered for one week, 1tablet/kg/day Placebo or Neophe was administered for one week, 1tablet/kg/day Blood Phe was determined 3 times Blood Phe was determined 3 times

30. E280K/E280K mg/dl  mol/L Before Neophezero 126.11566 zero 229.361761 zero 327.21632 Neophe24 hrs16.04962 72 hrs16960 96 hrs8.4504 1 week8.8528 Placebo 24 hrs 72 hrs 96 hrs 1 week 21.2 24.1 23.6 22.5 1272 1446 1416 1350

31. 299C/IVS12 nt1 g>a mg/dl  mol/L Before Neophezero 125.61536 zero 232.91974 zero 326.81608 Neophe24 hrs14.3858 72 hrs16.81008 96 hrs18.11086 1 week12.2732 Placebo24 hrs 72 hrs 96 hrs 1 week 19.2 20.6 24.8 23.7 1152 1236 1488 1422

32. F299C/- mg/dl  mol/L Before Neophezero 116.09965.4 zero 218.11086 zero 317.21032 NeoPhe24 hrs12720 72 hrs14.1846 96 hrs10.1606 1 week11.4684 Placebo 24 hrs 72 hrs 96 hrs 1 week 16.2 18.2 17.1 16.12 972 1092 1026 967.2

33. I65T/R408W mg/dl  mol/L Before Neophezero 124.11446 zero 223.01380 zero 321.11266 Neophe24 hrs12.8768 72 hrs11.2672 96 hrs12.9774 1 week13.5810 Placebo24 hrs 72 hrs 96 hrs 1 week 18.2 22.2 19.1 22.3 1092 1332 1146 1339

34. Zero µmol/l NeoPhe µmol/l Placebo µmol/l E280K/E280K 1653738.51350 F299C/IVS12ntgl>a 17069211422 F299C/- 1027.8712967.2 I65T/R408W 13647521339.2 Summary of Average Blood Phe

35. Summary of Double Blind Study in US

36. Conclusion LNAA can reduce blood Phe levels when given with meals LNAA can reduce blood Phe levels when given with meals Longer term double-blind placebo control studies are needed Longer term double-blind placebo control studies are needed Establishing the efficacy and safety of LNAA can improve treatment of PKU Establishing the efficacy and safety of LNAA can improve treatment of PKU

37. Double-Blind Study in Other Centers Brazil Brazil Russia Russia Ukraine Ukraine Italy Italy

38. EXPERIENCE IN BRAZIL WITH LARGE NEUTRAL AMINO ACIDS Cláudia Braga 1,2, Armando Fonseca 1,2, Maria de Fátima Santos de Oliveira 1, Maria Helena Tossman 1, Maria Célia Appel 1, Eduardo Vieira Neto 2 Associação de Pais e Amigos dos Excepcionais (APAE-Rio) 1 Diagnósticos Laboratoriais Especializados (DLE) 2 Rio de Janeiro, Brasil Apae-Rio

39. Methods We 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)

40. Case 1. RSL PKU mg% Enzimatic method 11,7mg%7,95mg%8,48mg%6,5mg% PKU average

41. Case 1. Phenylalanine Tandem mass spectrometry 44,5 %

42. Case 1. Tyrosine Tandem mass spectrometry

43. Case 2. TSA 6,9 mg%4,7 mg%6,5 mg%7,46mg% PKU mg% Enzimatic method PKU average 32%

44. Case 2. Phenylalanine Tandem mass espectrometry

45. Case 2. Tyrosine Tandem mass spectrometry

46. “ “Experience in Russia with Large Neutral Amino Acids” Peter Novikov Moscow Research Institute of Pediatrics and Child Surgery, Moscow, Russia

47. Russia LNAA Study TimePheTyr KHµmol/lmg/dlµmol/lmg/dl 0’635.410.5933.00.60 3 days 554.49.24242.04.40 322.25.3794.61.72 136.22.27110.02.00 102.61.7194.01.71

48. Time Time Phe PheTyr KNµmol/lmg/dlµmol/lmg/dl0’707.411.7942.90.78 3 days 607.210.12126.52.30 572.49.54159.52.91 585.69.7683.61.52 Russia LNAA STUDY

49. Time Phe PheTyr KAµmol/lmg/dlµmol/lmg/dl 0’718.811.9853.90.98 3 days 668.411.1491.31.66 523.28.72103.41.88 376.26.27108.31.97 Russia LNAA STUDY

50. Response Phe levels in PKU patients with NeoPhe administration (Russia) Patie nt Age Ge nde r 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 122FR408W/ R252W1261,19 782,637,9- 212MNot detected1264,2 626,0850,5- 311F R408W / R408W 1023,4 692,332,4- 412F R408W / R408W 1023,4 662,235,3903 519FR261X/R408W1143,8 933,1 *18,4- Mean s 15,21143,19739,29 35,3 903 * - The patient stopped to receipt NeoPhe because girl has complications

51. Conclusions Conclusions LNAA can decrease blood Phe levels in LNAA can decrease blood Phe levels in patients with classical PKU patients with classical PKU Decreasing of Blood Phe levels is accompanied by increased of blood Tyr levels Decreasing of Blood Phe levels is accompanied by increased of blood Tyr levels The grade of the decrease of Phe levels can be higher after the short period without LNAA The grade of the decrease of Phe levels can be higher after the short period without LNAA It will be necessary to check the different It will be necessary to check the different hypotheses of LNAA effects hypotheses of LNAA effects

52. Double-Blind Placebo study in Ukarine Elena Grechanina and Irene Novikova University of Kharkiv, Ukarine

53. 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

54. Large Neutral Amino Acids – a novel treatment for PKU Marcello Giovannini, Laura Fiori Department of Pediatrics, San Paolo Hospital, University of Milan, Italy

55. Week 1, on caps (phe mg% - mcM/L) Week 2, (no caps) Week 3, on caps (phe mg% - mcM/L) Basal16.7 - 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 / -

56. NeoPhe-Placebo study in Italy- Padova Alessandra Burlina and Alberto Burlina

57. Response to NeoPhe: Double-Blind Study PatientAgeBaselineNeoPhePlacebo % decrease MC261004787.3937.521.58 NV1559715755473.70 BL219261225 AL2010671375

58. Acknowledgement This study was supported in part by grants from Mid-Atlantic Connection for PKU and Allied Diseases (MACPAD) & South Texas Association for PKU and Allied Disease (STAPAD) This study was supported in part by grants from Mid-Atlantic Connection for PKU and Allied Diseases (MACPAD) & South Texas Association for PKU and Allied Disease (STAPAD) Generous supply of NeoPhe was given by PreKulab, Denmark Generous supply of NeoPhe was given by PreKulab, Denmark To all Centers in Brazil, Italy, Ukraine and Russia for their participation in the study To all Centers in Brazil, Italy, Ukraine and Russia for their participation in the study