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ICIEM 2006 Double Blind Placebo Control Trial in PKU with NeoPhe Reuben Matalon1, Kimberlee Michals-Matalon1, Alberto Burlina2, Alesandro Burlina2, Marcello Giovannini3, Laura Fiori3, Elena Grechanina4, Peter Novikov5, James Grady1, Stephen Tyring6, Flemming Guttler7 1University of Texas Medical Branch, 2University of Padova, 3University of Milan, 4University of Kharkiv, 5University of Moscow, 6University of Texas, 7Kennedy Institute Large Neutral Amino Acids (LNAA) Phenylalanine (Phe) Leucine Tyrosine Tryptophan Methionine Histidine Isoleucine Valine Threonine Transport of LNAA to the Brain Km mmol/L Phenylalanine (Phe) 0.12 Leucine 0.15 Tyrosine 0.16 Tryptophan 0.19 Methionine 0.19 Histidine 0.28 Isoleucine 0.33 Valine 0.63 Threonine 0.73 Pardridge, Inborn Errors of Metabolism in Humans. MTP Press, 1980. Km app 0.45 0.53 0.58 0.71 0.77 1.10 1.30 2.50 3.00 Andersen AE, Avins L LNAA injected to rat pups Phenylalanine hydroxylase was ihibited by parachlorophenylalanine Brain phenylalanine decreased 1976 Arch Neurology 33:684 Tyrosine in The Treatment of PKU Lou et al used Tyr 160 mg/kg in treated patients with PKU Increased attention span Increased dopamine synthesis 1987 Acta Paediatr Scand 76:560 Tyrosine in Treatment of PKU Pietz et al. used high dose tyrosine in adults with PKU and high blood Phe No difference in treated group vs placebo 1995 J Pediatr 127:936 Tryptophan in Treated PKU 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 1988 Dietary Phenylalanine and Brain Function. Birkhauser LNAA Supplementation in 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 Lysine was limiting amino acid 1995 J Inherit Metab Dis 18:127 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 Absolute and apparent Km values of neutral amino acids for the neutral amino acid transporter in the BBB (Partridge, 1980)0 Amino acid Typical Km App Km plasma level (mM) (mM) (mM) LNAA’s Phe 0.05 0.12 0.45 Leu 0.10 0.15 0.53 Tyr Trp Met 0.09 0.10 0.04 0.16 0.16 0.19 0.58 0.71 0.77 Isoleu Val Thr 0.07 0.14 0.19 0.33 0.63 0.73 1.3 2.5 3.0 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 His Arg 0.05 0.10 0.28 0.09 1.1 0.40 Lys 0.30 0.10 0.25 LNAA Transport in Intestinal Mucosa Km mmol/L Phenylalanine 1.0 Leucine 2.0 Valine 3.0 Methionine 5.0 Histidine 6.0 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. Amino acid inhibition of Phe transport in Caco-2cells – 10uM Phe in buffer applied to monolayers in presence of 1 mM concentration of each amino acid Inhibitor LNAA’s % inhibition Leu Tyr Trp 55% 45% 36% Basis Aa’s Lys His 50% 33% Hidalgo Biochem Biophys. Acta 1008: 5-30a (1990) PKU Mice on NeoPhe phe mg/dl Mice Control NeoPhe 78- 80- 83- 86- 159- 162- F 577 F 579 F 582 23.8 23.7 28.8 21.4 19.4 25.1 28.4 21.9 22.2 24.9 33.1 20.9 18 8.3 10.3 11 14.8 8.3 F 584 23.8 30.7 7.8 12.4 30 25.3 PKU Mice on NeoPhe phe mg/dl Mice Control NeoPhe 78- 80- 83- 86- 159- 162- F 585 F 586 F 588 20.6 23.2 21.6 21.7 25.7 21.7 24.4 21.2 24.2 19.8 21.9 24.4 8.5 13.5 10.9 12.3 11.3 10.9 Avg each time pt Avg all Pre-LNAA Avg all Post-LNAA 23.6 24.1 11.3 23.9 23.6 25.1 11 11.6 Pre- and Post-LNAA Blood Phe Levels 35 Pre-LNAA Post-LNAA Blood Phe [mg/dl] 30 25 20 15 10 5 0 1 2 3 4 5 6 Russia LNAA STUDY Time Phe Tyr KA 0’ 3 days µmol/l 718.8 668.4 mg/dl 11.98 11.14 µmol/l 53.9 91.3 mg/dl 0.98 1.66 3 days 3 days 523.2 376.2 8.72 6.27 103.4 108.3 1.88 1.97 Russia LNAA STUDY Time Phe Tyr KN µmol/l mg/dl µmol/l mg/dl 0’ 707.4 11.79 42.9 0.78 3 days 3 days 3 days 607.2 572.4 585.6 10.12 9.54 9.76 126.5 159.5 83.6 2.30 2.91 1.52 Russia LNAA Study Time Phe Tyr KH µmol/l mg/dl µmol/l mg/dl 0’ 635.4 10.59 33.0 0.60 3 days 554.4 9.24 242.0 4.40 3 days 322.2 5.37 94.6 1.72 3 days 136.2 2.27 110.0 2.00 3 days 102.6 1.71 94.0 1.71 Response of Blood Phe to LNAA Ukraine 25 20 15 10 5 0 21 yo girl µmol/l 0 24h 21 14.5 1260 870 72hr 1 wk 2 wk 3 wk 4 wk 16 7.9 960 474 12.7 762 7.1 13 426 780 Response on Phe on LNAA Ukraine 25 20 15 10 5 0 0 36hr 72h 1wk 12 yo boy 20 13.8 12.75 10.4 µmol/l 1200 828 765 624 USA LNAA STUDY Time Phe Tyr GDL µmol/l mg/dl µmol/l mg/dl 0’ 2 days 4 days 1290.6 1198.2 115.8 21.51 19.97 1.93 69.8 73.7 140.25 1.27 1.34 2.55 KM 0’ 1540.2 25.67 30.8 0.56 8 days 883.8 14.37 53.8 0.98 0’ 2 days 1978.2 1608.6 32.97 26.81 68.7 207.35 1.25 3.77 USA LNAA STUDY Time Phe Try ES µmol/l mg/dl µmol/l mg/dl 0’ 1375.8 22.93 31.9 0.58 4-7 days 767.4 12.79 121.5 2.12 0’ 965.4 16.09 58.8 1.07 2 days 828.6 13.81 156.2 2.84 RC US Blood Phe and Tyr NeoPhe Patient K 1 Week µmol/L (mg) Control phe 1978.1(32.97) 1139.6 (25.66) 1456.2 (24.27) tyr 1.25 0.62 0.62 NeoPhe phe tyr µmol/L (mg) 1356.0 (22.6) 1308 (21.8) 1146 (19.1) 24% reduction 5.0 4.1 3.82 US Blood Phe and Tyr NeoPhe Patient G 1 Week µmol/L (mg) Control phe mg/dl tyr NeoPhe phe mg/dl tyr 1560 (26.0) 0.92 953 (15.89) 4.35 1764 (29.4) 1.9 505 (8.43) 3.32 56% reduction US Blood Phe and Tyr NeoPhe Patient K 1 Week µmol/L (mg) Control phe 1978.1(32.97) 1139.6 (25.66) 1456.2 (24.27) tyr 1.25 0.62 0.62 NeoPhe phe tyr µmol/L (mg) 1356.0 (22.6) 1308 (21.8) 1146 (19.1) 24% reduction 5.0 4.1 3.82 US Blood Phe and Tyr NeoPhe Patient G 1 Week µmol/L (mg) Control phe mg/dl tyr NeoPhe phe mg/dl tyr 1560 (26.0) 0.92 953 (15.89) 4.35 1764 (29.4) 1.9 505 (8.43) 3.32 56% reduction Figure 1. Blood Phe Response to 0.5g/kg NeoPhe in Patients with PKU 2000 1800 1600 IVS12nt1g>a/R261Q IVS12nt1g>a/Y356X IVS12ntg>a/IVS10nt11g>a E280K/R408W IVS12nt1g>a/IVS12nt1g>a R261Q/R408W R408W/R408W IVS4ntg>t/R408W umol/L 1400 1200 1000 800 R408W/R408W E280K/E280K F299C/IVS12nt1g>a I65T/R408W F299C/unk 600 400 200 0 Zero Time 1 week Blood Phe Paired t-test: p=0.001 Figure 2. Blood Phe Response to 1.0 g/kg NeoPhe in Patients with PKU 1800 1600 1400 umol/L 1200 IVS12nt1g>a/R261Q IVS12nt1g>a/Y356X IVS12nt1g>a/IVS10nt11g>a E280K/R408W IVS12nt1g>a/IVS12nt1g>a ND R408W/R252W 1000 800 600 400 200 0 Zero Time 1 week Blood Phe Paired t-test: p=0.006 Double-Blind Placebo Control on patients in the US Patients were genotyped Baseline Phenylalanine was determined 3 times Placebo or Neophe was administered for one week, 1tablet/kg/day Blood Phe was determined 3 times mg/dl mmol/L zero 1 26.1 1566 zero 2 29.36 1761 zero 3 27.2 1632 24 hrs 16.04 962 72 hrs 16 960 96 hrs 8.4 504 1 week 8.8 528 24 hrs 72 hrs 96 hrs 1 week 21.2 24.1 23.6 22.5 1272 1446 1416 1350 E280K/E280K Before Neophe Neophe Placebo mg/dl mmol/L zero 1 25.6 1536 zero 2 32.9 1974 zero 3 26.8 1608 24 hrs 14.3 858 72 hrs 16.8 1008 96 hrs 18.1 1086 1 week 12.2 732 24 hrs 72 hrs 96 hrs 1 week 19.2 20.6 24.8 23.7 1152 1236 1488 1422 299C/IVS12 nt1 g>a Before Neophe Neophe Placebo mg/dl mmol/L zero 1 16.09 965.4 zero 2 18.1 1086 zero 3 17.2 1032 24 hrs 12 720 72 hrs 14.1 846 96 hrs 10.1 606 1 week 11.4 684 24 hrs 72 hrs 96 hrs 1 week 16.2 18.2 17.1 16.12 972 1092 1026 967.2 F299C/Before Neophe NeoPhe Placebo mg/dl mmol/L zero 1 24.1 1446 zero 2 23.0 1380 zero 3 21.1 1266 24 hrs 12.8 768 72 hrs 11.2 672 96 hrs 12.9 774 1 week 13.5 810 24 hrs 72 hrs 96 hrs 1 week 18.2 22.2 19.1 22.3 1092 1332 1146 1339 I65T/R408W Before Neophe Neophe Placebo Summary of Average Blood Phe Zero µmol/l NeoPhe µmol/l Placebo µmol/l E280K/E280K 1653 738.5 1350 F299C/IVS12ntgl>a 1706 921 1422 1027.8 712 967.2 1364 752 1339.2 F299C/- I65T/R408W Summary of Double Blind Study 1800 1600 micromol/L 1400 1200 E280K/E280K 1000 F299C/IVS12ntg1>a 800 F299C/- 600 I65T/R408W 400 200 0 zero 1 week On Neo Phe 2 week Placebo Conclusion LNAA can reduce blood Phe levels when given with meals Longer term double-blind placebo control studies are needed Establishing the efficacy and safety of LNAA can improve treatment of PKU 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) Generous supply of NeoPhe was given by PreKulab, Denmark