Advances in UCD treatment and management of UCDs Brendan Lee M.D.,Ph.D. Howard Hughes Medical Institute Department of Molecular and Human Genetics Baylor College of Medicine
Nitrogen and the urea cycle Food Ammonia Urea Breakdown of body proteins (stress)
Why a cycle Four synthetic steps and two degradative steps to transfer one nitrogen from ammonia and one nitrogen from aspartate to the two nitrogens in urea Ornithine is always regenerated No net synthesis or production of urea cycle intermediates Mitochondrial and plasma membrane transporters important
Why a urea cycle Eliminate 40-45% of food protein nitrogen that is not needed for growth Provide important intermediates for other biochemical pathways (Arginine) Net synthesis of intermediates from the gut and kidneys
Pathogenesis of UCDs Carbamyl Phosphate + Ornithine X Citrulline X X X AMMONIA Arginine X Argininosuccinic Acid
Urea Cycle Disorders: Treatment Food Restrict Protein intake Buphenyl Ammonul Benzoate Ammonia Urea Breakdown of body proteins (stress) Arginine Citrulline
Alternative route disposal of nitrogen Sodium Phenylbutyrate X UREA CYCLE Phenylacetate + Glutamine Glycine + Benzoate Phenylacetylglutamine Hippurate URINARY EXCRETION
Arginine/Citrulline Supplementation Carbamyl Phosphate + Ornithine X Citrulline X X X ASPARTATE AMMONIA Arginine X Argininosuccinic Acid
What is difficult to manage? Frequent hyperammonemia Treatment non-adherence Dietary protein restriction Medication intolerance Feeding aversion Poor growth and development
Case One Referral for difficult management 3 year old female diagnosed with partial OTCD by DNA Presented with recurrent vomiting Multiple GI evaluations Treated with protein restriction, Buphenyl, citrulline, carnitine Repeated hospitalizations 1-3/month (NH 3 < 200 µm) Feeding aversion; complex feeding regimen
AMINO ACID CONC 0-1 YR 2-18 YR (um) RANGE RANGE o-phosphoserine 5 0-22 0-15 o-phosphoethanolamine 0 0-10 0-32 Taurine 154 0-189 5-127 Aspartic Acid 9 1-42 1-21 Threonine 179 20-210 34-161 Serine 158 56-188 57-169 Asparagine 61 12-72 7-82 Glutamic Acid 120 13-133 3-89 Glutamine 599 238-842 266-746 Proline 279 59-299 39-332 Glycine 407 104-344 92-346 Alanine 924 148-420 103-528 Citrulline 106 2-41 6-38 2-Amino-n-butyric Acid 9 1-31 3-34 Argininosuccinic Acid 0 0 0 Valine 63 50-242 82-293 Cystine 31 1-49 7-43 Methionine 21 9-45 7-34 Isoleucine 19 10-86 16-89 Leucine 26 30-142 35-164 Tyrosine 66 20-96 19-100 Phenylalanine 46 23-79 25-82 Homocystine (free) 0 < 1 < 1 Ornithine 102 5-129 5-100 Tryptophan 18 0-94 0-81 Lysine 179 53-201 41-225 l-methylhistidine 0 0-5 0-14 Histidine 89 37-97 38-103 3-Methylhistidine 1 0-8 0-12 Arginine 175 42-132 18-127 Albumin 4.1 g/dl (3.7 5.5) Prealbumin 20 mg/dl (18 44)
AMINO ACID CONC 0-1 YR 2-18 YR (um) RANGE RANGE o-phosphoserine 8 0-22 0-15 o-phosphoethanolamine 74 0-10 0-32 Taurine 95 0-189 5-127 Aspartic Acid 8 1-42 1-21 Threonine 207 20-210 34-161 Serine 129 56-188 57-169 Asparagine 39 12-72 7-82 Glutamic Acid 167 13-133 3-89 Glutamine 600 238-842 266-746 Proline 189 59-299 39-332 Glycine 319 104-344 92-346 Alanine 725 148-420 103-528 Citrulline 115 2-41 6-38 2-Amino-n-butyric Acid 10 1-31 3-34 Argininosuccinic Acid 0 0 0 Valine 106 50-242 82-293 Cystine 35 1-49 7-43 Methionine 13 9-45 7-34 Isoleucine 21 10-86 16-89 Leucine 28 30-142 35-164 Tyrosine 58 20-96 19-100 Phenylalanine 41 23-79 25-82 Homocystine (free) 0 < 1 < 1 Ornithine 102 5-129 5-100 Tryptophan 16 0-94 0-81 Lysine 177 53-201 41-225 l-methylhistidine 0 0-5 0-14 Histidine 87 37-97 38-103 3-Methylhistidine 0 0-8 0-12 Arginine 152 42-132 18-127
Natural history of UCDs Infancy Childhood Adolescence Adulthood Growth rate Protein tolerance Susceptibility to hyperammonemia Development Spasticity, liver dysfunction, hypertension
Natural History Honeymoon period in early infancy Difficulties with control at the end of the first year and during rapid growth (puberty) More severe with NAGS, CPS, OTC null activity patients Increased protein tolerance with age Emerging correlations
The honeymoon period Rapid growth and protein utilization Restricted environmental exposures Simpler dietary regimens Environmental Switch from breast milk Introduction of solid foods Exposure to infectious agents
Pathogenesis Hyperammonemia Hyperglutaminemia? Elevated arginine in argininemia Elevated argininosuccinic acid in ASA Arginine deficiency Overzealous protein restriction Branched chain amino acid depletion Unique enzyme functions?
Catabolic stress vs. dietary nonadherence Magnitude of nitrogen utilization from peripheral vs. central sources Getting sick is worse then eating too much Eating too little is worse then eating too much Prevention of catabolic stress Clinical and subclinical infection Strenuous and prolonged exercise Feeding aversion and G-button
Effects of pharmacologic therapy β-oxidation rate Gastrointestinal effects Metabolic effects Arginine, citrulline, carbamylglutamate, citric acid, carnitine
Essential Amino Acids Branched Chain Amino Acids Leucine, Valine, Isoleucine Phenylalanine Lysine Methionine Threonine Tryptophan
Buphenyl Benzoate
Branched Chain Amino Acids and UCDs UCD patients treated with protein restriction and PB have low BCAAs in the face of protein sufficiency Control subjects given PAB have selective decrease of BCAAs Supplementation with BCAAs may enable better titration of protein restriction How does BCAA flux affect protein synthesis and nitrogen transfer in UCD?
BCAA supplementation in UCD Compare BCAA with other essential amino acids and other parameters of protein sufficiency vs. insufficiency Tolerance of protein restriction with BCAA supplementation? BCAA and Buphenyl for better management and/or decrease risk of catabolism?
Management of Urea Cycle Disorders: Rationale for Nutritional Support Restrict dietary protein Prevent total body protein catabolism Use essential AA mix to provide 50% protein prescription Mix of essential amino acids, protein-free formula, and cow s milk formula Supplement with BCAA to allow further titration of protein restriction
Laboratory evaluation Useful laboratories in the clinic visit Useful laboratories in the hospitalization Diurnal variation Evaluation at steady state
Currency of nitrogen transfer Sources Breakdown of dietary protein Breakdown of endogenous protein Glutamine Alanine Glycine Essential amino acids Ammonia
Psychosocial factors Access to healthcare Laboratory studies Dietary services Metabolic expertise Family participation Educational background
Caveats of treatment Stoichiometric conversion of medications G-button GI side effects Strict control of caloric and protein intake Growth requires essential amino acids
Long Term Correction of Urea Cycle Disorders Role for liver transplantation OTCD CPS ASL Gene replacement therapy
Liver Transplantation for UCDs Neonatal onset CPS, OTC Cirrhosis associated with ASL Difficult medical management (ASS?) Risk - benefit assessment Access to medical/surgical expertise Outcome Psycho-socioeconomic factors Risks of hyperammonemia vs. risks of surgery and immunosuppression
UNOS Transplant Registry January 1988 January 2004 113 patients with UCDs underwent OLT Type of liver 63% received cadveric whole livers 19% received cadaveric partial/reduced livers 12% received cadaveric split livers 6% received living related donor livers 5 year post-olt survival was 85% for UCD subtypes Overall retransplantation rate 7%
Table 2. Griffiths Developmental Scale Results Subj Visit Age (mo) Overall* Gross Motor Personal- Social Lang. Fine Motor Perf. Pract. Reas. 1 1 24 51 69 50 52 50 50 5mo 2 34 70 94 58 71 71 58 CPS 3 49 83 98 73 94 61 90 82 4 63 89 89 95 98 70 83 102 2 1 28 86 100 96 73 77 82 11mo 2 41 80 117 78 63 68 73 76 OTC 3 50 76 100 60 68 84 76 82 4 68 86 90 90 73 87 93 81 3 1 11 56 48 70 65 67 72 4mo 2 20 51 45 63 59 78 82 CPS 3 33 47 34 47 42 52 61 4 DECEASED 1 1 48 71 69 82 51 82 35mo 2 58 68 69 83 48 69 OTC 3 71 82 90 76 82 87 4 88 88 88 86 88 86 * developmental scale results are standardized ratios, where mean =100, standard deviation = 10.8 94 83 87 98 55 73 79
Nitric oxide and the urea cycle Ornithine ArgI Arginine ASL NOS Citrulline + Nitric Oxide Argininosuccinic Acid
Nitrogen transfer through the urea cycle Diet (0.4 gm/kg/d protein) +/- Medication Day 1 Day 2 Day 3 CBC NH 3 PAA Infusion:[ 15 N]Gln & [ 18 O][ 13 C]Urea 0h 4h 8h 12h Measure NH 3, PAA [ 15 N]Gln [ 15 N]Urea [ 18 O]Urea
Acknowledgements Baylor College of Medicine Susan Carter R.N., Alyssa Tran, Brendan Lanpher, M.D., Fernando Scaglia, M.D., Nicola Brunetti, M.D.,Ph.D. Farook Jahoor, Ph.D., Juan Marini, Ph.D., Peter Garlick, Ph.D., William O Brien, Ph.D. GCRC, MRDRC, DDC UCD RDCRC NIH NIDDKD, NICHD Ucyclyd Pharma O Malley Foundation NUCDF and families
Helper-Dependent Adenoviral Vector Gene Therapy Reduction in chronic hepatic toxicity and adaptive immune response Increased duration of transgene expression Increased capacity for genomic DNA and tissue-specific genomic promoters and enhancers Increased capacity for multiple transgenes and regulatory switches
Urinary Orotic Acid (mm/mol creatinine) 250 200 150 100 50 0 PBS PEPCK hotc PEPCK hotc-wpre Human OTC cdna PEPCK promoter LITR+Ψ * * * * * * * * * 0 2 4 6 8 10 12 14 16 18 20 22 24 Weeks After Treatment WPRE RITR+E4 A. Mian et. al. Molecular Therapy 2004
Increased histochemical liver OTC activity in treated mice Wildtype Saline HDV-hOTC HDV-hOTC- WPRE 5 weeks 25 weeks A. Mian et. al. Molecular Therapy 2004
Dose response: Increasing the expression of hotc and effects on urinary orotic acid 5X10 11 vp/kg 1X10 12 vp/kg PEPCK hotc WPRE vs. PEPCK hotc WPRE HCR Nicola Brunetti
FG vs HD-Ad time course: Platelets Dose: 2 x 10 12 vp/kg Each time point represents a group of 5 mice Viraj Mane
Reduced Thrombocytopenia in TNFα Null Mice * * * * Viraj Mane
Effects of TNFα Ab pretreatment * Viraj Mane
Efficiency of Ad-Mediated Hepatic Transduction Following Systemic Injection Nunes et al., 1999 Portal vein injection into rhesus Morral et al., 2001 Peripheral vein injection into baboon Brunetti-Pierri et al. 2004 Peripheral vein injection into baboon 1 x 10 12 5 x 10 12 1 x 10 11 1.2 x 10 12 5.6 x 10 12 1 x 10 13 1.2 x 10 13 1.1 x 10 13 Conclusions: Dose (vp/kg) % transduction 1x10 13 100% 5x10 12 1x10 12 ~ 50% <<1% 5x10 11 undetectable Lethal human dose = 6x10 11 vp/kg
Dose response: Hydrodynamic expression of HDV Ad hotc and effects on urinary orotic acid 140 120 mmol/mol Cr 100 80 60 40 20 1x10e12 conv 5x10e11 conv 5x10e11 Hydro 1x10e11 Hydro 0 0 2 4 6 8 10 weeks after injection Doses in vp/kg Conv = conventional injection Hydro = hydrodynamic injection Nicola Brunetti
Phil Ng
Acute toxicity current approaches Threshold effect Local delivery and recirculation Saturation of nonspecific targets Increased gene expression Innate immune response Pegylation Pharmacologic blockade TNFα, complement, antibody
Acknowledgements William McCormack Gabriele Toietta Asad Mian Vincenzo Cerullo Viraj Mane Mike Seiler Nicola Brunetti Philip Ng Arthur Beaudet Jose Lopez Milton Finegold Lucio Pastore Lali K. Medine-Kauwe Timothy Nichols Anthony McDonagh Steven Pipe NIH NIDDKD, NICHD, BCM MRDDRC