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

Ammonia Urea

Food

Breakdown of body proteins (stress)

Nitrogen and the urea cycle

Why a cycle• Four synthetic steps and two degradative

steps to transfer one nitrogen from ammonia and one nitrogen from aspartateto 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

Arginine

CarbamylPhosphate

CitrullineOrnithine

ArgininosuccinicAcid

XX

XX

+

XAMMONIA

Urea Cycle Disorders: Treatment

Ammonia Urea

Food

Breakdown of body proteins (stress)

Restrict Protein intake

BuphenylAmmonulBenzoate

ArginineCitrulline

Alternative route disposal of nitrogen

Phenylacetate + Glutamine

Phenylacetylglutamine

Glycine + Benzoate

Hippurate

UREA CYCLE

URINARY EXCRETION

Sodium PhenylbutyrateX

Arginine/Citrulline Supplementation

Arginine

CarbamylPhosphate

CitrullineOrnithine

ArgininosuccinicAcid

XX

XX

+

AMMONIA

X ASPARTATE

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 (NH3 < 200 µm)

• Feeding aversion; complex feeding regimen

AMINO ACID CONC 0-1 YR 2-18 YR(uM) RANGE RANGE

o-Phosphoserine 5 0-22 0-15o-Phosphoethanolamine 0 0-10 0-32Taurine 154 0-189 5-127Aspartic Acid 9 1-42 1-21Threonine 179 20-210 34-161Serine 158 56-188 57-169Asparagine 61 12-72 7-82Glutamic Acid 120 13-133 3-89Glutamine 599 238-842 266-746Proline 279 59-299 39-332Glycine 407 104-344 92-346Alanine 924 148-420 103-528Citrulline 106 2-41 6-382-Amino-n-butyric Acid 9 1-31 3-34Argininosuccinic Acid 0 0 0Valine 63 50-242 82-293Cystine 31 1-49 7-43Methionine 21 9-45 7-34Isoleucine 19 10-86 16-89Leucine 26 30-142 35-164Tyrosine 66 20-96 19-100Phenylalanine 46 23-79 25-82Homocystine (free) 0 < 1 < 1Ornithine 102 5-129 5-100Tryptophan 18 0-94 0-81Lysine 179 53-201 41-225l-Methylhistidine 0 0-5 0-14Histidine 89 37-97 38-1033-Methylhistidine 1 0-8 0-12Arginine 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 0-15o-Phosphoethanolamine 74 0-10 0-32Taurine 95 0-189 5-127Aspartic Acid 8 1-42 1-21Threonine 207 20-210 34-161Serine 129 56-188 57-169Asparagine 39 12-72 7-82Glutamic Acid 167 13-133 3-89Glutamine 600 238-842 266-746Proline 189 59-299 39-332Glycine 319 104-344 92-346Alanine 725 148-420 103-528Citrulline 115 2-41 6-382-Amino-n-butyric Acid 10 1-31 3-34Argininosuccinic Acid 0 0 0Valine 106 50-242 82-293Cystine 35 1-49 7-43Methionine 13 9-45 7-34Isoleucine 21 10-86 16-89Leucine 28 30-142 35-164Tyrosine 58 20-96 19-100Phenylalanine 41 23-79 25-82Homocystine (free) 0 < 1 < 1Ornithine 102 5-129 5-100Tryptophan 16 0-94 0-81Lysine 177 53-201 41-225l-Methylhistidine 0 0-5 0-14Histidine 87 37-97 38-1033-Methylhistidine 0 0-8 0-12Arginine 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%

79988688868888884

81938773909086684

102837098958989634

* developmental scale results are standardized ratios, where mean =100, standard deviation = 10.8

73878782769082713OTC

5583694883696858235mo

9482518269714811 ♀

DECEASED4

615242473447333CPS

8278596345512024mo

7267657048561113 ♂

827684686010076503OTC

76736863781178041211mo

82777396100862812 ♂

82906194739883493CPS

5871715894703425mo

5050525069512411♂

Pract. Reas.Perf.Fine

MotorLang.Personal-Social

Gross Motor

Overall*Age (mo)VisitSubj

Table 2. Griffiths Developmental Scale Results

Nitric oxide and the urea cycle

ArginineCitrulline

+Nitric Oxide

NOS

Argininosuccinic Acid

Ornithine

ArgI

ASL

Day 1 Day 2 Day 3

Diet (0.4 gm/kg/d protein)+/- Medication

0h 4h 8h 12h

Infusion:[15N]Gln& [18O][13C]Urea

CBCNH3PAA

NH3, PAA[15N]Gln[15N]Urea[18O]Urea

Measure

Nitrogen transfer through the urea cycle

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

0

50

100

150

200

250

Weeks After Treatment

Urin

ary

Oro

ticA

cid

(mM

/mol

cre

atin

ine)

0 2 4 6 8 10 12 14 16 18 20 22 24* * * * ¶ ¶ * * * **

§ ‡ ‡ ‡‡ ‡ ‡¥ ¥ ¥

A. Mian et. al. Molecular Therapy 2004

PBSPEPCK hOTCPEPCK hOTC-WPRE

♦

PEPCK promoterHuman OTC cDNA

LITR+Ψ RITR+E4

WPRE

5 weeks

25 weeks

Wildtype Saline

HDV-hOTC

HDV-hOTC-

WPRE

Increased histochemical liver OTC activity in treated mice

A. Mian et. al. Molecular Therapy 2004

Dose response: Increasing the expression of hOTC and effects

on urinary orotic acid

Nicola Brunetti

5X1011 vp/kg

1X1012 vp/kg

PEPCK hOTC WPRE vs. PEPCK hOTC WPRE HCR

FG vs HD-Ad time course: Platelets

Dose: 2 x 1012 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

1 x

1013

1 x

1012

Morral et al., 2001 Peripheral vein injection into baboon

Nunes et al., 1999 Portal vein injection into rhesus

1.1

x 10

135.

6 x

1012

Brunetti-Pierri et al. 2004 Peripheral vein injection into baboon

1.2

x 10

131.

2 x

1012

5 x

1012

1 x

1011

% transductionDose (vp/kg)

undetectable5x1011

<<1%1x1012

~ 50%5x1012

100%1x1013

Conclusions:

Lethal human dose = 6x1011 vp/kg

Efficiency of AdEfficiency of Ad--Mediated Hepatic Transduction Following Systemic Mediated Hepatic Transduction Following Systemic InjectionInjection

0

20

40

60

80

100

120

140

0 2 4 6 8 10

weeks after injection

mm

ol/m

ol C

r 1x10e12 conv

5x10e11 conv

5x10e11 Hydro

1x10e11 Hydro

Doses in vp/kgConv = conventional injectionHydro = hydrodynamic injection

Dose response: Hydrodynamic expression of HDV Ad hOTC and effects

on urinary orotic acid

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

AcknowledgementsWilliam McCormackGabriele ToiettaAsad Mian

Vincenzo CerulloViraj ManeMike SeilerNicola Brunetti

Philip NgArthur Beaudet Jose LopezMilton Finegold

Lucio Pastore

Lali K. Medine-Kauwe

Timothy Nichols

Anthony McDonagh

Steven Pipe

NIH NIDDKD, NICHD, BCM MRDDRC