Efficacy and safety of a synthetic biotic for treatment of phenylketonuria: a phase 2 clinical trial.

Despite available treatment options, many patients with phenylketonuria (PKU) cannot achieve target plasma phenylalanine (Phe) levels1. We previously modified Escherichia coli Nissle 1917 to metabolize Phe in the gut after oral administration (SYNB1618) and designed a second strain (SYNB1934) with enhanced activity of phenylalanine ammonia lyase2,3. In a 14-day open-label dose-escalation study (Synpheny-1, NCT04534842 ), we test a primary endpoint of change from baseline in labeled Phe (D5-Phe AUC0-24; D5-Phe area under the curve (AUC) over 24 hours after D5-Phe administration) in plasma after D5-Phe challenge in adult participants with screening Phe of greater than 600 µM. Secondary endpoints were the change from baseline in fasting plasma Phe and the incidence of treatment-emergent adverse events. A total of 20 participants (ten male and ten female) were enrolled and 15 completed the study treatment. Here, we show that both strains lower Phe levels in participants with PKU: D5-Phe AUC0-24 was reduced by 43% from baseline with SYNB1934 and by 34% from baseline with SYNB1618. SYNB1934 led to a decrease in fasting plasma Phe of 40% (95% CI, -52, -24). There were no serious adverse events or infections. Four participants discontinued because of adverse events, and one withdrew during the baseline period. We show that synthetic biotics can metabolize Phe in the gut, lower post-prandial plasma Phe levels and lower fasting plasma Phe in patients with PKU.

Complications of the Low Phenylalanine Diet for Patients with Phenylketonuria and the Benefits of Increased Natural Protein.

Phenylketonuria (PKU) is an inherited disorder in which phenylalanine (Phe) is not correctly metabolized leading to an abnormally high plasma Phe concentration that causes profound neurologic damage if left untreated. The mainstay of treatment for PKU has centered around limiting natural protein in the diet while supplementing with medical foods in order to prevent neurologic injury while promoting growth. This review discusses several deleterious effects of the low Phe diet along with benefits that have been reported for patients with increased natural protein intake while maintaining plasma Phe levels within treatment guidelines.

Anaplerotic therapy in propionic acidemia.

BACKGROUND: Propionic acidemia is a rare metabolic disorder caused by a deficiency of propionyl- CoA carboxylase, the enzyme converting propionyl-CoA to methylmalonyl-CoA that subsequently enters the citric acid cycle as succinyl-CoA. Patients with propionic acidemia cannot metabolize propionic acid, which combines with oxaloacetate to form methylcitric acid. This, with the defective supply of succinyl-CoA, may lead to a deficiency in citric acid cycle intermediates. PURPOSE: The objective of this study was to determine whether supplements with glutamine (400mg/kg per day), citrate (7.5mEq/kg per day), or ornithine α-ketoglutarate (400mg/kg per day) (anaplerotic agents that could fill up the citric acid cycle) would affect plasma levels of glutamine and ammonia, the urinary excretion of Krebs cycle intermediates, and the clinical outcome in 3 patients with propionic acidemia. METHODS: Each supplement was administered daily for four weeks with a two week washout period between supplements. The supplement that produced the most favorable changes was supplemented for 30 weeks following the initial study period and then for a 2 year extension. RESULTS: The urinary excretion of the Krebs cycle intermediates, α-ketoglutarate, succinate, and fumarate increased significantly compared to baseline during citrate supplementation, but not with the other two supplements. For this reason, citrate supplements were continued in the second part of the study. The urinary excretion of methylcitric acid and 3-hydroxypropionic acid did not change with any intervention. No significant changes in ammonia or glutamine levels were observed with any supplement. However, supplementation with any anaplerotic agents normalized the physiological buffering of ammonia by glutamate, with plasma glutamate and alanine levels significantly increasing, rather than decreasing with increasing ammonia levels. No significant side effects were observed with any therapy and safety labs (blood counts, chemistry and thyroid profile) remained unchanged. Motor and cognitive development was severely delayed before the trial and did not change significantly with therapy. Hospitalizations per year did not change during the trial period, but decreased significantly (p<0.05) in the 2years following the study (when citrate was continued) compared to the 2years before and during the study. CONCLUSIONS: These results indicate that citrate entered the Krebs cycle providing successful anaplerotic therapy by increasing levels of the downstream intermediates of the Krebs cycle: α-ketoglutarate, succinate and fumarate. Citrate supplements were safe and might have contributed to reduce hospitalizations in patients with propionic acidemia.

Evidence-based treatment of guanidinoacetate methyltransferase (GAMT) deficiency.

BACKGROUND: Guanidinoacetate methyltransferase (GAMT) deficiency causes cerebral creatine deficiency. Patients can have autistic behavior, seizures, intellectual disability, and severe speech delay. The goal of therapy is to increase creatine while reducing potentially neurotoxic guanidinoacetate concentrations. Here we evaluate how different therapies affect plasma guanidinoacetate levels in patients with GAMT deficiency. METHODS: Retrospective analysis of data from five new patients with GAMT deficiency (four with delays and seizures, one diagnosed at birth). RESULTS: The four symptomatic patients had decreased brain creatine by magnetic resonance spectroscopy and three also had abnormal globi pallidi by MRI. GAMT sequencing identified four previously reported mutations and one novel missense mutation (c.233T>A/p.V78E). Treatment with creatine (250-1000 mg/kg/day), ornithine (100-800 mg/kg/day), and sodium benzoate (50-135 mg/kg/day) supplements along with dietary protein restriction (0.8-1.5 g/kg/day) improved seizures and development with all patients becoming verbal. The patient treated at birth remains developmentally normal. Reduction in glycine and increase in ornithine levels significantly decreased plasma guanidinoacetate, with glycine levels being the best predictor of guanidinoacetate levels. In contrast, arginine levels were not significantly correlated with plasma guanidinoacetate. CONCLUSIONS: Our results show that supplements of creatine, sodium benzoate (to reduce glycine) and ornithine reduce guanidinoacetate levels in patients with GAMT deficiency (dietary therapy was not evaluated in our study). Normal development with early therapy renders GAMT deficiency an ideal candidate for inclusion in newborn screening panels.

Glutaric acidemia type 1: outcomes before and after expanded newborn screening.

Glutaric acidemia type 1 (GA-1) is an autosomal recessive disorder of lysine, hydroxylysine, and tryptophan metabolism. Patients may present with brain atrophy, macrocephaly, and acute dystonia secondary to striatal degeneration typically triggered by an infection, fever, and/or dehydration. This disorder is identified on expanded newborn screening by increased glutarylcarnitine. We evaluated the outcome of 19 patients with GA-1. Ten patients were diagnosed by newborn screening and 9 were diagnosed clinically. DNA testing in 12 patients identified 15 different mutations in the glutaryl-CoA dehydrogenase gene. Plasma glutarylcarnitine and urinary 3-hydroxyglutaric acid were elevated in all patients. However, only 10 of 17 patients who underwent urine organic acid analysis were high excretors of glutaric acid. Levels of glutarylcarnitine in plasma correlated with the urinary excretion of glutaric and 3-hydroxyglutaric acid, but not with clinical outcome. Plasma lysine was also significantly correlated with urinary glutaric acid, but not with urinary 3-hydroxyglutaric acid. Brain magnetic resonance imaging in all patients showed wide Sylvian fissures before treatment, which normalized by 4 years of age in treated patients. The occurrence of three adverse outcomes (oral motor function, ambulatory capability, and dystonic movements) was on average reduced by 75% (relative risk 0.25 to 0.28) in patients identified by newborn screening compared to patients diagnosed before newborn screening (Fisher's exact test; p=0.0055 for oral motor function and ambulatory capability; p=0.023 for dystonic movements). Newborn screening is effective in the prevention of complications in patients with GA-1 when coupled with treatment strategies.

Correlation of age-specific phenylalanine levels with intellectual outcome in patients with phenylketonuria.

Patients with treated phenylketonuria (PKU) can have subtle deficits in intellect, academic skills, and executive functioning. This study evaluates the relationship between intellectual outcome and concentration/variation in blood phenylalanine (Phe) during specific developmental periods (0-6 years, 7-12 years, >12 years) in our patients with PKU. Verbal comprehension, perceptual reasoning, and processing speed were used as measures of intelligence. Data were collected from 55 patients receiving treatment at the University of Utah Metabolic Clinic. Yearly median Phe levels increased and mean number of blood Phe samples decreased as patients aged. Yearly median blood Phe from 0-6 and 7-12 years were inversely associated with perceptual reasoning abilities using linear regression. Additionally, increased blood Phe concentration negatively impacted specific areas of verbal comprehension abilities for those 0-6 years of age (p = 0.001). Variation of Phe levels around the mean (assessed as standard deviation) in each patient was associated with diagnostic (highest pretreatment) Phe levels and yearly median Phe levels (p < 0.001 for both), but did not significantly impact intelligence in our group of patients. Frequent blood Phe monitoring from 7-12 years significantly reduced the probability of yearly median Phe exceeding 360 µM (p = 0.005). Our data show that compliance with treatment in patients with PKU affects both the concentration and variation of blood Phe levels, and may have a greater impact on verbal comprehension and perceptual reasoning skills during the first 12 years of life when compared the influence beyond 12 years.

Progressive cerebral vascular degeneration with mitochondrial encephalopathy.

MELAS (mitochondrial encephalopathy with lactic acidosis and stroke-like episodes) is a maternally inherited disorder characterized by recurrent cerebral infarctions that do not conform to discreet vascular territories. Here we report on a patient who presented at 7 years of age with loss of consciousness and severe metabolic acidosis following vomiting and dehydration. She developed progressive sensorineural hearing loss, myopathy, ptosis, short stature, and mild developmental delays after normal early development. Biochemical testing identified metabolites characteristic of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (hexanoylglycine and suberylglycine), but also severe lactic acidemia (10-25 mM) and, in urine, excess of lactic acid, intermediates of the citric cycle, and marked ketonuria, suggesting mitochondrial dysfunction. She progressed rapidly to develop temporary cortical blindness. Brain imaging indicated generalized atrophy, more marked on the left side, in addition to white matter alterations consistent with a mitochondrial disorder. Magnetic resonance angiography indicated occlusion of the left cerebral artery with development of collateral circulation (Moyamoya syndrome). This process worsened over time to involve the other side of the brain. A muscle biopsy indicated the presence of numerous ragged red fibers. Molecular testing confirmed compound heterozygosity for the common mutation in the MCAD gene (985A>G) and a second pathogenic mutation (233T>C). MtDNA testing indicated that the muscle was almost homoplasmic for the 3243A>T mutation in tRNALeu, with a lower mutant load (about 50% heteroplasmy) in blood and skin fibroblasts. These results indicate that mitochondrial disorders may be associated with severe vascular disease resulting in Moyamoya syndrome. The contribution of the concomitant MCAD deficiency to the development of the phenotype in this case is unclear.

Metabolic changes associated with hyperammonemia in patients with propionic acidemia.

Propionic acidemia is an autosomal recessive disorder caused by deficiency of propionyl CoA carboxylase. Affected patients can develop severe hyperammonemia, whose causative mechanism is unknown. In this study, we monitored changes in metabolic parameters associated with hyperammonemia in patients with propionic acidemia. Levels of ammonia were correlated with plasma levels of individual amino acids and carnitine and with urinary organic acids. Significance of correlations was determined with analysis of variance. Hyperammonemia positively correlated with an increase in branched-chain amino acids (leucine and isoleucine) and a decrease in glutamine/glutamate and esterified carnitine. The urinary excretion of methylcitric acid, formed by the combination of propionic acid with oxaloacetate from the Krebs cycle, increased while that of citric acid decreased with hyperammonemia. These results suggest that in propionic acidemia, hyperammonemia is triggered by catabolism with the accumulation of propionic acid derivatives. The decrease of the plasma levels of glutamine/glutamate with hyperammonemia in patients with propionic acidemia indicates that the mechanism producing hyperammonemia differs from that in urea cycle defects. The increase in methylcitric acid and decline in citric acid urinary excretion suggest that hyperammonemia in propionic acidemia might be related to inability to maintain adequate levels of glutamine precursors through a dysfunctional Krebs cycle.