Clinical experience with glycerol phenylbutyrate in 20 patients with urea cycle disorders at a UK paediatric centre.

In urea cycle disorders (UCDs) ammonia scavenger drugs, usually sodium-based, have been the mainstay of treatment. Increasingly, glycerol phenylbutyrate (GPB, Ravicti®) is being used but scant real-world data exist regarding clinical outcomes. A retrospective study of UCD patients initiated on or switched to GPB was performed at a UK centre. Data on population characteristics, treatment aspects, laboratory measurements, and clinical outcomes were collected before and after patients started GPB with a sub-group analysis undertaken for patients with ≥12 months of data before and after starting GPB. UCDs included arginosuccinate synthetase deficiency (n = 8), arginosuccinate lyase deficiency (n = 6), ornithine carbamoyltransferase deficiency (n = 3), and carbamoyl phosphate synthetase 1 deficiency (n = 3). In the sub-group analysis (n = 11), GPB resulted in lower plasma ammonia (31 vs. 41 µmol/L, p = 0.037), glutamine (670 vs. 838 µmol/L, p = 0.002), annualised hyperammonaemic episodes (0.2 vs. 1.9, p = 0.020), hospitalisations (0.5 vs. 2.2, p = 0.010), and hyperammonaemic episodes resulting in hospitalisation (0.2 vs. 1.6, p = 0.035) reflecting changes seen in the whole group. Overall, patients exposed to sodium and propylene glycol levels above UK daily limits reduced by 78% and 83% respectively. Mean levels of branched chain amino acids, haemoglobin, and white cell count were unchanged. Two adverse drug reactions (pancytopenia, fatigue/appetite loss) resolved without GPB discontinuation. Patients/families preferred GPB for its lower volume, greater palatability and easier administration. GPB appeared to improve biochemical measures and clinical outcomes. The causes are multi-factorial and are likely to include prolonged action of GPB and its good tolerability, even at higher doses, facilitating tighter control of ammonia.

Direct replacement of oral sodium benzoate with glycerol phenylbutyrate in children with urea cycle disorders.

Long-term management of urea cycle disorders (UCDs) often involves unlicensed oral sodium benzoate (NaBz) which has a high volume and unpleasant taste. A more palatable treatment is licenced and available (glycerol phenylbutyrate [GPB], Ravicti) but guidance on how to transition patients from NaBz is lacking. A retrospective analysis of clinical and biochemical data was performed for eight children who transitioned from treatment with a single ammonia scavenger, NaBz, to GPB at a single metabolic centre; UCDs included arginosuccinic aciduria (ASA) (n = 5), citrullinaemia type 1 (n = 2) and carbamoyl phosphate synthetase I deficiency (CPS1) (n = 1). Patients transitioned either by gradual transition over 1-2 weeks (n = 3) or direct replacement of NaBz with GPB (n = 5). Median initial dose of GPB was 8.5 mL/m2/day based on published product information; doses were revisited subsequently in clinic and titrated individually (range 4.5-11 mL/m2/day). Pre-transition and post-transition mean ammonia levels were 37 µmol/L (SD 28 µmol/L) and 29 µmol/L (SD 22 µmol/L), respectively (p = 0.09), and mean glutamine levels were 664 µmol/L (SD 225 µmol/L) and 598 µmol/L (SD 185 µmol/L), respectively (p = 0.24). There were no reductions in levels of branched chain amino acids. No related adverse drug reactions were reported. Patients preferred GPB because of its lower volume and greater palatability. Direct replacement of NaBz with GPB maintained metabolic control and was simple for the health service and patients to manage. A more cautious approach with additional monitoring would be warranted in brittle patients and patients whose ammonia levels are difficult to control.

Lentiviral Hematopoietic Stem Cell Gene Therapy Rescues Clinical Phenotypes in a Murine Model of Pompe Disease.

Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.

Regulation of glycine metabolism by the glycine cleavage system and conjugation pathway in mouse models of non-ketotic hyperglycinemia.

Glycine abundance is modulated in a tissue-specific manner by use in biosynthetic reactions, catabolism by the glycine cleavage system (GCS), and excretion via glycine conjugation. Dysregulation of glycine metabolism is associated with multiple disorders including epilepsy, developmental delay, and birth defects. Mutation of the GCS component glycine decarboxylase (GLDC) in non-ketotic hyperglycinemia (NKH) causes accumulation of glycine in body fluids, but there is a gap in our knowledge regarding the effects on glycine metabolism in tissues. Here, we analysed mice carrying mutations in Gldc that result in severe or mild elevations of plasma glycine and model NKH. Liver of Gldc-deficient mice accumulated glycine and numerous glycine derivatives, including multiple acylglycines, indicating increased flux through reactions mediated by enzymes including glycine-N-acyltransferase and arginine: glycine amidinotransferase. Levels of dysregulated metabolites increased with age and were normalised by liver-specific rescue of Gldc expression. Brain tissue exhibited increased abundance of glycine, as well as derivatives including guanidinoacetate, which may itself be epileptogenic. Elevation of brain tissue glycine occurred even in the presence of only mildly elevated plasma glycine in mice carrying a missense allele of Gldc. Treatment with benzoate enhanced hepatic glycine conjugation thereby lowering plasma and tissue glycine. Moreover, administration of a glycine conjugation pathway intermediate, cinnamate, similarly achieved normalisation of liver glycine derivatives and circulating glycine. Although exogenous benzoate and cinnamate impact glycine levels via activity of glycine-N-acyltransferase, that is not expressed in brain, they are sufficient to lower levels of glycine and derivatives in brain tissue of treated Gldc-deficient mice.

Modelling of neutrophil dynamics in children receiving busulfan or treosulfan for haematopoietic stem cell transplant conditioning.

AIMS: Busulfan and treosulfan are cytotoxic agents used in the conditioning regime prior to paediatric haematopoietic stem cell transplantation (HSCT). These agents cause suppression of myeloid cells leaving patients severely immunocompromised in the early post-HSCT period. The main objectives were: (i) to establish a mechanistic pharmacokinetic-pharmacodynamic (PKPD) model for the treatment and engraftment effects on neutrophil counts comparing busulfan and treosulfan-based conditioning, and (ii) to explore current dosing schedules with respect to time to HSCT. METHODS: Data on 126 patients, 72 receiving busulfan (7 months-18 years, 5.1-47.0 kg) and 54 treosulfan (4 months-17 years, 3.8-35.8 kg), were collected. In total, 8935 neutrophil count observations were recorded during the study period in addition to drug concentrations to develop a mechanistic PKPD model. Absolute neutrophil count profiles were modelled semimechanistically, accounting for transplant effects and differing set points pre- and post-transplant. RESULTS: PK were best described by 2-compartment models for both drugs. The Friberg semimechanistic neutropenia model was applied with a linear model for busulfan and a maximum efficacy model for treosulfan describing drug effects at various stages of neutrophil maturation. System parameters were consistent across both drugs. The HSCT was represented by an amount of progenitor cells enhancing the neutrophils' proliferation and maturation compartments. Alemtuzumab was found to enhance the proliferative rate under which the absolute neutrophil count begin to grow after HSCT. CONCLUSION: A semimechanistic PKPD model linking exposure to either busulfan or treosulfan to the neutrophil reconstitution dynamics was successfully built. Alemtuzumab coadministration enhanced the neutrophil proliferative rate after HSCT. Treosulfan administration was suggested to be delayed with respect to time to HSCT, leaving less time between the end of the administration and stem cell infusion.

Proposed Therapeutic Range of Treosulfan in Reduced Toxicity Pediatric Allogeneic Hematopoietic Stem Cell Transplant Conditioning: Results From a Prospective Trial.

Treosulfan is given off-label in pediatric allogeneic hematopoietic stem cell transplant. This study investigated treosulfan's pharmacokinetics (PKs), efficacy, and safety in a prospective trial. Pediatric patients (n = 87) receiving treosulfan-fludarabine conditioning were followed for at least 1 year posttransplant. PKs were described with a two-compartment model. During follow-up, 11 of 87 patients died and 12 of 87 patients had low engraftment (≤ 20% myeloid chimerism). For each increase in treosulfan area under the curve from zero to infinity (AUC(0-∞) ) of 1,000 mg hour/L the hazard ratio (95% confidence interval) for mortality increase was 1.46 (1.23-1.74), and the hazard ratio for low engraftment was 0.61 (0.36-1.04). A cumulative AUC(0-∞) of 4,800 mg hour/L maximized the probability of success (> 20% engraftment and no mortality) at 82%. Probability of success with AUC(0-∞) between 80% and 125% of this target were 78% and 79%. Measuring PK at the first dose and individualizing the third dose may be required in nonmalignant disease.

Argininosuccinic aciduria fosters neuronal nitrosative stress reversed by Asl gene transfer.

Argininosuccinate lyase (ASL) belongs to the hepatic urea cycle detoxifying ammonia, and the citrulline-nitric oxide (NO) cycle producing NO. ASL-deficient patients present argininosuccinic aciduria characterised by hyperammonaemia, multiorgan disease and neurocognitive impairment despite treatment aiming to normalise ammonaemia without considering NO imbalance. Here we show that cerebral disease in argininosuccinic aciduria involves neuronal oxidative/nitrosative stress independent of hyperammonaemia. Intravenous injection of AAV8 vector into adult or neonatal ASL-deficient mice demonstrates long-term correction of the hepatic urea cycle and the cerebral citrulline-NO cycle, respectively. Cerebral disease persists if ammonaemia only is normalised but is dramatically reduced after correction of both ammonaemia and neuronal ASL activity. This correlates with behavioural improvement and reduced cortical cell death. Thus, neuronal oxidative/nitrosative stress is a distinct pathophysiological mechanism from hyperammonaemia. Disease amelioration by simultaneous brain and liver gene transfer with one vector, to treat both metabolic pathways, provides new hope for hepatocerebral metabolic diseases.

Mutations in PROSC Disrupt Cellular Pyridoxal Phosphate Homeostasis and Cause Vitamin-B6-Dependent Epilepsy.

Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, functions as a cofactor in humans for more than 140 enzymes, many of which are involved in neurotransmitter synthesis and degradation. A deficiency of PLP can present, therefore, as seizures and other symptoms that are treatable with PLP and/or pyridoxine. Deficiency of PLP in the brain can be caused by inborn errors affecting B6 vitamer metabolism or by inactivation of PLP, which can occur when compounds accumulate as a result of inborn errors of other pathways or when small molecules are ingested. Whole-exome sequencing of two children from a consanguineous family with pyridoxine-dependent epilepsy revealed a homozygous nonsense mutation in proline synthetase co-transcribed homolog (bacterial), PROSC, which encodes a PLP-binding protein of hitherto unknown function. Subsequent sequencing of 29 unrelated indivduals with pyridoxine-responsive epilepsy identified four additional children with biallelic PROSC mutations. Pre-treatment cerebrospinal fluid samples showed low PLP concentrations and evidence of reduced activity of PLP-dependent enzymes. However, cultured fibroblasts showed excessive PLP accumulation. An E.coli mutant lacking the PROSC homolog (ΔYggS) is pyridoxine sensitive; complementation with human PROSC restored growth whereas hPROSC encoding p.Leu175Pro, p.Arg241Gln, and p.Ser78Ter did not. PLP, a highly reactive aldehyde, poses a problem for cells, which is how to supply enough PLP for apoenzymes while maintaining free PLP concentrations low enough to avoid unwanted reactions with other important cellular nucleophiles. Although the mechanism involved is not fully understood, our studies suggest that PROSC is involved in intracellular homeostatic regulation of PLP, supplying this cofactor to apoenzymes while minimizing any toxic side reactions.

Glycine decarboxylase deficiency causes neural tube defects and features of non-ketotic hyperglycinemia in mice.

Glycine decarboxylase (GLDC) acts in the glycine cleavage system to decarboxylate glycine and transfer a one-carbon unit into folate one-carbon metabolism. GLDC mutations cause a rare recessive disease non-ketotic hyperglycinemia (NKH). Mutations have also been identified in patients with neural tube defects (NTDs); however, the relationship between NKH and NTDs is unclear. We show that reduced expression of Gldc in mice suppresses glycine cleavage system activity and causes two distinct disease phenotypes. Mutant embryos develop partially penetrant NTDs while surviving mice exhibit post-natal features of NKH including glycine accumulation, early lethality and hydrocephalus. In addition to elevated glycine, Gldc disruption also results in abnormal tissue folate profiles, with depletion of one-carbon-carrying folates, as well as growth retardation and reduced cellular proliferation. Formate treatment normalizes the folate profile, restores embryonic growth and prevents NTDs, suggesting that Gldc deficiency causes NTDs through limiting supply of one-carbon units from mitochondrial folate metabolism.

Urine analysis of glucose tetrasaccharide by HPLC; a useful marker for the investigation of patients with Pompe and other glycogen storage diseases.

A high performance liquid chromatography method, adapted from an established urinary sugars method, has been developed for the analysis of a tetraglucose oligomer (Glc(4)) in urine. Pompe disease results from defects in the activity of lysosomal acid α-glucosidase (GAA) with patients typically excreting increased amounts of Glc(4). Rapid determination of GAA in dried blood spots is now possible. However, enzymatic analysis is unable to discriminate between patients with Pompe disease and those individuals harbouring pseudo deficiency mutations. This method was able to quantify Glc(4) levels in all patients analysed with an established diagnosis of Pompe disease, and all controls analysed had Glc(4) levels below the limit of detection for this method. Importantly the method was able to discriminate between an individual known to harbour a pseudo Pompe mutation and patients with Pompe disease, providing a useful supporting test to enzymatic analysis. Sequential measurement of urinary Glc(4) has been proposed to monitor the effects of enzyme replacement therapy (ERT). We observed a clear decrease in Glc(4) levels following commencement of treatment in three patients studied. Additionally, raised levels of Glc(4) were observed in patients with glycogen storage disease (GSD) type Ia and type III suggesting that this method may have applications in other GSDs.

Multicentre age-related reference intervals for cerebrospinal fluid serine concentrations: implications for the diagnosis and follow-up of serine biosynthesis disorders.

The disorders of serine biosynthesis are a group of inborn errors of metabolism characterised by congenital microcephaly, seizures and severe psychomotor retardation. Although these disorders are rare the prompt recognition of serine deficiency is important as these disorders are treatable. The diagnosis is based on decreased concentrations of serine in cerebrospinal fluid (CSF). It has previously been reported that CSF serine concentrations are inversely associated with age. However, accurate age-related reference intervals have not been generated which has contributed to cases not being identified. In a multicentre study involving 9 different laboratories a total of 424 CSF serine results were obtained. Regression based analyses were performed to calculate age-specific reference intervals. Lower reference intervals for subjects aged 1week, 1month, 6months, 1year, 3years and 15years were 35.0, 31.0, 26.0, 24.0, 21.0 and 17.0µmol/L respectively. Assessment of CSF serine concentrations in 11 patients (aged 1day to 13years) previously diagnosed with disorders of serine biosynthesis (serine concentrations ranging from 5 to 18µmol/L) were clearly decreased compared to our age-related reference intervals and would have correctly identified all cases, thus enabling prompt treatment. However, if age had not been taken into consideration a reference interval of 12.6-69.4µmol/L would be obtained for the combined data set and would have resulted in 2 cases being missed. In conclusion, appropriate age-related reference intervals for CSF serine should be used to diagnose patients with inborn errors of serine biosynthesis.