Clinical effect and safety profile of pegzilarginase in patients with arginase 1 deficiency.

Hyperargininemia in patients with arginase 1 deficiency (ARG1-D) is considered a key driver of disease manifestations, including spasticity, developmental delay, and seizures. Pegzilarginase (AEB1102) is an investigational enzyme therapy which is being developed as a novel arginine lowering approach. We report the safety and efficacy of intravenously (IV) administered pegzilarginase in pediatric and adult ARG1-D patients (n = 16) from a Phase 1/2 study (101A) and the first 12 weeks of an open-label extension study (102A). Substantial disease burden at baseline included lower-limb spasticity, developmental delay, and previous hyperammonemic episodes in 75%, 56%, and 44% of patients, respectively. Baseline plasma arginine (pArg) was elevated (median 389 µM, range 238-566) on standard disease management. Once weekly repeat dosing resulted in a median decrease of pArg of 277 µM after 20 cumulative doses (n = 14) with pArg in the normal range (40 to 115 µM) in 50% of patients at 168 hours post dose (mean pegzilarginase dose 0.10 mg/kg). Lowering pArg was accompanied by improvements in one or more key mobility assessments (6MWT, GMFM-D & E) in 79% of patients. In 101A, seven hypersensitivity reactions occurred in four patients (out of 162 infusions administered). Other common treatment-related adverse events (AEs) included vomiting, hyperammonemia, pruritus, and abdominal pain. Treatment-related serious AEs that occurred in five patients were all observed in 101A. Pegzilarginase was effective in lowering pArg levels with an accompanying clinical response in patients with ARG1-D. The improvements with pegzilarginase occurred in patients receiving standard treatment approaches, which suggests that pegzilarginase could offer benefit over existing disease management.

Sodium phenylbutyrate improved the clinical state in an adult patient with arginase 1 deficiency.

An adult female patient was diagnosed with arginase 1 deficiency (ARG1-D) at 4 years of age, and had been managed with protein restriction combined with sodium benzoate therapy. Though the treatment was successful in ameliorating hyperammonemia, hyperargininemia persisted. After being under control with a strict restriction of dietary protein, severe fall of serum albumin levels appeared and her condition became strikingly worsened. However, after sodium phenylbutyrate (NaPB) therapy was initiated, the clinical condition and metabolic stability was greatly improved. Current management of ARG1-D is aimed at lowering plasma arginine levels. The nitrogen scavengers, such as NaPB can excrete the waste nitrogen not through the urea cycle but via the alternative pathway. The removal of nitrogen via alternative pathway lowers the flux of arginine in the urea cycle. Thereby, the clinical complications due to insufficient amount of protein intake can be prevented. Thus, NaPB therapy can be expected as a useful therapeutic option, particularly in patients with ARG1-D.

Lipid nanoparticle-targeted mRNA therapy as a treatment for the inherited metabolic liver disorder arginase deficiency.

Arginase deficiency is caused by biallelic mutations in arginase 1 (ARG1), the final step of the urea cycle, and results biochemically in hyperargininemia and the presence of guanidino compounds, while it is clinically notable for developmental delays, spastic diplegia, psychomotor function loss, and (uncommonly) death. There is currently no completely effective medical treatment available. While preclinical strategies have been demonstrated, disadvantages with viral-based episomal-expressing gene therapy vectors include the risk of insertional mutagenesis and limited efficacy due to hepatocellular division. Recent advances in messenger RNA (mRNA) codon optimization, synthesis, and encapsulation within biodegradable liver-targeted lipid nanoparticles (LNPs) have potentially enabled a new generation of safer, albeit temporary, treatments to restore liver metabolic function in patients with urea cycle disorders, including ARG1 deficiency. In this study, we applied such technologies to successfully treat an ARG1-deficient murine model. Mice were administered LNPs encapsulating human codon-optimized ARG1 mRNA every 3 d. Mice demonstrated 100% survival with no signs of hyperammonemia or weight loss to beyond 11 wk, compared with controls that perished by day 22. Plasma ammonia, arginine, and glutamine demonstrated good control without elevation of guanidinoacetic acid, a guanidino compound. Evidence of urea cycle activity restoration was demonstrated by the ability to fully metabolize an ammonium challenge and by achieving near-normal ureagenesis; liver arginase activity achieved 54% of wild type. Biochemical and microscopic data showed no evidence of hepatotoxicity. These results suggest that delivery of ARG1 mRNA by liver-targeted nanoparticles may be a viable gene-based therapeutic for the treatment of arginase deficiency.

Metal ions-induced stability and function of bimetallic human arginase-I, a therapeutically important enzyme.

Recent studies have highlighted the therapeutic importance of bimetallic human arginase-I against hyperargininemia and L-arginine auxotrophic cancers. The longer retention of catalytic activity of the Co2+-enzyme than that of the Mn2+ in human serum is associated with its enhanced therapeutic potential. To understand the basis of this and also to explore the role of a bimetallic center as well as the role of individual metal ions in the stability, we performed a detailed biochemical and biophysical investigation. The thermodynamic and kinetic stabilities of both the holo proteins are found to be significantly higher than the apo form, indicating that an intact bimetallic centre is vital for the enhanced stability of the holo proteins. The Co2+-protein is found to be more stable than that of the Mn2+, which might explain its longer retention of activity observed in the serum. Mutational studies demonstrated that the metal ions are individually crucial for both the enhanced stability and catalytic activity. Furthermore, we investigated the underlying mechanism for the effect of heat activation on the holo protein for higher catalytic activity, which is not yet known for arginases. Our data reveal that heat activation significantly increases the stability of the holo protein through a metal-induced increase in the helical content leading to the formation of a kinetically competent enzyme. Thus, the present study provides an in-depth insight into the significance of heat activation and the role of metal ions in human arginase, which may be useful for better understanding of its therapeutic use.

Human recombinant arginase enzyme reduces plasma arginine in mouse models of arginase deficiency.

Arginase deficiency is caused by deficiency of arginase 1 (ARG1), a urea cycle enzyme that converts arginine to ornithine. Clinical features of arginase deficiency include elevated plasma arginine levels, spastic diplegia, intellectual disability, seizures and growth deficiency. Unlike other urea cycle disorders, recurrent hyperammonemia is typically less severe in this disorder. Normalization of plasma arginine levels is the consensus treatment goal, because elevations of arginine and its metabolites are suspected to contribute to the neurologic features. Using data from patients enrolled in a natural history study conducted by the Urea Cycle Disorders Consortium, we found that 97% of plasma arginine levels in subjects with arginase deficiency were above the normal range despite conventional treatment. Recently, arginine-degrading enzymes have been used to deplete arginine as a therapeutic strategy in cancer. We tested whether one of these enzymes, a pegylated human recombinant arginase 1 (AEB1102), reduces plasma arginine in murine models of arginase deficiency. In neonatal and adult mice with arginase deficiency, AEB1102 reduced the plasma arginine after single and repeated doses. However, survival did not improve likely, because this pegylated enzyme does not enter hepatocytes and does not improve hyperammonemia that accounts for lethality. Although murine models required dosing every 48 h, studies in cynomolgus monkeys indicate that less frequent dosing may be possible in patients. Given that elevated plasma arginine rather than hyperammonemia is the major treatment challenge, we propose that AEB1102 may have therapeutic potential as an arginine-reducing agent in patients with arginase deficiency.

Hyperargininemia: 7-month follow-up under sodium benzoate therapy in an Italian child presenting progressive spastic paraparesis, cognitive decline, and novel mutation in ARG1 gene.

BACKGROUND: Hyperargininemia due to mutations in ARG1 gene is an autosomal recessive inborn error of metabolism caused by a defect in the final step of the urea cycle. Common clinical presentation is a variable association of progressive spastic paraparesis, epilepsy, and cognitive deficits. METHODS: We describe the clinical history of an Italian child presenting progressive spastic paraparesis, carrying a new homozygous missense mutation in the ARG1 gene. A detailed clinical, biochemical, and neurophysiological follow-up after 7 months of sodium benzoate therapy is reported. RESULTS: Laboratory findings, gait abnormalities, spastic paraparesis, and electroencephalographic and neurophysiological abnormalities remained quite stable over the follow-up. Conversely, a mild cognitive deterioration has been detected by means of the neuropsychologic assessment. CONCLUSIONS: Further longitudinal studies by means of longer follow-up and using clinical, biochemical, radiological, and neurophysiological assessments are needed in such patients to describe natural history and monitor the effects of treatments.

Treatment of arginase deficiency revisited: guanidinoacetate as a therapeutic target and biomarker for therapeutic monitoring.

Hyperargininaemia is a disorder of the last step of the urea cycle. It is an autosomal recessive disease caused by deficiency of liver arginase-1 and usually presents later in childhood with progressive neurological symptoms including marked spasticity. In contrast with other urea cycle disorders, hyperammonaemia is not usually present but can be a feature. A number of guanidine compounds may accumulate in the blood and cerebrospinal fluid of these patients, which could play an important pathophysiological role. Guanidinoacetate is of particular interest as a well-known potent epileptogenic compound in guanidinoacetate methyltransferase deficiency. We found markedly elevated guanidinoacetate levels in a patient with arginase deficiency, which dropped significantly in response to dietary and medical treatment. Measurement of guanidinoacetate and other guanidino compounds may, therefore, be important for therapeutic monitoring in arginase deficiency.

Lethal phenotype in conditional late-onset arginase 1 deficiency in the mouse.

Human arginase deficiency is characterized by hyperargininemia and infrequent episodes of hyperammonemia, which lead to neurological impairment with spasticity, loss of ambulation, seizures, and severe mental and growth retardation; uncommonly, patients suffer early death from this disorder. In a murine targeted knockout model, onset of the phenotypic abnormality is heralded by weight loss at around day 15, and death occurs typically by postnatal day 17 with hyperargininemia and markedly elevated ammonia. This discrepancy between the more attenuated juvenile-onset human disease and the lethal neonatal murine model has remained suboptimal for studying and developing therapy for the more common presentation of arginase deficiency. These investigations aimed to address this issue by creating an adult conditional knockout mouse to determine whether later onset of arginase deficiency also resulted in lethality. Animal survival and ammonia levels, body weight, circulating amino acids, and tissue arginase levels were examined as outcome parameters after widespread Cre-recombinase activation in a conditional knockout model of arginase 1 deficiency. One hundred percent of adult female and 70% of adult male mice died an average of 21.0 and 21.6 days, respectively, after the initiation of tamoxifen administration. Animals demonstrated elevated circulating ammonia and arginine at the onset of phenotypic abnormalities. In addition, brain and liver amino acids demonstrated abnormalities. These studies demonstrate that (a) the absence of arginase in adult animals results in a disease profile (leading to death) similar to that of the targeted knockout and (b) the phenotypic abnormalities seen in the juvenile-onset model are not exclusive to the age of the animal but instead to the biochemistry of the disorder. This adult model will be useful for developing gene- and cell-based therapies for this disorder that will not be limited by the small animal size of neonatal therapy and for developing a better understanding of the characteristics of hyperargininemia.

Arginase deficiency with new phenotype and a novel mutation: contemporary summary.

In areas without expanded newborn screening, instead of presenting neonatally, patients with arginase deficiency typically present with spastic paraplegia in early childhood. Diagnosis of this rare neurometabolic disease poses the first challenge because it is often misdiagnosed as cerebral palsy during initial stages. We describe arginase deficiency in a 20-year-old woman with spastic paraplegia, progressive dystonia, dementia, peripheral neuropathy, epilepsy, liver cirrhosis, and non-B/non-C hepatocellular carcinoma. A novel homozygous mutation NM_000045.2 (ARG1):c.673del (p.Arg225GlyfsX5) was detected. We suggest that all children presenting with progressive neurodegeneration or spastic paraplegia in the absence of risk factors for cerebral palsy should be screened for inborn errors of metabolism, including arginase deficiency. For monitoring urea cycle defects, noninvasive imaging screening for liver fibrosis and hepatocellular carcinoma can help ensure early detection, with potential treatment implications.

Argocytes containing enzyme nanoparticles reduce toxic concentrations of arginine in the blood.

A method for incorporation of arginase nanoparticles into mouse erythrocytes has been developed and the possibility of reducing blood arginine concentration in animals with experimental hyperargininemia with arginase-loaded erythrocytes (argocytes) has been studied. Argocyte infusion to animals with hyperargininemia led to a rapid decrease in blood arginine concentration within 1 h and this effect of argocytes persisted for at least 4 h. This was paralleled by an increase in plasma concentrations of urea and ornithine. Hence, plasma arginine is hydrolyzed by arginase incorporated into argocytes; argocytes are functionally active and can serve as a defense system in pathological hyperargininemia, while the method developed by us can be regarded as a new nanobiotechnology for medicine and veterinary.

High plasma citrulline and arginine levels ensured by sustained-release citrulline supplementation in rats.

OBJECTIVE: Dietary-supplemented arginine has been shown to have positive effects on cardiovascular disease, but several drawbacks exist and could potentially be avoided by using L-citrulline, since it is recycled to L-arginine. However, citrulline is very rapidly metabolized. We therefore developed a sustained-release form of citrulline and evaluated its metabolic behavior in rats. METHODS: Male Sprague Dawley rats were divided into three groups: receiving "empty microcapsule" (control group), 1 g/kg/d immediate-release citrulline (IR citrulline group), or 1 g/kg/d sustained-release citrulline (SR citrulline group). Citrulline was given each day at 9 a.m. after blood samples for 9 d, and on day 10, blood samples were drawn every 4 h to study the decrease in plasma amino acid concentrations. RESULTS: SR citrulline led to a sustained increase in citrullinemia and argininemia compared to IR citrulline, and on day 6 argininemia was significantly (P < 0.01) higher with SR compared to IR citrulline. Moreover, argininemia was significantly higher in the SR citrulline group than in controls throughout the study and SR citrulline maintained high argininemia and citrullinemia, at least over 12 h. CONCLUSION: This experimental study provides a strong rationale for using this new formulation for atherosclerosis treatment.