Inhibition of QDPR synergistically modulates intracellular tetrahydrobiopterin profiles in cooperation with methotrexate.

Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC50 of 0.72 µM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX.

Dysregulated BH4 metabolism facilitates immunosuppression in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive, malignant, and lethal cancers, displaying strong resistance to immunotherapy. In this issue of Cell Metabolism, a study by Liu et al. identifies tetrahydrobiopterin metabolic dysregulation as a key driver for the immunosuppressive PDAC environment in mouse and human.

Comparison of Cost Analysis in Patients with Tetrahydrobiopterin-Responsive and Non-Responsive Phenylketonuria in Turkey.

Phenylketonuria is an inherited metabolic disorder that leads to neurobehavioral dysfunction. The main treatment is a low-phenylalanine diet and/or the cofactor tetrahydrobiopterin. Regular outpatient follow-up care and measurement of the phenylalanine levels in the blood are required. We aimed to analyze the economic burden of phenylketonuria on families and the state. The patients with phenylketonuria were divided into three groups according to their treatment: a low-phenylalanine diet group (n = 50), a tetrahydrobiopterin group (n = 44), and a group taking tetrahydrobiopterin together with the diet (n = 25). A comparative cost analysis was carried out. The annual economic burden to the state was calculated to average EUR 18,801 ± 15,345 and was lowest in the diet group, then in the tetrahydrobiopterin group, and highest in the tetrahydrobiopterin + diet group (p < 0.001). Out-of-pocket costs amounted to EUR 1531 ± 1173 per year, and indirect losses averaged EUR 2125 ± 1930 per year for all families. The economic loss was significantly lower in the families taking tetrahydrobiopterin than in the other groups (p = 0.001). The combined use of medical nutrition and BH4 treatments has been shown to increase the economic burden on the state. Reimbursing low-protein products and increasing the number of patients eligible for financial allowances may reduce the economic burden on families.

Children and Adolescents with Early Treated Phenylketonuria: Cognitive Development and Fluctuations of Blood Phenylalanine Levels.

BACKGROUND: We assessed the relationship between the cognitive development of children and adolescents with phenylketonuria (PKU) and fluctuations in peripheral phenylalanine (Phe) levels. METHODS: We examined the neurocognitive performance of 33 children and adolescents with early treated PKU, of whom 18 were treated with sapropterin dihydrochloride, and 15 were on a classic diet. For 26 weeks, patients were assessed weekly for their blood phenylalanine (Phe) levels. Phe levels were analyzed for fluctuations indicated by the individual standard deviation. Fluctuations were compared to the standard deviation of 26 Phe level measurements before the study interval. We also assessed the concurrent IQ of the patients. This was repeated at one-, two-, and seven-year intervals. RESULTS: Full-scale IQ in patients treated with a classic diet did not change within the follow-up. In patients treated with Sapropterin dihydrochloride, however, there was a considerable gain in full-scale IQ. This was particularly true if blood Phe fluctuations increased in patients of this treatment group. CONCLUSIONS: Sapropterin dihydrochloride enhances Phe tolerance in patients with PKU. Increasing blood Phe fluctuations following enhanced Phe tolerance may indicate that the treatment not only allows patients to relax their Phe-restricted diet but also may support cognitive development in patients.

Nanoparticle-mediated delivery of tetrahydrobiopterin restores endothelial function in diabetic rats.

Endothelial dysfunction, underlying the vascular complications of diabetes and other cardiovascular disorders, may result from uncoupling of endothelial nitric oxide synthase (eNOS) activity due to decreased levels of tetrahydrobiopterin (BH4), a critical co-factor for eNOS. Some clinical trials attempting to deliver exogenous BH4 as a potential therapeutic strategy in vascular disease states have failed due to oxidation of BH4 in the circulation. We sought to develop a means of protecting BH4 from oxidation while delivering it to dysfunctional endothelial cells. Polymeric and solid lipid nanoparticles (NPs) loaded with BH4 were delivered by injection or oral gavage, respectively, to streptozotocin-induced diabetic rats. BH4 was measured in coronary endothelial cells and endothelium-dependent vascular reactivity was assessed in vascular rings. Lymphatic uptake of orally delivered lipid NPs was verified by sampling mesenteric lymph. BH4-loaded polymeric NPs maintained nitric oxide production by cultured endothelial cells under conditions of oxidative stress. BH4-loaded NPs, delivered via injection or ingestion, increased coronary endothelial BH4 concentration and improved endothelium-dependent vasorelaxation in diabetic rats. Pharmacodynamics assessment indicated peak concentration of solid lipid NPs in the systemic bloodstream 6 hours after ingestion, with disappearance noted by 48 hours. These studies support the feasibility of utilizing NPs to deliver BH4 to dysfunctional endothelial cells to increase nitric oxide bioavailability. BH4-loaded NPs could provide an innovative tool to restore redox balance in blood vessels and modulate eNOS-mediated vascular function to reverse or retard vascular disease in diabetes.

QDPR deficiency drives immune suppression in pancreatic cancer.

The relevance of biopterin metabolism in resistance to immune checkpoint blockade (ICB) therapy remains unknown. We demonstrate that the deficiency of quinoid dihydropteridine reductase (QDPR), a critical enzyme regulating biopterin metabolism, causes metabolite dihydrobiopterin (BH2) accumulation and decreases the ratio of tetrahydrobiopterin (BH4) to BH2 in pancreatic ductal adenocarcinomas (PDACs). The reduced BH4/BH2 ratio leads to an increase in reactive oxygen species (ROS) generation and a decrease in the distribution of H3K27me3 at CXCL1 promoter. Consequently, myeloid-derived suppressor cells are recruited to tumor microenvironment via CXCR2 causing resistance to ICB therapy. We discovered that BH4 supplementation is capable to restore the BH4/BH2 ratio, enhance anti-tumor immunity, and overcome ICB resistance in QDPR-deficient PDACs. Tumors with lower QDPR expression show decreased responsiveness to ICB therapy. These findings offer a novel strategy for selecting patient and combining therapies to improve the effectiveness of ICB therapy in PDAC.

Tetrahydrobiopterin metabolism attenuates ROS generation and radiosensitivity through LDHA S-nitrosylation: novel insight into radiogenic lung injury.

Genotoxic therapy triggers reactive oxygen species (ROS) production and oxidative tissue injury. S-nitrosylation is a selective and reversible posttranslational modification of protein thiols by nitric oxide (NO), and 5,6,7,8-tetrahydrobiopterin (BH4) is an essential cofactor for NO synthesis. However, the mechanism by which BH4 affects protein S-nitrosylation and ROS generation has not been determined. Here, we showed that ionizing radiation disrupted the structural integrity of BH4 and downregulated GTP cyclohydrolase I (GCH1), which is the rate-limiting enzyme in BH4 biosynthesis, resulting in deficiency in overall protein S-nitrosylation. GCH1-mediated BH4 synthesis significantly reduced radiation-induced ROS production and fueled the global protein S-nitrosylation that was disrupted by radiation. Likewise, GCH1 overexpression or the administration of exogenous BH4 protected against radiation-induced oxidative injury in vitro and in vivo. Conditional pulmonary Gch1 knockout in mice (Gch1fl/fl; Sftpa1-Cre+/- mice) aggravated lung injury following irradiation, whereas Gch1 knock-in mice (Gch1lsl/lsl; Sftpa1-Cre+/- mice) exhibited attenuated radiation-induced pulmonary toxicity. Mechanistically, lactate dehydrogenase (LDHA) mediated ROS generation downstream of the BH4/NO axis, as determined by iodoacetyl tandem mass tag (iodoTMT)-based protein quantification. Notably, S-nitrosylation of LDHA at Cys163 and Cys293 was regulated by BH4 availability and could restrict ROS generation. The loss of S-nitrosylation in LDHA after irradiation increased radiosensitivity. Overall, the results of the present study showed that GCH1-mediated BH4 biosynthesis played a key role in the ROS cascade and radiosensitivity through LDHA S-nitrosylation, identifying novel therapeutic strategies for the treatment of radiation-induced lung injury.

Exploring the therapeutic potential of tetrahydrobiopterin for heart failure with preserved ejection fraction: A path forward.

A large number of patients are affected by classical heart failure (HF) symptomatology with preserved ejection fraction (HFpEF) and multiorgan syndrome. Due to high morbidity and mortality rate, hospitalization and mortality remain serious socioeconomic problems, while the lack of effective pharmacological or device treatment means that HFpEF presents a major unmet medical need. Evidence from clinical and basic studies demonstrates that systemic inflammation, increased oxidative stress, and impaired mitochondrial function are the common pathological mechanisms in HFpEF. Tetrahydrobiopterin (BH4), beyond being an endogenous co-factor for catalyzing the conversion of some essential biomolecules, has the capacity to prevent systemic inflammation, enhance antioxidant resistance, and modulate mitochondrial energy production. Therefore, BH4 has emerged in the last decade as a promising agent to prevent or reverse the progression of disorders such as cardiovascular disease. In this review, we cover the clinical progress and limitations of using downstream targets of nitric oxide (NO) through NO donors, soluble guanylate cyclase activators, phosphodiesterase inhibitors, and sodium-glucose co-transporter 2 inhibitors in treating cardiovascular diseases, including HFpEF. We discuss the use of BH4 in association with HFpEF, providing new evidence for its potential use as a pharmacological option for treating HFpEF.

Mechanisms underlying the efficacy and limitation of dopa and tetrahydrobiopterin therapies for the deficiency of GTP cyclohydrolase 1 revealed in a novel mouse model.

Dopa and tetrahydrobiopterin (BH4) supplementation are recommended therapies for the dopa-responsive dystonia caused by GTP cyclohydrolase 1 (GCH1, also known as GTPCH) deficits. However, the efficacy and mechanisms of these therapies have not been intensively studied yet. In this study, we tested the efficacy of dopa and BH4 therapies by using a novel GTPCH deficiency mouse model, Gch1KI/KI, which manifested infancy-onset motor deficits and growth retardation similar to the patients. First, dopa supplementation supported Gch1KI/KI mouse survival to adulthood, but residual motor deficits and dwarfism remained. Interestingly, RNAseq analysis indicated that while the genes participating in BH4 biosynthesis and regeneration were significantly increased in the liver, no significant changes were observed in the brain. Second, BH4 supplementation alone restored the growth of Gch1KI/KI pups only in early postnatal developmental stage. High doses of BH4 supplementation indeed restored the total brain BH4 levels, but brain dopamine deficiency remained. While total brain TH levels were relatively increased in the BH4 treated Gch1KI/KI mice, the TH in the striatum were still almost undetectable, suggesting differential BH4 requirements among brain regions. Last, the growth of Gch1KI/KI mice under combined therapy outperformed dopa or BH4 therapy alone. Notably, dopamine was abnormally high in more than half, but not all, of the treated Gch1KI/KI mice, suggesting the existence of variable synergetic effects of dopa and BH4 supplementation. Our results provide not only experimental evidence but also novel mechanistic insights into the efficacy and limitations of dopa and BH4 therapies for GTPCH deficiency.

Serum Neopterin, Biopterin, Tryptophan, and Kynurenine Levels in Patients with Fabry Disease

Background: Fabry disease is characterized by the accumulation of globotriaosylceramide. Substrate accumulation in lysosomes is thought to trigger an inflammatory response and is responsible for progressive organ damage through the induction of autoimmunity. The levels of pteridine and kynurenine pathway metabolites increase when immune activation is observed and are employed to monitor several diseases and determine prognosis. Aims: To elucidate the effects of immune activation on the pathophysiology of Fabry disease and to investigate the potential utility of pteridine and kynurenine metabolites. Study Design: A prospective case-control study. Methods: In this study, 33 patients with Fabry disease and 33 age-and sex-matched healthy controls were included. Blood pteridine and kynurenine metabolites were studied in both groups. Organ involvement in Fabry disease and its correlation with the pteridine and kynurenine pathways were also investigated. Results: The patients' neopterin and biopterin levels and the tryptophan/kynurenine ratio were statistically higher than those of the healthy control group (p < 0.05). A statistically significant association was found between neopterin levels and hypertrophic cardiomyopathy, cardiac arrhythmias, and GFR values (p = 0.044, p = 0.021, and p = 0.030, respectively), tryptophan and corneal verticillate, hearing loss and tinnitus (p = 0.010, p = 0.009 and p = 0.046, respectively), and kynurenine levels and valvular heart disease (p = 0.020). Conclusion: From the onset of the disease, patients with Fabry disease exhibited elevated levels of inflammation and immune activation. Furthermore, inflammation and immune activation markers can be used as early disease biomarkers.

Photo-Induced Disproportionation-Mediated Photodynamic Therapy: Simultaneous Oxidation of Tetrahydrobiopterin and Generation of Superoxide Radicals.

We herein present an approach of photo-induced disproportionation for preparation of Type-I photodynamic agents. As a proof of concept, BODIPY-based photosensitizers were rationally designed and prepared. The photo-induced intermolecular electron transfer between homotypic chromophores leads to the disproportionation reaction, resulting in the formation of charged intermediates, cationic and anionic radicals. The cationic radicals efficiently oxidize the cellularimportant coenzyme, tetrahydrobiopterin (BH4 ), and the anionic radicals transfer electrons to oxygen to produce superoxide radicals (O2 - ⋅). One of our Type-I photodynamic agents not only self-assembles in water but also effectively targets the endoplasmic reticulum. It displayed excellent photocytotoxicity even in highly hypoxic environments (2 % O2 ), with a half-maximal inhibitory concentration (IC50 ) of 0.96 µM, and demonstrated outstanding antitumor efficacy in murine models bearing HeLa tumors.

Tetrahydrobiopterin (BH4) treatment stabilizes tyrosine hydroxylase: Rescue of tyrosine hydroxylase deficiency phenotypes in human neurons and in a knock-in mouse model.

Proteostatic regulation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, is crucial for maintaining proper brain neurotransmitter homeostasis. Variants of the TH gene are associated with tyrosine hydroxylase deficiency (THD), a rare disorder with a wide phenotypic spectrum and variable response to treatment, which affects protein stability and may lead to accelerated degradation, loss of TH function and catecholamine deficiency. In this study, we investigated the effects of the TH cofactor tetrahydrobiopterin (BH4) on the stability of TH in isolated protein and in DAn- differentiated from iPSCs from a human healthy subject, as well as from THD patients with the R233H variant in homozygosity (THDA) and R328W and T399M variants in heterozygosity (THDB). We report an increase in TH and dopamine levels, and an increase in the number of TH+ cells in control and THDA cells. To translate this in vitro effect, we treated with BH4 a knock-in THD mouse model with Th variant corresponding to R233H in patients. Importantly, treatment with BH4 significantly improved motor function in these mice, as demonstrated by increased latency on the rotarod test and improved horizontal activity (catalepsy). In conclusion, our study demonstrates the stabilizing effects of BH4 on TH protein levels and function in THD neurons and mice, rescuing disease phenotypes and improving motor outcomes. These findings highlight the therapeutic potential of BH4 as a treatment option for THDA patients with specific variants and provide insights into the modulation of TH stability and its implications for THD management.

BH4 as a Therapeutic Target for ADHD: Relevance to Neurotransmitters and Stress-Driven Symptoms.

Tetrahydrobiopterin (BH4) is a critical cofactor in a variety of metabolic pathways that have been linked to ADHD. There have been no previous studies utilizing BH4 as a supplement for ADHD. BH4 has been approved as a treatment for phenylketonuria (PKU). Individuals with PKU and ADHD appear to have low DA levels in common, suggesting that the hypodopaminergic state seen in both illnesses could be a relationship between the two. Clinical research involving supplementation of BH4 has shown low occurrence of adverse. In experiments, BH4 has also been found to have good blood-brain barrier permeability. BH4 also has the ability in scavenging ROS activity, which is an implication of stress and is seen in ADHD. BH4's significance in ADHD is reviewed in this paper because of its involvement in numerous neurodevelopmental metabolic pathways, and we anticipate that exogenous BH4 can be used to treat ADHD.

Genotypic variants of the tetrahydrobiopterin (BH4) biosynthesis genes in patients with hyperphenylalaninemia from different regions of Iran.

BACKGROUND: Hyperphenylalaninemia (HPA) is a metabolic disorder classified into phenylalanine-4-hydroxylase (PAH) and non-PAH deficiency. The latter is produced by mutations in genes involved in the tetrahydrobiopterin (BH4) biosynthesis pathway and DNAJC12 pathogenetic variants. The BH4 metabolism, including de novo biosynthesis involved genes (i.e., guanosine 5'-triphosphate cyclohydrolase I (GTPCH/GCH1), sepiapterin reductase (SR/SPR), 6-pyruvoyl-tetrahydropterin synthase (PTPS/PTS)), and two genes that play roles in cofactor regeneration pathway (i.e., dihydropteridine reductase (DHPR/QDPR) and pterin-4α-carbinolamine dehydratase (PCD/PCBD1)). The subsequent systemic hyperphenylalaninemia and monoamine neurotransmitter deficiency lead to neurological consequences. The high rate of consanguineous marriages in Iran substantially increases the incidence of BH4 deficiency. METHODS: We utilized the Sanger sequencing technique in this study to investigate 14 Iranian patients with non-PAH deficiency. All affected subjects in this study had HPA and no mutation was detected in their PAH gene. RESULTS: We successfully identified six mutant alleles in BH4-deficiency-associated genes, including three novel mutations: one in QDPR, one in PTS, and one in the PCBD1 gene, thus giving a definite diagnosis to these patients. CONCLUSION: In this light, appropriate patient management may follow. The clinical effect of reported variants is essential for genetic counseling and prenatal diagnosis in the patients' families and significant for the improvement of precision medicine.

Long-term comparative effectiveness of pegvaliase versus medical nutrition therapy with and without sapropterin in adults with phenylketonuria.

Phenylketonuria is characterized by intellectual disability and behavioral, psychiatric, and movement disorders resulting from phenylalanine (Phe) accumulation. Standard-of-care treatment involves a Phe-restricted diet plus medical nutrition therapy (MNT), with or without sapropterin dihydrochloride, to reduce blood Phe levels. Pegvaliase is an injectable enzyme substitution treatment approved for adult patients with blood Phe >600 µmol/L despite ongoing management. A previous comparative effectiveness analysis using data from the Phase 3 PRISM trials of pegvaliase (NCT01819727 and NCT01889862) and the Phenylketonuria Demographics, Outcomes and Safety Registry (PKUDOS; NCT00778206) suggested that pegvaliase was more effective at lowering mean blood Phe levels than sapropterin + MNT or MNT alone at 1 and 2 years of treatment. The current work augments and complements the previous analysis by including additional follow-up from the completed studies, robust methods reflecting careful consideration of issues with the distribution of Phe, and alternative methods for adjustment that are important for control of potential confounding in comparative effectiveness. Median blood Phe levels were lower, and median intact protein intakes were higher, in the pegvaliase group (n = 183) than in the sapropterin + MNT (n = 82) and MNT (n = 67) groups at Years 1, 2, and 3. In the pegvaliase group, median blood Phe levels decreased from baseline (1244 µmol/L) to Year 1 (535 µmol/L), Year 2 (142 µmol/L), and Year 3 (167 µmol/L). In the sapropterin + MNT group, median blood Phe levels decreased from baseline (900 µmol/L) to Year 1 (588 µmol/L) and Year 2 (592 µmol/L), and increased at Year 3 (660 µmol/L). In the MNT group, median blood Phe levels decreased slightly from baseline (984 µmol/L) to Year 1 (939 µmol/L) and Year 2 (941 µmol/L), and exceeded baseline levels at Year 3 (1157 µmol/L). The model-estimated proportions of participants achieving blood Phe ≤600 µmol/L were 41%, 100%, and 100% in the pegvaliase group at Years 1, 2, and 3, respectively, compared with 55%, 58%, and 38% in the sapropterin + MNT group and 5%, 16%, and 0% in the MNT group. The estimated proportions of participants achieving more stringent blood Phe targets of ≤360 µmol/L and ≤120 µmol/L were also higher in the pegvaliase group than in the other groups at Years 2 and 3. Overall, our results indicate that, compared with standard therapy, pegvaliase induces a substantial, progressive, and sustained decrease in blood Phe levels - to a much greater extent than sapropterin + MNT or MNT alone - which is expected to improve long-term outcomes in patients with phenylketonuria.

Tetrahydrobiopterin inhibitor-based antioxidant metabolic strategy for enhanced cancer ferroptosis-immunotherapy.

The induction of immunogenic ferroptosis in cancer cell is limited by the complex and delicate antioxidant system in the organism. Synergistic induction of oxidative damage and inhibition of the defensive redox system in tumor cells is critical to promote lethal accumulation of lipid peroxides and activate immunogenic death (ICD). To address this challenge, we present a multifunctional and dual-responsive layered double hydroxide (LDH) nanosheet to enhance immunogenic ferroptosis. The MTX-LDH@MnO2 nanoplatform is constructed by intercalating methotrexate (MTX) into LDH interlayers and electrostatically absorbing biomineralized ovalbumin (OVA)-MnO2 onto the LDH surface. Specifically, the released Mn2+ from the incorporated MnO2 triggers a Fenton-like reaction, leading to reactive oxygen species (ROS) accumulation, while the depletion of reduced glutathione (GSH) further intensifies oxidative stress, resulting in the induction of ferroptosis. MTX is released in response to the acidic environment of tumor cells and inhibits the regeneration of tetrahydrobiopterin (BH4), modulating the GTP cyclic hydrolase 1 (GCH1)/BH4 axis. MTX disrupts the antioxidant metabolic activity regulated by GCH1/BH4 axis and inhibits ROS consumption, further boosting the ferroptosis effect, which promoted the release of damage-associated molecular patterns (DAMPs) and triggered ICD in the tumor. This activation subsequently leads to significant antitumor immune reactions, including DCs maturation, infiltration of CD4+/CD8+ T cells and cytokines release. The redox-controllable nanoplatform demonstrates promising anticancer efficacy in a mouse breast model providing a novel strategy for cancer immunotherapy.

Relative Oral Bioavailability and Food Effects of Two Sepiapterin Formulations in Healthy Participants.

Sepiapterin is an orally administered drug in development for the treatment of phenylketonuria, an inborn error of metabolism characterized by the deficiency of the phenylalanine-metabolizing enzyme phenylalanine hydroxylase. This study characterized the pharmacokinetics, safety, and tolerability of 2 clinical sepiapterin formulations (Phase 1/2, Phase 3) and the effects of food on the pharmacokinetics of the Phase 3 formulation in healthy participants. In Part A, 18 participants were randomized to one of 2 treatment sequences, each with 4 dosing periods comprising a single dose (20 or 60 mg/kg) of the Phase 1/2 or the Phase 3 formulation with a low-fat diet. In Part B, 14 participants were randomized to one of 2 sequences, each comprising 4 dosing periods of a single dose (20 or 60 mg/kg) of the Phase 3 formulation under fed (high-fat) or fasted conditions. Following oral administration, sepiapterin was quickly absorbed and rapidly and extensively converted to tetrahydrobiopterin (BH4). BH4 was the major circulating active moiety. Under low-fat conditions, the Phase 3 formulation was bioequivalent to the Phase 1/2 formulation at 20 mg/kg, while slightly lower BH4 exposure (approximately 0.81×) for the Phase 3 formulation was observed at 60 mg/kg. BH4 exposure increased to approximately 1.7× under the low-fat condition and approximately 2.8× under the high-fat condition at a dose of either 20 or 60 mg/kg for the Phase 3 formulation, compared with the fasted condition. Both sepiapterin formulations were well tolerated, with no serious or severe adverse events reported. All treatment-emergent adverse events were mild or moderate in severity.

BH4 supplementation reduces retinal cell death in ischaemic retinopathy.

Dysregulation of nitric oxide (NO) production can cause ischaemic retinal injury and result in blindness. How this dysregulation occurs is poorly understood but thought to be due to an impairment in NO synthase function (NOS) and nitro-oxidative stress. Here we investigated the possibility of correcting this defective NOS activity by supplementation with the cofactor tetrahydrobiopterin, BH4. Retinal ischaemia was examined using the oxygen-induced retinopathy model and BH4 deficient Hph-1 mice used to establish the relationship between NOS activity and BH4. Mice were treated with the stable BH4 precursor sepiapterin at the onset of hypoxia and their retinas assessed 48 h later. HPLC analysis confirmed elevated BH4 levels in all sepiapterin supplemented groups and increased NOS activity. Sepiapterin treatment caused a significant decrease in neuronal cell death in the inner nuclear layer that was most notable in WT animals and was associated with significantly diminished superoxide and local peroxynitrite formation. Interestingly, sepiapterin also increased inflammatory cytokine levels but not microglia cell number. BH4 supplementation by sepiapterin improved both redox state and neuronal survival during retinal ischaemia, in spite of a paradoxical increase in inflammatory cytokines. This implicates nitro-oxidative stress in retinal neurones as the cytotoxic element in ischaemia, rather than enhanced pro-inflammatory signalling.

Predictors of eventual requirement of phenylalanine-restricted diet in young infants with phenylalanine hydroxylase deficiency initially managed with sapropterin monotherapy.

BACKGROUND: Phenylalanine (Phe)-restricted diet is associated with lower quality of life for patients with phenylketonuria (PKU), and a concern for caregivers of recently-diagnosed infants. Sapropterin is an oral drug used as an alternative or adjunct to dietary treatment. We have observed that some of the young infants initially managed successfully with sapropterin monotherapy have required dietary treatment in long-term follow-up. We aimed to determine the baseline factors associated with future initiation of dietary treatment in these patients. METHODS: Data were obtained retrospectively from the medical records of 80 PKU patients started on sapropterin monotherapy before 3 months of age between 2011 and 2021. RESULTS: The patients were followed for a median of 3.9 years (Q1-Q3: 2.5-5.75 years). Sapropterin was tapered down and discontinued in 5 patients (6.3%) as their Phe levels remained below 360 µmol/L without treatment. Sapropterin monotherapy was sufficient in 62 patients (77.5%), while 13 (16.2%) required dietary treatment. Phe and tyrosine (Tyr) levels, and Phe:Tyr ratios differed significantly among the patients maintained on sapropterin monotherapy and those started on dietary treatment, but the Phe:Tyr ratio at diagnosis was the most important independent baseline variable (OR: 1.61, 95% CI: 1.15-2.27, p = 0.006), with Phe:Tyr ratio at diagnosis >5.25 associated with dietary treatment (sensitivity: 90.0%, specificity: 81.8%). Genotypic phenotype value (GPV), unavailable at baseline, was also associated with dietary treatment (median GPV 9.2 vs. 3.8, p = 0.006), but some genotypes were not specific to the final treatment modality. DISCUSSION: We propose that the Phe:Tyr ratio at diagnosis is an important indicator to predict dietary requirement in young infants initially managed with sapropterin monotherapy.

Urinary neopterin and biopterin indicate that inflammation has a role in autism spectrum disorder.

Inflammation is thought to be involved in the pathogenesis of autism spectrum disorder (ASD). Pteridine metabolites are biomarkers of inflammation that increase on immune system activation. In this study, we investigated the urinary pteridine metabolites in ASD patients as a possible biomarker for immune activation and inflammation. This observational, cross-sectional, prospective study collected urine samples from 212 patients with ASD and 68 age- and sex-matched healthy individuals. Urine neopterin (NE) and biopterin (BIO) levels were measured. Patients who had chronic disorders, active infection at the time of sampling, or high C-reactive protein levels were excluded. The urine NE and BIO concentrations were determined by high-performance liquid chromatography. The ratios of both NE and BIO to creatinine (CRE) were used to standardise the measurements. The NE/CRE and NE/BIO levels were significantly higher in ASD patients than controls. Univariate and multivariate models revealed a significant increase in NE/CRE and NE/BIO in ASD patients. There was a significant relationship between the NE/BIO [average area under the curve (AUC) = 0.717; range: 0.637-0.797] and NE/CRE (average AUC = 0.756; range: 0.684-0.828) ratios, which distinguished individuals with ASD from controls. The elevated NE/CRE and NE/BIO ratios suggest that inflammation and T cell-mediated immunity are involved in the pathophysiology of autism. NE/BIO could serve as a diagnostic inflammatory marker in the pathogenesis of ASD.