Complete correction of murine phenylketonuria by selection-enhanced hepatocyte transplantation.

BACKGROUND AND AIMS: Hepatocyte transplantation for genetic liver diseases has several potential advantages over gene therapy. However, the low efficiency of cell engraftment has limited its clinical implementation. This problem could be overcome by selectively expanding transplanted donor cells until they replace enough of the liver mass to achieve therapeutic benefit. We previously described a gene therapy method to selectively expand hepatocytes deficient in cytochrome p450 reductase (Cypor) using acetaminophen (APAP). Because Cypor is required for the transformation of APAP to a hepatotoxic metabolite, Cypor-deficient cells are protected from toxicity and are able to expand following APAP-induced liver injury. Here, we apply this selection system to correct a mouse model of phenylketonuria by cell transplantation. APPROACH AND RESULTS: Hepatocytes from a wild-type donor animal were edited in vitro to create Cypor deficiency and then transplanted into phenylketonuric animals. Following selection with APAP, blood phenylalanine concentrations were fully normalized and remained stable following APAP withdrawal. Cypor-deficient hepatocytes expanded from < 1% to ~14% in corrected animals, and they showed no abnormalities in blood chemistries, liver histology, or drug metabolism. CONCLUSIONS: We conclude that APAP-mediated selection of transplanted hepatocytes is a potential therapeutic for phenylketonuria with long-term efficacy and a favorable safety profile.

Cardiac phenotype in adolescents and young adults with long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) deficiency.

PURPOSE: Long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency (LCHADD) is a rare fatty acid oxidation disorder characterized by recurrent episodes of metabolic decompensation and rhabdomyolysis, as well as retinopathy, peripheral neuropathy, and cardiac involvement, such as infantile dilated cardiomyopathy. Because LCHADD patients are surviving longer, we sought to characterize LCHADD-associated major cardiac involvement in adolescence and young adulthood. METHODS: A retrospective cohort of 16 adolescent and young adult participants with LCHADD was reviewed for cardiac phenotype. RESULTS: Major cardiac involvement occurred in 9 of 16 participants, including sudden death, out-of-hospital cardiac arrest, acute cardiac decompensations with heart failure and/or in-hospital cardiac arrest, end-stage dilated cardiomyopathy, and moderate restrictive cardiomyopathy. Sudden cardiac arrest was more common in males and those with a history of infant cardiomyopathy. CONCLUSION: The cardiac manifestations of LCHADD in adolescence and early adulthood are complex and distinct from the phenotype seen in infancy. Life-threatening arrhythmia occurs at substantial rates in LCHADD, often in the absence of metabolic decompensation or rhabdomyolysis. The potential risk factors identified here-male sex and history of infant cardiomyopathy-may hint at strategies for risk stratification and possibly the prevention of these events.

Evaluating change in diet with pegvaliase treatment in adults with phenylketonuria: Analysis of phase 3 clinical trial data.

Phenylketonuria (PKU), a genetic disorder characterized by phenylalanine hydroxylase (PAH) deficiency and phenylalanine (Phe) accumulation, is primarily managed with a protein-restricted diet and PKU-specific medical foods. Pegvaliase is an enzyme substitution therapy approved for individuals with PKU and uncontrolled blood Phe concentrations (>600 µmol/L) despite prior management. This analysis assessed the effect of pegvaliase on dietary intake using data from the Phase 3 PRISM-1 (NCT01819727), PRISM-2 (NCT01889862), and 165-304 (NCT03694353) clinical trials. Participants (N = 250) had a baseline diet assessment, blood Phe ≥600 µmol/L, and had discontinued sapropterin; they were not required to follow a Phe-restricted diet. Outcomes were analyzed by baseline dietary group, categorized as >75%, some (>0% but ≤75%), or no protein intake from medical food. At baseline, mean age was 29.1 years, 49.2% were female, mean body mass index was 28.4 kg/m2, and mean blood Phe was 1237.0 µmol/L. Total protein intake was stable up to 48 months for all 3 baseline dietary groups. Over this time, intact protein intake increased in all groups, and medical protein intake decreased in those who consumed any medical protein at baseline. Of participants consuming some or >75% medical protein at baseline, 49.1% and 34.1% were consuming no medical protein at last assessment, respectively. Following a first hypophenylalaninemia (HypoPhe; 2 consecutive blood Phe measurements <30 µmol/L) event, consumption of medical protein decreased and consumption of intact protein increased. Substantial and sustained Phe reductions were achieved in all 3 baseline dietary groups. The probability of achieving sustained Phe response (SPR) at ≤600 µmol/L was significantly greater for participants consuming medical protein versus no medical protein in an unadjusted analysis, but no statistically significant difference between groups was observed for probability of achieving SPR ≤360 or SPR ≤120 µmol/L. Participants with alopecia (n = 49) had longer pegvaliase treatment durations, reached HypoPhe sooner, and spent longer in HypoPhe than those who did not have alopecia. Most (87.8%) had an identifiable blood Phe drop before their first alopecia episode, and 51.0% (n = 21/41) of first alopecia episodes with known duration resolved before the end of the HypoPhe episode. In conclusion, pegvaliase treatment allowed adults with PKU to lower their blood Phe, reduce their reliance on medical protein, and increase their intact and total protein intake. Results also suggest that HypoPhe does not increase the risk of protein malnutrition in adults with PKU receiving pegvaliase.

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.

State-of-the-art 2023 on gene therapy for phenylketonuria.

Phenylketonuria (PKU) or hyperphenylalaninemia is considered a paradigm for an inherited (metabolic) liver defect and is, based on murine models that replicate all human pathology, an exemplar model for experimental studies on liver gene therapy. Variants in the PAH gene that lead to hyperphenylalaninemia are never fatal (although devastating if untreated), newborn screening has been available for two generations, and dietary treatment has been considered for a long time as therapeutic and satisfactory. However, significant shortcomings of contemporary dietary treatment of PKU remain. A long list of various gene therapeutic experimental approaches using the classical model for human PKU, the homozygous enu2/2 mouse, witnesses the value of this model to develop treatment for a genetic liver defect. The list of experiments for proof of principle includes recombinant viral (AdV, AAV, and LV) and non-viral (naked DNA or LNP-mRNA) vector delivery methods, combined with gene addition, genome, gene or base editing, and gene insertion or replacement. In addition, a list of current and planned clinical trials for PKU gene therapy is included. This review summarizes, compares, and evaluates the various approaches for the sake of scientific understanding and efficacy testing that may eventually pave the way for safe and efficient human application.

Early diagnosis and treatment by newborn screening (NBS) or family history is associated with improved visual outcomes for long-chain 3-hydroxyacylCoA dehydrogenase deficiency (LCHADD) chorioretinopathy.

Long chain 3-hydroxyacyl-CoA dehydrogenase (LCHADD) is the only fatty acid oxidation disorder to develop a progressive chorioretinopathy resulting in vision loss; newborn screening (NBS) for this disorder began in the United States around 2004. We compared visual outcomes among 40 participants with LCHADD or trifunctional protein deficiency diagnosed symptomatically to those who were diagnosed via NBS or a family history. Participants completed ophthalmologic testing including measures of visual acuity, electroretinograms (ERG), fundal imaging, contrast sensitivity, and visual fields. Records were reviewed to document medical and treatment history. Twelve participants presented symptomatically with hypoglycemia, failure to thrive, liver dysfunction, cardiac arrest, or rhabdomyolysis. Twenty eight were diagnosed by NBS or due to a family history of LCHADD. Participants diagnosed symptomatically were older but had similar percent males and genotypes as those diagnosed by NBS. Treatment consisted of fasting avoidance, dietary long-chain fat restriction, MCT, C7, and/or carnitine supplementation. Visual acuity, rod- and cone-driven amplitudes on ERG, contrast sensitivity scores, and visual fields were all significantly worse among participants diagnosed symptomatically compared to NBS. In mixed-effects models, both age and presentation (symptomatic vs. NBS) were significant independent factors associated with visual outcomes. This suggests that visual outcomes were improved by NBS, but there was still lower visual function with advancing age in both groups. Early diagnosis and treatment by NBS is associated with improved visual outcomes and retinal function compared to participants who presented symptomatically. Despite the impact of early intervention, chorioretinopathy was greater with advancing age, highlighting the need for novel treatments.

Resting and total energy expenditure of patients with long-chain fatty acid oxidation disorders (LC-FAODs).

The basis of medical nutrition therapy for patients with LC-FAODs is to provide adequate energy to maintain anabolism and prevent catabolism. In practice, energy needs are estimated based on formulas derived from normal populations but it is unknown if energy expenditure among patients with LC-FAODs is similar to the normal population. We measured resting energy expenditure (REE), total energy expenditure (TEE) and body composition in 31 subjects with LC-FAODs ranging in age from 7 to 64 years. Measured REE was lower than estimated REE by various prediction equations and measured TEE was lower than estimated TEE. It is possible that the lower energy expenditure based on prediction formulas from the normal population is due to differences in body composition; we compared body composition to normal data from the 2017-18 National Health and Nutrition Examination Survey (NHANES). Fat free mass and fat mass was similar between subjects with an LC-FAOD and NHANES normal data suggesting no difference in body composition. We then compared measured REE and TEE to normal published data from the Dietary Reference Intakes (DRI). Measured REE and TEE were significantly lower among subjects with LC-FAODs compared to normal published energy expenditure data. Our results suggests patients with a LC-FAOD exhibit a lower REE and therefore actually have a slightly lower TEE than estimated. Current prediction equations may overestimate energy expenditure of patients with a LC-FAOD.

Biochemical and behavioural profile of NTBC treated Tyrosinemie type 1 mice.

BACKGROUND: Tyrosinemia type 1 (HT1) is a rare metabolic disorder caused by a defect in the tyrosine catabolic pathway. Since HT1 patients are treated with NTBC, outcome improved and life expectancy greatly increased. However extensive neurocognitive and behavioural problems have been described, which might be related to treatment with NTBC, the biochemical changes induced by NTBC, or metabolites accumulating due to the enzymatic defect characterizing the disease. OBJECTIVE: To study the possible pathophysiological mechanisms of brain dysfunction in HT1, we assessed blood and brain LNAA, and brain monoamine neurotransmitter metabolite levels in relation to behavioural and cognitive performance of HT1 mice. DESIGN: C57BL/6 littermates were divided in three different experimental groups: HT1, heterozygous and wild-type mice (n = 10; 5 male). All groups were treated with NTBC and underwent cognitive and behavioural testing. One week after behavioural testing, blood and brain material were collected to measure amino acid profiles and brain monoaminergic neurotransmitter levels. RESULTS: Irrespective of the genetic background, NTBC treatment resulted in a clear increase in brain tyrosine levels, whereas all other brain LNAA levels tended to be lower than their reference values. Despite these changes in blood and brain biochemistry, no significant differences in brain monoamine neurotransmitter (metabolites) were found and all mice showed normal behaviour and learning and memory. CONCLUSION: Despite the biochemical changes, NTBC and genotype of the mice were not associated with poorer behavioural and cognitive function of the mice. Further research involving dietary treatment of FAH-/- are warranted to investigate whether this reveals the cognitive impairments that have been seen in treated HT1 patients.

Modeling the cognitive effects of diet discontinuation in adults with phenylketonuria (PKU) using pegvaliase therapy in PAH-deficient mice.

Existing phenylalanine hydroxylase (PAH)-deficient mice strains are useful models of untreated or late-treated human phenylketonuria (PKU), as most contemporary therapies can only be initiated after weaning and the pups have already suffered irreversible consequences of chronic hyperphenylalaninemia (HPA) during early brain development. Therefore, we sought to evaluate whether enzyme substitution therapy with pegvaliase initiated near birth and administered repetitively to C57Bl/6-Pahenu2/enu2 mice would prevent HPA-related behavioral and cognitive deficits and form a model for early-treated PKU. The main results of three reported experiments are: 1) lifelong weekly pegvaliase treatment prevented the cognitive deficits associated with HPA in contrast to persisting deficits in mice treated with pegvaliase only as adults. 2) Cognitive deficits reappear in mice treated with weekly pegvaliase from birth but in which pegvaliase is discontinued at 3 months age. 3) Twice weekly pegvaliase injection also prevented cognitive deficits but again cognitive deficits emerged in early-treated animals following discontinuation of pegvaliase treatment during adulthood, particularly in females. In all studies, pegvaliase treatment was associated with complete correction of brain monoamine neurotransmitter content and with improved overall growth of the mice as measured by body weight. Mean total brain weight however remained low in all PAH deficient mice regardless of treatment. Application of enzyme substitution therapy with pegvaliase, initiated near birth and continued into adulthood, to PAH-deficient Pahenu2/enu2 mice models contemporary early-treated human PKU. This model will be useful for exploring the differential pathophysiologic effects of HPA at different developmental stages of the murine brain.

Management of early treated adolescents and young adults with phenylketonuria: Development of international consensus recommendations using a modified Delphi approach.

BACKGROUND: Early treated patients with phenylketonuria (PKU) often become lost to follow-up from adolescence onwards due to the historical focus of PKU care on the pediatric population and lack of programs facilitating the transition to adulthood. As a result, evidence on the management of adolescents and young adults with PKU is limited. METHODS: Two meetings were held with a multidisciplinary international panel of 25 experts in PKU and comorbidities frequently experienced by patients with PKU. Based on the outcomes of the first meeting, a set of statements were developed. During the second meeting, these statements were voted on for consensus generation (≥70% agreement), using a modified Delphi approach. RESULTS: A total of 37 consensus recommendations were developed across five areas that were deemed important in the management of adolescents and young adults with PKU: (1) general physical health, (2) mental health and neurocognitive functioning, (3) blood Phe target range, (4) PKU-specific challenges, and (5) transition to adult care. The consensus recommendations reflect the personal opinions and experiences from the participating experts supported with evidence when available. Overall, clinicians managing adolescents and young adults with PKU should be aware of the wide variety of PKU-associated comorbidities, initiating screening at an early age. In addition, management of adolescents/young adults should be a joint effort between the patient, clinical center, and parents/caregivers supporting adolescents with gradually gaining independent control of their disease during the transition to adulthood. CONCLUSIONS: A multidisciplinary international group of experts used a modified Delphi approach to develop a set of consensus recommendations with the aim of providing guidance and offering tools to clinics to aid with supporting adolescents and young adults with PKU.

Effects of fasting, feeding and exercise on plasma acylcarnitines among subjects with CPT2D, VLCADD and LCHADD/TFPD.

BACKGROUND: The plasma acylcarnitine profile is frequently used as a biochemical assessment for follow-up in diagnosed patients with fatty acid oxidation disorders (FAODs). Disease specific acylcarnitine species are elevated during metabolic decompensation but there is clinical and biochemical heterogeneity among patients and limited data on the utility of an acylcarnitine profile for routine clinical monitoring. METHODS: We evaluated plasma acylcarnitine profiles from 30 diagnosed patients with long-chain FAODs (carnitine palmitoyltransferase-2 (CPT2), very long-chain acyl-CoA dehydrogenase (VLCAD), and long-chain 3-hydroxy acyl-CoA dehydrogenase or mitochondrial trifunctional protein (LCHAD/TFP) deficiencies) collected after an overnight fast, after feeding a controlled low-fat diet, and before and after moderate exercise. Our purpose was to describe the variability in this biomarker and how various physiologic states effect the acylcarnitine concentrations in circulation. RESULTS: Disease specific acylcarnitine species were higher after an overnight fast and decreased by approximately 60% two hours after a controlled breakfast meal. Moderate-intensity exercise increased the acylcarnitine species but it varied by diagnosis. When analyzed for a genotype/phenotype correlation, the presence of the common LCHADD mutation (c.1528G > C) was associated with higher levels of 3-hydroxyacylcarnitines than in patients with other mutations. CONCLUSIONS: We found that feeding consistently suppressed and that moderate intensity exercise increased disease specific acylcarnitine species, but the response to exercise was highly variable across subjects and diagnoses. The clinical utility of routine plasma acylcarnitine analysis for outpatient treatment monitoring remains questionable; however, if acylcarnitine profiles are measured in the clinical setting, standardized procedures are required for sample collection to be of value.

A novel Pah-exon1 deleted murine model of phenylalanine hydroxylase (PAH) deficiency.

Phenylalanine hydroxylase (PAH) deficiency, colloquially known as phenylketonuria (PKU), is among the most common inborn errors of metabolism and in the past decade has become a target for the development of novel therapeutics such as gene therapy. PAH deficient mouse models have been key to new treatment development, but all prior existing models natively express liver PAH polypeptide as inactive or partially active PAH monomers, which complicates the experimental assessment of protein expression following therapeutic gene, mRNA, protein, or cell transfer. The mutant PAH monomers are able to form hetero-tetramers with and inhibit the overall holoenzyme activity of wild type PAH monomers produced from a therapeutic vector. Preclinical therapeutic studies would benefit from a PKU model that completely lacks both PAH activity and protein expression in liver. In this study, we employed CRISPR/Cas9-mediated gene editing in fertilized mouse embryos to generate a novel mouse model that lacks exon 1 of the Pah gene. Mice that are homozygous for the Pah exon 1 deletion are viable, severely hyperphenylalaninemic, accurately replicate phenotypic features of untreated human classical PKU and lack any detectable liver PAH activity or protein. This model of classical PKU is ideal for further development of gene and cell biologics to treat PKU.

Pegvaliase for the treatment of phenylketonuria: Results of the phase 2 dose-finding studies with long-term follow-up.

BACKGROUND: Phenylketonuria (PKU) is characterized by a deficiency in phenylalanine hydroxylase (PAH) that may lead to elevated blood phenylalanine (Phe) and significant neurocognitive and neuropsychological comorbidities. Pegvaliase (PALYNZIQ®, BioMarin Pharmaceutical Inc.) is a PEGylated recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL), which converts Phe to trans-cinnamic acid and ammonia, and was approved in May 2018 in the United States and in May 2019 in the European Union for decreasing blood Phe levels in adults with PKU with blood Phe levels >600 µmol/L. The efficacy and safety of pegvaliase was assessed in two phase 2 dose-finding studies in adults with PKU (PAL-002, NCT00925054, and PAL-004, NCT01212744). Participants completing these studies could enroll in a long-term extension study (PAL-003, NCT00924703). METHODS: Participants in PAL-002 received pegvaliase 0.001, 0.003, 0.01, 0.03, or 0.1 mg/kg weekly for 8 weeks, then continued treatment for a further 8 weeks with dose and/or frequency adjusted to achieve blood Phe concentrations of 60 to 600 µmol/L. Participants in PAL-004 received pegvaliase 0.001 to 0.4 mg/kg 5 days/week for 13 weeks, with modifications made to the starting dose in response to safety and/or efficacy, followed by 3 additional weeks of follow-up assessments. The maximum allowable daily dose in both studies was 1.0 mg/kg/day (5.0 mg/kg/week). Participants who completed any of the phase 2 studies (PAL-002; PAL-004; or a third phase 2 study, 165-205) were eligible to enroll in an open-label, multicenter, long-term extension study (PAL-003, NCT00924703). RESULTS: Thirty-seven of the 40 enrolled participants completed PAL-002 and 15 of the 16 enrolled participants completed PAL-004. Mean blood Phe at baseline was 1311.0 (standard deviation [SD] 354) µmol/L in PAL-002 and 1482.1 (SD 363.5) µmol/L in PAL-004. Mean blood Phe did not substantially decrease with pegvaliase treatment in PAL-002 (-206.3 [SD 287.1] µmol/L at Week 16) or PAL-004 (-410.8 [SD 653.7] µmol/L at Week 13). In PAL-004, mean blood Phe dropped from baseline by 929.1 µmol/L (SD 691.1) by Week 2; subsequent to dose modifications and interruptions, this early decrease in mean Phe level was not sustained. With increased pegvaliase dose and duration in PAL-003, mean blood Phe levels steadily decreased from baseline, with mean reductions by Week 120 of 68.8% (SD 44.2%) in PAL-002 participants and 75.9% (SD 32.4%) in PAL-004 participants. All participants in PAL-002 and PAL-004 reported ≥1 adverse event (AE), with higher exposure-adjusted event rates in PAL-004. The majority of AEs were mild (87.2% in PAL-002, 86.7% in PAL-004) or moderate (12.4% in PAL-002, 13.3% in PAL-004). The most commonly reported AEs in PAL-002 were injection site reaction (50.0% of participants), headache (42.1%), injection site erythema (36.8%), nausea (34.2%), and arthralgia (29.0%), and in PAL-004 were arthralgia (75.0%), headache (62.5%), dizziness (56.3%), injection site erythema (56.3%), and injection site reaction (50.0%). CONCLUSIONS: In two phase 2 dose-finding studies, pegvaliase did not lead to substantial blood Phe reductions. Higher and more frequent pegvaliase dosing in PAL-004 led to a substantial initial drop in blood Phe, but an increase in the number of hypersensitivity AEs and dose reductions or interruptions. With increased dose and duration of treatment in PAL-003, mean blood Phe reduction was substantial and sustained, and the frequency of hypersensitivity AEs decreased and stabilized. Together, these studies led to the development of an induction-titration-maintenance regimen that has been approved for pegvaliase, with patients starting at a low weekly dose that gradually increases in dose and frequency until they achieve a standard non-weight-based daily maintenance dose. This regimen has been tested in a third phase 2 study, as well as in two successful phase 3 studies of pegvaliase.

Cardiac tissue citric acid cycle intermediates in exercised very long-chain acyl-CoA dehydrogenase-deficient mice fed triheptanoin or medium-chain triglyceride.

Anaplerotic odd-chain fatty acid supplementation has been suggested as an approach to replenish citric acid cycle intermediate (CACi) pools and facilitate adenosine triphosphate (ATP) production in subjects with long-chain fatty acid oxidation disorders, but the evidence that cellular CACi depletion exists and that repletion occurs following anaplerotic substrate supplementation is limited. We exercised very long-chain acyl-CoA dehydrogenase-deficient (VLCAD-/-) and wild-type (WT) mice to exhaustion and collected cardiac tissue for measurement of CACi by targeted metabolomics. In a second experimental group, VLCAD-/- and WT mice that had been fed chow prepared with either medium-chain triglyceride (MCT) oil or triheptanoin for 4 weeks were exercised for 60 minutes. VLCAD-/- mice exhibited lower succinate in cardiac muscle at exhaustion than WT mice suggesting lower CACi in VLCAD-/- with prolonged exercise. In mice fed either MCT or triheptanoin, succinate and malate were greater in VLCAD-/- mice fed triheptanoin compared to VLCAD-/- animals fed MCT but lower than WT mice fed triheptanoin. Long-chain odd acylcarnitines such as C19 were elevated in VLCAD-/- and WT mice fed triheptanoin suggesting some elongation of the heptanoate, but it is unknown what proportion of heptanoate was oxidized vs elongated. Prolonged exercise was associated with decreased cardiac muscle succinate in VLCAD-/- mice in comparison to WT mice. VLCAD-/- fed triheptanoin had increased succinate compared to VLCAD-/- mice fed MCT but lower than WT mice fed triheptanoin. Cardiac CACi were higher following dietary ingestion of an anaplerotic substrate, triheptanoin, in comparison to MCT.

Evidence- and consensus-based recommendations for the use of pegvaliase in adults with phenylketonuria.

PURPOSE: Phenylketonuria (PKU) is a rare metabolic disorder that requires life-long management to reduce phenylalanine (Phe) concentrations within the recommended range. The availability of pegvaliase (PALYNZIQ™, an enzyme that can metabolize Phe) as a new therapy necessitates the provision of guidance for its use. METHODS: A Steering Committee comprising 17 health-care professionals with experience in using pegvaliase through the clinical development program drafted guidance statements during a series of face-to-face meetings. A modified Delphi methodology was used to demonstrate consensus among a wider group of health-care professionals with experience in using pegvaliase. RESULTS: Guidance statements were developed for four categories: (1) treatment goals and considerations prior to initiating therapy, (2) dosing considerations, (3) considerations for dietary management, and (4) best approaches to optimize medical management. A total of 34 guidance statements were included in the modified Delphi voting and consensus was reached on all after two rounds of voting. CONCLUSION: Here we describe evidence- and consensus-based recommendations for the use of pegvaliase in adults with PKU. The manuscript was evaluated against the Appraisal of Guidelines for Research and Evaluation (AGREE II) instrument and is intended for use by health-care professionals who will prescribe pegvaliase and those who will treat patients receiving pegvaliase.

Long-term safety and efficacy of glycerol phenylbutyrate for the management of urea cycle disorder patients.

INTRODUCTION: Glycerol phenylbutyrate (GPB) is currently approved for use in the US and Europe for patients of all ages with urea cycle disorders (UCD) who cannot be managed with protein restriction and/or amino acid supplementation alone. Currently available data on GPB is limited to 12 months exposure. Here, we present long-term experience with GPB. METHODS: This was an open-label, long-term safety study of GPB conducted in the US (17 sites) and Canada (1 site) monitoring the use of GPB in UCD patients who had previously completed 12 months of treatment in the previous safety extension studies. Ninety patients completed the previous studies with 88 of these continuing into the long-term evaluation. The duration of therapy was open ended until GPB was commercially available. The primary endpoint was the rate of adverse events (AEs). Secondary endpoints were venous ammonia levels, number and causes of hyperammonemic crises (HACs) and neuropsychological testing. RESULTS: A total of 45 pediatric patients between the ages of 1 to 17 years (median 7 years) and 43 adult patients between the ages of 19 and 61 years (median 30 years) were enrolled. The treatment emergent adverse events (TEAE) reported in ≥10% of adult or pediatric patients were consistent with the TEAEs reported in the previous safety extension studies with no increase in the overall incidence of TEAEs and no new TEAEs that indicated a new safety signal. Mean ammonia levels remained stable and below the adult upper limit of normal (<35 µmol/L) through 24 months of treatment in both the pediatric and adult population. Over time, glutamine levels decreased in the overall population. The mean annualized rate of HACs (0.29) established in the previously reported 12-month follow-up study was maintained with continued GPB exposure. CONCLUSION: Following the completion of 12-month follow-up studies with GPB treatment, UCD patients were followed for an additional median of 1.85 (range 0 to 5.86) years in the present study with continued maintenance of ammonia control, similar rates of adverse events, and no new adverse events identified.

Higher dietary protein intake preserves lean body mass, lowers liver lipid deposition, and maintains metabolic control in participants with long-chain fatty acid oxidation disorders.

Medical nutrition therapy for long-chain fatty acid oxidation disorders (LC-FAODs) currently emphasizes fasting avoidance, restricted dietary long-chain fatty acid intake, supplementation with medium chain triglycerides, and increased carbohydrate intake. We hypothesize that increasing dietary protein intake relative to carbohydrate intake would preserve metabolic control yet induce physical benefits including reduced hepatic lipogenesis. Therefore, we compared two dietary approaches with similar fat intake but different carbohydrate to protein ratios in participants diagnosed with LC-FAODs. Thirteen participants were enrolled and randomized into either a high-protein (PRO) or a high-carbohydrate (CHO) diet for 4 months. Baseline and 4-month assessments included body composition, ectopic lipid deposition, and resting energy expenditure. End of study assessments also included total energy expenditure, metabolic responses to oral feedings, and whole-body fatty acid oxidation capacity. At the end of the dietary intervention, both groups had similar energy expenditure, fat and glucose oxidation rates, and glucolipid responses to mixed meal and oral glucose loads. Neither dietary group experienced worsening symptoms related to their LC-FAOD. Compared to the CHO group, the PRO group exhibited increased blood levels of short-chain acylcarnitines, reduced intrahepatic lipid content, and maintained lean body mass while the CHO group lost lean mass. In patients with LC-FAODs, increasing protein intake maintained metabolic control, reduced liver fat without risk of metabolic decompensation, and helped preserve lean body mass. We propose that a modest increase in dietary protein along with fasting avoidance and fat restriction may improve body composition and energy expenditure in patients with LC-FAODs.

Blood phenylalanine reduction corrects CNS dopamine and serotonin deficiencies and partially improves behavioral performance in adult phenylketonuric mice.

Central nervous system (CNS) deficiencies of the monoamine neurotransmitters dopamine and serotonin have been implicated in the pathophysiology of neuropsychiatric dysfunction in human phenylketonuria (PKU). In this study, we confirmed the occurrence of brain dopamine and serotonin deficiencies in association with severe behavioral alterations and cognitive impairments in hyperphenylalaninemic C57BL/6-Pahenu2/enu2 mice, a model of human PKU. Phenylalanine-reducing treatments, including either dietary phenylalanine restriction or liver-directed gene therapy, initiated during adulthood were associated with increased brain monoamine content along with improvements in nesting behavior but without a change in the severe cognitive deficits exhibited by these mice. At euthanasia, there was in Pahenu2/enu2 brain a significant reduction in the protein abundance and maximally stimulated activities of tyrosine hydroxylase (TH) and tryptophan hydroxylase 2 (TPH2), the rate limiting enzymes catalyzing neuronal dopamine and serotonin synthesis respectively, in comparison to levels seen in wild type brain. Phenylalanine-reducing treatments initiated during adulthood did not affect brain TH or TPH2 content or maximal activity. Despite this apparent fixed deficit in striatal TH and TPH2 activities, initiation of phenylalanine-reducing treatments yielded substantial correction of brain monoamine neurotransmitter content, suggesting that phenylalanine-mediated competitive inhibition of already constitutively reduced TH and TPH2 activities is the primary cause of brain monoamine deficiency in Pahenu2 mouse brain. We propose that CNS monoamine deficiency may be the cause of the partially reversible adverse behavioral effects associated with chronic HPA in Pahenu2 mice, but that phenylalanine-reducing treatments initiated during adulthood are unable to correct the neuropathology and attendant cognitive deficits that develop during juvenile life in late-treated Pahenu2/enu2 mice.

Pegvaliase for the treatment of phenylketonuria: A pivotal, double-blind randomized discontinuation Phase 3 clinical trial.

INTRODUCTION: Pegvaliase is a recombinant Anabaena variabilis phenylalanine ammonia lyase (PAL) enzyme under investigation for treatment of adult phenylketonuria (PKU). This manuscript describes results of a randomized discontinuation trial (RDT) designed to evaluate the effects of pegvaliase treatment on blood phenylalanine (Phe) and neuropsychiatric outcomes in adults with PKU. METHODS: PRISM-2 is a 4-part, Phase 3 study that enrolled adults with PKU receiving pegvaliase treatment (initiated in a prior Phase 2 or Phase 3 study). The RDT, Part 2 of PRISM-2, was an 8-week trial that evaluated change in blood Phe concentrations, neuropsychiatric and neurocognitive measures, and safety outcomes in PRISM-2 participants who had achieved at least a 20% blood Phe reduction from pre-treatment baseline with pegvaliase treatment. Participants were randomized 2:1 to either continue pegvaliase (20 mg/day or 40 mg/day) or switch to matching placebo. RESULTS: The pooled pegvaliase group enrolled 66 participants and each placebo group enrolled 14 participants. The primary endpoint of change in blood Phe concentration from RDT entry to RDT Week 8 was met with clinically meaningful and statistically significant differences between the pegvaliase and placebo groups. Mean (SD) blood Phe at the beginning of the RDT when all participants were receiving pegvaliase was 563.9 µM (504.6) in the group assigned to the 20 mg/day placebo group (n = 14), 508.2 µM (363.7) in those assigned to the 40 mg/day placebo group (n = 14), and 503.9 µM (520.3) in those assigned to continue pegvaliase treatment (n = 58). At Week 8 of the RDT, the least squares mean change (95% confidence interval) in blood Phe was 949.8 µM (760.4 to 1139.1) for the 20 mg/day placebo group and 664.8 µM (465.5 to 864.1) for the 40 mg/day placebo group in comparison to 26.5 µM (-68.3 to 121.3) for the pooled (20 mg/day and 40 mg/day) pegvaliase group (P < 0.0001 for pooled pegvaliase group vs each placebo group). Adverse events (AEs) were usually lower in the pooled placebo group when compared to the pooled pegvaliase group. The most common AEs for the pooled pegvaliase and pooled placebo groups were arthralgia (13.6% and 10.3%, respectively), headache (12.1% and 24.1%), anxiety (10.6% and 6.9%), fatigue (10.6% and 10.3%), and upper respiratory tract infection (1.5% and 17.2%). CONCLUSION: Mean blood Phe reduction was sustained in the pegvaliase group, while placebo groups had mean blood Phe concentration increase toward pre-treatment baseline levels. Results from this study confirmed the efficacy of pegvaliase in maintaining reduced blood Phe concentrations with a manageable safety profile for most participants.

Pharmacokinetics of glycerol phenylbutyrate in pediatric patients 2 months to 2 years of age with urea cycle disorders.

INTRODUCTION: Glycerol phenylbutyrate (GPB) is approved in the US and EU for the chronic management of patients ≥2 months of age with urea cycle disorders (UCDs) who cannot be managed by dietary protein restriction and/or amino acid supplementation alone. GPB is a pre-prodrug, hydrolyzed by lipases to phenylbutyric acid (PBA) that upon absorption is beta-oxidized to the active nitrogen scavenger phenylacetic acid (PAA), which is conjugated to glutamine (PAGN) and excreted as urinary PAGN (UPAGN). Pharmacokinetics (PK) of GPB were examined to see if hydrolysis is impaired in very young patients who may lack lipase activity. METHODS: Patients 2 months to <2 years of age with UCDs from two open label studies (n = 17, median age 10 months) predominantly on stable doses of nitrogen scavengers (n = 14) were switched to GPB. Primary assessments included traditional plasma PK analyses of PBA, PAA, and PAGN, using noncompartmental methods with WinNonlin™. UPAGN was collected periodically throughout the study up to 12 months. RESULTS: PBA, PAA and PAGN rapidly appeared in plasma after GPB dosing, demonstrating evidence of GPB cleavage with subsequent PBA absorption. Median concentrations of PBA, PAA and PAGN did not increase over time and were similar to or lower than the values observed in older UCD patients. The median PAA/PAGN ratio was well below one over time, demonstrating that conjugation of PAA with glutamine to form PAGN did not reach saturation. Covariate analyses indicated that age did not influence the PK parameters, with body surface area (BSA) being the most significant covariate, reinforcing current BSA based dosing recommendations as seen in older patients. CONCLUSION: These observations demonstrate that UCD patients aged 2 months to <2 years have sufficient lipase activity to adequately convert the pre-prodrug GPB to PBA. PBA is then converted to its active moiety (PAA) providing successful nitrogen scavenging even in very young children.