Co-ingestion of glutamine and leucine synergistically promotes mTORC1 activation.

Leucine (Leu) regulates protein synthesis and degradation via activation of mammalian target of rapamycin complex 1 (mTORC1). Glutamine (Gln) synergistically promotes mTORC1 activation with Leu via glutaminolysis and Leu absorption via an antiporter. However, Gln has also been shown to inhibit mTORC1 activity. To resolve this paradox, we aimed to elucidate the effects of Gln on Leu-mediated mTORC1 activation. We administered Leu, Gln, tryptophan, Leu + Gln, or Leu + tryptophan to mice after 24-h fasting. The mice were then administered puromycin to evaluate protein synthesis and the gastrocnemius muscle was harvested 30 min later. Phosphorylated eukaryotic initiation factor 4E-binding protein 1, 70-kDa ribosomal protein S6 kinase 1, and Unc-51 like kinase 1 levels were the highest in the Leu + Gln group and significantly increased compared with those in the control group; however, Gln alone did not increase the levels of phosphorylated proteins. No difference in glutamate dehydrogenase activity was observed between the groups. Leu concentrations in the gastrocnemius muscle were similar in the Leu-intake groups. Our study highlights a novel mechanism underlying the promotive effect of Gln on Leu-mediated mTORC1 activation, providing insights into the pathway through which amino acids regulate muscle protein metabolism.

The oral protease inhibitor (PF-07321332) protects Syrian hamsters against infection with SARS-CoV-2 variants of concern.

There is an urgent need for potent and selective antivirals against SARS-CoV-2. Pfizer developed PF-07321332 (PF-332), a potent inhibitor of the viral main protease (Mpro, 3CLpro) that can be dosed orally and that is in clinical development. We here report that PF-332 exerts equipotent in vitro activity against the four SARS-CoV-2 variants of concerns (VoC) and that it can completely arrest replication of the alpha variant in primary human airway epithelial cells grown at the air-liquid interface. Treatment of Syrian Golden hamsters with PF-332 (250 mg/kg, twice daily) completely protected the animals against intranasal infection with the beta (B.1.351) and delta (B.1.617.2) SARS-CoV-2 variants. Moreover, treatment of SARS-CoV-2 (B.1.617.2) infected animals with PF-332 completely prevented transmission to untreated co-housed sentinels.

Oral Nirmatrelvir for High-Risk, Nonhospitalized Adults with Covid-19.

BACKGROUND: Nirmatrelvir is an orally administered severe acute respiratory syndrome coronavirus 2 main protease (Mpro) inhibitor with potent pan-human-coronavirus activity in vitro. METHODS: We conducted a phase 2-3 double-blind, randomized, controlled trial in which symptomatic, unvaccinated, nonhospitalized adults at high risk for progression to severe coronavirus disease 2019 (Covid-19) were assigned in a 1:1 ratio to receive either 300 mg of nirmatrelvir plus 100 mg of ritonavir (a pharmacokinetic enhancer) or placebo every 12 hours for 5 days. Covid-19-related hospitalization or death from any cause through day 28, viral load, and safety were evaluated. RESULTS: A total of 2246 patients underwent randomization; 1120 patients received nirmatrelvir plus ritonavir (nirmatrelvir group) and 1126 received placebo (placebo group). In the planned interim analysis of patients treated within 3 days after symptom onset (modified intention-to treat population, comprising 774 of the 1361 patients in the full analysis population), the incidence of Covid-19-related hospitalization or death by day 28 was lower in the nirmatrelvir group than in the placebo group by 6.32 percentage points (95% confidence interval [CI], -9.04 to -3.59; P<0.001; relative risk reduction, 89.1%); the incidence was 0.77% (3 of 389 patients) in the nirmatrelvir group, with 0 deaths, as compared with 7.01% (27 of 385 patients) in the placebo group, with 7 deaths. Efficacy was maintained in the final analysis involving the 1379 patients in the modified intention-to-treat population, with a difference of -5.81 percentage points (95% CI, -7.78 to -3.84; P<0.001; relative risk reduction, 88.9%). All 13 deaths occurred in the placebo group. The viral load was lower with nirmatrelvir plus ritonavir than with placebo at day 5 of treatment, with an adjusted mean difference of -0.868 log10 copies per milliliter when treatment was initiated within 3 days after the onset of symptoms. The incidence of adverse events that emerged during the treatment period was similar in the two groups (any adverse event, 22.6% with nirmatrelvir plus ritonavir vs. 23.9% with placebo; serious adverse events, 1.6% vs. 6.6%; and adverse events leading to discontinuation of the drugs or placebo, 2.1% vs. 4.2%). Dysgeusia (5.6% vs. 0.3%) and diarrhea (3.1% vs. 1.6%) occurred more frequently with nirmatrelvir plus ritonavir than with placebo. CONCLUSIONS: Treatment of symptomatic Covid-19 with nirmatrelvir plus ritonavir resulted in a risk of progression to severe Covid-19 that was 89% lower than the risk with placebo, without evident safety concerns. (Supported by Pfizer; ClinicalTrials.gov number, NCT04960202.).

Leucine supplementation during late gestation globally alters placental metabolism and nutrient transport via modulation of the PI3K/AKT/mTOR signaling pathway in sows.

In a previously published study we reported that sow dietary leucine supplementation during late pregnancy significantly improved newborn piglet birth weight by stimulating protein synthesis in the longissimus dorsi muscle. However, there is still limited knowledge as to whether leucine can exert its effects on the placenta, one of the most important temporal organs during pregnancy, to promote maternal-fetal nutrient supply and thus contribute to fetal intrauterine development. Therefore, we tested this hypothesis in the present study. In total, 150 sows at day 90 of gestation were divided into three groups and fed with either a control diet (CON), CON + 0.4% Leu or CON + 0.8% Leu, respectively, until parturition. Placental metabolomics, full spectrum amino acids and nutrient transporters were systematically analyzed after sample collection. The results indicated that Leu supplementation led to an altered placental metabolism with an increased number of metabolites related to glycolysis and the oxidation of fatty acids, as well as elevated levels of amino acid accumulation in the placenta. In addition, nutrient transporters of amino acids, glucose and fatty acids in the placenta were globally up-regulated and several enzymes related to energy metabolism, including hexokinase, succinate dehydrogenase, lactated hydrogenase, glycogen phosphorylase and hydroxyacyl-CoA-dehydrogenase, were also significantly increased with no change observed in the antioxidative status of those groups with Leu supplementation. Furthermore, the phosphorylation of PI3K, Akt, and mTOR was enhanced in the placenta of sows undergoing Leu treatment. Collectively, we concluded that supplementing the diets of sows with Leu during late gestation globally altered placental metabolism and promoted maternal-fetus nutrient transport (amino acids, glucose, and fatty acids) via modulation of the PI3K/Akt/mTOR signaling pathway.

Effects of dietary leucine and valine levels on growth performance, glycolipid metabolism and immune response in Tilapia GIFT Oreochromis niloticus.

An 8-week feeding trial was performed to evaluate the effects of dietary leucine (Leu) and valine (Val) levels on growth performance, glycolipid metabolism and immune response in Oreochromis niloticus. Fish (15.23 ± 0.05 g) were randomly fed four diets containing two Leu levels (1.2% and 2.3%) and two Val levels (0.7% and 1.4%) as a 2 × 2 experimental design (LL-LV, LL-HV, HL-LV and HL-HV). Compared with LL-LV group, the growth parameters (final weight, daily growth coefficient (DGC) and growth rate per metabolic body weight (GRMBW)), feed conversion rate (FCR), the activities of intestinal amylase, lipase, creatine kinase (CK) and Na+, K+-ATPase, liver NAD+/NADH ratio, as well as the expression of SIRT1, GK, PK, FBPase, PPARα, CPT IA, ACO and IL10 all increased significantly in the HL-LV group; however, in the high Val group, final weight, DGC, GRMBW, intestinal enzyme activities, as well as the expression of PEPCK, SREBP1, FAS, IL8 and IL10 of the HL-HV group were significantly lower than those of the LL-HV group, while the opposite was true for the remaining indicators. Significant interactions between dietary Leu and Val were observed in final weight, DGC, GRMBW, plasma IL1ß and IL6 levels, intestinal amylase and CK activities, liver NAD+/NADH ratio, as well as the expression of SIRT1, PK, PEPCK, FBPase, SREBP1, FAS, PPARα, CPT IA, ACO, NF-κB1, IL1ß, IL6 and IL10. The highest values of growth parameters, intestinal enzyme activities and expression of SIRT1, FBPase, PPARα, CPT IA and ACO were observed in the HL-LV group, while the opposite was true for the expression of SREBP1, FAS, PPARα, NF-κB1, IL1ß and IL6. Overall, our findings indicated that dietary Leu and Val can effect interactively, and fish fed with diets containing 2.3% Leu with 0.7% Val had the best growth performance and hepatic health status of O. niloticus.

Safety and Efficacy of Acetyl-DL-Leucine in Certain Types of Cerebellar Ataxia: The ALCAT Randomized Clinical Crossover Trial.

Importance: Cerebellar ataxia is a neurodegenerative disease impairing motor function characterized by ataxia of stance, gait, speech, and fine motor disturbances. Objective: To investigate the efficacy, safety, and tolerability of the modified essential amino acid acetyl-DL-leucine in treating patients who have cerebellar ataxia. Design, Setting, and Participants: The Acetyl-DL-leucine on Cerebellar Ataxia (ALCAT) trial was an investigator-initiated, multicenter, double-blind, randomized, placebo-controlled, clinical crossover trial. The study was conducted at 7 university hospitals in Germany and Austria between January 25, 2016, and February 17, 2017. Patients were aged at least 18 years and diagnosed with cerebellar ataxia of hereditary (suspected or genetically confirmed) or nonhereditary or unknown type presenting with a total score of at least 3 points on the Scale for the Assessment and Rating of Ataxia (SARA). Statistical analysis was performed from April 2018 to June 2018 and January 2020 to March 2020. Interventions: Patients were randomly assigned (1:1) to receive acetyl-DL-leucine orally (5 g per day after 2 weeks up-titration) followed by a matched placebo, each for 6 weeks, separated by a 4-week washout, or vice versa. The randomization was done via a web-based, permuted block-wise randomization list (block size, 2) that was stratified by disease subtype (hereditary vs nonhereditary or unknown) and site. Main Outcomes and Measures: Primary efficacy outcome was the absolute change of SARA total score from (period-dependent) baseline to week 6. Results: Among 108 patients who were randomly assigned to sequence groups (54 patients each), 55 (50.9%) were female; the mean (SD) age was 54.8 (14.4) years; and the mean (SD) SARA total score was 13.33 (5.57) points. The full analysis set included 105 patients (80 patients with hereditary, 25 with nonhereditary or unknown cerebellar ataxia). There was no evidence of a difference in the mean absolute change from baseline to week 6 in SARA total scores between both treatments (mean treatment difference: 0.23 points [95% CI, -0.40 to 0.85 points]). Conclusions and Relevance: In this large multicenter, double-blind, randomized, placebo-controlled clinical crossover trial, acetyl-DL-leucine in the investigated dosage and treatment duration was not superior to placebo for the symptomatic treatment of certain types of ataxia. The drug was well tolerated; and ALCAT yielded valuable information about the duration of treatment periods and the role of placebo response in cerebellar ataxia. These findings suggest that further symptom-oriented trials are needed for evaluating the long-term effects of acetyl-DL-leucine for well-defined subgroups of cerebellar ataxia. Trial Registration: EudraCT 2015-000460-34.

An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.

The worldwide outbreak of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic. Alongside vaccines, antiviral therapeutics are an important part of the healthcare response to countering the ongoing threat presented by COVID-19. Here, we report the discovery and characterization of PF-07321332, an orally bioavailable SARS-CoV-2 main protease inhibitor with in vitro pan-human coronavirus antiviral activity and excellent off-target selectivity and in vivo safety profiles. PF-07321332 has demonstrated oral activity in a mouse-adapted SARS-CoV-2 model and has achieved oral plasma concentrations exceeding the in vitro antiviral cell potency in a phase 1 clinical trial in healthy human participants.

Amino Acid Profile in Malnourished Patients with Liver Cirrhosis and Its Modification with Oral Nutritional Supplements: Implications on Minimal Hepatic Encephalopathy.

Low plasma levels of branched chain amino acids (BCAA) in liver cirrhosis are associated with hepatic encephalopathy (HE). We aimed to identify a metabolic signature of minimal hepatic encephalopathy (MHE) in malnourished cirrhotic patients and evaluate its modification with oral nutritional supplements (ONS) enriched with ß-Hydroxy-ß-methylbutyrate (HMB), a derivative of the BCAA leucine. Post hoc analysis was conducted on a double-blind placebo-controlled trial of 43 individuals with cirrhosis and malnutrition, who were randomized to receive, for 12 weeks, oral supplementation twice a day with either 220 mL of Ensure® Plus Advance (HMB group, n = 22) or with 220 mL of Ensure® Plus High Protein (HP group, n = 21). MHE evaluation was by psychometric hepatic encephalopathy score (PHES). Compared to the HP group, an HMB-specific treatment effect led to a larger increase in Val, Leu, Phe, Trp and BCAA fasting plasma levels. Both treatments increased Fischer's ratio and urea without an increase in Gln or ammonia fasting plasma levels. MHE was associated with a reduced total plasma amino acid concentration, a reduced BCAA and Fischer´s ratio, and an increased Gln/Glu ratio. HMB-enriched ONS increased Fischer´s ratio without varying Gln or ammonia plasma levels in liver cirrhosis and malnutrition, a protective amino acid profile that can help prevent MHE.

Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19.

COVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

Supplement-based nutritional strategies to tackle frailty: A multifactorial, double-blind, randomized placebo-controlled trial.

BACKGROUND: Sarcopenia plays a central role in the development of frailty syndrome. Nutrition and exercise are cornerstone strategies to mitigate the transition to frailty; however, there is a paucity of evidence for which dietary and exercise strategies are effective. OBJECTIVE: This large, multifactorial trial investigated the efficacy of different dietary strategies to enhance the adaptations to resistance training in pre-frail and frail elderly. METHODS: This was a single-site 16-week, double-blind, randomized, placebo-controlled trial conducted at the Clinical Hospital, School of Medicine - University of São Paulo, Sao Paulo, Brazil. Four integrated, sub-investigations were conducted to compare: 1) leucine vs. placebo; 2) whey vs. soy vs. placebo; 3) creatine vs. whey vs. creatine plus whey vs. placebo; 4) women vs. men in response to whey. Sub-investigations 1 to 3 were conducted in women, only. Two-hundred participants (154 women/46 men, mean age 72 ± 6 years) underwent a twice-a-week, resistance training program. The main outcomes were muscle function (assessed by dynamic and isometric strength and functional tests) and lean mass (assessed by DXA). Muscle cross-sectional area, health-related quality of life, bone and fat mass, and biochemical markers were also assessed. RESULTS: We observed that leucine supplementation was ineffective to improve muscle mass and function. Supplementation with whey and soy failed to enhance resistance-training effects. Similarly, supplementation with neither whey nor creatine potentiated the adaptations to resistance training. Finally, no sex-based differences were found in response to whey supplementation. Resistance exercise per se increased muscle mass and function in all sub-investigations. There were no adverse effects. CONCLUSION: Neither protein (whey and soy), leucine, nor creatine supplementation enhanced resistance training-induced adaptations in pre-frail and frail elderly, regardless of sex. These findings do not support the notion that some widely used supplement-based interventions can add to the already potent effects of resistance exercise to counteract frailty-related muscle wasting and dynapenia. CLINICAL TRIAL REGISTRY: NCT01890382; https://clinicaltrials.gov/ct2/show/NCT01890382. DATA SHARING: Data described in the manuscript will be made available upon request pending application.

Safety, Tolerability, and Biologic Activity of AXA1125 and AXA1957 in Subjects With Nonalcoholic Fatty Liver Disease.

INTRODUCTION: AXA1125 and AXA1957 are novel, orally administered endogenous metabolic modulator compositions, specifically designed to simultaneously support multiple metabolic and fibroinflammatory pathways associated with nonalcoholic fatty liver disease (NAFLD). This study assessed safety, tolerability, and biologic activity of AXA1125 and AXA1957 in NAFLD. METHODS: In this multicenter, 16-week, placebo-controlled, single-blind, randomized clinical study in subjects with NAFLD stratified by type 2 diabetes, AXA1125 24 g, AXA1957 13.5 g or 20.3 g, or placebo was administered twice daily. Key metabolism (MRI-proton density fat fraction [MRI-PDFF] and homeostasis model assessment of insulin resistance [HOMA-IR]) and fibroinflammation markers (alanine aminotransferase [ALT], corrected T1 [cT1], keratin-18 [K-18] M65, and N-terminal type III collagen propeptide [Pro-C3]) were evaluated. Safety outcomes included adverse events and standard laboratory assessments. RESULTS: Baseline characteristics of the 102 enrolled subjects, including 40 with type 2 diabetes, were consistent with presumed nonalcoholic steatohepatitis. AXA1125 showed consistently greater biologic activity than AXA1957 or placebo. Week 16 changes from baseline with AXA1125 vs placebo: MRI-PDFF -22.9% vs -5.7%, HOMA-IR -4.4 vs +0.7, ALT -21.9% vs -7.2%, K-18 M65 -13.6% vs +20.1%, cT1 -69.6 vs +18.3 ms (P < 0.05), and Pro-C3 -13.6% vs -3.6%. Week 16 changes from baseline with AXA1957 20.3 g: MRI-PDFF -8.1%, HOMA-IR +8.4, ALT -20.7%, K-18 M65 6.6%, cT1 -34.7 ms, and Pro-C3 -15.6%. A greater proportion of subjects treated with AXA1125 achieved clinically relevant thresholds: ≥30% MRI-PDFF, ≥17-IU/L ALT, and ≥80-ms cT1 reductions at week 16. Study products were safe and well tolerated with stable lipid and weight profiles. DISCUSSION: Both compositions showed multitargeted activity on relevant NAFLD pathways. AXA1125 demonstrated the greatest activity over 16 weeks, warranting continued clinical investigation in nonalcoholic steatohepatitis subjects.

Epigenetic regulation of intestinal peptide transporter PEPT1 as a potential strategy for colorectal cancer sensitization.

Human intestinal peptide transporter PEPT1 is commonly repressed in human colorectal cancer (CRC), yet its relationship with sensitivity to the common CRC treatment ubenimex has not previously been elucidated. In this study, we confirmed PEPT1 suppression in CRC using real-time quantitative polymerase chain reaction and western blotting and then investigated the underlying epigenetic pathways involved using bisulfite sequencing, chromatin immunoprecipitation, siRNA knockdown, and reporter gene assays. We found that PEPT1 transcriptional repression was due to both DNMT1-mediated DNA methylation of the proximal promoter region and HDAC1-mediated histone deacetylation, which blocked P300-mediated H3K18/27Ac at the PEPT1 distal promoter. Finally, the effects of the epigenetic activation of PEPT1 on the CRC response to ubenimex were evaluated using sequential combination therapy of decitabine and ubenimex both in vitro and in xenografts. In conclusion, epigenetic silencing of PEPT1 due to increased DNMT1 and HDAC1 expression plays a vital role in the poor response of CRC to ubenimex.

Development and Characterization of Excipient Enhanced Growth (EEG) Surfactant Powder Formulations for Treating Neonatal Respiratory Distress Syndrome.

This study aimed to develop and characterize a spray-dried powder aerosol formulation of a commercially available surfactant formulation, Survanta® intratracheal suspension, using the excipient enhanced growth (EEG) approach. Survanta EEG powders were prepared by spray drying of the feed dispersions containing Survanta® (beractant) intratracheal suspension, hygroscopic excipients (mannitol and sodium chloride), and a dispersion enhancer (l-leucine or trileucine) in 5 or 20% v/v ethanol in water using the Buchi Nano Spray Dryer B-90 HP. Powders were characterized for primary particle size, morphology, phospholipid content, moisture content, thermal properties, moisture sorption, and surface activity. The aerosol performance of the powders was assessed using a novel low-volume dry powder inhaler (LV-DPI) device operated with 3-mL volume of dispersion air. At both ethanol concentrations, in comparison to trileucine, l-leucine significantly reduced the primary particle size and span and increased the fraction of submicrometer particles of the Survanta EEG powders. The l-leucine-containing Survanta EEG powders exhibited good aerosolization performance with ≥ 88% of the mass emitted (% nominal) after 3 actuations from the modified LV-DPI device. In addition, l-leucine-containing powders had a low moisture content (< 3% w/w) with transition temperatures close to the commercial surfactant formulation and retained their surface tension reducing activity after formulation processing. A Survanta EEG powder containing l-leucine was developed which showed efficient aerosol delivery from the modified LV-DPI device using a low dispersion air volume.

Successful treatment by on-demand glecaprevir and pibrentasvir for hepatitis C flare during R-CHOP in patients with diffuse large B-cell lymphoma: a case report.

BACKGROUND: In patients with hepatitis C virus (HCV) and malignant lymphoma, hepatitis C flare during R-CHOP can result in discontinuation of treatment. However, appropriate therapeutic strategies for managing hepatitis C flare during R-CHOP have not been established, and this issue is complicated by conflicting results regarding the use of direct-acting antivirals in patients with uncontrolled malignancies. CASE PRESENTATION: We report the first case of effective and safe treatment with on-demand 8-week glecaprevir and pibrentasvir for hepatitis C flare during R-CHOP in a patient with diffuse large B-cell lymphoma (DLBCL). The patient completed five additional courses of R-CHOP without hepatic toxicity. A complete response of DLBCL and a sustained virological response were observed at 24 weeks after glecaprevir and pibrentasvir completion. CONCLUSION: On-demand, direct-acting antivirals could be a novel strategy for managing hepatitis C flare during R-CHOP.