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Enteroendocrine cells (EECs) are sensory cells in the intestinal epithelium that respond to ingested nutrients and microbial products in the gut lumen by releasing over 30 different hormones and neurotransmitters. These signaling molecules travel throughout the body to influence a wide variety of metabolic functions, including glucose metabolism, satiety, and gut motility. EECs are typically divided into subtypes based on their predominant hormone. As enteroendocrine hormones have different and sometimes antagonistic metabolic effects, this subdivision enables fine-tuned control of metabolism in response to a variety of stimuli. While many aspects of EEC development and physiology have been defined, the mechanisms underlying EEC subtype-specific development and physiologic adaptations to diet and microbiota remain largely unknown. To investigate these questions, we recently established zebrafish as a model system for studying EEC physiology. The optical transparency of larval zebrafish enables live imaging to observe EEC adaptations in vivo and in real time. We used single-cell RNA sequencing to profile sorted intestinal epithelial cells from zebrafish larvae and identified secretory lineages including 7 EEC subtypes that we identified by expression of EEC hormones and transcription factors. Many zebrafish EEC subtypes we identified correlate with subtypes described in the mammalian small intestine, suggesting an evolutionary preserved role for EEC subtypes in regulating animal physiology. We also conducted LC-MS/MS peptidomics on EECs sorted from adult zebrafish intestine to reveal extensive homology in EEC hormone peptide sequence and post-translational processing. Using this information, we have begun to create transgenic lines to label and manipulate each of the EEC subtypes. For example, we identify a single EEC subtype that expresses genes encoding ghrelin, somatostatin, and glucose dependent insulinotropic peptides. Peptidomic analysis reveals striking conservation in their posttranslational processing including octanoylated and decanoylated isoforms of the ghrelin peptide. We have generated a transgenic line using the ghrelin gene and are using it to test how the number, distribution, and function of ghrelin positive cells vary as a function of diet and microbial colonization status. Establishment of a toolkit for zebrafish EEC subtype biology will provide an important alternative to the prevailing mammalian models and help unravel their unique developmental programs, physiologic functions, and adaptations to environmental variables. NIH R01-DK093399 and the Gordon & Betty Moore Foundation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

This study (ALVOPAD FIRST) assessed bioequivalence, safety, and immunogenicity of AVT02, an adalimumab biosimilar, compared with reference product adalimumab (EU- and US-approved Humira®). Healthy subjects (N = 392) were randomized 1:1:1 to receive one 40 mg dose of AVT02, EU-reference product, or US-reference product subcutaneously. An interim analysis was planned when ~30 subjects per arm had completed the study, to optimize final sample size. The primary PK parameters were Cmax, AUC0-t, and AUC0-inf. Bioequivalence was demonstrated if the 90% confidence intervals (CI) for the ratio of geometric means for the primary pharmacokinetic (PK) parameters were all contained within the prespecified margins of 80% and 125%. Safety and immunogenicity were assessed until Day 64. The 90% CI for the ratio of geometric means for the primary PK parameters, based on Fisher’s Combination test analysis, were all contained within the prespecified bioequivalence margins of 80% and 125%, supporting the demonstration of bioequivalence between AVT02 and both EU- and US-reference product. The safety and immunogenicity profiles were comparable across all three treatment arms. PK bioequivalence was supported between AVT02, US-licensed- and EU-approved-reference product adalimumab. Similar safety and immunogenicity were also demonstrated. The trial is registered at ClinicalTrials.gov (CT.gov identifier: NCT03849313).

Background Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal disease characterized by multiple symptoms such as progressive cerebellar ataxia and cognitive decline. The modified amino acid N-acetyl-leucine has been associated with positive symptomatic and neuroprotective, disease-modifying effects in various studies, including animal models of NPC, observational clinical case studies, and a multinational, rater-blinded Phase IIb clinical trial. Here, we describe the development of a study protocol (Sponsor Code “IB1001-301”) for the chronic treatment of symptoms in adult and pediatric patients with NPC. Methods This multinational double-blind randomized placebo-controlled crossover Phase III study will enroll patients with a genetically confirmed diagnosis of NPC patients aged 4 years and older across 16 trial sites. Patients are assessed during a baseline period and then randomized (1:1) to one of two treatment sequences: IB1001 followed by placebo or vice versa. Each sequence consists of a 12-week treatment period. The primary efficacy endpoint is based on the Scale for the Assessment and Rating of Ataxia, and secondary outcomes include cerebellar functional rating scales, clinical global impression, and quality of life assessments. Discussion Pre-clinical as well as observational and Phase IIb clinical trials have previously demonstrated that IB1001 rapidly improved symptoms, functioning, and quality of life for pediatric and adult NPC patients and is safe and well tolerated. In this placebo-controlled cross-over trial, the risk/benefit profile of IB1001 for NPC will be evaluated. It will also give information about the applicability of IB1001 as a therapeutic paradigm for other rare and common neurological disorders. Trial registrations: The trial (IB1001-301) has been registered at www.clinicaltrials.gov (NCT05163288) and www.clinicaltrialsregister.eu (EudraCT: 2021-005356-10).

Funder: Foundation for Liver Research, Funder: Royal Society and a CAMS-UK fellowship, The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.

Introduction: It has been suggested that the gut microbiome of patients with inflammatory bowel disease (IBD) is unable to ferment dietary fibre. This project explored the in vitro effect of fibre fermentation on production of short-chain fatty acids (SCFA) and on microbiome composition. Methods: Faecal samples were collected from 40 adults (>16 y) with IBD (n = 20 with Crohn’s disease and n = 20 with ulcerative colitis) in clinical remission and 20 healthy controls (HC). In vitro batch culture fermentations were carried out using as substrates maize starch, apple pectin, raftilose, wheat bran, α cellulose and a mixture of these five fibres. SCFA concentration (umol/g) was quantified with gas chromatography and microbiome was profiled with 16S rRNA sequencing. Results: Fibre fermentation did not correct the baseline microbial dysbiosis or lower diversity seen in either patients with CD or UC. For all fibres, up to 51% of baseline ASVs or genera changed in abundance in HC. In patients with IBD, fermentation of fibre substrates had no effect on species or genera abundance. Production of SCFA varied among the different fibre substrates but this was not different between the two IBD groups and compared to HC after either 5 or 24 h fermentation. Conclusions: Despite extensive microbial dysbiosis, patients with IBD have a similar capacity to ferment fibre and release SCFA as HC. Fibre supplementation alone may be unlikely to restore to a healthy status the compositional shifts characteristic of the IBD microbiome.

Background AVT02 (adalimumab) is a proposed biosimilar to Humira®. AVT02 is produced at a 100 mg/mL concentration with a citrate-free formulation. Objectives The aim of this study was to compare the efficacy, safety and immunogenicity of AVT02 versus Humira® in subjects with moderate to severe chronic plaque psoriasis. Methods This double-blind, randomised, parallel group, active control study of adult subjects compared (at a 1:1 ratio) AVT02 with originator adalimumab 80 mg subcutaneously in Week 1, then 40 mg every other week. At Week 16, subjects who had received originator adalimumab were re-randomised at a 1:1 ratio to continue receiving originator adalimumab, or to switch to AVT02, every other week until Week 48, with final efficacy endpoint at Week 50. Subjects who initially received AVT02 continued to receive AVT02 from Week 16 to Week 48. The primary endpoint was percentage improvement in Psoriasis Area and Severity Index (PASI) score at Week 16. Secondary efficacy endpoints included percentage improvement in PASI score at additional timepoints, change from baseline in Dermatology Life Quality Index (DLQI) score and number and percentage of subjects achieving static Physician’s Global Assessment (sPGA) responses of ‘clear’ or ‘almost clear’. Additional secondary endpoints included comparison of adverse event profiles, anti-drug antibodies and neutralising antibodies, and serum trough levels of adalimumab at steady state. Results A total of 413 subjects were randomised (205 to AVT02 and 208 to originator). The percentage improvement in PASI score at Week 16 was 91.6% for AVT02-treated subjects and 89.6% for originator adalimumab. The 90% confidence intervals for the primary endpoint were within the pre-defined equivalence margin of ±10% (90% CI − 0.76 to 5.29; 95% CI − 1.34 to 5.88), and a comparable pattern for DLQI score (11.4-point and 10.6-point improvement in AVT02-treated and originator adalimumab-treated groups, respectively) and sPGA (90.5% in both groups achieving ‘clear’ or ‘almost clear’) at Week 16 supported the assessment. Efficacy persisted through Week 50 of the study in all treatment groups, including those who switched from originator adalimumab to AVT02, for percent improvement in PASI score, quality-of-life assessment and sPGA. The safety, tolerability and immunogenicity profiles between AVT02 and originator adalimumab were similar at Week 16, and this persisted in the switched and continued groups through Week 50. Conclusion Objective and subjective measures of efficacy supported the evaluation of biosimilarity between AVT02 and originator adalimumab at Week 16 and until Week 50, in switched and continued treatment groups. AVT02 was safe and well tolerated, with a safety and immunogenicity profile similar to that observed in originator adalimumab with no clinically meaningful difference between the two. Clinical Trial Registration EudraCT: 2017-003367-35; ClinicalTrials.gov: NCT03849404. Supplementary Information The online version contains supplementary material available at 10.1007/s40259-021-00502-w.

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7–10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses.

518 Background: Eryaspase, asparaginase encapsulated in red blood cells is an investigational product under development. The encapsulated asparaginase induces the degradation of asparagine and glutamine, crucial for cancer cell growth and survival. An earlier Phase 2b study in patients with advanced pancreatic cancer showed an improvement in overall survival (OS) and progression free survival (PFS) with eryaspase plus chemotherapy. Methods: TRYbeCA-1 was a randomized, open-label Phase 3 trial of eryaspase combined with chemotherapy in patients with advanced adenocarcinoma of the pancreas who have progressed on only one prior line of systemic anti-cancer therapy. Patients were randomized in a 1:1 ratio to gemcitabine/nab-paclitaxel or irinotecan/fluorouracil (5FU) therapy (depending on first-line received) with or without eryaspase, administered as IV infusion on Day 1 and Day 15 of each 4-week cycle. Key eligibility criteria included progression on or following first-line systemic treatment, ECOG performance status 0 or 1, stage III-IV disease, documented evidence of disease progression, available tumor tissue and adequate organ function. The primary endpoint was OS. A total of 412 events were required for 90% power to detect a treatment effect hazard ratio (HR) of 0.725 at a two-sided significance level of 5%. Results: A total of 512 patients were included. Baseline characteristics were well balanced between the treatment arms. The study did not meet the OS primary endpoint [HR: 0.92 (95% confidence interval (CI), 0.76-1.11), p-value 0.375]. The median OS for patients treated with eryaspase plus chemotherapy was 7.5 mo (95% CI, 6.5-8.3), compared to 6.7 mo (95% CI, 5.4-7.5) for chemotherapy alone. There was a trend of nominal OS benefit in 107 patients treated with eryaspase and irinotecan-5FU compared to 109 patients in control subgroup, with a median OS of 8.0 mo versus 5.7 mo, respectively [HR: 0.81 (95% CI: 0.60- 1.09)]. Treatment effect was consistent across various prognosis factors. Median PFS was 3.7 mo vs. 3.5 mo in the eryaspase and control arms, respectively [HR: 0.89 (95% CI: 0.73-1.07), p-value 0.215]. Disease control rate was 57.6% and 49.0% (p-value 0.047) in the eryaspase and control arms, respectively. The most common adverse events were in the eryaspase arm were asthenia, diarrhea, and anemia (Grade 3-4: 16.9%, 7.66% and 17.3%, respectively). Eryaspase did not appear to enhance toxicity of chemotherapy. Conclusion: This large prospective study did not meet it primary endpoint of improving OS in patients treated with eryaspase. The addition of eryaspase demonstrated nevertheless a well-tolerated profile and an encouraging survival benefit in the irinotecan/5FU subgroup, warranting further investigation. Clinical trial information: NCT03665441.

Purpose To assess the magnitude of regional response to respiratory therapeutic agents in the lungs by using treatment response mapping (TRM) with hyperpolarized gas magnetic resonance (MR) imaging. TRM was used to quantify regional physiologic response in adults with asthma who underwent a bronchodilator challenge. Materials and Methods This study was approved by the national research ethics committee and was performed with informed consent. Imaging was performed in 20 adult patients with asthma by using hyperpolarized helium 3 (3He) ventilation MR imaging. Two sets of baseline images were acquired before inhalation of a bronchodilating agent (salbutamol 400 μg), and one set was acquired after. All images were registered for voxelwise comparison. Regional treatment response, ΔR(r), was calculated as the difference in regional gas distribution (R[r] = ratio of inhaled gas to total volume of a voxel when normalized for lung inflation volume) before and after intervention. A voxelwise activation threshold from the variability of the baseline images was applied to ΔR(r) maps. The summed global treatment response map (ΔRnet) was then used as a global lung index for comparison with metrics of bronchodilator response measured by using spirometry and the global imaging metric percentage ventilated volume (%VV). Results ΔRnet showed significant correlation (P < .01) with changes in forced expiratory volume in 1 second (r = 0.70), forced vital capacity (r = 0.84), and %VV (r = 0.56). A significant (P < .01) positive treatment effect was detected with all metrics; however, ΔRnet showed a lower intersubject coefficient of variation (64%) than all of the other tests (coefficient of variation, ≥99%). Conclusion TRM provides regional quantitative information on changes in inhaled gas ventilation in response to therapy. This method could be used as a sensitive regional outcome metric for novel respiratory interventions. © RSNA, 2017 Online supplemental material is available for this article.

International audience; PURPOSE: This Phase IIb (NCT02195180) open-label study evaluated erythrocyte-encapsulated asparaginase (eryaspase) in combination with chemotherapy in second-line advanced pancreatic adenocarcinoma.METHODS: Eligible patients were randomized 2:1 to either eryaspase in combination with gemcitabine or mFOLFOX6 (eryaspase arm), or to gemcitabine or mFOLFOX6 alone (control arm). Co-primary endpoints were overall survival (OS) and progression-free survival (PFS) in patients with low asparagine synthetase (ASNS) expression. Secondary endpoints included OS and PFS in the entire population.RESULTS: 141 patients were randomized (eryaspase arm, n = 95; control arm, n = 46). Median OS and PFS in patients with low ASNS expression were 6.2 months (95% CI, 5.1-8.8) in the eryaspase arm versus 4.9 months (3.1-7.1) in the control arm (HR, 0.63; 95% CI, 0.39-1.01; P = 0.056) and 2.0 months (95% CI, 1.8-3.4) in the eryaspase arm versus 1.8 months (1.4-3.8) in the control arm (HR, 0.67; 95% CI, 0.40-1.12; P = 0.127), respectively. In the entire population, median OS and PFS for the eryaspase arm versus control were 6.0 months versus 4.4 months (HR, 0.60; P = 0.008) and 2.0 months versus 1.6 months (HR, 0.56; 95% CI, 0.37-0.84; P = 0.005), respectively. The combination of eryaspase and chemotherapy was well tolerated. The most frequent Grade 3/4 adverse events in the eryaspase arm (n = 93) were gamma-glutamyltransferase increase (16 [17.2%]), neutropenia (12 [12.9%]), and physical health deterioration (12 [12.9%]).CONCLUSION: Eryaspase in combination with chemotherapy is associated with improvements in OS and PFS, irrespective of ASNS expression in second-line advanced pancreatic adenocarcinoma. A Phase III trial is underway.

Richard Kay, senior research associate at the Wellcome Trust MRC Institute of Metabolic Science at Cambridge University (UK), discusses how mass spectrometry and triple quadrupole systems are being used to study the peptidome of people with diabetes and post-bariatric surgery.