Safety, tolerability, and efficacy of NLY01 in early untreated Parkinson's disease: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Converging lines of evidence suggest that microglia are relevant to Parkinson's disease pathogenesis, justifying exploration of therapeutic agents thought to attenuate pathogenic microglial function. We sought to test the safety and efficacy of NLY01-a brain-penetrant, pegylated, longer-lasting version of exenatide (a glucagon-like peptide-1 receptor agonist) that is believed to be anti-inflammatory via reduction of microglia activation-in Parkinson's disease. METHODS: We report a 36-week, randomised, double-blind, placebo-controlled study of NLY01 in participants with early untreated Parkinson's disease conducted at 58 movement disorder clinics in the USA. Participants meeting UK Brain Bank or Movement Disorder Society research criteria for Parkinson's disease were randomly allocated (1:1:1) to one of two active treatment groups (2·5 mg or 5·0 mg NLY01) or matching placebo, based on a central computer-generated randomisation scheme using permuted block randomisation with varying block sizes. All participants, investigators, coordinators, study staff, and sponsor personnel were masked to treatment assignments throughout the study. The primary efficacy endpoint for the primary analysis population (defined as all randomly assigned participants who received at least one dose of study drug) was change from baseline to week 36 in the sum of Movement Disorder Society Unified Parkinson's Disease Rating Scale (MDS-UPDRS) parts II and III. Safety was assessed in the safety population (all randomly allocated participants who received at least one dose of the study drug) with documentation of adverse events, vital signs, electrocardiograms, clinical laboratory assessments, physical examination, and scales for suicidality, sleepiness, impulsivity, and depression. This trial is complete and registered at ClinicalTrials.gov, NCT04154072. FINDINGS: The study took place between Jan 28, 2020, and Feb 16, 2023. 447 individuals were screened, of whom 255 eligible participants were randomly assigned (85 to each study group). One patient assigned to placebo did not receive study treatment and was not included in the primary analysis. At 36 weeks, 2·5 mg and 5·0 mg NLY01 did not differ from placebo with respect to change in sum scores on MDS-UPDRS parts II and III: difference versus placebo -0·39 (95% CI -2·96 to 2·18; p=0·77) for 2·5 mg and 0·36 (-2·28 to 3·00; p=0·79) for 5·0 mg. Treatment-emergent adverse events were similar across groups (reported in 71 [84%] of 85 patients on 2·5 mg NLY01, 79 [93%] of 85 on 5·0 mg, and 73 [87%] of 84 on placebo), with gastrointestinal disorders the most commonly observed class in active groups (52 [61%] for 2·5 mg, 64 [75%] for 5·0 mg, and 30 [36%] for placebo) and nausea the most common event overall (33 [39%] for 2·5 mg, 49 [58%] for 5·0 mg, and 16 [19%] for placebo). No deaths occurred during the study. INTERPRETATION: NLY01 at 2·5 and 5·0 mg was not associated with any improvement in Parkinson's disease motor or non-motor features compared with placebo. A subgroup analysis raised the possibility of motor benefit in younger participants. Further study is needed to determine whether these exploratory observations are replicable. FUNDING: D&D Pharmatech-Neuraly.

Synthesis, Optimization, and Biological Evaluation of Corrinated Conjugates of the GLP-1R Agonist Exendin-4.

Corrination is the conjugation of a corrin ring containing molecule, such as vitamin B12 (B12) or B12 biosynthetic precursor dicyanocobinamide (Cbi), to small molecules, peptides, or proteins with the goal of modifying pharmacology. Recently, a corrinated GLP-1R agonist (GLP-1RA) exendin-4 (Ex4) has been shown in vivo to have reduced penetration into the central nervous system relative to Ex4 alone, producing a glucoregulatory GLP-1RA devoid of anorexia and emesis. The study herein was designed to optimize the lead conjugate for GLP-1R agonism and binding. Two specific conjugation sites were introduced in Ex4, while also utilizing various linkers, so that it was possible to identify Cbi conjugates of Ex4 that exhibit improved binding and agonist activity at the GLP-1R. An optimized conjugate (22), comparable with Ex4, was successfully screened and subsequently assayed for insulin secretion in rat islets and in vivo in shrews for glucoregulatory and emetic behavior, relative to Ex4.

Recombinant Expression and Stapling of a Novel Long-Acting GLP-1R Peptide Agonist.

Owing to their pleiotropic metabolic benefits, glucagon-like peptide-1 receptor (GLP-1R) agonists have been successfully utilized for treating metabolic diseases, such as type 2 diabetes and obesity. As part of our efforts in developing long-acting peptide therapeutics, we have previously reported a peptide engineering strategy that combines peptide side chain stapling with covalent integration of a serum protein-binding motif in a single step. Herein, we have used this strategy to develop a second generation extendin-4 analog rigidified with a symmetrical staple, which exhibits an excellent in vivo efficacy in an animal model of diabetes and obesity. To simplify the scale-up manufacturing of the lead GLP-1R agonist, a semisynthesis protocol was successfully developed, which involves recombinant expression of the linear peptide followed by attachment of a polyethylene glycol (PEG)-fatty acid staple in a subsequent chemical reaction step.

A once-monthly GLP-1 receptor agonist for treatment of diabetic cats.

There is growing evidence that peptidic glucagon-like peptide-1 receptor agonists (GLP-1RA), such as exenatide, may provide useful therapeutic options for treatment of feline diabetes. However, because such drugs are administered subcutaneously, it is desirable that they be long-acting and not require frequent injections. We have developed a chemically controlled delivery system to support half-life extension of peptidic therapeutics. Here, the peptide is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection of the microspheres, the peptide is slowly released from the depot to the systemic circulation. Using this technology, we developed a delivery system that supports once-monthly administration of a stable exenatide analog, [Gln28]exenatide, in rodents (Schneider, et al, ACS Chem Biol 12, 2107 to 2116, 2017). The purposes of the present study were a) to demonstrate pharmacokinetic and pharmacodynamic similarities of the deamidation-sensitive GLP-1RA exenatide and the closely related, more stable [Gln28]exenatide and b) to develop a long-acting GLP-1RA in cats. The results show that exenatide and [Gln28]exenatide injected intravenously or subcutaneously at 10 µg/kg have nearly identical pharmacokinetics in the cat-both having elimination half-lives of ∼40 min-but subcutaneously administered [Gln28]exenatide has superior bioavailability-93% for [Gln28]exenatide vs 52% for exenatide. The results also show that exenatide and [Gln28]exenatide have similar insulinotropic activities in the cat during a high-dose intravenous glucose tolerance test; they increased the area under the curve (AUC) for insulin to a similar extent but had no effect on glucose AUC. Finally, subcutaneous injection of a microsphere-[Gln28]exenatide conjugate containing an appropriate self-cleaving linker in the cat provides plasma [Gln28]exenatide with a half-life of about 40 d vs 40 min with the injected free peptide. Hence, the large body of information available for exenatide can be used to facilitate clinical development of [Gln28]exenatide as a treatment for feline diabetes, and the microsphere-[Gln28]exenatide conjugate is quite suitable for once-monthly subcutaneous administration of the peptide in the cat.

Exendin-4 Derivatives with an Albumin-Binding Moiety Show Decreased Renal Retention and Improved GLP-1 Receptor Targeting.

The glucagon-like peptide-1 receptor (GLP-1R) is highly and specifically expressed on the pancreatic ß-cells. It plays an important role in glucose metabolism as well as in ß-cell-derived diseases like diabetes, insulinoma, or congenital and adult hyperinsulinemic hypoglycemia. Radiolabeled exendin-4, a ligand of GLP-1R, has routinely been used in clinics to image insulinomas. However, its major drawback is the high kidney accumulation. Here, we show that the addition of an albumin-binding moiety (ABM) to radiolabeled exendin-4 results in a significant reduction of kidney uptake while retaining its high affinity and specificity to GLP-1R. The four tested peptides were shown to have high affinity to the GLP-1 receptor (IC50 of 3.7 ± 0.6 to 15.1 ± 0.8 nM). The radiolabeled derivatives were taken up into cells efficiently, internalizing between 39 ± 2 and 56 ± 2% after 2 h. Thus, the derivatives with ABM outperformed the reference peptide with its IC50 of 22.5 ± 2.9 nM and internalization of 41 ± 4%. Stability in human blood plasma was slightly enhanced by the addition of the albumin binder. In biodistribution studies, the radioligands exhibited an improved target-to-kidney ratio in comparison to the reference peptide of up to seven-fold. This was confirmed qualitatively in single-photon-emission computed tomography (SPECT)/CT imaging. This study demonstrated in vitro and in vivo that the addition of an ABM to radiolabeled exendin-4 strongly decreased kidney accumulation while retaining affinity to GLP-1R. Thus, exendin-4 derivatives with an albumin-binding moiety could present a viable class of diagnostic tracers for the detection of insulinomas and other GLP-1R-positive tissue in clinical application.

An Albumin Sandwich Enhances in Vivo Circulation and Stability of Metabolically Labile Peptides.

The effectiveness of numerous molecular drugs is hampered by their poor pharmacokinetics. Different from previous approaches with limited effectiveness, most recently, emerging high-affinity albumin binding moieties (ABMs) for in vivo hitchhiking of endogenous albumin opens up an avenue to chaperone small molecules for long-acting therapeutics. Although several FDA-approved fatty acids have shown prolonged residence and therapeutic effect, an easily synthesized, water-soluble, and high-efficiency ABM with versatile drug loading ability is urgently needed to improve the therapeutic efficacy of short-lived constructs. We herein identified an ideal bivalent Evans blue derivative, denoted as N(tEB)2, as a smart ABM-delivery platform to chaperone short-lived molecules, through both computational modeling screening and efficient synthetic schemes. The optimal N(tEB)2 could reversibly link two molecules of albumin through its two binding heads with a preferable spacer, resulting in significantly extended circulation half-life of a preloaded cargo and water-soluble. Notably, this in situ dimerization of albumin was able to sandwich peptide therapeutics to protect them from proteolysis. As an application, we conjugated N(tEB)2 with exendin-4 for long-acting glucose control in a diabetic mouse model, and it was superior to both previously tested NtEB-exendin-4 (Abextide) and the newly FDA-approved semaglutide, which has been arguably the best commercial weekly formula so far. Hence, this novel albumin binder has excellent clinical potential for next-generation biomimetic drug delivery systems.

Antidiabetic Effect of Abextide, a Long-Acting Exendin-4 Analogue in Cynomolgus Monkeys.

Abextide, synthesized by conjugating an albumin-binding moiety-truncated Evans blue-to glucagon-like peptide 1 receptor (GLP-1R) agonist exendin-4, shows extended drug release and enhanced hypoglycemic effect in diabetic mice. The aim of this study is to evaluate the pharmacodynamics of Abextide in nonhuman primates. Two batches of elderly cynomolgus monkeys with naturally occurring diabetes are used for this study. During the whole experiment period, no abnormalities such as swelling at the injection site, lethargy, or hypoglycemia are observed in all animals. The monkeys in the Abextide group lose appetite after drug administration and then recover over time. In the single dose treatment, at day 1 and day 3 after treatment, decreased plasma glucose level is observed in the Abextide-treated group but not in placebo or Albiglutide-treated group. For monkeys that receive two doses of drug, the blood glucose level in all subjects in Abextide group decreases rapidly upon drug administration and return to a plateau by day 3. A similar pattern of response is seen after the second dose administration. The delayed drug release and hypoglycemic effect of Abextide make it potentially useful as an antidiabetic drug for weekly subcutaneous administration.

Exendin-4 from Heloderma suspectum venom: From discovery to its latest application as type II diabetes combatant.

Type II diabetes mellitus (T2DM) is a chronic non-communicable disease due to abnormal insulin actions causing uncontrolled hyperglycaemia. The treatment for T2DM, for instance, metformin and incretin mimetic, mainly focuses on the restoration of insulin sensitivity and secretion. Exendin-4 is a short incretin-mimetic peptide consisting of 39 amino acids. It is discovered in the venom of Heloderma suspectum as a full agonist for the glucagon-like peptide 1 (GLP-1) receptor and produces insulinotropic effects. It is more resistant to enzymatic degradation by dipeptidyl-peptidase-4 and has a longer half-life than the endogenous GLP-1; thus, it is further developed as an incretin hormone analogue used to treat T2DM. The helical region of the peptide first interacts with the extracellular N-terminal domain (NTD) of GLP-1 receptor while the C-terminal extension containing the tryptophan cage further enhances its binding affinity. After binding to the NTD of the receptor, it may cause the receptor to switch from its auto-inhibited state of the receptor to its auto-activated state. Exendin-4 enhances the physiological functions of ß-cells and the up-regulation of GLP-1 receptors, thus reducing the plasma glucose levels. Moreover, exendin-4 has also been found to ameliorate neuropathy, nephropathy and ventricular remodelling. The therapeutic effects of exendin-4 have also been extrapolated into several clinical trials. Although exendin-4 has a reasonable subcutaneous bioavailability, its half-life is rather short. Therefore, several modifications have been undertaken to improve its pharmacokinetics and insulinotropic potency. This review focuses on the pharmacology of exendin-4 and the structure-function relationships of exendin-4 with GLP-1 receptor. The review also highlights some challenges and future directions in the improvement of exendin-4 as an anti-diabetic drug.

Chemical chaperone-conjugated exendin-4 as a cytoprotective agent for pancreatic ß-cells.

Endoplasmic reticulum stress has been considered a major cause of pancreatic ß-cell dysfunction and apoptosis leading to diabetes. Glucagon-like peptide-1 receptor activation and chemical chaperones have been known to reduce endoplasmic reticulum stress and improve ß-cell function and survival. The purpose of this study was to prepare and evaluate the chemical chaperone tauroursodeoxycholic acid-conjugated exendin-4 as a protective agent for pancreatic ß-cells. Mono-tauroursodeoxycholic acid-Lys27-exendin-4 conjugate (TUM1-Ex4) showed better receptor binding affinity than other conjugates with strong in vitro insulinotropic activity in rat pancreatic ß-cells and in vivo hypoglycemic activity in type 2 diabetic db/db mice. In INS-1 cells under endoplasmic reticulum stress induced by thapsigargin, TUM1-Ex4 promoted cell survival in a dose-dependent manner. In western blot analysis, TUM1-Ex4 reduced the expression of the endoplasmic reticulum stress marker GRP78 and phosphorylation of the translation initiation factor eIF2α. These results reveal that TUM1-Ex4 accelerates translational recovery and contributes to ß-cell protection and survival. The present study indicates that the chemical chaperone-coupled glucagon-like peptide-1 receptor agonist is a feasible therapeutic strategy to enhance ß-cell function and survival.

α-Helix or ß-Turn? An Investigation into N-Terminally Constrained Analogues of Glucagon-like Peptide 1 (GLP-1) and Exendin-4.

Peptide agonists acting on the glucagon-like peptide 1 receptor (GLP-1R) promote glucose-dependent insulin release and therefore represent important therapeutic agents for type 2 diabetes (T2D). Previous data indicated that an N-terminal type II ß-turn motif might be an important feature for agonists acting on the GLP-1R. In contrast, recent publications reporting the structure of the full-length GLP-1R have shown the N-terminus of receptor-bound agonists in an α-helical conformation. To reconcile these conflicting results, we prepared N-terminally constrained analogues of glucagon-like peptide 1 (GLP-1) and exendin-4 and evaluated their receptor affinity and functionality in vitro; we then examined their crystal structures in complex with the extracellular domain of the GLP-1R and used molecular modeling and molecular dynamics simulations for further investigations. We report that the peptides' N-termini in all determined crystal structures adopted a type II ß-turn conformation, but in vitro potency varied several thousand-fold across the series. Potency correlated better with α-helicity in our computational model, although we have found that the energy barrier between the two mentioned conformations is low in our most potent analogues and the flexibility of the N-terminus is highlighted by the dynamics simulations.

A vitamin B12 conjugate of exendin-4 improves glucose tolerance without associated nausea or hypophagia in rodents.

AIMS: While pharmacological glucagon-like peptide-1 receptor (GLP-1R) agonists are FDA-approved for treating type 2 diabetes mellitus (T2DM) and obesity, a major side effect is nausea/malaise. We recently developed a conjugate of vitamin B12 (B12) bound to the GLP-1R agonist exendin-4 (Ex4), which displays enhanced proteolytic stability and retention of GLP-1R agonism. Here, we evaluate whether the conjugate (B12-Ex4) can improve glucose tolerance without producing anorexia and malaise. MATERIALS AND METHODS: We evaluated the effects of systemic B12-Ex4 and unconjugated Ex4 on food intake and body weight change, oral glucose tolerance and nausea/malaise in male rats, and on intraperitoneal glucose tolerance in mice. To evaluate whether differences in the profile of effects of B12-Ex4 vs unconjugated Ex4 are the result of altered CNS penetrance, rats received systemic injections of fluorescein-Ex4 (Flex), Cy5-B12 or Cy5-B12-Ex4 and brain penetrance was evaluated using confocal microscopy. Uptake of systemically administered Cy5-B12-Ex4 in insulin-containing pancreatic beta cells was also examined. RESULTS: B12-Ex4 conjugate improves glucose tolerance, but does not elicit the malaise and anorexia produced by unconjugated Ex4. While Flex robustly penetrates into the brain (dorsal vagal complex, paraventricular hypothalamus), Cy5-B12 and Cy5-B12-Ex4 fluorescence were not observed centrally, supporting an absence of CNS penetrance, in line with observed reduction in CNS-associated Ex4 side effects. Cy5-B12-Ex4 colocalizes with insulin in the pancreas, suggesting direct pancreatic action as a potential mechanism underlying the hypoglycaemic effects of B12-Ex4. CONCLUSION: These novel findings highlight the potential clinical utility of B12-Ex4 conjugates as possible future T2DM therapeutics with reduced incidence of adverse effects.