Conjugation of a peptide to an antibody engineered with free cysteines dramatically improves half-life and activity.

The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.

A novel long-acting oxyntomodulin analogue eliminates diabetes and obesity in mice.

Oxyntomodulin (OXM) was identified as a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist to suppress appetite, increase energy expenditure, and induce body weight loss in obese humans. However, the activities of native OXM to activate GCGR and GLP-1R in vitro were much weaker than the natural ligands. To address this gap, structural modifications were adopted and novel OXM analogues were obtained through chimeric peptide sequence design. One specific analogue with enhanced and balanced GCGR/GLP-1R activations was chemically conjugated with polyethylene glycol (PEG) to achieve sustained release in vivo. This PEGylated analogue was further explored pharmacologically in db/db and diet-induced obese (DIO) mice models. Chronic weekly administration significantly induced hypoglycemic effects and body weight loss with dose dependency, along with normalized adiposity, lipid metabolism, and liver steatosis. Based on its profiles in vitro and in vivo, the analogue has the great potential to develop as a novel anti-diabetic and/or anti-obese candidate. As observed more insulin stimulation and improved insulin resistance, it may be also explored for the treatment of nonalcoholic steatohepatitis (NASH) in the future.

Design and characterization of novel oxyntomodulin derivatives with potent dual GLP-1/glucagon receptor activation and prolonged antidiabetic effects.

AIMS: To investigate the combination of dimerization and PEGylation to enhance the receptor activation and in vivo stability of Oxyntomodulin (OXM). MAIN METHODS: All LDM peptides were produced by using standard method of solid phase synthesis. The in vitro effects of LDM peptides were assessed by glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GcgR) binding test and Proteolytic stability test. Subsequently, saline, Liraglutide and three doses of LDM-3 treated groups were subjected to the evaluation of aute and long-term efficacy. KEY FINDINGS: Five long-acting OXM conjugates, termed LDM-1 to LDM-5, were designed using cysteine (Cys)-specific modification reaction including the activated PEG, bisMal-NH2, and OXM-Cys, and all prepared with high purity. LDM-3 exhibited greater GLP-1R and GcgR activation and ameliorative serum stability. In addition, LDM-3 was identified with enhanced insulinotropic and glycemic abilities in the gene knockout mice. The prolonged glucose-lowering effects of the LDM-3 were proved by hypoglycemic duration test and multiple oral glucose tolerance tests (OGTTs) in the diet-induced obesity (DIO) mice. Furthermore, the pharmacokinetic tests in Sprague Dawley (SD) rat and cynomolgus monkey exhibited the lifespans of LDM-3 at 90 nmol·kg-1 were 101.5 h and 119.4 h, respectively. Nevertheless, consecutive 8-week administration of LDM-3 improved the cumulative body weight gain, food intake, % HbA1c, glucose tolerance and the pancreatic of the obese mice. SIGNIFICANCE: LDM-3, as a dual GLP-1R and GcgR agonist, holds potential to be developed as a promising therapeutic candidate for both diabetes and obesity.

New Generation Oxyntomodulin Peptides with Improved Pharmacokinetic Profiles Exhibit Weight Reducing and Anti-Steatotic Properties in Mice.

Oxyntomodulin (OXM) is an intestinal peptide hormone that activates both glucagon-like peptide-1 (GLP-1) and glucagon (GCG) receptors. The natural peptide reduces body weight in obese subjects and exhibits direct acute glucoregulatory effects in patients with type II diabetes. However, the clinical utility of OXM is limited due to its lower in vitro potency and short in vivo half-life. To overcome these issues, we developed stapled, long-acting, and highly potent OXM analogs with balanced activities at both GLP-1 and GCG receptors. The lead molecule O14 exhibits potent and long-lasting effects on glucose control, body weight loss, and reduction of hepatic fat reduction in DIO mice. Importantly, O14 significantly reversed hepatic steatosis; reduced liver weight, total cholesterol, and hepatic triglycerides; and improved markers of liver function in a nonalcoholic steatohepatitis (NASH) mouse model. A symmetrical version of the peptide was also shown to be more efficacious and long-lasting in controlling glucose than semaglutide and the clinical candidate cotadutide in wild-type mice, highlighting the utility of our designs of the dual agonist as a potential new therapy for diabetes and liver diseases.

Controlling the bioactivity of a peptide hormone in vivo by reversible self-assembly.

The use of peptides as therapeutic agents is undergoing a renaissance with the expectation of new drugs with enhanced levels of efficacy and safety. Their clinical potential will be only fully realised once their physicochemical and pharmacokinetic properties have been precisely controlled. Here we demonstrate a reversible peptide self-assembly strategy to control and prolong the bioactivity of a native peptide hormone in vivo. We show that oxyntomodulin, a peptide with potential to treat obesity and diabetes, self-assembles into a stable nanofibril formulation which subsequently dissociates to release active peptide and produces a pharmacological effect in vivo. The subcutaneous administration of the nanofibrils in rats results in greatly prolonged exposure, with a constant oxyntomodulin bioactivity detectable in serum for at least 5 days as compared to free oxyntomodulin which is undetectable after only 4 h. Such an approach is simple, cost-efficient and generic in addressing the limitations of peptide therapeutics.

Pulmonary delivery of anorectic oxyntomodulin in rats: food intake suppression, reduced body weight gain and pharmacokinetics.

BACKGROUND: Oxyntomodulin (OXM1-37) is an anorectic gut-secreting peptide with a promise to treat obesity, but its needle-free delivery has yet to be successful. RESULTS: Pulmonary delivery of OXM1-37, but not its C-terminal octapeptides, caused dose-related, transient 4-6 h food intake suppression in rats. At 0.5 mg/kg, its 30-38% food intake suppression led to 46% reduction in body weight gain by day 8. Its lung absorption was fast, elevating the systemic level rapidly, yet the bioavailability was low at 13%. In the brain, twofold neuronal c-fos activation was seen in the hypothalamus arcuate nucleus and brainstem area postrema. CONCLUSION: Pulmonary delivery is a promising needle-free systemic delivery option for OXM1-37 to treat obesity, as enabling effective lung absorption and brain interaction.

Chemical polysialylation: design of conjugated human oxyntomodulin with a prolonged anorexic effect in vivo.

Recombinant gut hormone oxyntomodulin (OXM) is known to act as a satiety signal in human subjects and has therapeutic potential as an appetite controlling agent. The only form of this hormone that has a prospective use is a modified one, because native OXM has a very short half-life in vivo. Conjugation of OXM and the natural hydrophilic polymer polysialic acid (PSA) may significantly improve its half-life. Chemical polysialylation in vitro was used to create a long-acting form of OXM, the polysialic acid-oxyntomodulin (PSA-OXM) conjugate. The conjugation site was identified using mass shift comparative analysis of Asp-N proteolytic digests. The anorexic effect of the conjugate was tested on the lean, fasted mouse model. A two-stage purification technique was developed to obtain a homogeneous PSA-OXM conjugate, suitable for in vivo testing. The N-terminal backbone primary amino group was found to be the only point of conjugation. The conjugate obtained was resistant to the DPP-IV protease. A single injection of PSA-OXM at 15 µmol/kg dose was sufficient to maintain a steady decrease in food consumption for 8 h (P < 0.05). The length of the anorexic effect achieved is comparable to other long-acting derivatives of OXM but it requires a much higher dose for administration. It is expected that site-directed attachment of the PSA chain to the inner residues of OXM, away from the site of interaction with receptors, would produce a compound with a higher specific activity but comparable stability in the bloodstream. The conjugation technique used may be used to create OXM derivatives and other related hormones to obtain long-lasting variants, with improved suitability for clinical use.

Determination of oxyntomodulin, an anorectic polypeptide, in rat plasma using 2D-LC-MS/MS coupled with ion pair chromatography.

Polypeptide therapeutics present a challenge for quantitative analysis when using immunoassays or recently, liquid chromatography-tandem mass spectrometry because of their structural similarities to endogenous proteins and peptides in plasma. In this assay, a Waters Oasis® mixed-mode anion exchange (MAX) microelution modified solid phase extraction (SPE) method coupled with two-dimensional reversed phase ion pair chromatography-tandem mass spectrometry was used for the validation and analysis of oxyntomodulin in rat plasma. Oxyntomodulin (OXM) and its isotope labeled internal standard were extracted from rat plasma and analyzed with a chromatographic run time of 8 min. Modified SPE, two-dimensional liquid chromatography coupled with 3-nitrobenzyl alcohol as a mobile phase additive, and monitoring of multiply charged SRM transitions (+7 charge state) of OXM were necessary to achieve a lower limit of quantification of 1 ng/mL. The method was validated with a linear range of 1-1000 ng/mL, with average R² of 0.992, and reversed calculated residuals between -8.6% and 6.0%. Precision and accuracy for inter- and intra-day were determined to be ±17%. Following a complete validation, the method was applied to show utility using rat plasma samples that were intravenously dosed with oxyntomodulin.

Investigation of structure-activity relationships of Oxyntomodulin (Oxm) using Oxm analogs.

Oxyntomodulin (Oxm) is an intestinal peptide that inhibits food intake and body weight in rodents and humans. These studies used peptide analogs to study aspects of structure and function of Oxm, and the sensitivity of parts of the Oxm sequence to degradation. Analogs of Oxm were synthesized and studied using receptor binding and degradation studies in vitro. Their effects on food intake and conditioned taste avoidance were measured in vivo in rodents. Oxm breakdown by the enzyme dipeptidyl peptidase IV (DPPIV) was demonstrated in vitro and in vivo. In vitro degradation was reduced and in vivo bioactivity increased by inhibitors of DPPIV. Modifications to the N terminus of Oxm modulated binding to the glucagon-like peptide (GLP)-1 receptor and degradation by DPPIV. Modifications to the midsection of Oxm modulated binding to the GLP-1 receptor and degradation by neutral endopeptidase. These modifications also altered bioactivity in vivo. The C-terminal octapeptide of Oxm was shown to contribute to the properties of Oxm in vitro and in vivo but was not alone sufficient for the effects of the peptide. Elongation and acylation of the C terminus of Oxm altered GLP-1 receptor binding and duration of action in vivo, which may be due to changes in peptide clearance. An Oxm analog was developed with enhanced pharmaceutical characteristics, with greater potency and longevity with respect to effects on food intake. These studies suggest that Oxm is a potential target for antiobesity drug design.