A Hydrogel-Microsphere Drug Delivery System That Supports Once-Monthly Administration of a GLP‑1 Receptor Agonist

We have developed a chemically controlled very long-acting delivery system to support once-monthly administration of a peptidic GLP-1R agonist. Initially, the prototypical GLP-1R agonist exenatide was covalently attached to hydrogel microspheres by a self-cleaving β-eliminative linker; after subcutaneous injection in rats, the peptide was slowly released into the systemic circulation. However, the short serum exenatide half-life suggested its degradation in the subcutaneous depot. We found that exenatide undergoes deamidation at Asn²⁸ with an <i>in vitro</i> and <i>in vivo</i> half-life of approximately 2 weeks. The [Gln²⁸]­exenatide variant and exenatide showed indistinguishable GLP-1R agonist activities as well as pharmacokinetic and pharmacodynamic effects in rodents; however, unlike exenatide, [Gln²⁸]­exenatide is stable for long periods. Two different hydrogel-[Gln²⁸]­exenatide conjugates were prepared using β-eliminative linkers with different cleavage rates. After subcutaneous injection in rodents, the serum half-lives for the released [Gln²⁸]­exenatide from the two conjugates were about 2 weeks and one month. Two monthly injections of the latter in the Zucker diabetic fatty rat showed pharmacodynamic effects indistinguishable from two months of continuously infused exenatide. Pharmacokinetic simulations indicate that the delivery system should serve well as a once-monthly GLP-1R agonist for treatment of type 2 diabetes in humans

Bifunctional PEGylated Exenatide-Amylinomimetic Hybrids to Treat Metabolic Disorders: An Example of Long-Acting Dual Hormonal Therapeutics

Peptide hybrids (phybrids) comprising covalently linked peptide hormones can leverage independent biological pathways for additive or synergistic metabolic benefits. PEGylation of biologics offers enhanced stability, duration, and reduced immunogenicity. These two modalities can be combined to produce long-acting therapeutics with dual pharmacology and enhanced efficacy. Compound <b>10</b> is composed of an exenatide (AC2993) analogue, AC3174, and an amylinomimetic, davalintide (AC2307), with an intervening 40 kD PEG moiety. It displayed dose-dependent and prolonged efficacy for glucose control and body weight reduction in rodents with superior <i>in vitro</i> and <i>in vivo</i> activities compared to those of a side-chain PEGylated phybrid <b>6</b>. In diet-induced obese (DIO) rats, the weight-loss efficacy of <b>10</b> was similar to that of a combination of PEG-parents <b>3</b> and <b>4</b>. A single dose of <b>10</b> elicited sustained body weight reduction in DIO rats for at least 21 days. Compound <b>10</b>’s terminal half-life of ∼27 h should translate favorably to weekly dosing in humans

<i>In Vitro</i> Activity, Metabolic Stability, and Plasma Glucose in OGTT.

<p>Stability results (measured as plasma AUC 0–5 h; pancreatin mix AUC 0–2 h)) are expressed as a percentage of peptide remaining versus stable control peptide ± SEM. Replicates within each assay n = 3, except for exenatide and [Leu¹⁴]exenatide-ABP where n = 2.</p

PK profile of orally dosed peptides in monkeys.

<p>[Leu¹⁴]exenatide-ABD (▾) and [Leu¹⁴]exenatide-ABP (▿), n = 3. Data are presented as mean ± SEM.</p

Four-week chronic dosing of peptides in diabetic <i>Lep^ob/ob</i> Mice.

<p>(A) effects on HbA1c (B) effects on body weight and (C) effects on food intake. Vehicle (□), [Leu¹⁴]exenatide-ABD (▾) and exenatide (▪). Data are presented as mean ± SEM, n = 10. *p<0.05 vs. vehicle, #p<0.05 vs. exenatide infusion by ANOVA with Tukey’s test.</p

Names and Sequences of Compounds.

<p>Names and Sequences of Compounds.</p

Binding Constants for Albumin Interactions at 25°C.

<p>The number in parentheses represents the standard deviation in the last significant digit.</p

Functional activity at the GLP-1 receptor.

<p>GLP-1 (7–36) (▪), [Leu¹⁴]exenatide-ABD (▾) and [Leu¹⁴]exenatide-ABP (▿). GLP-1 (7–36) was used as a reference standard in the assay. The assay was run in quadruplicates and data are presented as mean ± SD. Abbreviations: Fluorescence (F).</p

PK profile of intravenously dosed peptides in rats and monkeys.

<p>(A) rats, n = 3–4 and (B) monkeys, n = 3. Exenatide (▪), [Leu¹⁴]exenatide-ABD (▾) and [Leu¹⁴]exenatide-ABP (▿). Data are presented as mean ± SEM.</p

Improved Glucose Control and Reduced Body Weight in Rodents with Dual Mechanism of Action Peptide Hybrids

<div><p>Combination therapy is being increasingly used as a treatment paradigm for metabolic diseases such as diabetes and obesity. In the peptide therapeutics realm, recent work has highlighted the therapeutic potential of chimeric peptides that act on two distinct receptors, thereby harnessing parallel complementary mechanisms to induce additive or synergistic benefit compared to monotherapy. Here, we extend this hypothesis by linking a known anti-diabetic peptide with an anti-obesity peptide into a novel peptide hybrid, which we termed a phybrid. We report on the synthesis and biological activity of two such phybrids (AC164204 and AC164209), comprised of a glucagon-like peptide-1 receptor (GLP1-R) agonist, and exenatide analog, AC3082, covalently linked to a second generation amylin analog, davalintide. Both molecules acted as full agonists at their cognate receptors <i>in vitro</i>, albeit with reduced potency at the calcitonin receptor indicating slightly perturbed amylin agonism. In obese diabetic <i>Lep^ob/Lep</i>^<i>ob</i> mice sustained infusion of AC164204 and AC164209 reduced glucose and glycated haemoglobin (Hb_A1c) equivalently but induced greater weight loss relative to exenatide administration alone. Weight loss was similar to that induced by combined administration of exenatide and davalintide. In diet-induced obese rats, both phybrids dose-dependently reduced food intake and body weight to a greater extent than exenatide or davalintide alone, and equal to co-infusion of exenatide and davalintide. Phybrid-mediated and exenatide + davalintide-mediated weight loss was associated with reduced adiposity and preservation of lean mass. These data are the first to provide <i>in vivo</i> proof-of-concept for multi-pathway targeting in metabolic disease via a peptide hybrid, demonstrating that this approach is as effective as co-administration of individual peptides.</p> </div