In vitro and in vivo stability and pharmacokinetic profile of unacylated ghrelin (UAG) analogues

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Abstract

Ghrelin, an endocrine hormone predominantly produced by the stomach, exists in acylated and unacylated forms in the circulation. Unacylated ghrelin (UAG), the more abundant form in blood, possesses similar, independent or opposite physiological actions as acylated ghrelin (AG). AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG6–13), and its full (AZP531) and partially (AZP533) cyclised derivatives, exhibit the same pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We investigated the stability of these three fragments in vitro in human blood samples and in vivo after subcutaneous and intravenous injection in rats and dogs using liquid chromatography–mass spectrometry. In both species, AZP502 is rapidly degraded generating two major metabolites. Partial cyclisation of AZP502 and acylation at its N-terminus (AZP533 peptide) improves its stability in human plasma in vitro. Full cyclisation of AZP502 (AZP531 peptide) also completely protects the peptide from peptidase degradation in vitro in human blood samples. Moreover this cyclisation strongly improves the stability and the bioavailability of this peptide in vivo in both dogs and rats (mean bioavailability of 10–15% and 85–95% for AZP502 and AZP531 respectively). Taken together these results support the rationale for developing AZP531 as a long-acting UAG analogue for subcutaneous injection for the treatment of type 2 diabetes mellitus and other metabolic disorders.

Graphical abstract

Pharmacokinetic profile of unacylated ghrelin (UAG) analogues, AZP502 (Ser-Pro-Glu-His-Gln-Arg-Val-Gln-NH2) and AZP531 (cyclo(-Ser-Pro-Glu-His-Gln-Arg-Val-Gln)), following subcutaneous (rectangle) and intravenous (diamond) administration in rats. Mean bioavailability from the PK analysis in the rat was 11.7% for AZP502 and 96.5% for AZP531 showing the difference in the aliphatic and cyclised forms of the peptide.

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Introduction

The stability of peptides in plasma is a major obstacle to their use as therapeutic drugs. In general, unmodified peptides have short plasma half-lives due to a combination of rapid renal clearance and their predisposition for enzymatic degradation (Rauh et al., 2007, Vergote et al., 2008). Moreover, peptides generally have poor bioavailability in tissues and organs. Therefore, in order to be used as drugs, peptides tend to be modified so that their proteolytic degradation is diminished and their bioavailability is increased. A wide variety of chemical modifications of the peptide backbone (i.e. use of non-natural amino acids, cyclisation, D-amino acid substitution, N-terminal amidation, etc.) may be performed. However, these modifications are useful only if they are strategically introduced to maximise stability towards enzymatic degradation and bioavailability, while at the same time preserving or even enhancing the potency of the bioactive peptide. Historically, the quantitation of peptides in plasma has been performed using immunological based approaches. However, these techniques can result in an inability to distinguish between intact and degraded forms. The use of traditional liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) and selected reaction monitoring (SRM) based analyses allows highly specific monitoring of peptides, and can enable simultaneous quantitation of both parent and metabolite peptides (Rauh et al., 2007). Furthermore, the development of quantitative LC–MS/MS methodologies are extremely rapid compared with immunological techniques, and are therefore well suited for determining the stability of peptides both in vitro and in vivo during the early stages of product development.

Ghrelin is a 28-amino acid peptide hormone that is present in both acylated and unacylated forms in plasma (Kojima et al., 1999, Van der Lely et al., 2004). Acylation of the peptide at Ser3 is required for its ability to activate the growth hormone secretagogue receptor type 1a (GHS-R1a), and stimulate secretion of growth hormone (GH) from the pituitary (Sun et al., 2004). In addition to its GH secretagogue activity, acylated ghrelin (AG) stimulates food intake, increases body weight, adiposity and gastric motility and induces insulin resistance and hyperglycemia (Tschöp et al., 2000, Wren et al., 2000, Wren et al., 2001, Broglio et al., 2001, Broglio et al., 2008, Tong et al., 2010). Unacylated ghrelin (UAG) represents by far the most abundant form of circulating ghrelin and was initially considered an inactive form of ghrelin since at physiological concentrations it is devoid of GHS-R1a-binding affinity. However, there is growing evidence that UAG is a biologically active peptide with specific activities through yet undetermined receptor(s), sometimes supporting, but most often counteracting and opposing the effects of AG (Delhanty and van der Lely, 2011, Kumar et al., 2010). Hence, while AG and UAG promote pancreatic β-cell survival (Granata et al., 2006, Granata et al., 2007), UAG has been reported to counteract AG-induced insulin resistance and hyperglycemia (Broglio et al., 2004, Gauna et al., 2004), to suppress AG-induced glucose output by primary hepatocytes (Gauna et al., 2005) and, in contrast to AG, to promote glucose and free fatty acid uptake by cardiomyocytes, myotubes or adipocytes (Lear et al., 2010). UAG also exerts beneficial cardiovascular effects (Baldanzi et al., 2002, Bedendi et al., 2003, Kleinz et al., 2006) and improves vascular remodeling (Togliatto et al., 2010).

In order to determine the minimal amino acid domain of UAG responsible of its anti-diabetogenic potential, Granata et al. (in press) has tested the pharmacological properties of different UAG fragments. They showed that the UAG fragment 6-13 (AZP502) and its cyclic analogues (partial cyclic and N-terminus acylated (AZP533) and full cyclic (AZP531)) exert survival effects on pancreatic β-cells and human pancreatic islets which are comparable to those of UAG, their parent molecule. Moreover, AZP502 inhibits streptozotocin-induced diabetes in rats and reduces oxidative stress and senescence in human circulating angiogenic cells. These data suggest that UAG analogues have potential as therapeutic leads to treat type 2 diabetes.

The determination of peptide stability in human plasma in vitro and after in vivo administration in animal species constitutes a powerful screening assay for selecting stable peptides for further development. The aims of this study were to determine the stability of UAG analogues (AZP502, AZP531, AZP533) and their metabolites in human plasma in vitro, and to determine the pharmacokinetic parameters of these peptides following single administration to rats and dogs, animal species commonly used to test toxicity of drug products during preclinical studies. State of the art SRM based LC–MS/MS methods were used for these analyses. The results of this study should allow the selection of pharmacologically active UAG analogues as therapeutic leads in the development of a treatment for type 2 diabetes and other metabolic disorders.

Section snippets

Chemicals

AZP502, AZP531 and AZP533 (Fig. 1) were synthesised by Bachem (Bubendorf, Switzerland). Acetonitrile, methanol and bovine serum albumin (BSA) were purchased from Fisher Scientific (Loughborough, UK). Formic acid, SigmaFast (EDTA free) protease inhibitor cocktail, and ammonium formate were purchased from Sigma Aldrich (St Louis Mo, USA). Ultra-pure (18.2 MΩ) water was prepared using a Triple Red system (Long Crendon, UK).

Acetonitrile peptide extraction methodology

Our previously described extraction method, modified slightly to be more

Stability in human plasma in vitro of UAG analogues

The relative levels of the UAG analogues (AZP502, AZP531 or AZP533) in the plasma extracts from the stability experiment were determined using SRM analysis (Fig. 2). The relative levels of each peptide at each time point were compared against the value obtained at the t = 0 min time point.

Discussion

The short half-life of peptides in vivo is the major factor preventing their use as therapeutic drugs. Their low in vivo stability mostly results from proteolytic degradation. Furthermore, their rapid renal clearance and their limited bioavailability can also be limiting factors. Because peptides are mainly degraded by proteases and peptidases, peptide delivery is the bottleneck in the development of new peptide therapeutic compounds. To increase peptide half-life, many strategies involving

Conclusion

We describe pharmacodynamics properties of AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG6–13), and its full (AZP531) and partially (AZP533) cyclised derivatives. Of these compounds it has been reported that they exhibit an equal pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We observed that the full cyclic peptide, AZP531, showed a better stability and bioavailability both in vitro and in vivo than the linear peptide

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