desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon: Long-acting peptide-based PEGylated and acylated glucagon receptor antagonists with potential antidiabetic activity

European Journal of Pharmacology 709 (2013) 43–51

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Endocrine pharmacology

desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon: Long-acting peptide-based PEGylated and acylated glucagon receptor antagonists with potential antidiabetic activity Nigel Irwin n, Zara J. Franklin, Finbarr P.M. O'Harte SAAD Centre for Pharmacy and Diabetes, School of Biomedical Sciences, University of Ulster, Coleraine, Northern Ireland BT52 1SA, UK

art ic l e i nf o

a b s t r a c t

Article history: Received 9 January 2013 Received in revised form 19 March 2013 Accepted 24 March 2013 Available online 3 April 2013

Glucagon is hormone secreted from the pancreatic alpha-cells that is involved in blood glucose regulation. As such, antagonism of glucagon receptor signalling represents an exciting approach for treating diabetes. To harness these beneficial metabolic effects, two novel glucagon analogues, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon, has been evaluated for potential glucagon receptor antagonistic properties. Both novel peptides were completely resistant to enzymatic breakdown and significantly (P o0.05 to P o0.001) inhibited glucagon-mediated elevations of cAMP production in glucagon receptor transfected cells. Similarly, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon effectively antagonised glucagon-induced increases of insulin secretion from BRIN BD11 cells. When administered acutely to normal, high fat fed or ob/ob mice, both analogues had no significant effects on overall blood glucose or plasma insulin levels when compared to saline treated controls. However, desHis1Glu9-glucagon-[mPEG] significantly (Po 0.05) annulled glucagon-induced increases in blood glucose and plasma insulin levels in normal mice and had similar non-significant tendencies in high fat and ob/ob mice. In addition, desHis1Glu9(Lys30PAL)-glucagon effectively (Po 0.05 to Po 0.001) antagonised glucagon-mediated elevations of blood glucose levels in high fat fed and ob/ob mice, but was less efficacious in normal mice. Further studies confirmed the significant persistent glucagon receptor antagonistic properties of both novel enzyme-resistant analogues 4 h post administration in normal mice. These studies emphasise the potential of longer-acting peptide-based glucagon receptor antagonists, and particularly acylated versions, for the treatment of diabetes. & 2013 Elsevier B.V. All rights reserved.

Keywords: Glucagon Antagonist Insulin secretion High fat feeding ob/ob mice PEGylation Fatty acid derivatisation

1. Introduction Glucagon, a 29 amino acid peptide secreted from pancreatic alpha-cells, is the major counter regulatory hormone to insulin and plays a central role maintaining adequate glucose control (Jiang and Zhang, 2003; Dunning and Gerich, 2007). Thus, despite the widely accepted view that the pathophysiology of type 2 diabetes relates predominantly to insulin resistance and betacell dysfunction, numerous studies indicate that hyperglucagonaemia and lack of glucagon suppression following feeding are equally as critical (Dunning and Gerich, 2007; Unger and Orci, 1981). In addition, inappropriately elevated glucagon levels play a key role in the development of hyperglycaemia in type 2 diabetes (Bagger et al., 2011). Therefore, inhibition of glucagon signalling represents a potential new therapeutic approach for type 2 diabetes. Consistent with

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this, glucagon receptor knockout mice and mice treated with glucagon receptor antisense oligonucleotides exhibit improved fasting glucose, glucose tolerance and pancreatic beta-cell function (Gelling et al., 2003; Liang et al., 2004; Sloop et al., 2004). In addition, studies utilising prolonged administration of glucagon receptor neutralising monoclonal antibodies or small molecular weight glucagon receptor antagonists reveal similar metabolic improvements (Winzell et al., 2007; Gu et al., 2010; Mu et al., 2011; Xiong et al., 2012). However, given concerns regarding safety, tolerability and potential for induction of immune responses with the above approaches, peptide-based glucagon receptor antagonists may offer a more favourable therapeutic option (Unson et al., 1989; Franklin et al., 2012). In this regard, there are a number of reports describing and characterising peptide-based glucagon receptor antagonists. These studies focused on specific amino acid modifications to the native glucagon. As such, His1 and Asp9 are recognised as important amino acids involved in signal transduction, whereas Arg17, Arg18 and Asp21 are crucial for receptor binding (Unson et al., 1994, 1998; Sturm et al., 1998). Deletion of His1 in combination with

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N. Irwin et al. / European Journal of Pharmacology 709 (2013) 43–51

replacement of Asp9 for Glu9 yields an effective peptide-based glucagon receptor antagonist (Unson et al., 1989). Despite the promise of this peptide, such compounds are still susceptible to rapid enzymatic degradation and efficient renal filtration (Holst, 1991). Many studies have demonstrated that PEGylation or fatty acid derivatisation of regulatory peptides results in longer-acting analogues, largely by increased enzymatic stability and reduced renal clearance through binding to plasma proteins (Knudsen et al., 2000; Lee et al., 2006; Gault et al., 2008; Kerr et al., 2009, 2010). Building on these approaches to stabalise related regulatory hormones, we have developed two novel glucagon analogues, namely desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)glucagon. Both peptides are based on the well characterised glucagon receptor antagonist, desHis1Glu9-glucagon (Unson et al., 1989), with additional C-terminal modifications to prolong circulating half-life. The present study has assessed the in vitro and in vivo actions of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon. The results provide the experimental evidence that desHis1Glu9glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon may provide an effective means of treating type 2 diabetes.

2. Materials and methods

concentrations (10−11 to 10−6 M) of desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon in the presence and absence of stimulatory glucagon (10−7 M) in HBS buffer containing 1 mM IBMX. Medium was subsequently removed, cells lysed and cAMP levels in the lysate measured using a HTS chemiluminescent immunoassay kit (Millipore, Watford, England). For assessment of insulin-release, BRIN-BD11 cells were seeded into 24 well plates (105 cells per well) and allowed to attach overnight at 37 1C. The origin and characteristics of this insulin-secreting cell line are described elsewhere (McClenaghan et al., 1996). Prior to acute tests, cells were pre-incubated (40 min; 37 1C) in Krebs Ringer Bicarbonate (KRB) buffer (pH 7.4) supplemented with 0.5% (w/v) BSA and 1.1 mM glucose. Test incubations were performed in the presence of 5.6 mM glucose with a range of concentrations (10−12 to 10−6 M) of desHis1Glu9-glucagon-[mPEG] or desHis1Glu9 (Lys30PAL)-glucagon in the presence and absence of stimulatory glucagon (10−7 M). After incubation (20 min; 37 1C) buffer was removed from each well and aliquots (200 μl) stored at −20 1C prior to measurement of insulin by radioimmunoassay (Kerr et al., 2009).

2.4. Acute in vivo effects of glucagon, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon on glycaemic and insulinotropic responses

2.1. Peptide synthesis Native glucagon, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon were obtained from GL Biochem Ltd. (Shanghai, China). All peptides were characterized using matrixassisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry as described previously (Kerr et al., 2009). The theoretical molecular masses of native glucagon, desHis1Glu9glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon are 3482.8, 3503.8 and 3725.3; respectively, and the measured molecular masses were 3480.4, 3503.6 and 3726.8; respectively. 2.2. Degradation studies Native glucagon, desHis1Glu9-glucagon, desHis1Glu9-glucagon[mPEG] and desHis1Glu9(Lys30PAL)-glucagon were incubated at 37 1C in triethanolamine-HCL (pH 7.8) with purified porcine DPP-4 (5mU; Sigma-Aldrich, UK) for 0, 2, 8 and 24 h. Reactions were terminated by the addition of trifluoroacetic acid (TFA) in water (10 μl, 10%v/v). Reaction products were applied to a Synergi C-12 column (4.6
250 mm, Phenomonex, Cheshire, UK) and intact peptide separated from the major degradation products. The column was equilibrated with 0.12% (v/v) TFA/water at 1 ml/min using 0.1% (v/v) TFA in 70% acetonitrile/water. The concentration of acetonitrile in the eluting solvent was raised from 0 to 40% over 10 min, and from 40 to 100% over 30 min. Absorption was measured at 214 nm using a SpectraSystem UV2000 detector (Thermoquest Ltd., Manchester, UK). HPLC peak area data was used to calculate percentage intact peptide remaining at the time points recorded. 2.3. Effects of glucagon, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon on in vitro cAMP production and insulin secretion For cAMP measurements, glucagon receptor transfected HEK293GnT1-cells were harvested, seeded into 96 well plates (5
104 cells per well) and grown for 16 h. The origin and characteristics of this transfected cell line are described elsewhere (Unson et al., 1989). Cells were washed twice in Hanks Buffered Saline (HBS) buffer and incubated (20 min; 37 1C) with varying

The effects of native glucagon, desHis1Glu9-glucagon-[mPEG], desHis1Glu9(Lys30PAL)-glucagon, or a combination of glucagon with either desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)glucagon, was examined in 12–14 week-old normal NIH Swiss male mice, high fat fed NIH Swiss male mice and 12–14 week-old obese diabetic (ob/ob) mice. Experimental animals had free access to drinking water and standard rodent maintenance (10% fat, 30% protein and 60% carbohydrate, Trouw Nutrition, Cheshire, UK) or a high fat (45% fat, 35% carbohydrate and 20% protein, Special Diet Services, UK) diets as appropriate. Prior to commencement of studies, high-fat fed experimental animals were maintained on high fat diet from 6 weeks of age for 140 days. Obesity and insulin resistance were clearly manifested compared to mice maintained on normal laboratory diet as judged by body weight and plasma insulin analyses. Obese–diabetic (ob/ob) mice were derived from the colony originally maintained at Aston University (Bailey et al., 1982). All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. The animals were housed individually in an air-conditioned room at 2272 1C with a 12 h light: 12 h darkness cycle. A common experimental protocol was employed for all three species of mice. Thus, groups of mice (n ¼8) received an intraperitoneal (i.p.) injection of saline vehicle alone (0.9% (w/v) NaCl) or in combination with native glucagon, desHis1Glu9-glucagon[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each 25 nmol/kg body weight). In a second series of experiments, mice (n¼ 8) received an i.p. injection of glucagon alone (25 nmol/kg b w) or in combination with either desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each at 25 nmol/kg b w). A dose of 25 nmol/kg was chosen based pilot studies revealing ineffectiveness of glucagon analogues to annul glucagon mediated metabolic effects at doses of 0.25 and 2.5 nmol/kg [data no shown]. The intraperitoneal route was chosen as the method of drug delivery as this bypasses the stomach and intestines and so does not stimulate release of the plethora of peptides and hormones involved in glucose regulation. Thus, the sole effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)glucagon treatment can be more closely examined. Normal and high fat mice were fasted 4 h, and ob/ob mice 18 h, prior to peptide administration and blood glucose and plasma insulin

N. Irwin et al. / European Journal of Pharmacology 709 (2013) 43–51

2.5. Persistence of biological actions of desHis1Glu9-glucagon[mPEG] and desHis1Glu9(Lys30PAL)-glucagon in normal mice Groups (n ¼8) of 12–14 week-old normal NIH Swiss male mice received an i.p. injection of either saline vehicle (0.9%, w/v, NaCl), desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each at 25 nmol/kg b w) 2, 4 or 8 h prior to i.p. glucagon administration (25 nmol/kg b w). Mice were fasted 4 h prior to glucagon administration and blood glucose concentrations were measured immediately prior to and post glucagon injection at time points indicated on Fig. 6. 2.6. Biochemical analysis Blood samples were taken from the cut tip of the tail vein of conscious mice at times indicated in the figures. Blood glucose was measured directly using a hand-held Ascencia Contour blood glucose meter (Bayer Healthcare, UK). For insulin analysis, blood samples were collected into chilled fluoride/heparin glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Plasma was separated immediately by centrifugation (30 s at 13,000
g) using a Beckman microcentrifuge (Beckman Instruments, Galway, Ireland) and stored at −20 1C prior to insulin analysis. Insulin was measured using a modified dextran-coated charcoal radioimmunoassay as described previously (Flatt and Bailey, 1981). 2.7. Statistics Results are expressed as means 7S.E.M. and data compared using ANOVA, followed by a Student-Newman–Keuls post hoc test. Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer-generated program employing the trapezoidal rule with baseline subtraction. Groups of data from both were considered to be significantly different if P o0.05.

3.2. Inhibition of glucagon stimulated cAMP production and insulin secretion by desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon in vitro desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon had no significant effect on cAMP production (Fig. 1) or insulin secretion (Fig. 2) from HEK293GnT1- and BRIN-BD11 cells; respectively. In contrast, glucagon (10−7 M) induced a significant (Po0.001) increase in cellular cAMP and insulin output (Figs. 1 and 2). However, desHis1Glu9-glucagon-[mPEG] (10−11 to 10−6 M) and desHis1Glu9 (Lys30PAL)-glucagon (10−12 to 10−6 M) both significantly (Po0.05 to Po0.001) inhibited glucagon-mediated (10−7 M) elevations in cAMP production (Fig. 1), with IC50 values of 1.6
10−8 and 1.7
10−10 mol/l, respectively. Similarly, desHis1Glu9-glucagon-[mPEG] (10−12–10−6 M) significantly (Po0.001) inhibited glucagon (10−7 M) evoked augmentation of insulin secretion from BRIN-BD11 cells (Fig. 2A). However, at lower concentrations (10−12 and 10−11 M) of desHis1Glu9-glucagon[mPEG] in combination with 10−7 M glucagon, insulin secretion was significantly (Po0.05) increased compared to the analogue alone (Fig. 2A). Likewise, although desHis1Glu9(Lys30PAL)-glucagon significantly (P o0.05 to P o0.001) inhibited glucagon (10−7 M) induced elevations of insulin secretion, values were still substantially (Po 0.001) raised compared to the analogue alone at all concentrations examined (Fig. 2B).

0.4

Intracellular cAMP (pmol/l)

concentrations were assessed at the times indicated on Figs. 3–5. No adverse effects were noted following injection of peptides.

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0.3

0.2

** 0.1

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-10

-9

-8

-7

-6

-7

-6

Log10 [peptide] (M)

3. Results 3.1. In vitro metabolic stability of glucagon, desHis1Glu9-glucagon[mPEG] and desHis1Glu9(Lys30PAL)-glucagon

Table 1 Percentage intact peptide remaining after incubation with DPP IV. Peptide

Native glucagon desHis1Glu9-glucagon desHis1Glu9-glucagon-[mPEG] desHis1Glu9(Lys30PAL)-glucagon

% Intact peptide remaining after time (h) 0

2

8

24

100 100 100 100

95 75 100 100 100

36 73 100 100 100

8 72 100 100 100

Values represent the % intact parent peptide remaining relative to degradation products following incubation with DPP IV as determined from HPLC peak area data. The reactions were performed in triplicate and the means 7 S.E.M. values calculated.

0.4

Intracellular cAMP (pmol/l)

Table 1 shows that native glucagon was rapidly degraded by DPP IV with only 8 72% of the peptide remaining intact after a 24 h incubation. On the other hand, desHis1Glu9-glucagon, desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon remained fully intact after incubations of up to 24 h.

0.3

0.2

0.1

0.0

-11

-10

-9

-8

Log10 [peptide] (M) Fig. 1. Effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon alone and in combination with native glucagon on in vitro cAMP production. Glucagon receptor transfected HEK293GnT1-cells were exposed to various concentrations of (A) desHis1Glu9-glucagon-[mPEG] and (B) desHis1Glu9(Lys30PAL)glucagon in the absence or presence of stimulatory glucagon (10−7 M) for 20 min (n¼ 4). cAMP production was measured using ELISA. Values represent means7S.E.M. nn Po 0.01, nnnPo 0.001, nPo 0.05 compared to glucose alone. ΔPo 0.05, ΔΔPo 0.01, ΔΔΔ Po 0.001 compared with 10−7 M glucagon.

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6

4

6

Insulin secretion

(ng/10 cells/20 min)

8

2

0

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Insulin secretion

(ng/106 cells/ 20 min)

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6

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0

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-9

-8

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-6

Log 10 [peptide] (M) Fig. 2. Effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon alone and in combination with native glucagon on in vitro insulin secretion. BRIN BD11 cells were exposed to various concentrations of (A) desHis1Glu9glucagon-[mPEG] and (B) desHis1Glu9(Lys30PAL)-glucagon in the absence or presence of stimulatory glucagon (10−7 M) for 20 min (n¼ 8). Insulin secretion was measured using RIA. Values represent means 7 S.E.M. nnPo 0.01, nnnPo 0.001 compared to glucose alone. ΔPo 0.05, ΔΔPo 0.01, ΔΔΔPo 0.001 compared with 10−7 M glucagon. ΨPo 0.05, ΨΨΨP o0.001 compared with respective glucagon analogue alone.

3.3. Acute effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon alone and on the glucose and insulin stimulating actions of glucagon in vivo Native glucagon induced significant (Po 0.01 to P o0.001) elevations in blood glucose levels at 15, 30 and 60 min post injection in normal mice (Fig. 3A and C). This was corroborated by a significantly elevated (Po 0.01) overall glycaemic excursion compared to saline controls (Fig. 3A and C). Similarly, glucagon induced significant (P o0.05) increases in individual and overall insulin secretory responses in normal mice (Fig. 3B and D). Administration of desHis1Glu9-glucagon-[mPEG] or desHis1Glu9 (Lys30PAL)-glucagon alone was not associated with alterations in plasma insulin response in these mice (Fig. 3B and D). Similarly, blood glucose levels were not significantly different from saline controls following application of desHis1Glu9(Lys30PAL)-glucagon (Fig. 3C). Conversely, desHis1Glu9-glucagon-[mPEG] induced a modest (P o0.05) increase in blood glucose levels compared to controls at 30 min post injection, however there was no change in the overall glycaemic excursion (Fig. 3A). Moreover, desHis1Glu9glucagon-[mPEG] effectively annulled glucagon-induced increases in blood glucose and plasma insulin levels in normal mice (Fig. 3A and B). In addition, desHis1Glu9(Lys30PAL)-glucagon also partially blocked glucagon-mediated elevations of blood glucose and plasma insulin concentrations (Fig. 3C and D), but the overall

glycaemic excursion was still significantly (P o0.05) increased compared to saline controls (Fig. 3C). Metabolic responses in high fat fed mice were similar. Thus, native glucagon induced significant (Po0.05 to Po0.001) elevations of individual and overall blood glucose and plasma insulin levels compared to saline treated controls (Fig. 4A–D). In addition, administration of desHis1Glu9(Lys30PAL)-glucagon had no significant effect on glucose or insulin levels in high fat fed mice (Fig. 4C and D). desHis1Glu9-glucagon-[mPEG] administration did result in elevated (Po0.05) blood glucose at 15 and 30 min post injection compared to controls, but the overall glycaemic excursion was still similar to saline control high fat fed mice (Fig. 4A). Both glucagon analogues prevented glucagon-induced elevations of blood glucose in high fat fed mice (Fig. 4A and C). In addition, desHis1Glu9-glucagon-[mPEG] effectively thwarted glucagon-mediated increases in plasma insulin secretion (Fig. 4B). Similarly, desHis1Glu9(Lys30PAL)-glucagon had a tendency to reduce glucagon induced plasma insulin secretion, but this failed to reach significance (Fig. 4D). Finally, the effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon were assessed in ob/ob mice. Native glucagon application was again associated with elevations (P o0.05 to Po 0.001) in individual and overall blood glucose and plasma insulin concentrations compared to saline treated ob/ob controls (Fig. 5A–D). When administered alone, both glucagon analogues had small effects on blood glucose and plasma insulin levels (Fig. 5A–D). The only exception was a modest (P o0.05) increase in blood glucose levels 15 and 30 min post desHis1Glu9 (Lys30PAL)-glucagon injection (Fig. 5C). However, desHis1Glu9 (Lys30PAL)-glucagon completely annulled the elevated glucose and insulin responses associated with glucagon administration in ob/ob mice (Fig. 5C and D). In addition, whilst desHis1Glu9glucagon-[mPEG] blocked glucagon-induced increases in insulin secretion in ob/ob mice (Fig. 5B), it was unable to prevent the associated elevation of blood glucose concentrations (Fig. 5A). For comparative purposes, the glucagon-inhibitory actions of the parent peptide analogue, desHis1Glu9-glucagon, were evaluated using the same experimental protocol in ob/ob mice. However, desHis1Glu9-glucagon had no effect on glucagon-mediated elevations of glucose and insulin (data not shown), and thus follow up studies with this compound were not performed. 3.4. Persistence of biological effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon in normal mice Administration of desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon 2 h prior to a glucagon challenge significantly (P o0.05 to P o0.001) decreased individual and overall blood glucose levels when compared to saline treated controls (Fig. 6A and B). In addition, both analogues were equally as effective when administered four hours prior to the glucagon challenge in normal mice (Fig. 6C and D). However, administration of desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon eight hours prior to a glucagon load had no effect on glucagon-induced elevations of blood glucose concentrations, barring a modest (P o0.05) reduction at 15 min post glucagon injection with desHis1Glu9(Lys30PAL)-glucagon (Fig. 6E). This was highlighted by similar overall glycaemic responses in both peptide treated groups when compared to saline controls (Fig. 6F).

4. Discussion The pivotal role of dysregulated glucagon secretion and action in the pathophysiology of type 2 diabetes has led to renewed interest in the inhibition of glucagon signalling as a therapeutic option for diabetes (Bagger et al., 2011; Henquin et al., 2011). These

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N. Irwin et al. / European Journal of Pharmacology 709 (2013) 43–51

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0.8 0.6 0.4 0.2 0.0 0

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Fig. 3. Effects of native glucagon, desHis1Glu9-glucagon-[mPEG] (A,B) and desHis1Glu9(Lys30PAL)-glucagon (C,D) on glycaemic and plasma insulin responses in 4 h fasted normal mice. Blood glucose and plasma insulin concentrations were measured after i.p. administration of saline vehicle (0.9% (w/v) NaCl), desHis1Glu9-glucagon-[mPEG], desHis1Glu9(Lys30PAL)-glucagon and native glucagon (each at 25 nmol/kg b w) or a combination native glucagon (25 nmol/kg b w) with either desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each at 25 nmol/kg b w). Blood glucose and plasma insulin area under the curve (AUC) values for 0–105 min post injection are shown in insets. Values represent means 7S.E.M. for 8 mice. nPo 0.05, nnP o0.01, nnnPo 0.001 compared to saline control. ΔPo 0.05, ΔΔP o0.01, ΔΔΔP o0.001 compared to glucagon alone.

recent studies have focused on genetic elimination or chemical inhibition of glucagon receptor signaling using gene knockout, antisense oligonucleotides, monoclonal antibodies directed against the glucagon receptor and low molecular weight glucagon receptor antagonists (Gelling et al., 2003; Liang et al., 2004; Kim et al., 2008; Yan et al., 2009; Gu et al., 2010 Mu et al., 2011). However, the possible exploitation of peptide-based glucagon antagonists has been severely restricted due to efficient enzymatic catabolism and rapid renal filtration of such compounds (Holst, 1991). In spite of this, improved biological efficacy of other related regulatory peptides has been achieved through chemical modification to promote enzymatic stability and prolong biological half-life (Knudsen et al., 2000; Lee et al., 2006; Gault et al., 2008). Therefore, the effects of C-terminal fatty acid derivatisation or PEGylation of desHis1Glu9-glucagon, a recognised peptide-based glucagon antagonist (Unson et al., 1989), have been examined in the current study. Initially we established that desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon were fully resistant to enzymatic degradation, in contrast to the native peptide. However, it should be recognised that the parent peptide analogue, desHis1Glu9glucagon, was also enzymatically stable indicating that amino acid substitution, rather than secondary PEGylation or acylation, imparted DPP IV resistance. In this context, it is noteworthy that DPP IV is considered to be an extremely important enzyme involved in glucagon breakdown (Hinke et al., 2000). We then

revealed that both novel analogues lacked effect on cAMP production or insulin release in HEK293GnT1- and BRIN-BD11 cells; respectively. As expected, glucagon potently stimulated cAMP production and insulin secretion in a dose-dependent manner (Hussain et al., 2000). Subsequent studies confirmed the antagonistic properties of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon on glucagon-mediated biological action, by demonstrating that these compounds significantly inhibited glucagon induced elevations of cAMP generation and insulin release. Antagonistic effects on cAMP production were more prominent than corresponding actions on insulin secretion, suggesting that alternative signalling pathways may be involved in glucagon-mediated elevations of insulin secretion from BRIN BD11 cells (Rodgers, 2012). However, these data do accord with the supposition that the desHis1Glu9-glucagon derivatives are able to bind to, but not activate, the glucagon receptor (Unson et al., 1989). The maintenance of beneficial glucagon antagonistic in vitro biological activity, together with profound resistance to enzymatic degradation, supports the view that these analogues should have impressive bioactivity in vivo. To comprehensively assess the biological effects of both analogues we employed normal mice and mice with contrasting genetically- and dietary-induced pathophysiologies of obesity–diabetes, namely high fat fed and Aston obese diabetic (ob/ob) mice. Thus, high fat feeding results in progressive obesity, insulin resistance and features of the metabolic syndrome (Irwin et al., 2008)

N. Irwin et al. / European Journal of Pharmacology 709 (2013) 43–51

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Fig. 4. Effects of native glucagon, desHis1Glu9-glucagon-[mPEG] (A,B) and desHis1Glu9(Lys30PAL)-glucagon (C and D) on glycaemic and plasma insulin responses in 4 h fasted high fat fed mice. Blood glucose and plasma insulin concentrations were measured after i.p. administration of saline vehicle (0.9% (w/v) NaCl), desHis1Glu9-glucagon-[mPEG], desHis1Glu9(Lys30PAL)-glucagon and native glucagon (each at 25 nmol/kg b w) or a combination native glucagon (25 nmol/kg bw) with either desHis1Glu9-glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each at 25 nmol/kg b w). Blood glucose and plasma insulin area under the curve (AUC) values for 0–105 min post injection are shown in insets. Values represent means 7S.E.M. for eight mice. nPo 0.05, nnnPo 0.001 compared to saline control. ΔP o0.05, ΔΔP o 0.01, ΔΔΔP o0.001 compared to glucagon alone.

whereas, Aston ob/ob mice present with moderate hyperglycaemia and severe obesity due to the defective production of leptin (Bailey et al., 1982). Both novel glucagon analogues had nominal effects on blood glucose and plasma insulin concentrations in all animal models. However, there was slight contrast in their capacity to suppress glucagon action between each model. Thus, desHis1Glu9glucagon-[mPEG] effectively annulled glucagon action in normal mice, but there was only a non-significant tendency for similar actions in high fat and genetic models of obesity–diabetes. In contrast, desHis1Glu9(Lys30PAL)-glucagon had a strong, but nonsignificant tendency to antagonise overall glucagon mediated effects on glucose and insulin, as deduced from AUC values, in normal mice. However, in models of obesity–diabetes, desHis1Glu9(Lys30PAL)-glucagon completely thwarted the biological actions of glucagon. Nonetheless and importantly, there was a strong propensity for both peptides to annul glucagon action in all three animal models employed. Thus, desHis1Glu9(Lys30PAL)-glucagon appears more effective in experimental models of obesityrelated pathologies, where the agents are likely to be employed. These small, but significant, discrepancies between the in vivo efficacy of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9 (Lys30PAL)-glucagon are interesting however, and do require further detailed study, but may be related to differences in circulating active peptide concentrations and basal glucagon levels in each animal model employed. When injected acutely along with native glucagon in ob/ob mice, the parent peptide analogue, desHis1Glu9-glucagon, had no effect on annulling glucagon-

mediated elevations of blood glucose and plasma insulin, presumably reflecting efficient renal elimination of this compound. Thus, both PEGylation and fatty acid dervatisation are known to encourage binding to circulating plasma proteins, which extends circulating half-life but does reduce concentrations of free biologically active effective peptide (Green and Flatt, 2007). To further ascertain the pharmacodynamic properties of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9(Lys30PAL)-glucagon a delayed injection regime was employed in normal mice. Both analogues elicited glucagon receptor antagonistic actions for up to 4 h after initial administration, but this was not sustained beyond 8 h. This is consistent with other studies adopting similar approaches with chemically modified related regulatory peptides (Knudsen et al., 2000; Lee et al., 2006; Gault et al., 2008). It should be noted that the intraperitoneal route of administration was chosen in the current study, to avoid interference of data interpretation by the plethora of gut peptides released after oral delivery of glucose. Thus, we were able to examine the direct effects of desHis1Glu9-glucagon-[mPEG] and desHis1Glu9 (Lys30PAL)-glucagon on glucagon-mediated biological actions. However, ultimately therapeutic efficacy will be dependent their effectiveness to maintain glycaemic control following a meal. The relative differences between in vitro and in vivo efficacy, particularly in relation to desHis1Glu9(Lys30PAL)-glucagon, is intriguing and requires further detailed investigation. Glucagon receptor knockout mice have compensatory increased circulating levels of glucagon accompanied by pancreatic alpha cell

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Fig. 5. Effects of native glucagon, desHis1Glu9-glucagon-[mPEG] (A,B) and desHis1Glu9(Lys30PAL)-glucagon (C and D) on glycaemic and plasma insulin responses in 18 h fasted ob/ob mice. Blood glucose and plasma insulin concentrations were measured after i.p. administration of saline vehicle (0.9% (w/v) NaCl), desHis1Glu9-glucagon[mPEG], desHis1Glu9(Lys30PAL)-glucagon and native glucagon (each at 25 nmol/kg b w) or a combination native glucagon (25 nmol/kg b w) with either desHis1Glu9glucagon-[mPEG] or desHis1Glu9(Lys30PAL)-glucagon (each at 25 nmol/kg b w). Blood glucose and plasma insulin area under the curve (AUC) values for 0–105 min post injection are shown in insets. Values represent means 7 S.E.M. for 8 mice. nPo 0.05, nnP o 0.01, nnnPo 0.001 compared to saline control. ΔP o0.05, ΔΔPo 0.01, ΔΔΔPo 0.001 compared to glucagon alone. ΨΨPo 0.01, ΨΨΨP o 0.001 compared with desHis1Glu9-glucagon-[mPEG] alone.

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hypertrophy, and similar results have been shown in mice treated with glucagon receptor antisense oligonucleotides (Liang et al., 2004; Sloop et al., 2004). In contrast, chronic (82 days) treatment with a low molecular weight glucagon antagonist did not lead to detectable changes in islet cell morphology (Mu et al., 2011). Thus, a critical question regarding the current peptide-based glucagon antagonists is whether these compounds will induce similar compensatory responses. This issue can only be addressed by prolonged studies employing a sub-chronic dosing regime, which falls outside the remit of the current study. However, it should be noted that sustained inhibition of glucagon signalling may have additional benefits due to increases in circulating GLP-1 levels (Liang et al., 2004; Gu et al., 2010). This observation likely relates to the fact that glucagon and GLP-1 are encoded by the same preproglucagon gene (Drucker, 2001). Indeed, it is recognised that a number of conditions are associated with such a switch of proglucagon processing in alpha-cells, due to preferential expression of PC1/3 rather than PC2 (Wideman et al., 2007; Whalley et al., 2011). In conclusion, the current study reveals that desHis1Glu9glucagon-[mPEG] and particularly desHis1Glu9(Lys30PAL)-glucagon are potent and effective, longer-acting peptide-based glucagon receptor antagonists. Interestingly, glucagon receptor gene knockout was recently shown to prevent streptozotocin-induced insulin-deficient type 1 diabetes in mice (Lee et al., 2011), possibly opening up a new therapeutic avenue for compounds that inhibit glucagon signalling. Consequently, taken together there is encouraging evidence to support further longer-term studies, especially with desHis1Glu9(Lys30PAL)-glucagon, that will progress such peptide-based glucagon receptor antagonists for diabetes therapy.

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