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ENDOCRINE AND DIABETES
Departments of Metabolic Disorders Research (A.A.O., L.F.M., L.B.D., M.R.M., T.H.C., J.N.L., C.D.M.) and Research Technologies (T.M.B., K.J.L.), Bayer Pharmaceuticals Corporation, West Haven, Connecticut
Received May 11, 2007; accepted July 31, 2007.
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Abstract |
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The naturally occurring gut hormone PYY(3-36) is a nonselective agonist of the NPY receptor family (Small and Bloom, 2005
). PYY(3-36) reduces acute food intake in lean, DIO, ob/ob, and db/db mice, and in rats, rabbits, and monkeys, and daily administration of PYY(3-36) causes body weight loss in DIO and ob/ob mice, rats, and rabbits (Batterham et al., 2002; Challis et al., 2003; Cox and Randich, 2004; Halatchev et al., 2004; Pittner et al., 2004; Abbott et al., 2005; Chelikani et al., 2005; Koegler et al., 2005; Moran et al., 2005; Neary et al., 2005; Adams et al., 2006; Sileno et al., 2006; Vrang et al., 2006). PYY(3-36) does not reduce food intake in NPY2 knockout mice, or when it is coadministered with the NPY2 antagonist BIIE0246 in wild-type rats, suggesting that the anorexigenic effect of PYY(3-36) is mediated specifically through the NPY2 receptor (Batterham et al., 2002; Abbott et al., 2005). In addition to the anorexigenic effect associated with NPY2 receptor activation, PYY(3-36) also activates the NPY1 and NPY5 receptors. The nonselective nature of PYY(3-36) is undesirable, because NPY1 and NPY5 agonists stimulate feeding in rodents (Hu et al., 1996; Kanatani et al., 2000b; Mullins et al., 2001).
The properties of PYY(3-36) in animal models of feeding and obesity make NPY2 receptor modulation an attractive mechanism for the therapeutic management of obesity. Indeed, intravenous and nasal administration of PYY(3-36) to humans reduces caloric intake (Batterham et al., 2002, 2003; Degen et al., 2005). However, the effect of PYY(3-36) is short-lived, and it may be as short as 4 h in humans (Brandt et al., 2004; http:www.nastech.com). Therefore, PYY(3-36) suffers from a short physiological duration of action and the potential for NPY1 and NPY5 receptor activation to undermine the reduction of food intake. It is desirable, then, to discover a selective, long-acting NPY2 receptor agonist of potential superior therapeutic benefit to PYY(3-36). In particular, a NPY2-selective mechanism of action will avoid potential appetite stimulation due to NPY1 and NPY5 activation as well as the possibility of other potential effects associated with the activation of the NPY1 and NPY5 receptors, and an extended duration of action will reduce dosing frequency and potentially improve patient compliance.
We have described previously an approach to attain long-acting, selective NPY2 peptide agonists using PYY as a peptide scaffold (DeCarr et al., 2007a; Lumb et al., 2007). NPY1 and NPY5 receptor affinity was abrogated at the expense of NPY2 affinity by N-terminal deletions of residues from PYY to arrive at a peptide core corresponding to residues 25 to 36 of human PYY. NPY2 affinity was then restored with N-terminal nonpeptidic modifications while maintaining selectivity against the NPY1 and NPY5 receptors. Members of the resulting series of peptides exhibit greater selectivity for the NPY2 receptor than previously described peptide agonists (DeCarr et al., 2007a). Use of N-terminal nonpeptidic modifications that incorporate a thiol for site-specific PEGylation led to the identification of a second series of NPY2 agonists that contains peptide 1 (Table 1) (Lumb et al., 2007). Once-daily subcutaneous dosing of peptide 1 over 7 days in DIO mice results in a significant body-weight reduction of 7% (Lumb et al., 2007).
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Here, we present a substantially more efficacious peptide resulting from structure-activity relationship studies of the peptide core of peptide 1. Peptide 2 reduces food intake in both lean mice and rats. The effect is mitigated in lean mice by coadministration with the selective NPY2 antagonist BIIE0246, suggesting that peptide 2 functions via activation of the NPY2 receptor. Once-daily dosing for 40 days results in a significant and sustained reduction in body weight in DIO mice. In addition, metabolic risk factors associated with glucose metabolism are improved following once-daily dosing in DIO mice. Therefore, peptide 2 constitutes a promising therapeutic for the management of obesity.
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Materials and Methods |
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) was purchased from Bachem Biosciences (King of Prussia, PA) (catalog number H-3546). BIIE0246 (Doods et al., 1999) and L-152,804 (Kanatani et al., 2000a) were purchased from Tocris Cookson Inc. (Ellisville, MO) (catalog numbers 1700 and 1382, respectively).
Peptide Synthesis. Peptides were synthesized with solid-phase, 9-fluorenylmethoxycarbonyl chemistry on Rink Amide resin with an ABI 433A synthesizer (Applied Biosystems, Foster City, CA) as described previously (Lumb et al., 2007). The N terminus was derivatized with 2-mercaptonicotinic acid (Aldrich Chemical Co., Milwaukee, WI) (catalog number 419702) in peptides 1 and 2, or with 2-methythionicotinic acid (Aldrich Chemical Co.) (catalog number 523127) to generate peptide 3. Purification was by reversed phase C18 HPLC and a linear water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Purity (>98%) was confirmed with analytical C18 and cation-exchange HPLC. Identity was confirmed with electrospray mass spectrometry, and in each case, the expected and observed masses agreed to within 1 Da. The peptide sequence was confirmed with electrospray tandem mass spectrometry.
Polyethylene Glycol-Conjugation. Peptides were cross-linked to mPEG conjugated to maleimide via the thiol group of the N-cap. Peptides were incubated with a 2-fold molar excess of 20-kDa mPEG derivatized with 
-methoxy-
-(3-[3-maleimido-1-oxopropyl]amino propyl polyoxyethylene) (catalog number ME-200MA; NOF Corporation, Tokyo, Japan) in 100 mM Tris, pH 8, for 2 h. The PEGylated peptide was purified with cation-exchange HPLC using a SP-5PW column (TosaHaas, Philadelphia, PA) equilibrated in 10 mM HCl, 20% methanol using a linear NaCl gradient. The product was dialyzed against water and lyophilized. Purity (>97%), including the absence of free peptide, was confirmed with analytical C18 and cation-exchange HPLC.
Preparation of PEG-Cys. Cys was used to block the maleimide moiety of the mPEG reagent for use as a control for in vivo studies. A 2-fold molar excess of Cys and 20-kDa mPEG (NOF ME-200MA) were incubated overnight in Tris, pH 7.4, at room temperature, and then they were dialyzed against water and lyophilized.
Peptide Concentration. Peptide amount was determined with amino acid analysis performed by the W. M. Keck Foundation Biotechnology Facility, Yale University (New Haven, CT).
Receptor Binding and Activation Assays. Assays using human receptors were performed as described previously (DeCarr et al., 2007a). NPY2 receptor [35S]GTP
S incorporation assays and 125I-PYY displacement assays were performed in a scintillation proximity assay format with membrane prepared from KAN-TS cells as a source of endogenous human NPY2 receptor. NPY1 and NPY5 receptor 125I-PYY displacement assays were performed in a filter-binding format using membranes prepared from SK-N-MC cells as a source of endogenous human NPY1 receptor or human embryonic kidney 293 cells expressing recombinant human NPY5 receptor.
Mouse and rat NPY2 receptor 125I-PYY competition-binding assays were performed in a filter-binding format with membrane pellets obtained from whole mouse brain homogenates and rat hippocampus. Endogenous NPY1 and NPY4 receptors were blocked with 1 µM (Pro30,Tyr32,Leu34)-neuropeptide Y(28-36), and the NPY5 receptor was blocked with 1 µM L-152,804 (Leban et al., 1995; Kanatani et al., 2000a). Membrane pellets were suspended in ice-cold buffer A (50 mM HEPES, pH 7.4, 10 mM CaCl2, and 5 mM MgCl2) and immediately used for determining protein content with the Bradford assay (Bio-Rad, Hercules, CA) and for binding assays. Increasing concentrations of peptide were incubated with 75 pM (rat) or 200 pM 125I-PYY (mouse) (PerkinElmer Life and Analytical Sciences, Boston, MA) and membrane (35 µg of rat and 50 µg of mouse protein) in buffer A containing 0.1% bovine serum albumin at a final volume of 200 µl for 2 h at room temperature. Nonspecific binding was defined with 1 µM PYY. The binding reactions were terminated by filtration through pretreated (0.05% bovine serum albumin in buffer A) Millipore HV filter plates (Millipore Corporation, Billerica, MA) using a vacuum manifold. Filters were washed three times with 200 µl of ice-cold buffer A, and filters dried overnight. MicroScint-O (20–30 µl; PerkinElmer Life and Analytical Sciences) was added to each well, and radioactivity bound to the filters was measured using a Wallac 1450 MicroBeta Trilux liquid scintillation counter (PerkinElmer Wallac, Gaithersburg, MD). Data were fit to a single-site binding model with Prism 3.0.3 (GraphPad Software Inc., San Diego, CA).
Fasted-Refed Feeding Studies in C57BL/6 Lean Mice. Lean C57BL/6 male mice (Taconic Farms, Germantown, NY) were acclimated for a minimum of a week with controlled temperature and humidity on a 12-h light/dark cycle. Mice were housed in pairs in cages with a grid floor with water and food (standard chow pellet diet) continuously available. A study included 20 mice per treatment group with an average body weight of 22 g. The mice were fasted overnight (18 h) with water available during the dark phase, and they were dosed subcutaneously with peptide in USP saline. Control groups were dosed with USP saline or PEG-Cys in USP saline. Preweighed food was provided 30 min after dosing.
Fasted-Refed Feeding Studies in Wistar Rats. Lean Wistar male rats (Harlan, Indianapolis, IN) were acclimated for a minimum of a week with controlled temperature and humidity on a 12-h light/dark cycle. Rats were housed singly in cages with a grid floor with water and food (standard chow powder diet) continuously available. A study included 10 rats per treatment group with an average body weight of 264 g (SD 14 g). The rats were fasted overnight (18 h) with water available during the dark phase, and they were dosed subcutaneously with peptide in USP saline. Control groups were dosed with USP saline or PEG-Cys in USP saline. Preweighed food was provided 6 h after dosing (reflecting the longer plasma Tmax of peptide 2 in the rat compared with the mouse).
Body-Weight Studies in DIO Mice. Male C57BL/6 mice (Taconic Farms) were fed a high-fat diet containing 45% calories from fat (rodent diet D12451 [GenBank] ; Research Diets, Inc., New Brunswick, NJ) for 20 to 22 weeks, and they had an average body weight over 5 SDs greater than mice fed a standard chow pellet diet (5% calories from fat). A study included 10 mice per treatment group. The mice used for the 14-day dose-response study had an average initial body weight of 46.9 g (SD 0.9 g), and the mice used for the 40-day study had an average initial body weight of 48.5 g (SD 0.6 g). Mice were kept in standard animal rooms under controlled temperature and humidity and a 12/12-h light dark cycle. Water and food were continuously available. Animals were adapted to the grid floors for 4 days, and they were dosed with vehicle (either USP saline or PEG-Cys in USP saline) for a further 4 days before recording of 2 days of baseline body weight and 24-h food and water consumption. Mice were assigned to treatment groups based upon their body weight such that the initial mean and standard error of the mean of body weight were similar. Animals were administered peptide in USP saline subcutaneously every day before the dark phase, and body weight and food and water consumption were measured.
Glucose Metabolism Parameters. Male DIO mice were dosed for 11 days with 0.94 µmol/kg peptide 2 or PEG-Cys as described above. The mice had an average initial body weight of 45.4 g (SD 0.2 g). Blood samples were collected from the tail of fed mice and analyzed with an Ascensia glucose meter (Bayer Healthcare, Tarrytown, NY) on day 10 of treatment. Food was removed 5 h into the feeding phase, and the animals were fasted for 14 h before blood collection using retro-orbital bleeding for insulin analysis. The IPGTT was performed 2 h after retro-orbital bleeding following intraperitoneal administration of 2 g/kg glucose, and tail blood glucose was measured at 30-min intervals for 3 h. Insulin was measured by enzyme-linked immunosorbent assay (Alpco Diagnostics, Windham, NH).
Statistics. The significance of differences was evaluated by analysis of variance followed by Fisher's protected least significant difference test post hoc analysis (StatView; SAS Institute, Cary, NC).
Regulatory Approvals. All procedures involving animals were reviewed and approved by the Bayer Animal Use and Care Committee, and all experiments were performed in accordance with relevant guidelines and regulations.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Use of a longer peptide core corresponding to residues 13 to 36 of human PYY to generate peptide 2 increases the in vitro potency 4-fold compared with peptide 1, with Ki and EC50 values of 9.1 ± 2.2 and 6.5 ± 1.6 nM, respectively (Fig. 1, A and B). Peptide 2 maintains selectivity against the human NPY1 and NPY5 receptors (Ki = 760 ± 70 and 630 ± 30 nM, respectively; Table 1). Peptide 2 binds rodent NPY2 receptors with similar affinity to the human receptor, with Ki values of 21 ± 2 and 31 ± 6 nM for the mouse (whole brain) and rat (hippocampus) NPY2 receptors, respectively (Table 1). Therefore, increasing the length of the peptide core improves in vitro receptor affinity while maintaining a high degree of specificity for the NPY2 receptor.
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; Lumb et al., 2007).
PYY(3-36) administered to lean mice at 0.94 µmol/kg elicits a significant reduction in cumulative food intake of 42% at 4 h (p < 0.05; Fig. 2). However, the anorexigenic effect of PYY(3-36) is not sustained, and cumulative food intake reduction after 24 h is only 9% (p > 0.05; Fig. 2). The results are in accordance with reports that PYY(3-36) reduces food intake in mice at doses over 25 nmol/kg (Batterham et al., 2002; Challis et al., 2003; Halatchev et al., 2004; Pittner et al., 2004; Neary et al., 2005; Adams et al., 2006; Vrang et al., 2006; DeCarr et al., 2007b; Lumb et al., 2007).
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Peptide 2 administered to lean mice at 0.94 µmol/kg exhibits a slower onset of food intake reduction and induces a substantial reduction in food intake of 60 ± 2% at 24 h (Fig. 2). In contrast to PYY(3-36), the reduction in food intake caused by peptide 2 is sustained and significant at 48 h (29 ± 2%) and 72 h (15 ± 1%). The reduction in food intake induced by peptide 2 is dependent on dose, and the lowest dose tested (0.0094 µmol/kg) is not efficacious (Fig. 2). Therefore, peptide 2 imparts superior in vivo anorexigenic efficacy with a longer duration of action than PYY(3-36) at a lower dose.
PEGylation Enhances In Vivo Activity. The contribution of PEGylation to in vivo efficacy and duration of action was evaluated using a non-PEGylated analog of peptide 2 in which the thiol group of the N-cap is modified with a methyl group (peptide 3) instead of PEG. In lean C57BL/6 mice, peptide 3 induces a significant reduction of food intake at 1 h at the highest tested dose of 3.1 µmol/kg that is mitigated at 4 h and does not persist to 24 h (Fig. 3). This low efficacy is in contrast to the potent PEGylated analog 2 (Fig. 3), which differs only in the modification with mPEG, indicating that PEGylation is required to impart an extended in vivo duration of action.
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). In the fasted-refed lean mouse model, subcutaneous administration of BIIE0246 at 11 µmol/kg (10 mg/kg) causes a statistically significant increase in food intake of 10% at 4 h (Fig. 4), as expected for a NPY2 antagonist. When the mice are pretreated with BIIE0246 for 15 min before peptide administration, the acute anorexigenic effects of both PYY(3-36) at 0.74 µmol/kg and peptide 2 at 0.94 µmol/kg are abrogated (Fig. 4). This result suggests that the mechanism of action of peptide 2 is mediated via NPY2 receptor stimulation.
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Acute Anorexigenic Activity in Lean Rats. The effects of peptide 2 and PYY(3-36) on feeding in the rat were evaluated with fasted-refed lean Wistar rats. Peptide 2 induces a dose-dependent reduction in food intake in the rat that persists to at least 48 h (Fig. 5). The reduction in food intake is lower in the rat than seen in the lean C57BL/6 mouse, with a 0.74-µmol/kg dose in the rat eliciting a 23 ± 4% reduction in food intake at 24 h compared with 40 ± 3% at 0.31 µmol/kg in the mouse (cf. Figs. 2 and 5). PYY(3-36) dosed at 2.74 µmol/kg also elicits a statistically significant reduction in food intake in the rat of 13 ± 4% at 4 h, although as in the mouse the anorexigenic effect of PYY(3-36) is short-lived and it does not persistent to 24 h (Fig. 5).
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Dose-Dependent Reduction on Body Weight in DIO Mice. The effects of once-daily administration of peptide 2 on body weight were evaluated in the DIO mouse (Fig. 6). Peptide 2 elicits a dose-dependent reduction in body weight, with a statistically significant loss of approximately 9% body weight over 14 days following dosing at 0.94 µmol/kg. In contrast, PYY(3-36) at 0.74 µmol/kg causes a modest, statistically significant 2.6% reduction in body weight over the same period. Efficacy is not observed for peptide 2 at the lowest dose shown (0.0094 µmol/kg).
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).
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In the second part of the study, animals were treated once daily with peptide 2 for 15 days, PEG-Cys for 13 days (i.e., peptide 2 was not administered), and finally, with peptide 2 for 12 days. During the first phase when animals were treated with peptide 2, essentially identical results were seen for the animals of the first part of the study in terms of body-weight loss and reduction of food and water intake (Fig. 7D). Upon withdrawal of peptide 2, the animals resumed untreated levels of feeding with a rebound or initial increase in food intake upon drug withdrawal (Fig. 7E). When peptide 2 was reintroduced, the effects on food and water intake and on body weight and feeding were essentially identical to those seen when the peptide was first administered (Fig. 7, D–F).
Adiponectin. An increase in adiponectin is a biomarker for decreased body fat. After 40 days of daily dosing, plasma adiponectin levels are increased significantly by 18% in the treated animals (20 ± 1 µg/ml) relative to the PEG-Cys control group (17 ± 1 µg/ml).
Glucose Metabolism. The effects on glucose metabolism were evaluated in DIO mice following 11 days of once-daily dosing at 0.94 µmol/kg. The animals experienced a 9% reduction in body weight (data not shown), consistent with the other two independent body-weight studies in DIO mice (Figs. 6 and 7). In the IPGTT, fasted mice treated with peptide 2 have enhanced glucose disposal compared with untreated mice (Fig. 8A), which translates to a 21% reduction in the glucose area under the curve over the time course of the measurements (Fig. 8B). In addition, fasting plasma insulin levels increased by 18% (Fig. 8C), and nonfasting plasma glucose levels decreased by 35% (Fig. 8D).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). The peptide functions via activation of the NPY2 receptor to reduce appetite in rodents (Batterham et al., 2002; Abbott et al., 2005), and food intake is reduced in humans following intravenous administration of PYY(3-36) (Batterham et al., 2002, 2003; Degen et al., 2005). PYY(3-36) is also an agonist of the NPY1 and NPY5 receptors that induces the undesired effect of feeding stimulation in rodents (Hu et al., 1996; Kanatani et al., 2000a; Mullins et al., 2001). Moreover, the duration of action of PYY(3-36) seems to be short in humans (Brandt et al., 2004). Therefore, a selective, long-acting NPY2 agonist is a desirable alternative to PYY(3-36) for obesity management.
Peptide 1 exemplifies a strategy to attain a series of selective NPY2 receptor peptide agonists (Table 1) (DeCarr et al., 2007a; Lumb et al., 2007). The structural features of 1 include 1) the N-terminal modifying group, which improves in vitro NPY2 receptor affinity; 2) the peptide core corresponding to residues 25 to 36 of human PYY, which provides the scaffold for NPY2 receptor selectivity; and 3) modification with 20-kDa mPEG to improve in vivo efficacy. The resulting peptide 1 induces a 7% reduction in body weight in DIO mice following subcutaneous daily dosing at 2.9 µmol/kg (Lumb et al., 2007).
To improve the in vivo efficacy of peptide 1, the length of the peptide core was increased to create peptide 2, because a longer peptide core exhibits greater in vitro potency at the NPY2 receptor (DeCarr et al., 2007a) and in vitro potency of a series of peptides related to peptide 1 correlates with in vivo reduction in food intake in mice (Lumb et al., 2007). Peptide 2 contains a peptide core corresponding to residues 13 to 36 of human PYY, the same N-terminal modification 2-mercaptonicotinic acid as peptide 1, and it exhibits an approximately 4-fold increase of in vitro potency over peptide 1.
Peptide 2 caused a significant 9% reduction in body weight in DIO mice in three independent studies, and the body-weight loss is sustained after 40 days of dosing. The loss in weight is recovered after a break in dosing in DIO mice, suggesting that tolerance is not developed to the weight-loss effect. Compared with peptide 1, peptide 2 elicits a higher level of body-weight loss (9% at 0.94 µmol/kg) than peptide 1 (7% at 2.9 µmol/kg; Lumb et al., 2007) at a 3-fold lower dose. Peptide 2 also induces a substantially greater level of body-weight reduction at a lower dose than the clinical candidate PYY(3-36) that acts via the same putative mechanism of NPY2 receptor activation.
Peptide 2 causes a substantial reduction in food intake that persists for at least 72 h in mice and at least 48 h in rats. The extended duration of action is in marked contrast to the short-lived effect of PYY(3-36), which is only 4 h in the mouse and rat. The extended duration of action is attributed to PEGylation, because the non-PEGylated analog peptide 3 exhibits efficacy in reducing food intake in the mouse only for a period of 2 to 4 h. Peptide 2 exhibits a longer half-life, longer Tmax, and higher Cmax in rodent plasma than PYY(3-36) (data not shown), suggesting that the improved pharmacodynamic profile of peptide 2 compared with PYY(3-36) is in part related to an extended in vivo lifetime and exposure.
Peptide 2 is envisaged to function through selective NPY2 receptor activation. Indeed, the anorexigenic effects of peptide 2 and PYY(3-36) in mice are blocked by the selective NPY2 antagonist BIIE0246, providing in vivo evidence that the mechanism of action of peptide 2 is via specific NPY2 receptor action.
Peptide 2 induces favorable improvements in metabolic parameters of DIO mice associated with risk for type II diabetes. Glucose disposal, plasma glucose, and insulin levels are all improved to levels expected upon treatment with marketed antidiabetic agents. This probably reflects the expected improvement in metabolic health following weight loss, although PYY(3-36) has been implicated in improving glucose disposal independently of food intake or body weight in C57BL/6 mice on a high-fat diet (van den Hoek et al., 2004), and it may contribute to glucose homeostasis (Boey et al., 2007).
The importance of PYY(3-36) as an antiobesity agent is controversial. A conglomerate of research groups have concluded that PYY(3-36) is not efficacious in the rat (Tschop et al., 2004; Boggiano et al., 2005). However, these investigators used much lower doses of PYY(3-36) (
0.1 µmol/kg) than used here (0.74–2.74 µmol/kg) or in other studies in rats in which efficacy was observed. Other factors such as stress may also influence the response to PYY(3-36) (Halatchev et al., 2004; Small and Bloom, 2005), and at least in the studies reported here, animals were acclimated to handling and subcutaneous dosing with either saline or PEG-Cys before dosing. Nonetheless, the simplest explanation for the apparent lack of consistent PYY(3-36) efficacy is the low doses used in the studies that generated negative results. However, human PYY(3-36) is particularly ineffective in the rat. Intravenous administration of PYY(3-36) to humans at 74 pmol/kg resulted in a 16% reduction in calorie intake over 24 h (Batterham et al., 2003), which is a large apparent efficacious dose difference of about 10,000-fold compared with the rat.
Peptide 2 induces a transient reduction in food intake that is accompanied by weight loss in the first 3 days of dosing. After 3 days, food intake returns to untreated levels, and yet the weight loss is maintained. PYY(3-36) also causes a transient reduction in food intake over the first 3 to 7 days that returns to untreated levels while maintaining weight loss in three different 7 to 56 day studies (Pittner et al., 2004; Adams et al., 2006; Vrang et al., 2006). Therefore, it seems possible that NPY2 agonists work initially via an anorexigenic effect that reduces food intake and leads to weight loss. The transient reduction in feeding induced by peptide 2 and PYY(3-36) may reflect NPY2 receptor desensitization, although arguing against this is the observation that the desired physiological effect (weight loss) is maintained. The maintenance of weight loss after the return to normal feeding levels also raises the possibility of an additional role for NPY2 beyond feeding cessation (Adams et al., 2006). In this regard, it is noteworthy that PYY(3-36) administration decreases the respiratory quotient in DIO mice, suggesting a potential role for NPY2 receptors in fat utilization (Adams et al., 2006), and PYY(3-36) may moderate food intake by action in areas other than the arcuate nucleus (Pittner et al., 2004). Other anorexigenic agents such as the small-molecule type 1 cannabinoid receptor antagonist rimonabant (SR141716) and a NPY5 receptor antagonist induce a transient reduction in food intake but a sustained reduction in body weight in rodents (Ravinet Trillou et al., 2003; Mashiko et al., 2007), indicating that the pattern of feeding behavior and weight loss seen is not a specific property of NPY2 agonists.
In conclusion, we have generated a selective NPY2 peptide agonist that outperforms the clinical candidate PYY(3-36) in reducing food intake in both lean mice and leans rats following a single dose, and in reducing body weight in DIO mice following once-a-day subcutaneous dosing. The peptide is long acting, with significant reductions in food intake observed at 48 h in both mice and rats following a single dose. Significant improvements in metabolic parameters representing risk factors for type II diabetes accompany the weight loss in DIO mice. The results provide preclinical evidence in two animal species that peptide 2 has potential for the management of obesity via selective activation of the NPY2 receptor.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: NPY, neuropeptide Y; PYY, peptide YY; DIO, diet-induced obese; PEGylated, polyethylene glycol-conjugated; PEGylation, polyethylene glycol-conjugation; HPLC, high-performance liquid chromatography; USP, United States Pharmacopeia; mPEG, monomethoxypolyethylene glycol; PEG, polyethylene glycol; GTPS, guanosine 5'-O-(3-thio)triphosphate; IPGTT, intraperitoneal glucose tolerance test; L-152,804, 5,5-dimethyl-2-(2,3,4,9-tetrahydro-3,3-dimethyl-1-oxo-1H-xanthen-9-yl)-1,3-cyclohexanedione; SR141716, 5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-N-(piperidin-1-yl)-1H-pyrazole-3-carboxamide; BIIE0246, N-[(1S)-4-[(aminoiminomethyl)amino]-1-[[[2-(3,5-dioxo-1,2-diphenyl-1,2,4-triazolidin-4-yl)ethyl]amino]carbonyl]butyl]-1-[2-[4-(6,11-dihydro-6-oxo-5H-dibenz[b,e]azepin-11-yl)-1-piperazinyl]-2-oxoethyl]-cyclopentaneacetamide formate.
Address correspondence to: Dr. Kevin J. Lumb, Merck Research Laboratories, West Point, PA 19486. E-mail: kevin_lumb{at}merck.com
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, and 
, p < 0.05 compared with columns 1, 3, and 5, respectively.
