Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2

Article pubs.acs.org/jmc

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2‑selective PEGylated Peptide Yoko Kanematsu-Yamaki, Naoki Nishizawa, Tomoko Kaisho, Hiroaki Nagai, Taisuke Mochida, Tomoko Asakawa, Hiroshi Inooka, Katsuko Dote, Hisashi Fujita, Kouta Matsumiya, Hideki Hirabayashi, Junichi Sakamoto, Tetsuya Ohtaki, Shiro Takekawa, and Taiji Asami* Pharmaceutical Research Division, Takeda Pharmaceutical Company, Ltd., Fujisawa, 251-8555, Japan S Supporting Information *

ABSTRACT: Neuromedin U (NMU) is a neuropeptide that mediates a variety of physiological functions via its receptors, NMUR1 and NMUR2. Recently, there has been an increased focus on NMU as a promising treatment option for diabetes and obesity. A short form of NMU (NMU-8) has potent agonist activity for both receptors but is metabolically unstable. Therefore, we designed and synthesized NMU-8 analogues modified by polyethylene glycol (PEG; molecular weight, 20 kDa; PEG20k) via a linker. 3-(2-Naphthyl)alanine substitution at position 19 increased NMUR2 selectivity of NMU-8 analogues with retention of high agonist activity. Compound 37, an NMUR2-selective PEG20k analogue containing piperazin-1-ylacetyl linker, exhibited a potent body weight-lowering effect with concomitant inhibition of food intake in a dose-dependent manner (body weight loss of 12.4% at 30 nmol/kg) by once-daily repeated dosing for 2 weeks in mice with diet-induced obesity.

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INTRODUCTION According to a 2014 WHO report, more than 1.9 billion adults aged 18 years and above were categorized as overweight, including 0.6 billion in obese adult category.1 Overall, the percentage of obese adults was about 13% (11% men and 15% women) of the world’s adult population in 2014. The population of overweight and obese has increased more than 2-fold at present compared to that in 1980. Obesity is a wellknown risk factor for metabolic diseases, including type 2 diabetes, dyslipidemia, and hypertension. It is one of the biggest health issues in the world currently. For more than 20 years, body weight control by diet and exercise has been regarded as the first-line therapy for the treatment of obesity and obesityrelated metabolic disorders. However, diet control and exercise therapy have failed to provide satisfactory results; therefore, development of alternative effective therapies is highly desired. Recently, increasing attention has been paid to anorectic peptides because of their antiobesity effects. Neuromedin U (NMU) is a neuropeptide, which was isolated and characterized in 1985 from the porcine spinal cord for inducing uterus muscle contraction.2 NMU neuropeptides are C-terminal amidated peptides composed of 25 amino acid residues in humans and swines, and 23 amino acid residues in rodents.3 NMU-8, an N-terminally truncated © 2017 American Chemical Society

peptide comprising eight amino acids, which is produced by cleavage at a pair of basic Arg−Arg residues, was identified in swines and dogs.4 In 2005, neuromedin S (NMS), a member of the NMU family of peptides comprising 36 amino acid residues, was isolated from the rat brain.5 The main features of NMU family peptides include a C-terminal amide structure with a highly conserved seven-amino-acid sequence in mammals, which plays a critical role in physiological functions.6 To date, two subtypes of NMU receptors have been identified: NMUR1 (GPR66/FM3),7−11 which is expressed in peripheral tissues such as the gastrointestinal tract (GIT), and NMUR2 (TGR1/FM4),10,12−14 which is expressed in the central nervous system (CNS), including the hypothalamus and spinal cord. NMU, which is widely distributed in the CNS and GIT, plays a role in a variety of physiological functions, such as stress response, reproduction, and cardiac function.3 NMU knockout mice were reported to have hyperphagia and obesity. Similarly, intracerebroventricularly administered NMU was reported to exert an anorectic effect; the effects of NMU on feeding behavior and energy consumption have attracted attention of researchers around the world.15,16 The NMU Received: March 2, 2017 Published: June 28, 2017 6089

DOI: 10.1021/acs.jmedchem.7b00330 J. Med. Chem. 2017, 60, 6089−6097

Journal of Medicinal Chemistry

Article

gene mutation was reported to cause obesity in humans;17 however, the effect of direct administration of NMU has not been investigated in clinical trials. C-terminal NMU analogues, NMU-8 and NMU-9, which contain eight and nine amino acid residues, respectively, are the smallest peptides that act as potent agonists.3 The sequences of NMU-8 and NMU-9 are highly conserved across species. The C-terminal seven−amino-acid-peptide is completely conserved in mammals. Over time, structure−activity relationship (SAR) studies on the heptapeptide have led to the discovery of potent, subtype−selective, and biologically stable NMUR agonists in vitro.18−20 Peripheral administration of rat NMU-23 was found to exert anorectic activity in mice,21 although the shorter peptide NMU8 did not reduce food intake.21 Interestingly, the native NMU has a short half-life of less than 5 min after subcutaneous injection.22 NMU-25 was also reported to show poor pharmacokinetic properties in mouse plasma with a calculated half-life of 8 min.23 The lack of the anorectic activity of NMU-8 was considered to be caused by its shorter half-life than rat NMU-23. These data led us to develop long-acting NMU-8 analogues and investigate their potential as antiobesity drugs. It is known that modification with a high-molecular-weight polyethylene glycol (PEG) moiety (PEGylation) is an effective strategy for improving the pharmacokinetic properties of peptides and proteins.24−26 Several PEGylated proteins and peptides, including NMU analogues, have been developed for clinical use.27 For example, an N-terminally PEGylated analogue of NMU-25 induced body weight loss and reduced blood glucose levels in diet-induced obese (DIO) mice.28−30 However, high molecular weight PEG moiety interferes with the interaction between ligand and receptor. In fact, NMUR1 and NMUR2 agonist activities of several PEGylated peptides consisting of seven or nine amino acid were drastically decreased.28−30 Immunoglobulin Fc fragment and human serum albumin (HSA) fusions are used to improve biodistribution and pharmacokinetics of peptides and proteins.31−33 For instance, HSA-conjugated NMU-25 showed prolonged and potent anorectic activity in DIO mice.34 Furthermore, modification with long-chain alkyl moiety, which interacts with plasma proteins, has been used to improve pharmacokinetic properties of peptides.35 NMU-25 analogues containing long-chain alkyl moiety were reported to have increased plasma stability and anorectic activity after acute subcutaneous dosing in lean mice.23 We tried to improve the pharmacokinetic profile of NMU-8 by introducing the PEG moiety and discovered that a PEGylated peptide (12), which was composed of 20 kDa PEG, piperazin-1-ylacetyl (PipAc) linker, and NMU-8, served as NMUR1 and NMUR2 dual agonist.36 Daily subcutaneous administration of 12 for 2 weeks exhibited a marked antiobesity effect in DIO mice. As described above, NMUR1 is highly expressed in gastrointestinal tissues and plays a role in gastrointestinal motility;37 therefore, NMUR1 activation may cause some adverse effects, such as diarrhea, whereas, central NMU activation via NMUR2 plays a role in body weight and energy balance regulation.38 Thus, selective NMUR2 agonists are expected to have superior profiles as antiobesity therapeutics compared to NMUR1 and NMUR2 dual agonist. In this study, NMUR2 selective agonists with potent body weight-lowering effect were designed and synthesized by modification with PEG moiety. First, NMUR2 selective non-PEGylated peptides were

obtained, and a suitable PEGylation site was identified. Next, NMUR2 agonist activity of PEGylated peptides was improved by conjugating them with amino acids and linker structures, followed by improvement of anorectic activity to obtain NMUR2 selective agonists with a potent body weight-lowering effect.

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RESULTS AND DISCUSSION Chemistry. All peptides were synthesized by using a standard 9-fluorenylmethoxycarbonyl (Fmoc)-based solid phase synthesis method. The obtained crude peptides were purified using preparative high-performance liquid chromatography (HPLC) to obtain a homogeneous product. PEGylation was performed by condensation of a carboxyl moiety of PEGylation reagent and amino moiety of peptide, or by reductive amination using aldehyde-type PEGylation reagent and sodium cyanoborohydride. The purity of each peptide analogue was verified by analytical reversed-phase (RP) HPLC, and the structures were confirmed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS). Biological Activities. All synthesized peptides were evaluated for their agonist activities by fluorometric imaging plate reader (FLIPR) assay, which has been previously used to determine the ligand-induced elevation of Ca2+ in Chinese hamster ovary (CHO) cells expressing human NMUR1 or NMUR2.7,12 The highest response was detected in the presence of peptide. The response is expressed as percentage, which was corrected to the highest concentration of the control peptide, NMU-8. Nonlinear regression of smoothed data was used to determine the potency (EC50) of peptides. Results of the in vitro screening are shown in Tables 1−5. PEGylated peptides with good agonist activities were further evaluated for their 16 h food intake inhibitory activities after a single administration at 100 or 600 nmol/kg in 9−10-week-old C57BL/6J mice (Table 5). Mono-amino Acid Substitution of NMU-8 Analogues. Comprehensive SAR of NMU-8 was investigated using a peptide with β-alanine (β-Ala) linker at the N-terminus of NMU-8. Table 1 shows β-Ala-NMU-8 analogues (3−11) that displayed improved NMUR2 agonist activity or selectivity. Among these, amino acid substitutions of Phe18 (3) and Val20 (7) were found to play a role in increasing the NMUR2 agonist activity in humans and mice. While peptides with these amino acids showed inadequate NMUR2 selectivity, a combination of amino acid substitutions was considered to increase it. Amino acid replacement at position 19, 21, or 22 markedly improved NMUR2 selectivity. Particularly, peptides with Glu19 (4) and 3(2-naphthyl)alanine19 [Nal(2)19] (5) showed high NMUR2 selectivity, indicating the utility of amino acid substitution at position 19 for the development of NMUR2 selective agonists. The linker structure also affected the agonist activity. NMUR2 agonist activity of PipAc analogue (2) was better than that of βAla analogue (1). Mono Amino Acid Substitution of PEGylated NMU-8 Analogues. PEGylated NMU-8 analogues with amino acid substitutions were synthesized for improving NMUR2 agonist activity and selectivity (Table 2). PEG moiety of 20 kDa was selected, because its effect on the antiobesity effect of NMU-8 analogues was previously reported in DIO mice.36 The introduction of PEG moiety in the peptide with Val20 (16), which exhibited moderate NMUR2 selectivity in the corresponding peptide 7, showed species-selective activity; good 6090


NMUR2

β-Ala PipAc β-Ala β-Ala β-Ala β-Ala β-Ala β-Ala β-Ala β-Ala β-Ala

agonist activity was only observed in mice. In contrast, peptides with Nal(2)19 (14), Cha19 (15), and Gln22 (17) substitutions maintained NMUR2 selectivity even after PEGylation of the corresponding peptides 5, 6, and 10, respectively. Interestingly, NMUR2 agonist activity of peptides with amino acid replacement at position 19 was affected relatively to a lesser extent by PEGylation, which suggests that position 19 is a suitable site to regulate receptor selectivity without steric hindrance by PEGylation. Nal(2)-substituted PEGylated peptide (14) possessed higher NMUR2 selectivity than that of non-PEGylated peptide (5). Compound 14 showed potent food intake inhibitory activity (−76%, for 16 h) at a subcutaneous dose of 600 nmol/kg in lean mice (Table 5). Preferable PEGylation Site of NMU-8 Analogues. The suitable PEGylation site for NMU-8 analogues was explored based on the SAR of analogues of Ac-[Nal(2)19]NMU-8 (Table 3). A Lys residue was introduced at positions 18 and 19 of 5, and the obtained peptides (18, 20) were modified with PEG at the ε-amino group of the side chain of Lys residue (19, 21). Furthermore, an analogue with Lys17 (22) at the N-terminus was synthesized, followed by PEGylation (23). The Lyssubstituted analogues maintained moderate NMUR2 agonist activity; however, agonist activity of PEGylated peptides was drastically decreased. Only 23 that had a PEG moiety at the side chain of Lys residue at position 17 showed high NMUR2 agonist activity and selectivity. Therefore, we used PEGylation at the N-terminus region of NMU-8 in the further synthesis. Combination of Amino Acid Substitutions in NMU-8 Analogues. A combination of amino acid substitutions was investigated for improving NMUR2 agonist activity and selectivity, and to obtain PEGylated peptides with potent anorectic activity (Table 4). NMU-8 analogues with amino acid replacement at multiple positions, which were expected to provide NMUR2 selectivity, were synthesized. These included N-methylalanine23 (MeAla23)-containing analogues, in which Pro23 was substituted with an Nα-substituted amino acid MeAla, in addition to Glu19-, Nal(2)19-, or Cha19-containing analogues. Compounds 24 and 25, having the combination of PipAc moiety and amino acid substitution at position 19 [Glu19, Nal(2)19], showed increased NMUR2 agonist activity and selectivity in mice compared to the corresponding β-Ala analogues (4, 5). Val20 substitution, which had a little effect on NMUR2 agonist activity (7), did not increase the activities of Glu19 and Cha19 analogues (26, 27). NMUR2 selective substitutions, 3-(4-pyridyl)alanine21 [Pya(4)21] and Gln22, increased NMUR2 selectivity of Cha19 and Nal(2)19 analogues (29−31) for human and mouse receptors compared with 6 (Cha19) and 5 [Nal(2)19], although their agonist activity was slightly decreased. Among these peptides, [Nal(2)19,Gln22] analogue (31) was >1000- and 100-times more selective for NMUR2 than NMUR1 in human and mouse, respectively. It indicated that the combination of amino acid substitutions at positions 19 and 22 play a synergistic role in NMUR2 selectivity. Combination of PipAc, Cha19, and Nle21 (28) showed high agonist activity in mouse, with EC50 in 10−11 M range, but relatively low selectivity in human. MeAla 23 analogues (32, 33) of 24 and 25 with good NMUR2 selectivity were synthesized to further improve agonist activity. Compounds 32 and 33 showed increased agonist activity with moderate NMUR2 selectivity. These peptides were expected to show potent in vivo activity after PEGylation. The introduction of linkers including 6-aminocaproic acid (Acp) (34) and O-(3aminopropyl)-O′-(N-diglycolyl-3-aminopropyl)diethylene gly-

EC50 values of agonist activities were determined as concentrations of peptide analogues that gave half-maximum [Ca2+] mobilizing activities (n = 2). The EC50 values of NMU-8 were averages of 15 tests (for human NMUR1/2) and 13 tests (for mouse NMUR1/2).

0.54 0.96 (0.631.4) 0.32 (0.250.40) 0.41 (0.350.49) >1000 7.8 (5.212) 1.3 (1.11.7) 0.31 (0.260.37) 1.0 (0.751.3) 0.73 (0.501.1) 1.9 (1.52.3) 0.77 (0.561.1)

NMUR1 AA23

Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Ala Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Gln Arg

AA22 AA21

Phe Phe Phe Phe Phe Phe Phe Phe Nle Pya(4) Phe Phe Leu Leu Leu Leu Leu Leu Leu Val Leu Leu Leu Leu

AA20 AA19

Phe Phe Phe Phe Glu Nal(2) Cha Phe Phe Phe Phe Phe Tyr Tyr Tyr Phe Tyr Tyr Tyr Tyr Tyr Tyr Tyr Tyr NMU-8 1 2 3 4 5 6 7 8 9 10 11

AA18 linker compound

Article

a

0.59 0.27 (0.240.31) 0.21 (0.160.26) 0.16 (0.0890.28) >1000 8.5 (5.912) 3.7 (2.55.5) 0.57 (0.410.80) 3.4 (2.84.1) 3.0 (2.34.0) 13 (1017) 0.69 (0.481.0)

NMUR1 NMUR2

0.40 0.69 (0.441.1) 0.22 (0.140.33) 0.28 (0.190.42) 1.2 (0.861.7) 0.37 (0.230.60) 0.47 (0.270.85) 0.15 (0.100.23) 0.43 (0.250.73) 0.31 (0.180.54) 0.39 (0.200.76) 0.41 (0.230.75)

mouse human

agonist activity [EC50, nM (95% confidence interval)]a

H-linker-AA18-AA19-AA20-AA21-AA22-AA23-Arg-Asn-NH2

Table 1. Agonist Activity of NMU-8 Analogues Substituted at Positions 18, 19, 20, 21, 22, and 23

0.18 0.085 (0.0630.12) 0.056 (0.0320.098) 0.029 (0.00880.095) 73 (43120) 0.50 (0.280.91) 0.21 (0.120.36) 0.088 (0.0610.13) 0.17 (0.140.20) 0.36 (0.230.56) 0.48 (0.240.95) 0.052 (0.0270.098)


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Table 2. Agonist Activity of PEGylated NMU-8 Analogues Substituted at Positions 18, 19, 20, 22, and 23 PEG20k-linker-AA18-AA19-AA20-Phe-AA22-AA23-Arg-Asn-NH2 agonist activity [EC50, nM (95% confidence interval)]a human

mouse

compound

linker

AA18

AA19

AA20

AA22

AA23

NMUR1

NMUR2

NMUR1

NMUR2

12 13 14 15 16 17

PipAc PipAc β-Ala β-Ala β-Ala β-Ala

Tyr Phe Tyr Tyr Tyr Tyr

Phe Trp Nal(2) Cha Phe Phe

Leu Leu Leu Leu Val Leu

Arg Arg Arg Arg Arg Gln

Pro Ala Pro Pro Pro Pro

8.3 (6.311) >880 210 (170260) 16 (1319) 14 (1218) 170 (130220)

39 (3247) 41 (2276) 4.5 (2.29.3) 6.0 (3.610) 26 (2035) 55 (28110)

23 (2028) >880 >1000 49 (2984) 46 (4053) >1000

9.0 (6.912) 31 (1951) 4.1 (1.99.3) 4.2 (2.37.6) 7.7 (5.012) 65 (39110)

a

EC50 values of agonist activities were determined as concentrations of peptide analogues that gave half-maximum [Ca2+] mobilizing activities (n = 2).

Table 3. Agonist Activity of NMU-8 Analogues Substituted at Positions 17, 18, and 19 with or without PEG Moiety R-AA17-AA18-AA19-Leu-Phe-Arg-Pro-Arg-Asn-NH2 agonist activity [EC50, nM (95% confidence interval)]a human compound

R

18 19 20

Ac Ac Ac

21 22

Ac Ac

23

Ac

AA17

AA18

AA19

NMUR1

Tyr Tyr Lys

Lys Lys(PEG20k) Nal(2)

Lys

Lys(PEG20k) Tyr

Nal(2) Nal(2)

Lys(PEG20k)

Tyr

Nal(2)

>1000 >1000 39 (25 59) >1000 17 (13 23) >1000

mouse

NMUR2

NMUR1

NMUR2

12 (9.316) >1000 3.4 (2.35.1)

83 (58120) >1000 38 (2167)

270 (230330) >1000 42 (2765)

210 (180260) 0.42 (0.220.79)

>1000 8.0 (5.212)

>1000 0.33 (0.120.90)

9.4 (6.713)

>1000

20 (1527)

a


Table 4. Agonist Activity of NMU-8 Analogues Substituted at Positions 19, 20, 21, 22, and 23 H-linker-Tyr-AA19-AA20-AA21-AA22-AA23-Arg-Asn-NH2 agonist activity [EC50, nM (95% confidence interval)]a human compound

linker

mouse

AA19

AA20

AA21

AA22

AA23

NMUR1

NMUR2

NMUR1

NMUR2

24 25 26 27 28 29 30 31 32

PipAc PipAc β-Ala β-Ala PipAc β-Ala β-Ala β-Ala PipAc

Glu Nal(2) Glu Cha Cha Cha Nal(2) Nal(2) Glu

Leu Leu Val Val Leu Leu Leu Leu Leu

Phe Phe Phe Phe Nle Pya(4) Pya(4) Phe Phe

Arg Arg Arg Arg Arg Arg Arg Gln Arg

Pro Pro Pro Pro Pro Pro Pro Pro MeAla

1.4 (1.02.0) 0.20 (0.150.25) 4.1 (2.56.7) 0.58 (0.350.94) 0.34 (0.200.61) 0.57 (0.321.0) 0.83 (0.591.2) 0.94 (0.701.3) 0.93 (0.681.3)

>1000 9.6 (6.913) >1000 7.4 (4.911) 5.8 (3.49.8) 28 (1941) >1000 >1000 160 (120200)

4.9 (2.59.8) 0.11 (0.0740.15) >1000 0.52 (0.300.89) 0.034 (0.0130.090) 1.1 (0.821.3) 4.8 (2.49.4) 9.7 (7.313) 2.4 (1.93.0)

33 34

PipAc Acp

Nal(2) Nal(2)

Leu Leu

Phe Phe

Arg Arg

MeAla MeAla

0.16 (0.120.22) 0.82 (0.203.4)

5.5 (3.29.3) 2.5 (0.5112)

0.075 (0.0510.11) 0.68 (0.182.7)

35

PEG(2)

Nal(2)

Leu

Phe

Arg

MeAla

>1000 9.5 (6.813) >1000 2.7 (2.43.1) 2.2 (1.72.9) 8.1 (5.911) 81 (55120) >1000 200 (160 250) 9.2 (6.513) 2.7 (0.65 11) 42 (3058)

0.74 (0.511.1)

23 (2027)

0.40 (0.290.57)

a


amino acid combination at positions 19, 21, 22, and 23 (Table 5). All PEGylated peptides (14, 36−43) showed full agonist activities for human and mouse NMUR2 (except 39 for mouse NMUR2). The anorectic effect of single dosing of PEGylated peptides at 100 or 600 nmol/kg was evaluated in lean C57BL/ 6J mice. Glu19 analogue containing PipAc linker (36) showed high NMUR2 selectivity; however, its agonist activity and

col [PEG(2)] (35) reduced NMUR2 agonist activity by several folds compared to PipAc-containing analogue (33). The NMUR2 selectivity of PEG(2) analogue was relatively high; however, Acp substitution showed little selectivity. Combination of Amino Acid Substitutions in PEGylated NMU-8 Analogues. NMUR2 agonist activity and selectivity of PEGylated peptides were investigated by the 6092


inhibition of food intake 16 h after administration were weaker than 14 [Nal(2)19 analogue with β-Ala linker]. In contrast, Nal(2)19 analogue (37) with improved NMUR2 selectivity exhibited the most potent anorectic activity in mice. Although [PipAc,Cha19,Nle21] analogue (38) displayed strong agonist activity for mouse NMUR2 and excellent anorectic effect, its NMUR2 selectivity was relatively low (15-fold vs NMUR1) in human. These results suggested that the combination of linker and amino acid substitutions at positions 19 and 21 impart the species difference observed in NMUR2 selectivity. The introduction of Pya(4)21 (39) or Gln22 (40) residue in 14 weakened the NMUR2 agonist activities of resulting peptides, particularly in mouse, which also decreased their anorectic activities. The introduction of MeAla23 in 36 decreased human NMUR2 agonist activity and selectivity of the resulting compound (41). A PEGylated peptide (42) containing Nal(2)19 and MeAla23 residues showed high NMUR2 agonist activity but slightly lower food intake suppression than 37. In non-PEGylated peptides, MeAla substitution for Pro at position 23 resulted in increased NMUR2 agonist activity; however, the effect of MeAla substitution was abrogated in PEGylated peptides. A PEGylated peptide (43) containing a flexible linker, PEG(2), showed weaker NMUR2 agonist activity and similar in vivo activity compared to 14. Compound 43 also showed moderate NMUR1 agonist activity in mice. Results showed that NMUR2 agonist activity, selectivity, and anorectic activity of a series of PEGylated peptides containing PipAc linker were superior to other linkers tested, for example, β-Ala, Acp, PEG(2). Moreover, the combination with Nal(2)19 substitution was the most preferable. This could be due to the cyclic structure of PipAc moiety, which could have contributed to the arrangement of peptide chain, PEG moiety, and receptor. NMUR2 agonist activity and selectivity of peptides with several amino acid substitutions, such as Pya(4)21, Gln22, MeAla23, were affected by the introduction of PEG moiety. These amino acid replacements did not show a synergistic effect with Nal(2)19 on NMUR2 agonist activity and selectivity; therefore, further optimization for substitutions between positions 19 and 23 would be needed to obtain more potent PEGylated peptide compared to 37. Pharmacokinetic Parameters of 37 in Mice. The pharmacokinetic parameters of [PipAc,Nal(2)19] analogue (37) were studied in male C57BL/6J mice. Compound 37 was injected intravenously and subcutaneously to three mice at a dose of 30 nmol/kg under fed condition. Pharmacokinetic parameters of 37 after intravenous administration were as follows: C5 min, 735 nmol/L; VdSS, 82.2 mL/kg; CLtotal, 18.1 mL/h/kg; MRT, 4.55 h; and AUC0−48h, 1660 nmol·h/L (Table 6, Figure S1). It indicated that clearance rate of 37 was very slow, suggesting that once-daily dosing is adequate. MRT was 10.9 h and AUC0−48h was 1720 nmol·h/L after subcutaneous administration. Bioavailability (F) of 37 was very high (103%), indicating its good subcutaneous penetration across the subcutaneous tissues. These results suggest that 37 is suitable for subcutaneous dosing in mice. Body Weight-Lowering Effect of 37 in DIO Mice. Body weight-lowering effect of once-daily administration of 37 was evaluated in C57BL/6J DIO mice. The mice were administered 10, 30, and 100 nmol/kg of 37 for 2 weeks (Figure 1). For comparison, an NMUR2 full agonist PEGylated peptide 13, peptide sequence of which is reported to show NMUR2 selectivity,30 was administered at 30 nmol/kg. Similar to the results obtained in lean mice, 37 significantly suppressed food

EC50 values of agonist activities were determined as concentrations of peptide analogues that gave half-maximum [Ca2+] mobilizing activities (n = 2). bPercentage inhibition of food intake 16 h after administration of peptide analogues at doses of 100 or 600 nmol/kg as compared to that after injection with saline vehicle in male C57BL/6J mice (n = 5 per group). (∗∗∗) p < 0.001 vs vehicle by Student’s t test. ($$) p < 0.01 vs vehicle by Aspin-Welch’s t test, (##) p < 0.01, (###) p < 0.001 vs vehicle by one-way ANOVA with post hoc Dunnett’s multiple comparison test. ND, not determined; ns, not significant by Aspin-Welch’s t test.

>1000 >1000 >1000 95 (65140) >1000 >1000 >1000 >1000 150 (110190) 4.5 (2.29.3) 9.2 (6.812) 8.8 (5.813) 5.9 (3.59.8) 45 (2872) 30 (1852) 27 (2234) 7.0 (4.411) 39 (15110) 210 (170260) >1000 >1000 89 (61130) >1000 >1000 >1000 >1000 >1000 Pro Pro Pro Pro Pro Pro MeAla MeAla MeAla Arg Arg Arg Arg Arg Gln Arg Arg Arg Nal(2) Glu Nal(2) Cha Nal(2) Nal(2) Glu Nal(2) Nal(2) β-Ala PipAc PipAc PipAc β-Ala β-Ala PipAc PipAc PEG(2) 14 36 37 38 39 40 41 42 43

Phe Phe Phe Nle Pya(4) Phe Phe Phe Phe

NMUR1 AA23 AA22 AA21 AA19 linker compound

Article

a

100

ND ND 51*** 42$$ ND 10 (ns) ND ND ND 76 22## 88### ND 33*** ND 30### 72### 69*** 4.1 (1.99.3) 29 (2141) 1.0 (0.801.4) 0.46 (0.220.94) >1000 69 (45110) 39 (2463) 1.3 (0.931.7) 16 (1221)

###

600 NMUR2

NMUR1

mouse human

agonist activity [EC50, nM (95% confidence interval)]a

PEG20k-linker-Tyr-AA19-Leu-AA21-AA22-AA23-Arg-Asn-NH2

Table 5. Agonist Activity of PEGylated NMU-8 Analogues Substituted at Positions 19, 21, 22, and 23

NMUR2

after bolus dosing (nmol/kg)

% food intake inhibitionb


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dose of 30 nmol/kg. Moreover, dose-dependent body weight reduction of 19.5% was observed at 100 nmol/kg. Furthermore, the body weight loss caused by 37 at 10 nmol/kg was comparable to that of 13 at 30 nmol/kg.

Table 6. Pharmacokinetic Parameters of 37 after Intravenous or Subcutaneous Administration in Micea compound 37 Intravenously C5 min (nmol/L) AUC0−48h (nmol-h/L) MRT (h) Vdss(mL/kg) CLtotal (mL/h/kg) Subcutaneously Cmax (nmol/L) Tmax (h) AUC0−48h (nmol-h/L) MRT (h) F (%)

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CONCLUSION NMUR2 selective PEGylated NMU-8 analogues with highly potent body weight-lowering and food intake inhibitory effects were designed and synthesized. The SAR studies of NMU-8 analogues showed that amino acid substitution at position 19 was effective to promote NMUR2 selectivity. For PEGylated peptides, Nal(2)19 replacement was the most preferable substitution for NMUR2 agonist activity and anorectic effect in mice. A 20 kDa PEG-modified analogue (37) containing PipAc linker showed the highest anorectic activity in lean mice.39 Once-daily repeated subcutaneous administration of 37 for 2-weeks showed highly potent anorectic and body weightlowering effects in DIO mice. A pharmacokinetic study indicated that the NMUR2 selective agonist (37) has potential to elucidate the physiological function of the NMU/NMUR2 system. Moreover, it also provides evidence for the application and therapeutic utility of 37 in diabetes and obesity.

735 1660 4.55 82.2 18.1 117 4.00 1720 10.9 103

a

Pharmacokinetic parameters after intravenous and subcutaneous administration of 37 at 30 nmol/kg were calculated from the mean (n = 3) plasma concentrations. See Experimental Section for further details.

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EXPERIMENTAL SECTION

Instruments and Materials. Solid phase syntheses were carried out by manual Fmoc solid phase peptide synthesis (SPPS), which included Fmoc cleavage with 20% piperidine/DMF (20 min) and Fmoc amino acid condensation reaction using N,N′-diisopropylcarbodiimide (DIPCDI)/1-hydroxyl-7-azabenzotriazole (HOAt) (4 equiv), or by using an automated peptide synthesizer [ABI 433A (Applied Biosystems, Foster City, CA, USA); as per the Fmoc/DCC/HOBt 0.25 mmol protocol)]. All final compounds without PEG moiety were purified to give ≥95% homogeneity in RP-HPLC analysis with UV detection at 210 nm using the following method: Phenomenex Kinetex 1.7 μm XB-C18 column (2.1 × 100 mm) (Torrance, CA, USA), linear gradient elution with eluents A/B = 95/5−45/55 (10 min), using 0.1% TFA in water as eluent A and 0.1% TFA-containing acetonitrile as eluent B, flow rate: 0.5 mL/min. All final compounds with PEG moiety were purified to give ≥95% homogeneity in RP-HPLC analysis with charged aerosol detection using the following method: Chromolith HighResolution RP-18 end-capped (4.6 × 100 mm) (Merck, Darmstadt, Germany), linear gradient elution with eluents A/B = 80/20−30/70 (12.5 min), using 0.1% TFA in water as eluent A and 0.1% TFA-containing acetonitrile as eluent B, flow rate of 2 mL/min. The identity of the peptides was confirmed by MALDI-TOF-MS on a Bruker autoflex speed system (Billerica, MA, USA). Commercially available amino acid derivatives and resins were purchased from Merck, Watanabe Chemical Industries (Hiroshima, Japan), Peptide Institute (Osaka, Japan), Bachem (Bubendorf, Switzerland), AnaSpec (Fremont, CA, USA), and Chem-Impex International (Wood Dale, IL, USA). Other reagents such as coupling and deprotection reagents were purchased from Wako Pure Chemical Industries (Osaka, Japan), Merck, Watanabe Chemical Industries, and Nacalai Tesque (Kyoto, Japan). Peperazin-1-yl-acetic acid and 2-(4-tert-butoxycarbonylpiperazin-1-yl)acetic acid were purchased from Merck and Fluorochem (Derbyshire, United Kingdom), respectively. ω-Aminocarboxylic acids were purchased from Watanabe Chemical Industries. Other reagents were purchased from Wako Pure Chemical Industries. General Procedure for Synthesis of NMU-8 Analogues. All peptides were synthesized in a manner similar to the following synthesis procedure for PipAc-[Nal(2)19]NMU(18−25) (25). To 14.5 mg (0.01 mmol) of Sieber Amide MBHA resin (0.69 mmol/g), Fmoc protected Asn(Trt), Arg(Pbf), Pro, Arg(Pbf), Phe, Leu, Nal(2), Tyr(tBu), and 2-(4-tert-butoxycarbonylpiperazin-1-yl)acetic acid were sequentially introduced using the standard Fmoc SPPS method (coupling: Fmoc-amino acid (5 equiv), 0.5 M HOAt/DMF (5 equiv), DIPCDI (5 equiv), 120 min; Fmoc deprotection: 20% piperidine/

Figure 1. Body weight-lowering and food intake inhibitory effect of 13 and 37 for 2 weeks in DIO mice. (A) body weight change, and (B) total food intake. Each peptide (13 at 30 nmol/kg; 37 at 10, 30, and 100 nmol/kg) was subcutaneously administered in 41-week-old DIO mice and the body weight and food intake were measured during the study (n = 6). (∗) p < 0.05; (∗∗) p < 0.01 vs vehicle by Student’s t test. (#) p < 0.025 vs vehicle by Williams test. ($) p < 0.05 vs vehicle by Aspin-Welch test.

intake in a dose-dependent manner, which also resulted in a marked body weight loss. Body weight lowering effect of 37 was sustained during the 2-week study and it exhibited a significant body weight loss (12.4%) at a minimum effective 6094



Article

(%) of test peptides are expressed as [(A − B)/(C − B)] × 100 [where increase of the intracellular Ca2+ concentration in test peptidetreated cells = A, vehicle-treated cells = B, and 1 μmol/L NMU8treated cells = C]. The EC50 values of all peptides (n = 2) were calculated using Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA) and are shown in Tables 1−5. Animal Studies. The care and use of the animals and the experimental protocols used in this study were approved by the Institutional Animal Care & Use Committee of Takeda Pharmaceutical Company Limited. The animals were maintained on a 12 h light/ dark cycle in a temperature-controlled facility (lights on at 07:00 AM), with tap water and chow provided ad libitum. Anorectic Effect in Lean Mice. Male C57BL/6J (9−10 week-old) mice purchased from CLEA Japan, Inc. (Tokyo, Japan) were divided into groups (n = 5) based on the data of body weight and food intake. Test compounds dissolved in saline were subcutaneously administered and the food intake was measured 16 h after the administration. The percentage of food intake inhibition compared to that of the vehicle group was calculated. Pharmacokinetics of 37 in Mice. Compound 37 was administered intravenously or subcutaneously to male C57BL/6J mice (n = 3) in fed condition at a dose of 30 nmol/kg. After administration, blood samples were collected at specified time points and centrifuged to obtain the plasma fraction. The plasma samples were deproteinized with methanol. After centrifugation, the supernatants were analyzed by liquid chromatography−tandem mass spectrometry (LC/MS/MS) to determine the plasma concentrations of samples after intravenous administration. Conversely, for samples after subcutaneous administration, the supernatant after deproteinization was transferred to a 96-well microtiter plate and evaporated to dryness under a steady stream of nitrogen. The residues were reconstituted with a mixture solution of acetonitrile and distilled water. An aliquot of this solution was injected into an LC/MS/MS to determine the plasma concentrations. The pharmacokinetic parameters of each group were assessed using moment analysis. The plasma concentration 5 min after injection (C5 min), area under the plasma concentration−time curve to the last time point (AUC0−48h), mean residence time (MRT), volume of distribution at steady state (Vdss), and total body clearance (CLtotal) were calculated for each mouse after intravenous administration. The maximum plasma concentration (Cmax), time to maximum plasma concentration (Tmax), AUC0−48h, MRT, and F for each mouse after subcutaneous administration were also calculated. Body Weight-Lowering Effect of 37 in DIO Mice. To induce obesity, C57BL/6J mice were fed a high fat diet (D12331; Research Diets Inc., NJ, USA) from 5 weeks of age to the end of the study. Male DIO C57BL/6J mice (41-weeks-old) were divided into groups (n = 6) based on the data of body weight, food intake, and plasma biochemical parameters. The test compound (37) dissolved in saline was subcutaneously administered once daily for 2 weeks. Body weight and food intake were monitored during the study. Statistics. Data are expressed as means + standard deviation (SD) or means ± SD in Figure 1. Statistical differences were analyzed using Student’s t test, Aspin−Welch’s t test, Williams test, one-tailed Dunnett’s test, or one-way ANOVA with post hoc Dunnett’s multiplecomparison test to compare among more than two groups, and dosedependency was analyzed using a one-tailed Williams test or Shirley− Williams test as indicated in the legends. P-values of

Potent Body Weight-Lowering Effect of a Neuromedin U Receptor 2

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