Incorporation of Monodisperse Oligoethyleneglycol Amino Acids into

Article pubs.acs.org/molecularpharmaceutics

Incorporation of Monodisperse Oligoethyleneglycol Amino Acids into Anticonvulsant Analogues of Galanin and Neuropeptide Y Provides Peripherally Acting Analgesics Liuyin Zhang,† Brian D. Klein,‡,§ Cameron S. Metcalf,§ Misty D. Smith,‡ Daniel R. McDougle,§ Hee-Kyoung Lee,† H. Steve White,‡ and Grzegorz Bulaj*,† †

Department of Medicinal Chemistry and ‡Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City, Utah 84108, United States § NeuroAdjuvants Inc., Salt Lake City, Utah 84108, United States S Supporting Information *

ABSTRACT: Delivery of neuropeptides into the central and/or peripheral nervous systems supports development of novel neurotherapeutics for the treatment of pain, epilepsy and other neurological diseases. Our previous work showed that the combination of lipidization and cationization applied to anticonvulsant neuropeptides galanin (GAL) and neuropeptide Y (NPY) improved their penetration across the blood−brain barrier yielding potent antiepileptic lead compounds, such as Gal-B2 (NAX 5055) or NPY-B2. To dissect peripheral and central actions of anticonvulsant neuropeptides, we rationally designed, synthesized and characterized GAL and NPY analogues containing monodisperse (discrete) oligoethyleneglycol-lysine (dPEG-Lys). The dPEGylated analogues Gal-B2-dPEG24, Gal-R2dPEG24 and NPY-dPEG24 displayed analgesic activities following systemic administration, while avoiding penetration into the brain. Gal-B2-dPEG24 was synthesized by a stepwise deprotection of orthogonal 4-methoxytrityl and allyloxycarbonyl groups, and subsequent on-resin conjugations of dPEG24 and palmitic acids, respectively. All the dPEGylated analogues exhibited substantially decreased hydrophobicity (expressed as logD values), increased in vitro serum stabilities and pronounced analgesia in the formalin and carrageenan inflammatory pain assays following systemic administration, while lacking apparent antiseizure activities. These results suggest that discrete PEGylation of neuropeptides offers an attractive strategy for developing neurotherapeutics with restricted penetration into the central nervous system. KEYWORDS: PEGylation, monodisperse oligoethyleneglycol amino acids, peripheral analgesics, nociception, pain, anticonvulsant neuropeptides, galanin, NPY

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INTRODUCTION PEGylation is a widely applied chemical modification toward peptides or proteins.1−6 PEGylation increases protein hydrodynamic radius, water solubility, chemical and metabolic stability and pharmacokinetic profiles while reducing protein immunogenicity and toxicity. However, the polydispersity of PEGylation can yield diverse PEGylated proteins or peptides.7,8 This challenge can be mitigated by the application of monodispersed (or discrete) oligoethyleneglycolation, also named dPEGylation strategy. This new PEGylation method has been studied in peptide drug development. The dPEGylated myoglobin, GFP and RGD peptides showed increased hydrophilicities and improved pharmacokinetic profiles.9−12 The dPEGylated antiTAG-72 diabody analogues (with dPEG12, dPEG24 or dPEG48) showed increased tumor uptake and decreased kidney uptake without losing immunoreactivity.13 The C-terminal dPEGylation of glucose-dependent insulinotropic peptide (with dPEG3) increased the peptide’s half-life against the dipeptidyl peptidase IV.14 The dPEGylation of Fmoc-Leu-enkephalin (with dPEG5) increased its antinociceptive activity after ip administration.15 © 2012 American Chemical Society

The dPEGylation improved the transdermal delivery of zidovudine and stavudine16 and the bioavailability of 18F-labeled stilbene analogues.17 Interestingly, dPEGylation proved useful in the development of peripherally active small molecular drug candidates, such as the naloxol analogue NKTR-118, which is currently under phase III clinical trial to reverse opioid-induced constipation.18 The dPEGylation was also applied to multimeric conjugates,19 dendrons,20−22 polymer brushes,23,24 precision polymers,25 space defined peptide-hydrogels,26 self-assembling dPEG-peptide conjugates,27 liposomal drug delivery systems,28 protein−protein interaction molecular tools,29 cell-penetrating peptides for cellular uptake mechanism studys30 and site-specific protein dPEGylation.31,32 Anticonvulsant neuropeptides offer attractive templates to engineer peptide-based therapeutics for many neurological Received: Revised: Accepted: Published: 574

April 26, 2012 December 14, 2012 December 21, 2012 December 21, 2012 dx.doi.org/10.1021/mp300236v | Mol. Pharmaceutics 2013, 10, 574−585

Molecular Pharmaceutics

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diseases.33 Previously, our group improved the central nervous system (CNS) bioavailability of neuropeptides galanin (GAL), neurotensin (NT), neuropeptide Y (NPY) and neuropeptide W (NPW) via a combination of lipidization and cationization that resulted in analogues that penetrated across the blood− brain barrier (BBB) yielding potent antiepileptic activities (Figure 1).34−41 Whereas GAL and NPY suppress epileptic

following systemic administration. To the best of our knowledge, this work describes for the first time an application of dPEGylation to dissect activities of neuropeptides that are mediated by their respective receptors expressed in PNS and CNS, resulting in analogues that offer first-in-class lead analgesic compounds.

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RESULTS Overview of the Experimental Strategy. The starting point for this project was our previous finding that an incorporation of lipoamino acids into GAL and NPY analogues yielded compounds that suppressed seizures in the brain.34−41 Here, we hypothesized that replacing lipidation with dPEGylation will restrict penetration of the neuropeptide analogues into the brain, while their activities in the PNS would be retained. Since our initial SAR studies focused on dPEGylation of a wellcharacterized lead compound Gal-B2, we first describe a rational design and synthesis of Gal-B2-dPEG24 that contains both a lipoamino acid and the dPEG24-lysine residue.34 In our subsequent efforts, we focused on neuropeptide analogues as peripherally acting drug leads for the treatment of pain. In this work, we describe two examples of neuropeptide-based analgesics, namely, GalR2 subtype preferring compound Gal-R2-dPEG24 and NPYbased NPY-dPEG24. Design and Chemical Synthesis of Gal-B2-dPEG24. As shown in Figure 1, to convert the BBB-penetrant lead compound Gal-B2 into a PNS-targeting analgesic compound, we first introduced dPEGylation at its C-terminus. Our previous results suggested that the Gal-B2 analogue, in which the palmitoyl group was replaced by PEG4 (CH3O(CH2CH2O)4CH2CO−), maintained some anticonvulsant activity in the 6 Hz (32 mA) seizure test when administrated via the ip route, but this analogue was less active than the lipoamino acid containing Gal-B2 analogue.34 Based on that observation and given a relatively high logD value (1.24) for Gal-B2, we selected dPEG24 to be coupled to an additional lysine residue at the C-terminus. The structure of the resulting galanin analogue Gal-B2-dPEG24 is shown in Figure 1. Two technical challenges were anticipated during the synthesis of Gal-B2-dPEG24: (1) the resin beads could become sticky after coupling the Lys-(palmitoyl)-OH during the synthesis of Gal-B2,41 and (2) the steric hindrance of the dPEG24 chain could decrease the yields during automatic peptide synthesis. To mitigate these potential problems, a low-load and highly crosslinked PEG resin (Clear resin) was selected. An apparent challenge of introducing chemical modifications to peptides with multiple functional groups is to use the most efficient orthogonal protection strategy during solid phase peptide synthesis (SPPS).64 From several protecting groups available for Nε-lysine, we selected allyloxycarbonyl (Aloc) and 4-methoxytrityl (Mmt) as the Fmoc orthogonal protecting groups.65,66 The Aloc group is usually removed with tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4) and scavengers, such as acetic acid (HAc)/N-methylmorpholine (NMM), whereas the Mmt group can be removed by HAc/trifluoroethanol (TFE) in CH2Cl2. Scheme 1 summarizes the synthesis of Gal-B2-dPEG24. The overall strategy was to couple dPEG24-acid prior to the conjugation of palmitic acid to avoid the adhesion of the resin upon lipidation. The Fmoc-Rink amide Clear resin was coupled sequentially with Fmoc-Lys(Mmt)-OH, Fmoc-Lys(Boc) and Fmoc-Lys(Aloc)-OH, followed by Fmoc deprotection with 20% piperidine in NMP. The remaining amino acids were assembled by the standard Fmoc peptide synthesis strategy. Then, the Mmt protecting group on the resin was removed with HAc/TFE/ CH2Cl2 (1/2/7 by volume). The deprotection process was

Figure 1. Design of an analgesic galanin analogue, Gal-B2-dPEG24, based on Gal-B2. Gal-B2 is a potent anticonvulsant drug lead that exhibits both analgesic and antiepileptic activities.34,39,66

seizures by binding to their respective receptors expressed within the brain,42−44 both peptides are also thought to produce analgesia through their receptors expressed in the peripheral nervous system (PNS).45−50 Galanin and its major receptors, subtype 1 (GalR1) and subtype 2 (GalR2), are expressed in sites of pain mediation outside the brain, including the dorsal root ganglion and the dorsal horn of the spinal cord.51 Both GalR1and GalR2-preferring agonists produce inhibitory effects on nociception.38,52−57 It is noteworthy that GalR3 may have less or no effects on peripheral antinociception activity, since low levels of GalR3 are found in both the dorsal root ganglion and spinal cord.58 Similarly, NPY and its major peripheral receptors involved in pain, Y1 and Y2, are expressed in sites of pain processing,45,59 and NPY has been shown to inhibit nociceptive transmission.60,61 Galanin analgesics that target both PNS and CNS could potentially produce centrally mediated side effects typical for anticonvulsant and analgesic drugs, such as drowsiness, dizziness and cognitive deficits. Thus, peripheral-selective analgesic peptide analogues are highly demanded to minimize the related CNS side effects.62 Strategies to develop peripherally acting drugs include forming zwitterions, increasing hydrophilicity or combining both characters to form amphiphilic compounds.63 Herein, we exploited the dPEGylation strategy to minimize and/or prevent penetration of the neuropeptide analogues into the CNS, testing the hypothesis that PNS-targeting GAL or NPY analogues would retain their analgesic properties, but lack any antiseizure activity 575

dx.doi.org/10.1021/mp300236v | Mol. Pharmaceutics 2013, 10, 574−585


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Scheme 1. Chemical Synthesis of Gal-B2-dPEG24a

a

Reagents and conditions: (a) Rink amide Clear resin:(Fmoc-amino acid/Pybop/DIPEA, 1/0.98/2), 1:5, 40 min; (b) HAc/TFE/CH2Cl2, 1/2/7, 10 min × 6; (c) Resin:(m-dPEG-24 acid/PyBop/HOBt/DIPEA, 1/0.98/1/2), 1:1.5, 24 h; (d) Resin:(Pd(PPh3)4 in HAc/NMM/CH2Cl2, 1/2/40), 1:2.8, 2 h; (e) Resin/(palmitic acid/PyBop/HOBt/DIPEA, 1/0.98/1/2), 1:1.5, 2 h; (f) Reagent K (TFA/phenol/water/thioanisole/ 1,2-dithioethane, 82.5/5/5/5/2.5), 3 h. Purity: >95%.

Table 1. Structures, Mass Spectrometry Data, LogD and Serum Stability Values (Half-Lives) of Galanin and NPY Analogues Described in This Study Gal-B2e Gal-B2-dPEG24 Gal-B5e Gal-R2-dPEG24 NPY-B2e NPY-dPEG24

sequencea

mass (MH+) calcd/expb

logDc

half-life (h)d

Sar-WTLNSAGYLLGPKK-Lys(Pal)-K-NH2 Sar-WTLNSAGYLLGPKK-Lys(Pal)-K-Lys(dPEG24)-NH2 WTLNSAGYLLGPKK-Lys(Pal)-K-NH2 WTLNSAGYLLGPKKKK-Lys(dPEG24)-NH2 Ac-YKK-Lys(Pal)-Ahx-ARHYINLITRQRY-NH2 Ac-YKK-Lys(dPEG24)-Ahx-ARHYINLITRQRY-NH2

2112.33/2112.33 3339.06/3338.90 2040.46/2040.13 3029.80/3029.80 2643.63/2643.70 3504.04/3504.13

1.24 ± 0.02 −0.24 ± 0.05 1.22 ± 0.02 −0.69 ± 0.08 1.76 ± 0.01 −0.76 ± 0.07

9.4 71.0 >10.0 21.8 3.0 14.0

Lys-Pal, Nε-palmitoyl-L-lysine; Lys-dPEG24, Nε-dPEG24-L-lysine; Sar, sarcosine; Ac, acetyl; Ahx, aminohexanoic acid. bData were collected from MALDI-TOF MS. cLogD values were determined by the shake-flask method in n-octanol/PBS (50/50), pH 7.4 at 25 °C. dSerum stability was determined by incubation of peptides in 25% rat serum at 37 °C. eData were reported in refs 34, 38 and 41.

a

identity was confirmed by MALDI-TOF mass spectrometry (Table 1). Partition Coefficient and Serum Stability of Gal-B2dPEG24. Our previous studies showed that chemically modified galanin analogues exhibited significant changes in both octanol/water partition coefficient (logD) and in vitro metabolic stability.34,36,41 Herein, we tested how dPEGylation affected these two important characteristics. The logD was determined using the shake-flask method as previously described.34,36,38,41 As shown in Table 1 and Figure 2, the hydrophobicity of Gal-B2-dPEG24, expressed by logD units, was significantly lower, as compared to those of Gal-B2 and Gal-(1−16). To evaluate the in vitro metabolic stability of Gal-B2dPEG24, we determined its half-life in buffered 25% rat blood serum at 37 °C, as previously described.34,36,41 The remaining amount of the analogue in the serum solution was quantified by HPLC, and the time course of its disappearance (Figure S2 in the Supporting Information) was used to calculate its half-life. Gal-B2-dPEG24 appeared very stable under these conditions with t1/2 > 24 h (Table 1).

monitored by the appearance of yellow color of Mmt cation solution. It is noteworthy that repeating the deprotection process several times (less than 10 min every time), instead of a single long deprotection process, efficiently reduced the reattachment of Mmt. The resin was then neutralized with 5% DIPEA/CH2Cl2 to free the Nε-amino group of Lys18, followed by the coupling of dPEG24-OH. The coupling process was monitored by the ninhydrin test. After the completion of the dPEGylation step, the Aloc group of Nε-Lys16 was removed by the application of Pd(P(Ph3))4 in HAc/NMM/CH2Cl2 (1/2/40). The trace palladium was removed by 0.02 M sodium diethyldithiocarbamate in DMF. The palmitic acid was conjugated after neutralizing the resin with DIPEA/CH2Cl2. The crude peptide Gal-B2dPEG24 was cleaved from the resin by reagent K and was purified by preparative HPLC. The purity of peptide was greater than 95% by analytical HPLC analyses (Figure S1 in the Supporting Information). Gal-B2-dPEG24 was quantified by measuring UV absorbance (λ = 279.8 nm, ε = 7000 cm−1 M−1), and its chemical 576



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Figure 2. Octanol/water partition coefficients (logD) of galanin analogues. LogD values were obtained from the average of three independent experiments via the shake-flask method using a 50/50 ratio of n-octanol/PBS, pH 7.4 at 25 °C. The amount of peptide in the aqueous phase was determined by peak area using analytical HPLC over a gradient ranging from 5 to 95% buffer B in 40 min. Results were obtained from the slopes of at least three independent experiments. The logD data for the unmodified galanin analogue Gal-(1−16) (GWTLNSAGYLLGPHAV-NH2) were previously published.34

In Vitro and in Vivo Pharmacological Characterization of Gal-B2-dPEG24. The common strategy to evaluate drugs’ peripheral selectivity is to compare the different pharmacological activities following central administration (i.e., icv) or peripheral administration (i.e., iv or sc).63 Three studies were performed to test the hypothesis that the dPEGylated Gal-B2 analogues preserved the analgesic activity of Gal-B2 following systemic administration, but hindered the CNS penetration of Gal-B2: (1) determination of galanin receptor binding ability, (2) determination of anticonvulsant activity following systemic and central administration, and (3) assessment of its analgesic activity following systemic administration. To assess the effect of dPEGylation of Gal-B2 on the binding affinities toward galanin receptors hGalR1 and hGalR2, we used time-resolved fluorescence-based competitive binding assays.34,41 As shown in Table 2 and Figures 3A and 3B, Table 2. Receptor Binding Affinities of dPEGylated Galanin Analogues toward Human GalR1 and GalR2 Receptors Ki (nM)

a

analogue

GalR1

GalR2

Gal-B2a Gal-B2-dPEG24 Gal-B5a Gal-R2-dPEG24

3.5 ± 1.0 1.3 ± 0.4 387 ± 123 242 ± 40

51.5 ± 34.4 13.5 ± 2.2 48.0 ± 11.3 60.4 ± 2.3

Data were reported in ref 34.

Figure 3. Representative binding curves obtained from competitive receptor binding studies with hGalR1 (A) and hGalR2 (B) membrane preparations for Gal-B2-dPEG24 (open circles) and Gal-R2-dPEG24 (filled squares). Europium-labeled galanin was used as the ligand for the assays. Each binding assay was performed in triplicate to generate one 10-point binding curve. Binding affinities (Ki) for the analogues are summarized in Table 2. Gal-B2-dPEG24 displayed higher affinity for hGalR1 compared to hGalR2, whereas Gal-R2-dPEG24 displayed higher affinity for hGalR2 compared to hGalR1.

Gal-B2-dPEG24 displayed low nanomolar affinities for both GalR1 and GalR2, with 10-fold higher binding affinity for GalR1. To evaluate the penetration of Gal-B2-dPEG24 into the brain, the anticonvulsant activity of the analogue was determined following ip and icv administrations using the 6 Hz (32 mA) seizure test in CF-1 mice, as described previously.34 Table 3 contains a summary of the results of this assay for each group tested, as well as the calculated area under the curve (AUC) for the time course of each test compound. As in our previous work, the BBB-penetrant analogue Gal-B2 displayed potent anticonvulsant

activity,34,39 whereas the peripherally active Gal-B2-dPEG24 showed no anticonvulsant effect following systemic administration. 577



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Table 3. Anticonvulsant Activities of Galanin Analoguesa

that a GalR2-specific analogue might display antinociceptive activity in inflammatory pain assays. We observed that Gal-R2dPEG24 showed similar activity in the formalin assay compared to that of Gal-B2-dPEG24 (Figure 4B); we therefore evaluated GalR2-dPEG24 in another established model of inflammatory pain; i.e., the carageenan model. In this model, localized inflammation is induced in mice by intraplantar injection of carrageenan,68,69 followed by assessment of paw withdrawal responses from thermal stimulation in the Hargreaves test.70−72 Figure 6 shows the analgesic activity of Gal-R2-dPEG24 in the carrageenan model. Mice were administered Gal-R2-dPEG24 at 4 and 8 mg/kg (ip) and tested 1 h later. The withdrawal latency difference (ipsilateral paw latency − contralateral paw latency) was significantly diminished at 8 mg/kg (Figure 6). Gal-R2-dPEG24 effectively reversed carrageenan-induced decreased latency. These data show that the peripherally acting GalR2 analogue, Gal-R2dPEG24, is analgesic in the carrageenan model of inflammatory pain. While Gal-R2-dPEG24 was less active than Gal-B2-dPEG24 in the formalin pain assay (Figure 4B), more studies are needed to determine whether these differences can be accounted for by the receptor subtype preferences and/or by their pharmacokinetic properties. Design and Characterization of dPEGylated NPY Analogue NPY-dPEG24. Our previous work suggested that a lipidated and truncated NPY analogue, namely, NPY-B2, could penetrate the BBB and be capable of producing potent anticonvulsant activity following ip administration.36 Here we tested the hypothesis that a redesigned analogue of NPY-B2, namely, NPY-dPEG24, might be an effective analgesic compound. As shown in Figure 7, we replaced the palmitoyl group in NPY-B2 with dPEG24, but otherwise the analogue structure was unaltered. The chemical synthesis of NPY-dPEG24 is described in detail in the Experimental Section. The replacement of palmitoyl with dPEG24 in NPY analogues significantly decreased the logD value by 2.5 units (Table 1). NPY-dPEG24 also displayed improved metabolic stability, as compared to that of NPY-B2. The in vitro activity of NPY-dPEG24 was evaluated in the neuropeptide Y2 receptor (NPY2R) functional assay. A concentration−response curve for NPY-dPEG24 was generated in CHO-K1 cells expressing human NPY2R. NPY-dPEG24 inhibited forskolin-mediated cyclic adenosine monophosphate (cAMP) production with a potency in the nanomolar range (EC50 = 39.4 ± 8.9 nM) as indicated in Figure 8, while the potency of NPY was in the low nanomolar range (EC50 = 2.5 ± 0.4 nM). The analgesic activity of NPY-dPEG24 was tested in the formalin and in the carrageenan inflammatory pain models. For the formalin test, mice were administered 4 and 8 mg/kg (ip) of NPY-dPEG24, and challenged with an injection of formalin after 2 h. While at the dose of 4 mg/kg, this compound had no apparent analgesic activity, the significant reduction in the acute phase of the nociceptive response was observed for the dose of 8 mg/kg (Figure 4B). For the carrageenan assay, mice were administered 2 and 4 mg/kg of NPY-dPEG24, and tested 2 h following ip administration. The paw withdrawal latency difference decreased at 2 mg/kg and was significantly diminished at 4 mg/kg (Figure 9). At a dose of 4 mg/kg the latency difference of the ipsilateral paw was significantly lower when compared to the contralateral paw. These data demonstrate that the peripherally acting NPY analogue, NPY-dPEG24, is analgesic in the carrageenan model of inflammatory pain. A restricted penetration of NPY-dPEG24 into the brain was confirmed by

anticonvulsant activityb analogue

admin meth 15 min 30 min

Gal-B2d ip (4 mg/kg) Gal-B2-dPEG24 ip (4 mg/kg) icv (1 nmol) icv (4 nmol) Gal-R2-dPEG24 ip (4 mg/kg) ip (8 mg/kg) icv (1 nmol) NPY-B2e ip (4 mg/kg) NPY-dPEG24 ip (8 mg/kg) icv (1 nmol)

3/4 0/4 0/4 3/4 0/4 1/4 1/4 1/4 1/4 2/4

4/4 0/4 1/4 4/4 0/4 0/4 1/4 3/4 1/4 2/4

1h

2h

4h

AUCc

4/4 0/4 3/4 3/4 0/4 0/4 2/4 3/4 0/4 2/4

4/4 0/4 0/4 1/4 0/4 0/4 2/4 0/4 0/4 1/4

0/4 0/4 0/4 0/4 0/4 1/4 1/4 0/4 0/4 0/4

16313 0 3938 6938 0 1688 9000 5250 750 6000

a

Anticonvulsant activity of the galanin analogues was tested in the 6 Hz (32 mA) seizure mouse model. The area under the curve (AUC) was calculated from the protected percentage (%) of mice plotted as a function of time. bNumber of protected mice out of 4 mice per group at different time points. cThe area under the curve values summarize the anticonvulsant time−response curves. The percent of animals protected at each time point was plotted against time, and the AUC values were calculated as described in ref 34. dData were published in ref 34. eData were published in ref 36.

However, this analogue retained anticonvulsant activity at 4 nmol when administered centrally. The analgesic properties of Gal-B2 in the mouse formalin assay were previously reported.56 Herein, Gal-B2-dPEG24 reduced both the acute and the inflammatory phases of the formalin assay at a dose of 4 mg/kg (ip, Figure 4). It is noteworthy that the same systemic dose did not produce anticonvulsant activity in the 6 Hz (32 mA) test (Table 3). Therefore, Gal-B2-dPEG24 exhibited pronounced analgesic activity at doses that do not produce any behavioral anticonvulsant activity effect. Design and Characterization of dPEGylated GalR2Preferring Analogue (Gal-R2-dPEG24). While GalR1-targeting analogues may exert undesirable effects in the peripheral system, such as hyperglycemia via activation of pancreatic GalR1,67 GalR2 targeting analogues are a potentially safer peripheral analgesic acting via the inhibition of primary afferent nociceptor activity.62 The next objective was therefore to re-engineer galanin receptor subtype preference into peripherally acting GAL analogues. The engineering subtype preference was based on our previous work with Gal-B5 (Table 1),38 in which the galanin analogues containing N-terminal Trp or N-methyl-Trp were GalR2-preferring, as compared to those analogues containing Gly or Sar residues. As summarized in Figure 5, we rationally designed a GalR2-preferring and peripherally acting galanin analogue, namely, Gal-R2-dPEG24, by removing the N-terminal Sar residue and the palmitoyl moiety. The chemical synthesis of Gal-R2dPEG24 is described in the Experimental Section. Gal-R2-dPEG24 was 1.9 logD units less hydrophobic, as compared to Gal-B5, while it retained the in vitro metabolic stability (Table 1). The receptor binding assay confirmed 4-fold preference for GalR2 over GalR1 (Ki 242 ± 40 nM (hGalR1), 60.4 ± 2.3 nM (hGalR2); Table 2 and Figure 3). The anticonvulsant results showed that this analogue (at two doses, 4 mg/kg and 8 mg/kg, ip) had minimal activity in the 6 Hz (32 mA) seizure test following systemic administration, while exhibiting the apparent anticonvulsant activity following icv injections, indicating restricted brain penetration (Table 3). Given the efficacy seen in Gal-B2-dPEG24 in suppressing inflammatory pain responses in the formalin assay, we proposed 578



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Figure 4. Analgesic activity of Gal-B2-dPEG24 in the formalin pain assay in mice (ip, injected 1 h prior to formalin injection in a paw). (A) Graph represents that the paw-licking (expressed in seconds) was monitored continuously for the first 2 min of each 5 min epoch until 40 min had elapsed. Duration of licking reflects acute pain response (phase I, at 0−10 min) and inflammatory pain response (phase II, at 10−40 min). Vehicle is 1% Tween in PBS. (B) Analgesic activities of various galanin analogues determined in the formalin pain assay following ip administration. Gal(1−16): mouse group number n = 4, vehicle is saline in PBS. All the other peptides: n = 8, vehicle is 1% Tween in PBS. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to the vehicle.

the treatment of inflammatory and neuropathic pain. For example, the endogenous analgesic activity of opioid analogues is mediated exclusively by peripheral opioid receptors in the later stage of inflammatory conditions.76 Peripheral sensitization is an important mechanism underlying neuropathic pain.77 Peripherally active small molecular drug candidates, such as opioid agonists morphine-6-glucuronide, loperamide (μ-), asimadoline (κ-), fedotozine (κ-), and the recently FDA-approved μ-opiod antagonists methylnatrexone and alvimopan, were reported.63

comparing the anticonvulsant activities of this compound following ip and icv injections (Table 3).

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DISCUSSION Recently, peripherally acting analgesics have been receiving more attraction as neurotherapeutics.73,74 Targeting primary afferent nociceptors to avoid untoward adverse CNS side effects has become an attractive direction for the discovery and delivery of safer analgesic drugs.75 Those analgesics are especially active for 579



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Figure 8. Functional characterization of NPY-dPEG24 by inhibition of forskolin-mediated cAMP production in cell-based assay. CHO-K1 cells transfected with human NPY2R were incubated in a 96-well format for 24 h. Increasing concentrations (up to 1 μM) of NPY-dPEG24 were coincubated with 20 μM forskolin at 37 °C for 30 min. The results are representative of three independent experiments performed in triplicate.

Figure 5. Design of analgesic galanin analogue Gal-R2-dPEG24. dPEGylation of the GalR-2 preferring anticonvulsant galanin analogue Gal-B5 yielded peripherally active analgesic Gal-R2-dPEG24.

Figure 9. Analgesic activity of NPY-dPEG24 in the mouse carrageenan model. The mean withdrawal latency difference (ipsilateral paw latency − contralateral paw latency) for each of the two doses of NPY-dPEG24 is shown. Vehicle is 1% Tween 20 in PBS. Mouse group number: vehicle, n = 12; 2 mg/kg, n = 8; 4 mg/kg, n = 8. ***P < 0.001, P value is the comparison of the withdrawal latency difference (ipsilateral paw latency − contralateral paw latency) for the vehicle treated mice versus NPYdPEG24 treated mice.

Figure 6. Analgesic activity of Gal-R2-dPEG24 in the mouse carrageenan model. The mean withdrawal latency difference (ipsilateral paw latency − contralateral paw latency) for each of the two doses of Gal-R2-dPEG24 is shown. Vehicle is 1% Tween 20 in PBS. Mouse group number: vehicle group, n = 19; 4 mg/kg group, n = 12; 8 mg/kg group, n = 12. **P < 0.01 compared with carrageenan control.

Galanin has been previously shown to reduce painful stimulation in a dose-dependent manner.51,79,80 Numerous galanin ligands have been shown to be analgesic.80 More recently, a GalR2-preferring agonist, Gal2-11, has been shown to reverse nerve injury induced allodynia.54 However, GalR2 ligands may also be pronociceptive.81−83 Therefore, the role of GalR2 in inflammatory pain is still largely unknown. Our results demonstrate that a GalR2-specific analogue increased withdrawal latency following localized administration of an algogen; therefore, these results support an analgesic role for GalR2 in inflammatory pain. Furthermore, the discrepancy between the pro- or antinociceptive effects of GalR2 agonists may be dependent on whether these agents are acting centrally or peripherally.54 The results presented herein suggest that a peripherally acting GalR2 analogue may be an effective analgesic. Similarly, NPY has been shown to play a neuromodulatory role in pain transmission. The analgesic activity of NPY-dPEG24 in the formalin assay was consistent with the previous report that NPY was active in the acute phase in this test following icv administration.84 NPY reduces signs of inflammatory and neuropathic pain.60 NPY also produces a long-lasting tonic inhibition of spinal nociception mediated by both Y1 and Y2 receptors.61 The role for the neuropeptide NPY system in the regulation of pain at the spinal

Figure 7. Design of analgesic NPY-dPEG24. NPY-dPEG24 was designed based on anticonvulsant NPY-B2 via the replacement of Lys-palmitoyl with dPEGylated Lys residue.

A few peptides with peripheral analgesic activities also was reported including CR845, CR665 (κ-, Cara Therapeutics Inc.),78 and LEF533.63 580



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(0.25 mL)/NMM (0.125 mL)/CH2Cl2 (5 mL) for 2 h. The resin was then washed with CH2Cl2 and 5% DIPEA/CH2Cl2 (v/v) to remove HAc, and sodium diethyldithiocarbamate solution in DMF (0.02 M) to remove palladium residues. The palmitic acid was conjugated to the resin using the same method as the dPEG coupling step. The peptide was cleaved from the resin with Reagent K (TFA/phenol/water/thioanisole/1,2-dithioethane 82.5:5:5:5:2.5 by volume), and was precipitated out of methyl tert-butyl ether.34 The crude peptide was purified with semipreparative HPLC (Vydac diphenyl column, 219TP101522). Buffer A (0.1% TFA in water) and buffer B (0.1% TFA, v/v, in 90% aqueous acetonitrile) were used to produce a linear gradient from 5 to 75% of buffer B over 50 min with a flow rate of 10 mL/min. The elution was monitored by UV detection at 220 nm. The purified analogues were quantified by measuring UV absorbance at 279.8 nm (molar absorbance coefficient ε = 7,000 cm−1 M−1). Peptide purification was monitored using an Alliance HPLC system with a linear gradient from 20% to 100% buffer B over 40 min. The purity of the final product was >95%. Syntheses of Gal-R2-dPEG24 and NPY-dPEG24. The GalR2-dPEG24 was synthesized via the coupling of FmocLys(Mmt)-OH on the Fmoc-Rink amide Clear resin before the assembling of peptide in the synthesizer. The N-terminal Trp was Boc-protected to facilitate the following dPEGylation step. In the synthesis of NPY-dPEG24, Fmoc-Lys(Aloc)-OH was coupled following the manual coupling of Fmoc-Ahx-OH; the N-terminus of the peptide was acetylated with acetic anhydride/ pyridine (1/20 by volume) after all amino acids were assembled on the resin. The Mmt group of Gal-R2-dPEG24 and the Aloc group of NPY-dPEG24 were deprotected with the same methods applied in the synthesis of Gal-B2-dPEG24; and then the m-dPEG-24 acid was coupled to give the target compounds. Partition Coefficient (LogD). The partition coefficient (logD) was determined by the shake-flask method. The peptide standard curves were first determined on RP-HPLC over a gradient ranging from 5 to 95% buffer B in 40 min; the great coefficients (r2) of Gal-B2-dPEG24, Gal-R2-dPEG24 and NPY-dPEG24 were 0.9998, 1.000 and 1.000, respectively, at the concentration of 80−400 μg/mL. A peptide sample (400 μg) was reconstituted in phosphatebuffered saline (PBS, 1 mL, pH 7.4). The equal volumes of peptide solution and n-octanol (saturated with water for 24 h) were mixed on a rotary mixer for 24 h. The aqueous layer was injected into HPLC, and the peptide concentration of the aqueous layer was calculated from the standard curve equation based on the HPLC peak integration of the aqueous layer. All assays were repeated at least three times. The logD value was calculated using the following equation:

level suggests that NPY analogues acting outside the brain may be effective tools for producing analgesia. In agreement with this, we show here that a peripherally selective NPY analogue, NPY-dPEG24, effectively reduces nociceptive responses in an inflammatory model of pain and supports the further evaluation of this analogue for the treatment of pain. This work extends a repertoire of systemically active analogues of anticonvulsant neuropeptides, such as GAL, NPY, NPW and NT.34−36,38,39,41 The three analogues described here, namely, Gal-B2-dPEG24, Gal-R2-dPEG24 and NPY-dPEG24, offer pharmacological tools that can be applied for reducing neuronal hyperexcitability that follows insults and injury on the peripheral nerves. Potential disease-modifying properties of anticonvulsant neuropeptides make these analogues useful to explore unique therapeutic strategies beyond symptomatic treatments.33 For example, judicious time- and sequence- dependent combinations of various anticonvulsant neuropeptides may deliver faster therapeutic outcomes, as compared to a monotherapy treatment. This work encourages further exploration of monodisperse oligoethyleneglycol residues toward more neuroactive peptides that mediate their actions in the central and peripheral nervous systems.

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EXPERIMENTAL SECTION General Synthetic Procedures. The Fmoc-Rink amide Clear resin was obtained from Peptide International Inc. The m-dPEG-24 acid was purchased from Quanta Biodesign Ltd. All the other Fmoc-amino acids were purchased from Chem-impex International Inc. Reagents and chemicals were obtained from Sigma-Aldrich Chemical Corporation and used without further purification. The automatic solid phase peptide synthesis was carried out in a Symphony peptide synthesizer (Protein Technologies Inc.). The manual coupling reactions were performed under N2 atmosphere, unless otherwise indicated. The HPLC mobile phases were buffer A, water (0.1% TFA), and buffer B, 90% acetonitrile in water (0.1% TFA). The peptide purification was carried out using a semipreparative diphenyl column (Vydac, 219TP101522) and a Waters 600 pump system equipped with a Waters 2487 dual wavelength detector (λ1 = 220 nm, λ2 = 280 nm). The purities of peptides and logD assays were determined on a Vydac diphenyl column (218TP54) in Waters Alliance 2695 system. Peptides were quantified on a Cary 50 Bio UV−visible spectrophotometer. The peptide metabolic stability assays were monitored using YMC ODSA S-5 120 Å column (AA12S052503WT) and Waters Alliance 2695 system. The metabolic stability assays were performed using an Eppendorf thermomixer. The peptide identities were verified by MALDI-TOF MS at the University of Utah Core Facility. Synthesis of Gal-B2-dPEG24. Fmoc-Lys(Mmt)-OH was manually coupled to the Fmoc-Rink amide Clear resin (0.4 meq, 50 μmol scale) by PyBop method (Fmoc-amino acid/PyBop/ DIPEA/resin (2:1.96:4:1, molar ratio), followed by the coupling of Fmoc-Lys(Boc)-OH, Fmoc-Lys(Aloc)-OH and all the other residues on automatic peptide synthesizer. The peptide N-terminus was conjugated with Boc-sarcosine. After coupling of all the amino acids, HAc/TFE/CH2Cl2 (1:2:7 by volume, 10 mL) was added to the resin and shaken for 10 min to remove the Mmt group. This Mmt deprotection process was repeated six times until the solution color changed from yellow to clear. The resin was then neutralized with 5% DIPEA/CH2Cl2. The m-dPEG-24 acid (75 μmol, 1.5-fold) was coupled to the resin using PyBop method (m-dPEG-24 acid/PyBop/HOBt/DIPEA (1:0.98:1:2 molar ratio)) for 24 h. The Aloc protection group was deprotected with Pd(PPh3)4 (0.16 g, 0.14 mmol) in HAc

logD = log([peptideoctanol ]/[peptideaqueous]) = log(([peptide] − [peptideaqueous])/[peptideaqueous])

Serum Stability Assay. The peptide metabolic stability was tested in 25% rat blood serum as previously described.34,36,38,41 The peptide, serum and Tris solution (tris(hydroxymethyl)aminomethane, 1 M) were constituted to the final concentration of peptide 20 μM, Tris 0.1 M, serum 25%. The sample at time 0 min (100 μL) was drawn just after the addition of serum to peptide−Tris solution, and then quenched with the quenching solution (trichloroacetic acid:isopropanol:water, 15:40:45 by volume, 50 μL). The quenched samples were incubated at −15 °C for 30 min, followed by centrifugation at 14,000 rpm for 5 min to remove serum proteins. The supernatants (100 μL) 581



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measured. Results were compared to those obtained from vehicle-treated control animals. The area under the curve (AUC) and subsequent percent of control for test compound-treated animal groups is determined using the GraphPad Prism. The Total AUC is calculated for both the test and control groups for both the acute and inflammatory phases. The AUC for individual animals for each phase is also calculated and converted to the percentage of total AUC of control. The average percentages and SEM for both the test compound treated and control groups are calculated. Carrageenan-Induced Thermal Hyperalgesia. A state of localized inflammation was induced in mice (25−35 g) by injecting carrageenan (25 μL, 2% λ-carrageenan in 0.9% NaCl) subcutaneously into the plantar surface of the right hind paw.68,69 The paw withdrawal responses from thermal stimulation were assessed according to previously described methods.70−72 Mice were placed in plexiglass chambers on top of a heated glass surface (30 °C). The thermal stimulation was applied with a projection bulb below the glass surface. The latency to paw withdrawal was measured from the onset of heat application until a full paw withdrawal occurred. Two measurements were obtained from each paw, with at least 1 min between assessments, and subsequently averaged to obtain the mean withdrawal latency of each paw. Experimental conditions, including animal habituation, glass plate temperature, and thermal stimulus intensity (35% of maximum), were optimized such that the baseline withdrawal latencies for contralateral (noninjected) and ipsilateral (carrageenan-injected) paws were 6−9 s and 2−4 s, respectively. Mice were treated with test compounds such that the time-topeak effect for each compound (1 and 2 h for Gal-R2-dPEG24 and NPY-dPEG24, respectively) coincided with the peak inflammatory effect of carrageenan (3 h). Vehicle-treated animals (1% Tween 20 in PBS) were also included with each group and tested at the comparative time points for Gal-R2-dPEG24 and NPYdPEG24, 1 and 2 h, respectively. The paw withdrawal latencies were measured 3 h following carrageenan administration, and either 1 or 2 h following analogue administration, as described above. All data are presented as means ± standard error. The mean withdrawal latencies for each paw in each group were compared, and the mean latency difference (ipsilateral paw latency minus contralateral paw latency)85 for each group is presented in Figure 9. Comparisons between multiple means were performed using a one-way ANOVA followed by a Newman−Keuls post hoc analysis. P < 0.05 was considered significant. Anticonvulsant Assay. Each analogue was administered to adult male CF-1 mice at doses of 4 mg/kg for ip injections, or 1 and 4 nmol per 5 μL injection volume for icv injections. Following drug administration, groups of mice (n = 4) received a 6 Hz (32 mA) corneal stimulation at one of five time points (15 min, 30 min, 1 h, 2 h, and 4 h). Mice not displaying a characteristic limbic seizure (jaw chomping, forelimb clonus, straub tail) were considered protected. The area under the curve (AUC) was calculated from the percentage of mice protected plotted as a function of time. Animal Care. Adult male CF-1 albino mice (26−35 g), obtained from Charles River (Portage, MI), were utilized for behavioral testing in the 6 Hz (32 mA) seizure test and the carrageenan-induced and formalin-induced nociception assays. The animals were allowed free access to food (Prolab RMH 3000) and water except during the short time they were removed from their cage for testing. The animals were housed, fed, and handled in a manner consistent with the recommendations in the

were injected into the RP-HPLC equipped with a YMC ODS-A S-5 120 Å column, eluted with a linear gradient of 5% to 60% buffer B over 55 min, monitored at 220 nm. The mixed solutions were incubated at 37 °C; and aliquots of different time points (up to 150 h) were drawn and the intact peptide concentrations were determined by RP-HPLC. All assays were repeated at least three independent experiments. The half-life was calculated based on this equation: t1/2 = [ln(50) − b]/m, where b is the y-intercept and m is the slope of the line. Receptor Binding Assay. A competitive fluorescence-based binding assay with the europium-labeled galanin was employed to determine binding affinities for the GalR1 and GalR2 receptors. The binding assays were performed on the AcroWell 96-well filter plates (Pall Life Sciences) using the purchased hGaR1 and hGalR2 receptor membrane preparations (Millipore and Perkin-Elmer), the europium-labeled galanin (Perkin-Elmer), and the DELFIA binding and wash buffers (Perkin-Elmer). The binding assays were performed in triplicate with 6 μg of the membrane protein (1.4 pmol/mg protein) and 2 nM of the europium-galanin, in a volume of 100 μL of the binding buffer (50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 25 μM ethylenediaminetetraacetic acid, and 0.2% bovine serum albumin). The galanin analogues were incubated at room temperature for 90 min, followed by 4 times of washing with the wash buffer (50 mM Tris-HCl, pH 7.5 and 5 mM MgCl2) using a vacuum manifold. The DELFIA enhancement solution (200 μL) was added, and the plates were incubated at room temperature for 30 min. The plates were read on a Victor plate reader (PerkinElmer) using a standard time-resolved fluorescence measurement for europium-based compounds (excitation at 340 nm, delay for 400 μs, and emission at 615 nm). The competition binding curves were analyzed with the GraphPad Prism software using a sigmoidal dose−response (variable slope) equation for nonlinear regression analysis. Inhibition of Forskolin-Mediated cAMP Production by NPY-dPEG24. The CHO-K1 cells transfected with human NPY2R (Discoverx, CA) were plated at 30,000 cells/well in a 96-well format. The inhibition of forskolin-mediated cAMP production was determined using the cAMP detection kit (DiscoveRx, Fremont, CA) according to the manufacturer’s instruction. The assay was performed in triplicate. Briefly, after 24 h of incubation, the medium was removed from the well, and increasing concentrations (up to 1 μM) of NPY-dPEG24 were coincubated with 20 μM of forskolin at 37 °C for 30 min. The amount of cAMP in the cell was determined measuring the luminescence on a Victor plate reader (Perkin-Elmer). The results were analyzed with the GraphPad Prism using the sigmoidal dose−response (variable slope) classical equation for nonlinear regression analysis. Three independent experiments were performed to generate the average EC50. Formalin Pain Assay. Test compounds were injected (ip) 1 h prior to the injection of 0.5% formalin (20 μL; 27 gauge needle) subdermally into the plantar region of the right hindpaw of mice. Formalin elicits a distinct biphasic behavioral profile characterized by the mouse licking the affected paw. Immediately following the injection the mouse licks the paw for about 10 min. This is phase I (acute) and is followed by a brief latent period where there is little behavioral activity. A more prolonged period of about 20 to 30 min of paw licking ensues, which constitutes phase II (inflammatory). Following the injection of the formalin each animal is observed for the first 2 min of 5 min epochs until 40 min has elapsed since the administration of the formalin. The cumulative length of licking for each 2 min time period is 582



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National Research Council Publication, “Guide for the Care and Use of Laboratory Animals.” All animals were euthanized in accordance with the Institute of Laboratory Resource policies on the humane care of laboratory animals.

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(8) Gaberc-Porekar, V.; Zore, I.; Podobnik, B.; Menart, V. Obstacles and pitfalls in the PEGylation of therapeutic proteins. Curr. Opin. Drug Discovery Dev. 2008, 11, 242−50. (9) Chen, X.; Park, R.; Hou, Y.; Khankaldyyan, V.; Gonzales-Gomez, I.; Tohme, M.; Bading, J. R.; Laug, W. E.; Conti, P. S. MicroPET imaging of brain tumor angiogenesis with 18F-labeled PEGylated RGD peptide. Eur. J. Nucl. Med. Mol. Imaging 2004, 31, 1081−9. (10) Gao, W.; Liu, W.; Christensen, T.; Zalutsky, M. R.; Chilkoti, A. In situ growth of a PEG-like polymer from the C terminus of an intein fusion protein improves pharmacokinetics and tumor accumulation. Proc. Natl. Acad. Sci. U.S.A 2010, 107, 16432−7. (11) Gao, W.; Liu, W.; Mackay, J. A.; Zalutsky, M. R.; Toone, E. J.; Chilkoti, A. In situ growth of a stoichiometric PEG-like conjugate at a protein’s N-terminus with significantly improved pharmacokinetics. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 15231−6. (12) Glaser, M.; Morrison, M.; Solbakken, M.; Arukwe, J.; Karlsen, H.; Wiggen, U.; Champion, S.; Kindberg, G. M.; Cuthbertson, A. Radiosynthesis and biodistribution of cyclic RGD peptides conjugated with novel [18F]fluorinated aldehyde-containing prosthetic groups. Bioconjugate Chem. 2008, 19, 951−7. (13) Li, L.; Crow, D.; Turatti, F.; Bading, J. R.; Anderson, A.-L.; Poku, E.; Yazaki, P. J.; Carmichael, J.; Leong, D.; Wheatcroft, M. P.; Raubitschek, A. A.; Hudson, P. J.; Colcher, D.; Shively, J. E. Sitespecific conjugation of monodispersed DOTA-PEGn to a thiolated diabody reveals the effect of increasing PEG size on kidney clearance and tumor uptake with improved 64-Copper PET imaging. Bioconjugate Chem. 2011, 22, 709−16. (14) Kerr, B. D.; Irwin, N.; Flatt, P. R.; Gault, V. A. Prolonged GIP receptor activation using stable mini-PEGylated GIP improves glucose homeostasis and beta-cell function in age-related glucose intolerance. Peptides 2009, 30, 219−25. (15) Shechter, Y.; Heldman, E.; Sasson, K.; Bachar, T.; Popov, M.; Fridkin, M. Delivery of neuropeptides from the periphery to the brain: studies with enkephalin. ACS Chem. Neurosci. 2010, 1, 399−406. (16) N’Da, D. D.; Breytenbach, J. C.; Breytenbach, J. W. Synthesis and in vitro human skin penetration of oligo- and polymeric ethylene glycol carbonates of zidovudine and stavudine. Arzneim. Forsch. 2010, 60, 575−82. (17) Zhang, W.; Oya, S.; Kung, M.-P.; Hou, C.; Maier, D. L.; Kung, H. F. F-18 Polyethyleneglycol stilbenes as PET imaging agents targeting Aβ aggregates in the brain. Nucl. Med. Biol. 2005, 32, 799−809. (18) Bentley, M. D.; Viegas, T. X.; Goodin, R. R.; Cheng, L.; Zhao, X. Chemically modified small molecules. US 7,786,133, 2010. (19) Mallikaratchy, P. R.; Ruggiero, A.; Gardner, J. R.; Kuryavyi, V.; Maguire, W. F.; Heaney, M. L.; McDevitt, M. R.; Patel, D. J.; Scheinberg, D. A. A multivalent DNA aptamer specific for the B-cell receptor on human lymphoma and leukemia. Nucleic Acids Res. 2011, 39, 2458−69. (20) Berna, M.; Dalzoppo, D.; Pasut, G.; Manunta, M.; Izzo, L.; Jones, A. T.; Duncan, R.; Veronese, F. M. Novel monodisperse peg-dendrons as new tools for targeted drug delivery: synthesis, characterization and cellular uptake. Biomacromolecules 2005, 7, 146−53. (21) Kozak, D.; Surawski, P.; Thoren, K. M.; Lu, C.-Y.; Marcon, L.; Trau, M. Improving the signal-to-noise performance of molecular diagnostics with PEG-lysine copolymer dendrons. Biomacromolecules 2009, 10, 360−5. (22) Sanclimens, G.; Shen, H.; Giralt, E.; Albericio, F.; Saltzman, M. W.; Royo, M. Synthesis and screening of a small library of proline-based biodendrimers for use as delivery agents. Biopolymers 2005, 80, 800−14. (23) Felipe, M. J. L.; Ponnapati, R. R.; Pernites, R. B.; Dutta, P.; Advincula, R. C. Synthesis and electrografting of dendron anchored oegylated surfaces and their protein adsorption resistance. ACS Appl. Mater. Interfaces 2010, 2, 3401−5. (24) Kizhakkedathu, J. N.; Janzen, J.; Le, Y.; Kainthan, R. K.; Brooks, D. E. Poly(oligo(ethylene glycol)acrylamide) brushes by surface initiated polymerization: effect of macromonomer chain length on brush growth and protein adsorption from blood plasma. Langmuir 2009, 25, 3794− 801.

ASSOCIATED CONTENT

S Supporting Information *

Figure S1 showing the HPLC chromatograms of Gal-B2-dPEG24. Figure S2 showing the metabolic stability studies of Gal-B2dPEG24 and Gal-R2-dPEG24. Figure S3 showing the metabolic stability study of NPY-dPEG24. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Department of Medicinal Chemistry, College of Pharmacy, University of Utah, 421 Wakara Way, Suite 360, Salt Lake City, Utah 84108, United States. E-mail: [email protected]. Phone: +1 801 581 4629. Fax: +1 801 581 7087. Notes

The authors declare the following competing financial interest(s): GB and HSW are scientific co-founders of NeuroAdjuvants. This COI has been disclosed in the manuscript.

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ACKNOWLEDGMENTS This work was supported by NIH Grant U01 NS 066991. ABBREVIATIONS USED Ahx, aminohexanoic acid; Aloc, allyloxycarbonyl; AUC, area under the curve; BBB, blood−brain barrier; cAMP, cyclic adenosine monophosphate; CNS, central nervous system; DIPEA, N,N-diisopropylethylamine; dPEG, discrete oligoethyleneglycol; GAL, galanin; GalR1, galanin receptor subtype 1; GalR2, galanin receptor subtype 2; ip, intraperitoneal; icv, intracerebroventricular; Lys-Pal, Nε-palmitoyl-L-lysine; LysdPEG24, Nε-dPEG24-L-lysine; Mmt, 4-methoxytrityl; NMM, Nmethylmorpholine; NPY, neuropeptide Y; NPW, neuropeptide W; PNS, peripheral nervous system; PyBop, (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate; Sar, sarcosine; SPPS, solid phase peptide synthesis; TFE, 2,2,2-trifluoroethanol; Y1, NPY receptor subtype 1; Y2, NPY receptor subtype 2

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REFERENCES

(1) Harris, J. M.; Chess, R. B. Effect of pegylation on pharmaceuticals. Nat. Rev. Drug Discovery 2003, 2, 214−21. (2) Knop, K.; Hoogenboom, R.; Fischer, D.; Schubert, U. Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives. Angew. Chem., Int. Ed. 2010, 49, 6288−308. (3) Roberts, M. J.; Bentley, M. D.; Harris, J. M. Chemistry for peptide and protein PEGylation. Adv. Drug Delivery Rev. 2002, 54, 459−76. (4) Ryan, S. M.; Mantovani, G.; Wang, X.; Haddleton, D. M.; Brayden, D. J. Advances in PEGylation of important biotech molecules: delivery aspects. Expert Opin. Drug Delivery 2008, 5, 371−83. (5) Veronese, F. M.; Pasut, G. PEGylation, successful approach to drug delivery. Drug Discovery Today 2005, 10, 1451−8. (6) Witt, K. A.; Huber, J. D.; Egleton, R. D.; Roberts, M. J.; Bentley, M. D.; Guo, L.; Wei, H.; Yamamura, H. I.; Davis, T. P. Pharmacodynamic and pharmacokinetic characterization of poly(ethylene glycol) conjugation to Met-enkephalin analog [d-Pen2,d-Pen5]-enkephalin (DPDPE). J. Pharmacol. Exp. Ther. 2001, 298, 848−56. (7) Fee, C. J.; Van Alstine, J. M. PEG-proteins: Reaction engineering and separation issues. Chem. Eng. Sci. 2006, 61, 924−39. 583



Article

(25) Hartmann, L.; Börner, H. G. Precision polymers: monodisperse, monomer-sequence-defined segments to target future demands of polymers in medicine. Adv. Mater. 2009, 21, 3425−31. (26) Wilson, M. J.; Liliensiek, S. J.; Murphy, C. J.; Murphy, W. L.; Nealey, P. F. Hydrogels with well-defined peptide-hydrogel spacing and concentration: impact on epithelial cell behavior. Soft Matter 2012, 8, 390−8. (27) Collier, J. H.; Messersmith, P. B. Self-Assembling polymer− peptide conjugates: nanostructural tailoring. Adv. Mater. 2004, 16, 907− 10. (28) Ansell, S. M.; Kojic, L. D.; Hankins, J. S.; Sekirov, L.; Boey, A.; Lee, D. K.; Bennett, A. R.; Klimuk, S. K.; Harasym, T. O.; Santos, N. D.; Semple, S. C. Application of oligo-(14-amino-3,6,9,12-tetraoxatetradecanoic acid) lipid conjugates as steric barrier molecules in liposomal formulations. Bioconjugate Chem. 1999, 10, 653−66. (29) Bach, A.; Chi, C. N.; Pang, G. F.; Olsen, L.; Kristensen, A. S.; Jemth, P.; Strømgaard, K. Design and synthesis of highly potent and plasma-stable dimeric inhibitors of the psd-95−NMDA receptor interaction. Angew. Chem., Int. Ed. 2009, 121, 9865−9. (30) Ziegler, A.; Seelig, J. Contributions of glycosaminoglycan binding and clustering to the biological uptake of the nonamphipathic cellpenetrating peptide WR9. Biochem. 2011, 50, 4650−64. (31) Mero, A.; Schiavon, M.; Veronese, F. M.; Pasut, G. A new method to increase selectivity of transglutaminase mediated PEGylation of salmon calcitonin and human growth hormone. J. Controlled Release 2011, 154, 27−34. (32) Mero, A.; Spolaore, B.; Veronese, F. M.; Fontana, A. Transglutaminase-mediated PEGylation of proteins: direct identification of the sites of protein modification by mass spectrometry using a novel monodisperse PEG. Bioconjugate Chem. 2009, 20, 384−9. (33) Robertson, C. R.; Flynn, S. P.; White, H. S.; Bulaj, G. Anticonvulsant neuropeptides as drug leads for neurological diseases. Nat. Prod. Rep. 2011, 28, 741−62. (34) Bulaj, G.; Green, B. R.; Lee, H. K.; Robertson, C. R.; White, K.; Zhang, L.; Sochanska, M.; Flynn, S. P.; Scholl, E. A.; Pruess, T. H.; Smith, M. D.; White, H. S. Design, synthesis, and characterization of high-affinity, systemically-active galanin analogues with potent anticonvulsant activities. J. Med. Chem. 2008, 51, 8038−47. (35) Green, B. R.; Smith, M.; White, K. L.; White, H. S.; Bulaj, G. Analgesic Neuropeptide W Suppresses Seizures in the Brain Revealed by Rational Repositioning and Peptide Engineering. ACS Chem. Neurosci. 2011, 2, 51−6. (36) Green, B. R.; White, K. L.; McDougle, D. R.; Zhang, L.; Klein, B.; Scholl, E. A.; Pruess, T. H.; White, H. S.; Bulaj, G. Introduction of lipidization-cationization motifs affords systemically bioavailable neuropeptide Y and neurotensin analogs with anticonvulsant activities. J. Pept. Sci. 2010, 16, 486−95. (37) Robertson, C. R.; Pruess, T. H.; Grussendorf, E.; White, H. S.; Bulaj, G. Generating orally active galanin analogues with analgesic activities. ChemMedChem 2012, 7, 903−9. (38) Robertson, C. R.; Scholl, E. A.; Pruess, T. H.; Green, B. R.; White, H. S.; Bulaj, G. Engineering galanin analogues that discriminate between GalR1 and GalR2 receptor subtypes and exhibit anticonvulsant activity following systemic delivery. J. Med. Chem. 2010, 53, 1871−5. (39) White, H. S.; Scholl, E. A.; Klein, B. D.; Flynn, S. P.; Pruess, T. H.; Green, B. R.; Zhang, L.; Bulaj, G. Developing novel antiepileptic drugs: characterization of NAX 5055, a systemically-active galanin analog, in epilepsy models. Neurotherapeutics 2009, 6, 372−80. (40) Zhang, L.; Bulaj, G. Converting peptides into drug leads by lipidation. Curr. Med. Chem. 2012, 19, 1602−18. (41) Zhang, L.; Robertson, C. R.; Green, B. R.; Pruess, T. H.; White, H. S.; Bulaj, G. Structural requirements for a lipoamino acid in modulating the anticonvulsant activities of systemically active galanin analogues. J. Med. Chem. 2009, 52, 1310−6. (42) Burgevin, M. C.; Loquet, I.; Quarteronet, D.; Habert-Ortoli, E. Cloning, pharmacological characterization, and anatomical distribution of a rat cDNA encoding for a galanin receptor. J. Mol. Neurosci. 1995, 6, 33−41.

(43) El Bahh, B.; Balosso, S.; Hamilton, T.; Herzog, H.; BeckSickinger, A. G.; Sperk, G.; Gehlert, D. R.; Vezzani, A.; Colmers, W. F. The anti-epileptic actions of neuropeptide Y in the hippocampus are mediated by Y and not Y receptors. Eur. J. Neurosci. 2005, 22, 1417−30. (44) Mazarati, A. M. Galanin and galanin receptors in epilepsy. Neuropeptides 2004, 38, 331−43. (45) Brumovsky, P.; Shi, T. S.; Landry, M.; Villar, M. J.; Hökfelt, T. Neuropeptide tyrosine and pain. Trends Pharmacol. Sci. 2007, 28, 93− 102. (46) Dray, A. Inflammatory mediators of pain. Br. J. Anaesth. 1995, 75, 125−31. (47) Gibbs, J. L.; Diogenes, A.; Hargreaves, K. M. Neuropeptide Y modulates effects of bradykinin and prostaglandin E2 on trigeminal nociceptors via activation of the Y1 and Y2 receptors. Br. J. Pharmacol. 2007, 150, 72−9. (48) Hökfelt, T.; Wiesenfeld-Hallin, Z.; Villar, M.; Melander, T. Increase of galanin-like immunoreactivity in rat dorsal root ganglion cells after peripheral axotomy. Neurosci. Lett. 1987, 83, 217−20. (49) Landry, M.; Åman, K.; Dostrovsky, J.; Lozano, A. M.; Carlstedt, T.; Spenger, C.; Josephson, A.; Wiesenfeld-Hallin, Z.; Hökfelt, T. Galanin expression in adult human dorsal root ganglion neurons: initial observations. Neuroscience 2003, 117, 795−809. (50) Zhang, X.; Ju, G.; Elde, R.; Hökfelt, T. Effect of peripheral nerve cut on neuropeptides in dorsal root ganglia and the spinal cord of monkey with special reference to galanin. J. Neurocytol. 1993, 22, 342− 81. (51) Lang, R.; Gundlach, A. L.; Kofler, B. The galanin peptide family: receptor pharmacology, pleiotropic biological actions, and implications in health and disease. Pharmacol. Ther. 2007, 115, 177−207. (52) Bartfai, T.; Lu, X.; Badie-Mahdavi, H.; Barr, A. M.; Mazarati, A.; Hua, X.-Y.; Yaksh, T.; Haberhauer, G.; Ceide, S. C.; Trembleau, L.; Somogyi, L.; Kröck, L.; Rebek, J. Galmic, a nonpeptide galanin receptor agonist, affects behaviors in seizure, pain, and forced-swim tests. Proc. Natl. Acad. Sci. U.S.A 2004, 101, 10470−5. (53) Flatters, S. J.; Fox, A. J.; Dickenson, A. H. Nerve injury induces plasticity that results in spinal inhibitory effects of galanin. Pain 2002, 98, 249−58. (54) Hulse, R. P.; Wynick, D.; Donaldson, L. F. Activation of the galanin receptor 2 in the periphery reverses nerve injury-induced allodynia. Mol. Pain 2011, 7, doi: 10.1186/1744-8069-7-26. (55) Hygge-Blakeman, K.; Brumovsky, P.; Hao, J. X.; Xu, X. J.; Hökfelt, T.; Crawley, J. N.; Wiesenfeld-Hallin, Z. Galanin over-expression decreases the development of neuropathic pain-like behaviors in mice after partial sciatic nerve injury. Brain Res. 2004, 1025, 152−8. (56) Scholl, E. A.; Smith, M. D.; Klein, B. D.; Flynn, S. P.; Pruess, T. H.; Green, B. R.; Zhang, L.; Bulaj, G. Anticonvulsant and analgesic profile of NAX-5055: a high affinity, metabolically stable, and blood-brain barrier penetrant galanin-based analog. In 39th Annual Society for Neuroscience Meeting, Washington, DC, 2009. (57) Wiesenfeld-Hallin, Z.; Bartfai, T.; Hökfelt, T. Galanin in sensory neurons in the spinal cord. Front. Neuroendocrinol. 1992, 13, 319−43. (58) Waters, S. M.; Krause, J. E. Distribution of galanin-1, -2 and -3 receptor messenger RNAs in central and peripheral rat tissues. Neuroscience 1999, 95, 265−71. (59) Gibson, S. J.; Polak, J. M.; Allen, J. M.; Adrian, T. E.; Kelly, J. S.; Bloom, S. R. The distribution and origin of a novel brain peptide, neuropeptide Y, in the spinal cord of several mammals. J. Comp. Neurol. 1984, 227, 78−91. (60) Smith, P. A.; Moran, T. D.; Abdulla, F.; Tumber, K. K.; Taylor, B. K. Spinal mechanisms of NPY analgesia. Peptides 2007, 28, 464−74. (61) Solway, B.; Bose, S. C.; Corder, G.; Donahue, R. R.; Taylor, B. K. Tonic inhibition of chronic pain by neuropeptide Y. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 7224−9. (62) Hulse, R. P.; Donaldson, L. F.; Wynick, D. Peripheral galanin receptor 2 as a target for the modulation of pain. Pain Res. Treat. 2012, 545386. (63) DeHaven-Hudkins, D. L.; Dolle, R. E. Peripherally restricted opioid agonists as novel analgesic agents. Curr. Pharm. Des. 2004, 10, 743−57. 584



Article

(84) Mellado, M. L.; Gibert-Rahola, J.; Chover, A. J.; Mico, J. A. Effect on nociception of intracerebroventricular administration of low doses of neuropeptide Y in mice. Life Sci. 1996, 58, 2409−14. (85) Jones, C. K.; Peters, S. C.; Shannon, H. E. Synergistic interactions between the dual serotonergic, noradrenergic reuptake inhibitor duloxetine and the non-steroidal anti-inflammatory drug ibuprofen in inflammatory pain in rodents. Eur. J. Pain 2007, 11, 208−15.

(64) Brunsveld, L.; Kuhlmann, J.; Alexandrov, K.; Wittinghofer, A.; Goody, R. S.; Waldmann, H. Lipidated ras and rab peptides and proteinssynthesis, structure, and function. Angew. Chem., Int. Ed. 2006, 45, 6622−46. (65) Lee, H. K.; Zhang, L.; Smith, M. D.; White, H. S.; Bulaj, G. Glycosylated neurotensin analogues exhibit sub-picomolar anticonvulsant potency in a pharmacoresistant model of epilepsy. ChemMedChem 2009, 4, 400−5. (66) Zhang, L.; Lee, H. K.; Pruess, T. H.; White, H. S.; Bulaj, G. Synthesis and applications of polyamine amino acid residues: improving the bioactivity of an analgesic neuropeptide, neurotensin. J. Med. Chem. 2009, 52, 1514−7. (67) Zorrilla, E. P.; Brennan, M.; Sabino, V.; Lu, X.; Bartfai, T. Galanin type 1 receptor knockout mice show altered responses to high-fat diet and glucose challenge. Physiol. Behav. 2007, 91, 479−85. (68) Gonzalez-Rodriguez, S.; Hidalgo, A.; Baamonde, A.; Menendez, L. Spinal and peripheral mechanisms involved in the enhancement of morphine analgesia in acutely inflamed mice. Cell. Mol. Neurobiol. 2010, 30, 113−21. (69) Kerr, B. J.; Gupta, Y.; Pope, R.; Thompson, S. W.; Wynick, D.; McMahon, S. B. Endogenous galanin potentiates spinal nociceptive processing following inflammation. Pain 2001, 93, 267−77. (70) Dirig, D. M.; Salami, A.; Rathbun, M. L.; Ozaki, G. T.; Yaksh, T. L. Characterization of variables defining hindpaw withdrawal latency evoked by radiant thermal stimuli. J. Neurosci. Methods 1997, 76, 183− 91. (71) Hargreaves, K.; Dubner, R.; Brown, F.; Flores, C.; Joris, J. A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 1988, 32, 77−88. (72) Hua, X. Y.; Svensson, C. I.; Matsui, T.; Fitzsimmons, B.; Yaksh, T. L.; Webb, M. Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia. Eur. J. Neurosci. 2005, 22, 2431−40. (73) Halina, M. Targeting of opioid-producing leukocytes for pain control. Neuropeptides 2007, 41, 355−63. (74) Stein, C.; Clark, J. D.; Oh, U.; Vasko, M. R.; Wilcox, G. L.; Overland, A. C.; Vanderah, T. W.; Spencer, R. H. Peripheral mechanisms of pain and analgesia. Brain Res. Rev. 2009, 60, 90−113. (75) Parada, C. A.; Tambeli, C. H.; Green, P. G.; Cairns, B. E. Primary Afferent Nociceptor as a Target for the Relief of Pain. Pain Res. Treat. 2012, 2012, doi: 10.1155/2012/348043. (76) Hua, S.; Cabot, P. J. Mechanisms of peripheral immune-cellmediated analgesia in inflammation: clinical and therapeutic implications. Trends Pharmacol. Sci. 2010, 31, 427−33. (77) Baron, R. Mechanisms of disease: neuropathic paina clinical perspective. Nat. Clin. Pract. Neurol. 2006, 2, 95−106. (78) Vanderah, T. W.; Largent-Milnes, T.; Lai, J.; Porreca, F.; Houghten, R. A.; Menzaghi, F.; Wisniewski, K.; Stalewski, J.; SueirasDiaz, J.; Galyean, R.; Schteingart, C.; Junien, J.-L.; Trojnar, J.; Rivière, P. J. M. Novel d-amino acid tetrapeptides produce potent antinociception by selectively acting at peripheral κ-opioid receptors. Eur. J. Pharmacol. 2008, 583, 62−72. (79) Crawley, J. N. Biological actions of galanin. Regul. Pept. 1995, 59, 1−16. (80) Mitsukawa, K.; Lu, X.; Bartfai, T. Galanin, galanin receptors and drug targets. Cell. Mol. Life Sci. 2008, 65, 1796−805. (81) Kerr, B. J.; Cafferty, W. B.; Gupta, Y. K.; Bacon, A.; Wynick, D.; McMahon, S. B.; Thompson, S. W. Galanin knockout mice reveal nociceptive deficits following peripheral nerve injury. Eur. J. Neurosci. 2000, 12, 793−802. (82) Liu, H.; Hökfelt, T. Effect of intrathecal galanin and its putative antagonist M35 on pain behavior in a neuropathic pain model. Brain Res. 2000, 886, 67−72. (83) Liu, H. X.; Brumovsky, P.; Schmidt, R.; Brown, W.; Payza, K.; Hodzic, L.; Pou, C.; Godbout, C.; Hökfelt, T. Receptor subtype-specific pronociceptive and analgesic actions of galanin in the spinal cord: selective actions via GalR1 and GalR2 receptors. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 9960−4. 585


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