Galanin-2 receptor agonists

Abstract

The present invention relates to chimeric proteins comprising (i) a galanin fragment consisting of residues 2-13 of galanin (Gal2-13) or residues 2-13 of galanin (Gal2-12), or a variant thereof, and (ii) an FC domain, as well as pharmaceutical compositions comprising these proteins, which are suitable for use in the treatment of pain.

Description

[Technical Field]

[0001] The present invention relates to galanin-2 receptor (GalR2) agonists and their use in the treatment of pain. [Background technology]

[0002] Pain is typically associated with injury or damage, causing defensiveness and immobilization of the affected area, resulting in unpleasant sensory and emotional experiences, yet also providing protection and promoting rapid healing. Chronic nerve injury or damage (also called neuropathy) in humans induces changes in many properties of the primary sensory neurons of the dorsal root ganglion (DRG) and their central connections, leading to the development of spontaneous pain, allodynia (the perception of pain with normally innocuous stimuli), hyperalgesia (an exaggerated response to any given painful stimulus), and enlargement of the receptive pain field (a much larger area of ​​the body "hurts" in relation to a relatively focused stimulus). These functional changes result in the development of a chronic neuropathic pain state that is often resistant to conventional analgesic therapy.

[0003] Galanin was first isolated from pig intestine in 1983. 1 Then, in 1987, a cDNA was cloned from a rat anterior pituitary library. 2 In adult rodents, primates, and humans, galanin is expressed at low levels in less than 5% of intact DRG neurons, which are primarily small-diameter C-fiber nociceptors. 3~5 Also, the main afferent terminals of the spinal cord (lamina II), dorsal horn interneurons 6 , and many brain regions thought to be important for pain processing 7、8 After various nerve injury models of neuropathic pain (NP), described below, galanin levels in the DRG are elevated 5- to 10-fold in rodents, primates, and humans, and the peptide is abundantly expressed in approximately 20% to 30% of sensory neurons. 9~11The increase in expression in the dorsal horn after axotomy was modest compared with the marked increase in the DRG. 12 , as well as axotomized sympathetic neurons 14 , which partly reflects increased anterograde transport of galanin from the cell body to the injury site. 13 There is a possibility.

[0004] Behavioral and electrophysiological studies in intact rats have shown that intrathecal administration of galanin at low doses has a stimulatory effect on nociception (the neuronal signaling that underlies the sensation of pain). 15、16 , with significant inhibition at high doses 17、18 Following nerve injury, when endogenous levels of galanin in the DRG are high, there is good agreement in the literature that the neuropeptide inhibits synaptic strength in many spinal cord preparations from terminally anesthetized animals. 19 To further define the role of galanin in NP regulation in awake, freely moving animals, two novel transgenic lines were generated and characterized. One line inducibly overexpressed galanin (approximately fourfold) in the DRG after nerve injury. 20 In the other line, galanin was constitutively and ectopically overexpressed (approximately 5-fold) both in intact DRG and after nerve injury. 21 We then modified the rat spared nerve injury (SNI) model of NP and applied it to wild-type mice, demonstrating that allodynia develops within 24 hours of lesion and persists for at least 6 months in all mouse strains tested so far. Both novel galanin-overexpressing strains showed a significant reduction in mechanical allodynia in the SNI model, lasting for at least 3 months. 21 , demonstrating that tachyphylaxis does not occur in the face of persistently elevated levels of galanin. A similar reduction in NP behavior was subsequently demonstrated in transgenic mice ectopically overexpressing galanin in the DRG under the control of the dopamine beta-hydroxylase promoter. 22More recently, a novel transgenic line using a binary transgenic tet-off system was shown to repressibly overexpress galanin in the DRG. 26 Phenotypic analysis revealed that allodynia was significantly attenuated when galanin was overexpressed, and increased after galanin suppression. This novel transgenic line demonstrates that the neuropeptide can reduce NP behavior, regardless of whether galanin expression is increased upon nerve injury or only after allodynia has been established. 23 In summary, the above animal data, using various pharmacological and genetic tools and various NP models, support an inhibitory role for galanin in pain processing in intact animals, particularly after nerve injury.

[0005] The physiological effects of galanin are mediated by activation of one or more of three G protein-coupled galanin receptor subtypes, designated GalR1, GalR2, and GalR3. All three receptors are G i / o It couples to and inhibits adenylyl cyclase. 24、25 , and GalR2 is G q / 11 It also transmits signals that activate phospholipase C (PLC) and protein kinase C (PKC) via 26 Studies using in-situ hybridization have shown that GalR1 and GalR2 mRNAs are expressed by 51% and 83% of adult rat DRG neurons, respectively. 27 , levels of both subtypes decrease after axotomy 28、29 In contrast, no changes in the expression of either subtype were observed in the dorsal horn of the spinal cord after axotomy. 30 More recently, we used a specific and sensitive semiquantitative RT-PCR (Taqman) assay to measure mouse GalR1 and GalR2 levels in the DRG 1 week after axotomy and demonstrated that expression was reduced by 37% and 28%, respectively, in wild-type (WT) animals. 31GalR3 expression in the spinal cord and DRG of rats and mice was very low as determined by RT-PCR. 31、32 , which cannot be detected using in situ hybridization 33 .

[0006] Previous analyses of the nociceptive phenotype of galanin receptor knockout (KO) animals have provided little information regarding NP. GalR1-KO mice show no change in the degree of allodynia and only a slight deficit in recovery rate in two different models of NP. 34、35 Recently, a developmental defect was described in a subset of nociceptors (pain-sensing neurons) in the DRG of GalR2-KO mice shortly after birth. 31 Although this precludes the analysis of the role that GalR2 plays in NP in adult animals, it nevertheless allows these mutant mice to be used as a tissue source for various GalR2-dependent functional assays. GalR3-KO animals have been described. 66 However, no significant NP phenotypes have been published.

[0007] To date, few high-affinity galanin receptor ligands have been described. Gal2-11 (also known as AR-M1896) has equal affinity for GalR2 and GalR3 and does not activate GalR1. 36、37 Attempts by many groups to generate GalR1 or GalR2 specific peptide agonists have been largely unsuccessful, and only "receptor-preferring" agonists have been described to date. 38、39 Furthermore, both galanin and Gal2-11 have half-lives of less than 10 minutes in plasma. 40、41 However, systemic administration is not possible. Intrathecal injection of galanin or Gal2-11 in the rat NP model demonstrates that the central effect of galanin is likely mediated by activation of GalR1 in the spinal cord. 36Recently, activation of either GalR1 or GalR2 in the dorsal horn of the spinal cord (either using a cocktail of pan-GalR1 / 2 agonists combined with a GalR2 antagonist, or using AR-M1896) has been shown to alter the electrophysiological properties of inhibitory neurons, compatible with antinociceptive roles for both receptor subtypes. 42 This conclusion is further strengthened by the demonstration that GalR2 activation directly inhibits calcium currents in cultured DRG neurons. 43 .

[0008] These reports provide a possible mechanism by which activation of GalR2 in the DRG and / or dorsal horn reduces NP. These findings are consistent with previous studies showing that modulation of calcium channels by gabapentinoids, ziconotide, and opioids all reduce NP in patients. More recently, using electrophysiology, we have shown that exogenous galanin dose-dependently alters the responses of mechano-nociceptive C-fiber afferents in both naive and nerve-injured animals, facilitating mechano-nociceptor activity at low concentrations and significantly inhibiting mechano-nociceptor activity at high concentrations. 44 Furthermore, using the galanin fragment Gal2-11, it was confirmed that the action of galanin is mediated by GalR2 activation. The inhibitory effect of peripheral GalR2 activation was further supported by the demonstration that after a nerve injury model of NP, mechanosensitive nociceptors in galanin-overexpressing transgenic mice had significantly higher thresholds than wild-type animals, which was associated with a marked reduction in spontaneous neuronal firing and C-fiber concentration in the spinal cord. 44 .

[0009] Pharmacological studies of galanin and its receptors have primarily involved the use of truncated and conjugated versions of the native peptide. Full-length galanin exhibits high (single-digit nM) affinity for GalR1, GalR2, and GalR3. Rat, human, and porcine galanin exhibit similarly high affinity for the human receptor. 48Residues 1–15 are completely conserved across all species, and this fragment binds with high affinity to both GalR1 and GalR2. 49 The study showed that Gal1-15, but not Gal1-29, induced a functional response in the dorsal hippocampus region of the brain. 50 , showing that this fragment preferentially activates the GalR1-GalR2 heterocomplex. 51 Gal1-10 is the shortest fragment shown to retain binding activity, and further truncation results in total loss of affinity. 52 Residues 17-29 show the highest sequence divergence between species, and this fragment failed to displace radiolabeled galanin in binding studies or demonstrate a functional response when tested in vivo. 53 This has led to speculation that the primary role of this portion of galanin is to protect the N-terminal region (e.g., residues 1–15) from proteolysis. 54 .

[0010] Removal of the N-terminal glycine of galanin results in selectivity for GalR2 over GalR1. 55 Further truncation of the peptide to a 10-residue fragment, Gal2-11, completely abolishes GalR1 activity (and affinity) while retaining GalR2 activity. 56 A number of chimeric galanin agonists have also been described. 57 , a fragment of galanin fused to spantide, neuropeptide Y, mastoparan, and bradykinin. M617, consisting of residues 1-13 of galanin fused to residues 2-9 of bradykinin with a glutamine at position 14, exhibits approximately 25-fold selectivity for GalR1 over GalR2. 58M1145, which exhibits 90-fold selectivity for GalR2, is composed of galanin residues 1-13 and GALP residues. Further focus on therapeutic intervention led to the generation of another subset of galanin analogs aimed at improving pharmacokinetic properties. Nax505-5 (Gal-B2), a 17-residue galanin fragment containing lipoamino acids and an additional C-terminal lysine residue, has been shown to exhibit improved serum stability, blood-brain barrier permeability, and anti-inflammatory properties in seizure and epilepsy models. 59 Nax409-9 (also called GalR2-dPEG24) is a monodisperse oligo-ethylene glycol-containing galanin analog encompassing galanin residues 2-13. 60 , which was generated as a peripherally restricted GalR2-preferring analog. This molecule showed efficacy in neuropathic pain models, but the biological effect lasted less than 4 hours. Two non-peptide galanin analogs, galnon, 61 and Galmic 62 have been described, both of which have low affinity and are not receptor subtype specific.

[0011] Although the above studies suggest that GalR2 agonism may have an effect on neuropathic pain in model systems, there are no reports of long-lasting GalR2 agonists with the specificity required to provide effective pain treatment. Summary of the Invention

[0012] The present inventors unexpectedly discovered that chimeric proteins comprising a galanin (Gal) fragment consisting of Gal residues 2-13 or variants thereof and the fragment crystallizable (FC) region of an immunoglobulin molecule exhibit long-lasting and highly selective GalR2 agonism in vivo compared to chimeric proteins comprising the FC region in combination with other Gal fragments.

[0013] A first aspect of the present invention is a chimeric protein comprising: (i) a galanin fragment consisting of residues 2-13 of galanin (Gal2-13), or residues 2-12 of galanin (Gal2-12), or a variant thereof; (ii) the FC region; The present invention provides a chimeric protein comprising:

[0014] Preferably, the chimeric protein comprises: (i) a galanin fragment consisting of residues 2 to 13 (Gal2-13) of galanin or a mutant thereof; (ii) the FC region; Includes:

[0015] Preferably, the galanin fragment of the chimeric protein of the first aspect consists of SEQ ID NO:9 or SEQ ID NO:10, most preferably SEQ ID NO:4.

[0016] A second aspect of the invention provides a nucleic acid encoding the chimeric protein of the first aspect.

[0017] A third aspect of the invention provides a vector comprising the nucleic acid of the second aspect.

[0018] A fourth aspect of the present invention provides a recombinant cell comprising the vector of the third aspect.

[0019] A fifth aspect of the present invention provides a pharmaceutical composition comprising the chimeric protein of the first aspect and a pharmaceutically acceptable excipient.

[0020] A sixth aspect of the invention provides a method for producing a pharmaceutical composition comprising mixing the chimeric protein of the first aspect with a pharmaceutically acceptable excipient.

[0021] A seventh aspect of the invention provides a chimeric protein of the first aspect for use in the treatment of the human or animal body.

[0022] An eighth aspect of the present invention provides a method for treating pain, comprising administering a therapeutically effective amount of the chimeric protein of the first aspect or the pharmaceutical composition of the fifth aspect.

[0023] A ninth aspect of the present invention provides the chimeric protein of the first aspect or the pharmaceutical composition of the fifth aspect for use in the treatment of pain.

[0024] A tenth aspect of the invention provides the use of the chimeric protein of the first aspect or the pharmaceutical composition of the fifth aspect in the manufacture of a medicament for the treatment of pain.

[0025] The pain according to the eighth, ninth and tenth aspects may be neuropathic pain (NP) or inflammatory pain.

[0026] Other aspects and embodiments of the invention are described in more detail below. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the relative potency of galanin and FC fusions in the GalR2 functional calcium imaging assay. [Figure 2] FIG. 1 shows in vitro profiling of FC fusions focusing on amino acids 12 and 13 using a β-arrestin assay. [Figure 3] FIG. 1 shows the binding affinity of Gal-FC fusions to human GalR2. [Figure 4] FIG. 1 shows the activity of Gal-FC fusions at the three known human galanin receptor subtypes. [Figure 5] FIG. 1 shows a summary of the effect of Ala substitution on the potency and selectivity of Gal2-13 for GalR2 versus GalR1. [Figure 6] FIG. 1 shows the effect of Gal2-30FC on pain threshold in a mouse CCI model of neuropathic pain. [Figure 7] FIG. 1 shows the effect of Gal2-13FC at doses ranging from 1 mg / kg to 30 mg / kg on pain threshold in a mouse CCI model of neuropathic pain. [Figure 8]FIG. 1 shows the effect of Gal2-13FC at doses ranging from 0.1 mg / kg to 1 mg / kg on pain threshold in a mouse CCI model of neuropathic pain. [Figure 9] FIG. 1 shows the effect of Gal2-13FC on weight bearing in the mouse CFA model of inflammatory pain. [Figure 10] FIG. 1 shows the duration of the effect of 1 mg / kg Gal2-13FC on pain threshold in the mouse CCI model of neuropathic pain. [Figure 11] FIG. 1 shows the effect of Gal2-13FC at doses ranging from 0.01 mg / kg to 0.1 mg / kg on pain threshold in a mouse CCI model of neuropathic pain. [Figure 12] FIG. 1 shows the effect of Gal2-13FC at doses ranging from 0.1 mg / kg to 3 mg / kg on pain threshold in a mouse chemotherapy-induced model of neuropathic pain. [Figure 13] FIG. 1 shows the effect of repeated administration of Gal2-13FC on pain threshold in a mouse CCI model of neuropathic pain. DETAILED DESCRIPTION OF THE INVENTION

[0028] The present invention relates to chimeric proteins comprising a fragment ("warhead") of galanin (Gal) linked to the Fc region. The fragment consists of Gal residues 2-13 or 2-12, or variants thereof. Unlike Gal2-11, these chimeric proteins are highly selective agonists of peripheral GalR2 and have a long duration of effect in vivo. Furthermore, because the chimeric proteins are restricted to the peripheral nervous system (i.e., do not cross the blood-brain barrier), they may exhibit reduced CNS-mediated side effects, such as sedation or addiction, compared to other analgesics. The chimeric proteins described herein may be useful in the treatment of pain, such as neuropathic pain or inflammatory pain.

[0029] Human galanin (Gene ID 51083) is a 30-mer neuropeptide with the reference amino acid sequence of NCBI database entry AAB20740.1. Mature Gal has the reference amino acid sequence of NP_057057.2 and corresponds to residues 33-62 of the Gal pre-pro-peptide, which can be encoded by the reference nucleotide sequence of NM_015973.4.

[0030] The Gal fragment of the chimeric protein specifically binds to Galanin Receptor 2 (GalR2). The Gal fragment may preferentially or selectively bind to GalR2 over Galanin Receptor 1 (GalR1) and Galanin Receptor 3 (GalR3). Human Galanin Receptor 2 (GalR2 Gene ID 8811) may have the reference amino acid sequence of database entry NP_003848.1 and may be encoded by the reference nucleotide sequence of database entry NM_003857.3.

[0031] Preferably, the Gal fragment of the chimeric protein consists of residues 2 to 13 of galanin, preferably human galanin, or a variant thereof. For example, the Gal fragment may consist of the amino acid sequence WTX1NSA X2YLLGX3, where X1, X2, and X3 are independently any amino acid (SEQ ID NO: 9).

[0032] Preferably, X1 is independently L or A, X2 is independently G or A, and X3 is independently P or A. For example, the Gal fragment may consist of SEQ ID NO:10.

[0033] In some preferred embodiments, the Gal fragment may consist of the amino acid sequence WTLNSAGYLLGP (SEQ ID NO: 4) or may be a variant thereof.

[0034] In one embodiment, the Gal fragment may consist of the amino acid sequence of WTLNSAGYLLG (SEQ ID NO: 49) or may be a variant thereof.

[0035] The Gal fragment is preferably located at the N-terminus of the chimeric protein.

[0036] The chimeric protein does not contain the full-length galanin, i.e., the chimeric protein does not contain the sequence GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS (SEQ ID NO: 15), GWTLNSAGYLLGPHAIDNHRSFHDKYGLA (SEQ ID NO: 16), or N-terminally truncated galanin, for example, the sequence WTLNSAGYLLGPHAVGNHRSFSDKNGLTS (SEQ ID NO: 57) or WTLNSAGYLLGPHAIDNHRSFHDKYGLA (SEQ ID NO: 17).

[0037] "Consisting of residues 2-13 of galanin" or similar means that the chimeric protein contains only residues 2-13 of Gal, e.g., the Gal fragment portion of the chimeric protein has a length of 12 amino acids. "Consisting of residues 2-12 of galanin" or similar means that the chimeric protein contains only residues 2-12 of Gal, e.g., the Gal fragment portion of the chimeric protein has a length of 11 amino acids. For example, the chimeric protein does not contain the sequence WTLNSAGYLLGPHA (SEQ ID NO: 58), WTLNSAGYLLGPH (SEQ ID NO: 59), or variants thereof.

[0038] The antibody fragment crystallizable region (FC region) may comprise the second and third constant domains (CH2 and CH3) of an immunoglobulin heavy chain. Suitable immunoglobulin FC regions for use in the chimeric proteins described herein are well known in the art and include the IgG FC region, preferably the FC region of IgG1 or IgG4. In some embodiments, the FC region of the chimeric proteins described herein may comprise the amino acid sequence of SEQ ID NO: 5 or may be a variant thereof.

[0039] In some embodiments, the Fc region may exhibit reduced immunogenicity and / or effector activity (e.g., reduced antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC) activity). For example, a chimeric protein may comprise an Fc region with reduced effector activity, e.g., an IgG4 Fc region, and / or may comprise an Fc region, such as an IgG1, IgG2, or IgG4 Fc region, that includes one or more amino acid mutations, such as substitutions, deletions, or insertions, that reduce or eliminate immunogenicity and / or effector activity. Suitable mutant FC regions are known in the art (e.g., WO 2004 / 110472, WO 2005 / 000892, WO 2005 / 113606, Wang et al (2018) Protein Cell 9 (1) 63-73, Saxena et al Front Immunol 2016 7 580, Lo et al; 2017 JBC 292 3900-3908, Jacobsen et al (2017) JBC 292 1865-1875).

[0040] The FC region is preferably located at the C-terminus of the chimeric protein. The Gal fragment may be connected via a linker, or more preferably, directly to the FC region.

[0041] Suitable linkers are well known in the art and include chemical linkers, more preferably peptidyl linkers.

[0042] The peptidyl linker may comprise a sequence of amino acid residues, for example, 3 to 15 amino acid residues, preferably 9 to 15 amino acid residues, more preferably 11 to 13 amino acid residues, and even more preferably about 12 amino acid residues. Any linker sequence may be employed. Preferably, the linker sequence is a heterologous sequence and non-immunogenic. Suitable linker amino acid sequences are well known in the art and may include the amino acid sequences AEAAAKEAAAKA (SEQ ID NO: 6), GGS (SEQ ID NO: 11), GGSGGS (SEQ ID NO: 12), GGSGGSGGSGGS (SEQ ID NO: 13), or PAPAPAPA (SEQ ID NO: 14), or a variant of any of these sequences.

[0043] The chimeric proteins described herein may comprise the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 8, or may be a variant of one of these sequences. In one embodiment, the chimeric proteins described herein may comprise the amino acid sequence of SEQ ID NO: 60 or SEQ ID NO: 25, or may be a variant of these sequences.

[0044] A chimeric protein, linker, binding moiety, or FC region described herein that is a variant of a reference sequence (such as the reference sequences described above) can have one or more amino acid residues changed relative to the reference sequence. For example, 50 or fewer amino acid residues, preferably 45 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, or 3 or fewer, 2 or 1, can be changed relative to the reference sequence. For example, a variant described herein can comprise a sequence of a reference sequence in which 50 or fewer, 45 or fewer, 40 or fewer, 30 or fewer, 20 or fewer, 15 or fewer, 10 or fewer, 5 or fewer, 3 or fewer, 2 or 1 amino acid residues have been changed relative to SEQ ID NO: 7 or SEQ ID NO: 8.

[0045] Amino acid residues in the reference sequence may be changed or mutated by insertion, deletion or substitution, preferably by substitution with a different amino acid residue. Such changes may be caused by one or more additions, insertions, deletions or substitutions of one or more nucleotides in the encoding nucleic acid.

[0046] A chimeric protein, linker, binding moiety, or FC region described herein that is a variant of a reference sequence may share at least 50% sequence identity, at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with the reference amino acid sequence. For example, a chimeric protein described herein may comprise an amino acid sequence that has at least 50% sequence identity with the reference amino acid sequence, or at least 55%, at least 60%, at least 65%, at least 70%, at least about 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:7 or SEQ ID NO:8.

[0047] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc., San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty of 4. Although the use of GAP may be preferred, other algorithms using commonly adopted default parameters may also be used, such as BLAST (using the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (using the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol. 147: 195-197), or the TBLASTN program of Altschul et al. (1990) mentioned above. In particular, the psi-Blast algorithm may be used (Nucl. Acids Res. (1997) 25 3389-3402). Sequence identity and similarity may also be determined using Genomequest™ software (Gene-IT, Inc., Worcester, Massachusetts, USA). Preferably, sequence comparisons are made over the entire length of the relevant sequences described herein.

[0048] The chimeric proteins described herein may further comprise one or more heterologous amino acid sequences in addition to the Fc domain, Gal fragment, and linker. For example, the chimeric proteins may further comprise one or more additional domains that improve the stability, pharmacokinetics, targeting, affinity, purification, and production properties of the chimeric proteins described herein.

[0049] The chimeric proteins described herein can be provided using synthetic or recombinant techniques standard in the art.

[0050] In some embodiments, the chimeric proteins described herein may be produced with an affinity tag, which may be useful, for example, for purification. An affinity tag is a heterologous peptide sequence that forms one member of a specific binding pair. A polypeptide containing a tag may be purified by binding of the other member of the specific binding pair to the polypeptide, for example, in an affinity column. For example, the tag sequence may form an epitope that is bound by an antibody molecule.

[0051] Suitable affinity tags include, for example, glutathione-S-transferase (GST), maltose binding domain (MBD), MRGS(H)6 (SEQ ID NO: 50), DYKDDDDK (FLAG™) (SEQ ID NO: 51), T7-,S-(KETAAAKFERQHMDS) (SEQ ID NO: 52), poly-Arg (R 5~6 ), poly-His(H 2~10 ), poly-Cys(C4) poly-Phe(F 11 ) Poly-Asp(D 5~16), SUMO tag (Invitrogen's Champion pET SUMO Expression System), Strept-tag II (WSHPQFEK) (SEQ ID NO: 53), c-myc (EQKLISEEDL) (SEQ ID NO: 54), influenza-HA tag (Murray, PJ et al (1995) Anal Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, DK et al (1991) FEBS Lett 283, 298-302), Tag.100 (Qiagen, a 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR, Santa Cruz Biotechnology Inc.) (SEQ ID NO: 55), and Cruz tag 22™ (MRDALDRLDRLA, Santa Cruz Biotechnology Inc.) (SEQ ID NO: 56). Known tag sequences are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533. In a preferred embodiment, a poly-His tag such as (H)6, His-SUMO tag (Invitrogen's Champion pET SUMO Expression System), or MRGS(H)6 can be used. The affinity tag sequence may be separated from the chimeric proteins described herein after purification, for example, using a site-specific protease.

[0052] In some embodiments, the chimeric proteins described herein can be coupled to a leader peptide for direct secretion of the chimeric protein from cells into the culture medium as a precursor chimeric protein. A variety of suitable leader peptides are known in the art, including SEQ ID NO: 3 or variants thereof. The leader peptide can be heterologous to the Gal fragment described herein, i.e., a non-galanin leader sequence. For example, the α-factor secretion signal or BiP leader sequence can be employed. The leader peptide is located at the N-terminus of the precursor chimeric protein. The Gal fragment can be located immediately adjacent to the N-terminal leader sequence in the precursor chimeric protein (i.e., the N-terminus of the binding moiety is directly linked to the C-terminus of the leader sequence). The leader peptide is then removed by post-translational processing after expression of the precursor to generate the chimeric protein.

[0053] The chimeric proteins described herein can be isolated, meaning free from contaminants such as other polypeptides and / or cellular components.

[0054] The chimeric proteins described herein may be selective agonists of GalR2 in vitro, i.e., they may activate GalR2 preferentially or selectively over GalR1 and / or GalR3. The in vitro activity of the chimeric proteins may be determined by any convenient method. For example, the effect of the chimeric proteins on GalR2 may be determined by measuring intracellular calcium mobilization, and the effect of the chimeric proteins on GalR1 activation may be determined by measuring cyclic AMP (cAMP). The effect of the chimeric proteins on both GalR1 and GalR2 may be determined by measuring β-arrestin (BA) recruitment, as described herein. Suitable assays are described in detail below.

[0055] The chimeric proteins may not be able to cross the BBB or penetrate the CNS in vivo and therefore may not be able to activate GalR2 in CNS neurons. Thus, the chimeric proteins may be selective agonists of peripheral GalR2, i.e., they may activate GalR2 in peripheral neurons but may not or substantially not activate GalR2 in CNS neurons. Because they do not activate GalR2 in CNS neurons, the chimeric proteins described herein may not exert sedative or other CNS-mediated effects in vivo.

[0056] The chimeric proteins described herein can exert an analgesic effect in vivo. The analgesic effect can be a peripheral analgesic effect, i.e., it can affect peripheral neurons but not CNS neurons. The in vivo activity of the chimeric protein can be determined by any convenient method. For example, it can be determined as the ability of the chimeric protein to reduce, inhibit, or reverse mechanical and / or thermal hyperalgesia or allodynia in a mouse chronic constriction injury (CCI) model, or complete Freund's adjuvant (CFA)-induced hyperalgesia or allodynia in a mouse model (Gould et al. Pain. 2000 Mar;85(1-2):301-3).

[0057] The chimeric proteins described herein may exhibit biological activity, such as analgesia, for extended periods of time, e.g., for more than 24 hours, more than 48 hours, more than 72 hours, or more than 96 hours, as measured in vivo, e.g., in a mouse model. Suitable models include the CCI model or the CFA-induced hyperalgesia and / or allodynia model, as described above. In some embodiments, the chimeric proteins may attenuate or reverse mechanical allodynia or hyperalgesia in the model system for at least 48 hours, at least 72 hours, or at least 96 hours. For example, the chimeric proteins described herein may reduce paw withdrawal in the CCI model or the CFA-induced hyperalgesia model for at least 48 hours, at least 72 hours, or at least 96 hours, compared to a control and / or gabapentin.

[0058] The chimeric protein may exhibit biological activity in vivo for a longer period than a control fusion protein, for example, a chimeric protein may exhibit biological activity for more than two-fold, three-fold, or four-fold longer than a control fusion protein in which the Gal2-13 fragment is replaced with the Gal2-30 fragment.

[0059] Another aspect of the present invention provides nucleic acids encoding the chimeric proteins described herein above and vectors comprising such nucleic acids.

[0060] Suitable vectors may be selected or constructed and contain appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes, and other sequences as needed. Preferably, the vector contains appropriate regulatory sequences that direct expression of the nucleic acid in mammalian cells. The vector may also contain sequences, such as an origin of replication, a promoter region, and a selectable marker, that allow its selection, expression, and replication in a bacterial host, such as E. coli.

[0061] Vectors may be plasmids, viruses, such as phages, or phagemids, as appropriate. For further details, see, for example, "Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press." Many known techniques and protocols for manipulating nucleic acids, for example, for creating nucleic acid constructs, mutagenesis, sequencing, introducing DNA into cells, and gene expression, are described in detail in "Current Protocols in Molecular Biology," Ausubel et al., eds., John Wiley & Sons, 1992.

[0062] The nucleic acids or vectors described herein can be introduced into a host cell.

[0063] Another aspect of the present invention provides a recombinant cell comprising a nucleic acid or vector that expresses the chimeric protein described above.

[0064] A variety of host cells suitable for producing recombinant chimeric proteins are known in the art. Suitable host cells include prokaryotic cells, particularly bacteria such as Escherichia coli and Lactococcus lactis cells, as well as eukaryotic cells, including mammalian cells such as CHO and CHO-derived cell lines (Lec cells), HeLa, COS, HEK293, and HEK-EBNA cells, amphibian cells such as Xenopus oocytes, insect cells such as Trichoplusia ni cells, Sf9, and Sf21, and yeast cells such as Pichia pastoris cells.

[0065] Techniques for introducing nucleic acids into cells are well established in the art, and any suitable technique can be employed depending on the specific situation. Suitable techniques for eukaryotic cells include calcium phosphate transfection, DEAE-Dextran, electroporation, liposome-mediated transfection, and transduction using retroviruses or other viruses, such as adenoviruses, AAV, lentiviruses, or vaccinia. Suitable techniques for bacterial cells include calcium chloride transformation, electroporation, and transfection using bacteriophages.

[0066] As is well known in the art, marker genes, such as antibiotic resistance or sensitivity genes, may be used to identify clones containing the nucleic acid of interest.

[0067] The introduced nucleic acid may be on an extrachromosomal vector within the cell, or the nucleic acid may be integrated into the genome of the host cell. Integration may be facilitated by inclusion of sequences within the nucleic acid or by the vector facilitating recombination with the genome, according to standard techniques.

[0068] Following introduction, the nucleic acid may be expressed to produce the encoded chimeric protein described herein. The host cell may be an in vitro host cell. In some embodiments, host cells (which may include cells that were actually transformed, but which are more likely to be the progeny of transformed cells) may be cultured in vitro under conditions for expression of the nucleic acid, such that the encoded serpin polypeptide is produced. If an inducible promoter is used, expression may require activation of the inducible promoter.

[0069] After production, the expressed polypeptide comprising or consisting of the chimeric protein may be isolated and / or purified. This may be achieved using any convenient method known in the art. Techniques for purifying recombinant polypeptides are well known in the art and include, for example, HPLC, FPLC, or affinity chromatography. In some embodiments, purification may be performed using an affinity tag for the polypeptide as described above.

[0070] Another aspect of the present invention provides a method for producing a chimeric protein described herein, comprising expressing a nucleic acid encoding the chimeric protein in a host cell and, optionally, isolating and / or purifying the chimeric protein so produced.

[0071] After production, the chimeric protein may be further investigated, for example, to determine its pharmacological properties and / or activity. Methods and tools for protein analysis are well known in the art.

[0072] The chimeric proteins described herein can be useful in therapeutic methods, as described herein. For example, the chimeric proteins can be administered to an individual for the treatment of pain.

[0073] While chimeric proteins may be administered alone, they are typically administered in the form of a pharmaceutical composition, which may contain at least one other component in addition to the chimeric protein. Thus, a pharmaceutical composition may contain, in addition to the chimeric protein itself, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by bolus, infusion, injection, or any other suitable route, as discussed below.

[0074] In some embodiments, the chimeric protein may be provided in lyophilized form for reconstitution prior to administration, for example, a lyophilized chimeric protein may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0075] For parenteral administration, e.g., subcutaneous or intravenous administration by injection, pharmaceutical compositions containing the chimeric proteins, nucleic acids, or cells described herein may be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity, and stability. Those skilled in the art are well able to prepare suitable solutions using isotonic vehicles such as sodium chloride solution, Ringer's solution, lactated Ringer's solution, etc. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be utilized as needed, including buffers such as phosphate, citrate and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum. Examples of suitable carriers, excipients include albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, such as Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

[0076] Pharmaceutical compositions and formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. Such methods include the step of bringing into association a chimeric protein described herein with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0077] Preferably, the chimeric proteins or compositions described herein may be administered by injection, either intravenously or subcutaneously.

[0078] Depending on the condition being treated, pharmaceutical compositions containing the chimeric proteins described herein may be administered alone or in combination with other treatments, either simultaneously or sequentially.

[0079] The chimeric proteins described herein may be used in methods of treatment of the human or animal body, including therapeutic and prophylactic or preventative treatments (e.g., treatment before the onset of a condition in an individual to reduce the risk of the condition occurring in the individual, delay its onset, or reduce its severity after onset). The methods of treatment may include administering a chimeric protein described herein to an individual in need thereof.

[0080] A method for treating pain can include administering a therapeutically effective amount of a chimeric protein or pharmaceutical composition described herein. Related aspects of the invention provide a chimeric protein or pharmaceutical composition for use in treating pain, and provide use of a chimeric protein or pharmaceutical composition described herein in the manufacture of a medicament for treating pain.

[0081] Pain includes neuropathic pain (NP), such as peripherally or centrally mediated neuropathic pain; inflammatory pain; nociceptive pain; or chronic pain.

[0082] In some embodiments, pain that may be treated as described herein may include symptoms of allodynia; hyperalgesia; spontaneous pain; or receptive field expansion. Causes of these pain-related conditions include neuropathy-related pain, such as diabetic peripheral neuropathy, arthritic pain, postherpetic neuralgia, trigeminal neuralgia, post-stroke pain, multiple sclerosis-related pain, idiopathic or post-traumatic neuropathy and mononeuritis, HIV-associated neuropathic pain, cancer-associated neuropathic pain, and carpal tunnel-associated neuropathic pain; hyperalgesia, e.g., opioid-induced hyperalgesia, sciatica, spinal cord injury-associated pain, complex regional pain syndrome, fibromyalgia-associated neuropathic pain, lumbar and neck pain, reflex sympathetic dystrophy, phantom limb pain, phantom limb syndrome, peripheral nerve or spinal cord trauma, and enlargement of the receptive field. These include pain caused by strangulation neuropathy, nerve amputation including surgery, Lissauer tract resection, limb amputation, stump pain, neuroma / tumor compression, arteriovenous malformation, vitamin B12 deficiency, diabetic neuropathy, alcoholic neuropathy, pain caused by side effects of anti-cancer and anti-AIDS therapy, pain associated with dental inflammation or infection (toothache), visceral pain, pain from chemical burns, pain from local or systemic infection, or pain caused by connective tissue diseases such as rheumatoid arthritis, Wallenberg's syndrome, systemic lupus erythematosus, multiple sclerosis, and polyarteritis nodosa.

[0083] The pain can be, for example, pain associated with cancer, surgery, visceral injury, headache, or trauma.

[0084] An individual suitable for such treatment may be a mammal, such as a rodent (e.g., guinea pig, hamster, rat, mouse), canine (e.g., dog), feline (e.g., cat), equine (e.g., horse), primate, ape (e.g., monkey or ape), monkey (e.g., marmoset, baboon), ape (e.g., gorilla, chimpanzee, orangutan, gibbon), or human.

[0085] In some preferred embodiments, the individual is a human. In other preferred embodiments, non-human mammals may be employed, particularly mammals traditionally used as models for demonstrating therapeutic efficacy in humans (e.g., murines, primates, porcines, canines, or lagomorphs).

[0086] Administration is typically in a "therapeutically effective amount" or a "prophylactically effective amount," which is sufficient to show benefit to the patient. Such benefit may be at least the alleviation of pain in the patient. The actual amount administered, as well as the rate and time-course of administration, will depend on the characteristics and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the dosing schedule, and other factors known to a physician.

[0087] The compositions may be administered alone or in combination with other treatments, for example with other analgesics (such as paracetamol, nonsteroidal anti-inflammatory drugs (NSAIDs), opioids), other antiepileptic drugs (such as gabapentin, pregabalin or carbamazepine), or antidepressants (such as amitriptyline or duloxetine), either simultaneously or sequentially depending on the condition of the individual being treated.

[0088] Prescribing treatment, e.g., determining dosage, is within the responsibility of general practitioners and other physicians and may depend on the severity and / or progression of the symptoms of the disease being treated. Appropriate dosages of therapeutic polypeptides are well known in the art (Ledermann JA et al. (1991) Int. J. Cancer 47: 659-664, Bagshawe KD et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). Depending on the type of drug being administered, specific dosages may be used that may be provided herein or in the Physician's Desk Reference (2003). Therapeutically effective or suitable doses of the chimeric proteins described herein may be identified by comparing their in vitro and in vivo activity in animal models. Methods are known for extrapolating effective dosages in mice and other test animals to humans. The exact dose will depend on many factors, including whether the chimeric proteins described herein are prophylactic or therapeutic, the size and location of the area to be treated, the exact characteristics of the chimeric proteins described herein, and the characteristics of any detectable label or other molecule attached to the chimeric proteins described herein.

[0089] An initial, higher loading dose may be administered, followed by one or more lower doses. This is the dose for a single treatment of an adult patient and may be adjusted proportionally for infants and toddlers. Treatment may be repeated daily, twice weekly, weekly, or monthly, at the physician's discretion. The treatment regimen for a given individual may depend on the pharmacokinetic and pharmacodynamic properties of the chimeric protein compositions described herein, the route of administration, and the characteristics of the condition being treated.

[0090] Treatment can be periodic, with the period between doses being about 12 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 96 hours or more, or one week or more. The chimeric proteins described herein have been shown to exert long-lasting effects in vivo. In some preferred embodiments, the period between doses can be one week or more, two weeks or more, or one month or more; for example, administration can be weekly, biweekly, or monthly.

[0091] Suitable formulations and routes of administration are described above.

[0092] Other aspects of the present invention provide for the use of the chimeric proteins described herein as analgesics, and the use of the chimeric proteins described herein as selective agonists of peripheral GalR2.

[0093] Other aspects and embodiments of the present invention provide for aspects and embodiments described above with the term "comprising" replaced by the term "consisting of," as well as aspects and embodiments described above with the term "comprising" replaced by the term "consisting essentially of."

[0094] It is understood that the present application discloses all combinations of any of the above aspects and embodiments described above with each other unless the context requires otherwise. Similarly, the present application discloses all combinations of preferred and / or optional features alone or with any of the other aspects unless the context requires otherwise.

[0095] Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to those skilled in the art upon reading this disclosure and, accordingly, are included within the scope of the present invention.

[0096] All documents and sequence database entries referred to herein are incorporated by reference in their entirety for all purposes.

[0097] As used herein, "and / or" is understood as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is understood as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, exactly as if each were individually presented herein. [Example]

[0098] experiment Materials and Methods cell culture CHO cell lines expressing human GalR1 and GalR2 were generated using the Flp-in system (Life Technologies, USA). GalR1 and GalR2 were cloned into pcDNA5 / FRT and transfected into CHO cells using Lipfoectamine 2000 (Life Technologies, USA) according to the manufacturer's guidelines. Cells were maintained in F12 Ham containing 10% (vol / vol) fetal bovine serum and 200 μg / ml hygromycin (both Life Technologies, USA). β-Arrestin assays were performed using PathHunter CHO-K1 GALR2 β-Arrestin cell line and PathHunter CHO-K1 GALR1 β-Arrestin cell line (DiscoverX, USA). Cells were maintained in DMEM / F12 containing 10% FBS, 300 μg / ml hygromycin, and 500 μg / ml Geneticin (both Life Technologies, USA).

[0099] Ligand Human full-length galanin (1-30), rat full-length galanin, rat galanin truncated 2-29, galanin truncated 1-15, galanin truncated 2-11, M617, M1145, and galnon were purchased from Tocris, UK. Ligands were typically resuspended in water to 1 mM. Galnon was resuspended in 100% DMSO to a final concentration of 10 mM. Nax409-9 was purchased from Cambridge Research Biochemicals, UK (see Table 1).

[0100] In vitro assays Calcium assay CHO GalR2 cells were seeded at 10,000 cells per well in black, clear-bottom plates (Corning, USA) and incubated overnight at 37°C with 5% CO2. The next day, cell culture medium was removed and replaced with Calcium 4 FLIPR dye (Molecular Devices, USA) and resuspended in HBSS containing 2 mM HEPES (Life Technologies, USA) and 0.1% BSA. The cells and dye were incubated at 37°C for 45 minutes and then allowed to equilibrate to room temperature for approximately 15 minutes. Ligands were prepared at 5x concentrations and added to the cells using FLIPR (Molecular Devices, USA). A baseline was typically obtained 20 seconds before ligand addition. Fluorescence was measured for 120 seconds at excitation 485 nM and emission 520 nM. For analysis, fluorescence was normalized to the baseline before addition and the peak fluorescence used for all quantifications.

[0101] cAMP assay The cAMP Dynamic 2 Kit (Cisbio International) was used as a competitive immunoassay to measure homogenous time-resolved fluorescence (HTRF). CHO GalR1 cells were seeded at 10,000 cells. The next day, the cell culture medium was removed and replaced with 5 μl of cell assay buffer (Dulbecco's phosphate-buffered saline (Gibco) by Life Technologies, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES, Sigma-Aldrich), 0.1% BSA (bovine serum albumin in DPBS from Sigma-Aldrich), and 0.5 mM 3-isobutyl-1-methylxanthene (IBMX, Life Technologies), a phosphodiesterase (PDE) inhibitor, to prevent cyclic AMP degradation. Forskolin (Cambridge University Hospital) in dilution buffer (DPBS solution, 10 mM HEPES, and 0.1% BSA) was then added. Cells were stimulated with 5 μl of the appropriate concentration of galanin agonist (Tocris, UK) containing galanin agonist (Tocris, UK) for 30 minutes at room temperature. The addition of detection reagents involved adding 5 μl of d2-conjugated cAMP, followed by 5 μl of anti-cAMP cryptate conjugate. The two reagents were not premixed; they were both prepared in cell lysis buffer and incubated for 1 hour in the dark at room temperature. Signals were quantified using a PHERAstar optimized for HTRF. Forskolin stimulation IC50 and fluorescence ratio (665 nm / 620 nm) were determined by nonlinear regression analysis.

[0102] β-arrestin assay PathHunter CHO-K1 GALR2 β-arrestin or PathHunter CHO-K1 GALR1 β-arrestin cells were typically seeded at 5,000 cells / well using Cell Plating Reagent 2 (DiscoverX, USA) and incubated overnight at 37°C with 5% CO2. Ligands were prepared in half-log dilutions using Cell Plating Reagent 2. Five microliters of each ligand was added to the cells, and the plate was incubated at 37°C for 90 minutes. The DiscoverX PathHunter Detection Kit (DiscoverX, USA) was added to the cells as described in the manufacturer's guidelines, and the plate was incubated at room temperature in the dark for 60 minutes. Chemiluminescent signals were measured using a PHERAstar FS (BMG, Germany).

[0103] Radioligand binding assay Cell membrane homogenates (4 μg of protein from CHO cells expressing the GalR2 receptor) were incubated in a buffer containing 25 mM Tris-HCl (pH 7.4), 10 mM MgCl2, and 0.5% BSA with 0.05 nM [ 125 The cells were incubated with [I] galanin. Nonspecific binding was measured in the presence of 1 μM porcine galanin. After incubation, samples were rapidly filtered under vacuum through glass fiber filters (GF / B, Packard) presoaked in 0.3% PEI and rinsed several times with ice-cold 50 mM Tris-HCl using a 96-sample cell harvester (Unifilter, Packard). After drying the filters, radioactivity was counted in a scintillation counter (Topcount, Packard) using a scintillation cocktail (Microscint 0, Packard). Results are expressed as percent inhibition of control radioligand-specific binding. The standard reference compound was porcine galanin, which was tested at several concentrations in each experiment to determine its IC 50 to obtain the competitive curves that are created.

[0104] GalR3 reporter gene assay GalR3 activity was measured along with GalR1 and GalR2 using a luciferase reporter assay. The reporter and receptor plasmids were transiently transfected into target cell lines. The reporter plasmids were coupled to luciferase transcripts and expressed specific G proteins (G αi Use SRE;G αq The target receptors contained serum response elements (SREs) or nuclear factor of activated T cells response elements (NFAT-REs), which induce luciferase expression upon activation (using the NFAT-RE). The target receptors were continuously expressed after cell transfection. For GALR1 and GALR3 screening, genetically engineered HEK293 Gqi cells were transiently transfected with SRE reporter and receptor (GALR1 and GALR3) plasmids. For GALR2 screening, HEK293 cells were transiently transfected with NFAT-RE reporter and receptor (GALR2) plasmids. Cells were treated with galanin receptor agonists (3 μM to 95 pM, half-log dilutions) for 5 hours. Luciferase activity was analyzed using the Bright-Glo luciferase assay (Promega) on an EnSpire plate reader.

[0105] In vivo assay Chronic Constriction Injury (CCI) Model of Chronic Pain The chronic constriction injury (CCI) model of neuropathic pain involves the unilateral placement of four loose ligatures around the left sciatic nerve, spaced 1 mm apart, at mid-thigh level (Chaplan SR, et al. J Neurosci Methods. 1994 Jul; 53(1):55-63). This procedure results in the development of hyperalgesia, allodynia, and spontaneous pain (ectopic action potentials), which can be measured using mechanical and thermal behavioral assessments. This model is believed to mimic some of the symptoms and pathogenesis of neuropathic pain observed in the clinic (Bennett GJ, Xie YK. Pain. 1988 33(1):87-107; Field MJ, et al. Pain. 1999 Nov; 83(2):303-11). The mouse CCI model of neuropathic pain differs from the rat CCI model in that there are three ligatures around the sciatic nerve instead of four.

[0106] Control group: PBS, 10 mL / kg Reference group: pregabalin (15 mg / kg po) Animals: Naive male C57B16 mice were 6 weeks old on arrival and were acclimated to the treatment room in home cages with free access to food and water. Environmental conditions: Animals were housed in groups of 2-4 in standard caging and laboratory conditions with a 12 / 12 light / dark cycle and free access to food (5CR4, Purina) and water (except while in the test box). Environmental enrichment was provided from the day of arrival and changed every 3 days to prevent autotomy.

[0107] Static mechanical tactile allodynia: Static mechanical (tactile) allodynia was assessed by measuring the pain threshold using calibrated (force; g) von-Frey monofilaments (Touch-Test Sensory Evaluator; Scientific Marketing Associates) applied to the plantar surface of the hind paw. Animals were placed in individual Perspex boxes on a raised metal mesh for 30–40 min before testing. A series of graded von Frey hairs (0.07 g, 0.16 g, 0.4 g, 0.6 g, and 1 g) were applied sequentially using a 1-second on, 1-second off protocol repeated 10 times. Each hair was applied perpendicular to the center of the ventral surface of the paw until it bent slightly. The force applied to the animal's hind paw to elicit a response in 5 out of 10 trials was recorded as the paw pain threshold (PWT).

[0108] Baseline PWT was assessed on three consecutive days (days -2, -1, and 0), and the mean of the last two readings was used as the baseline pain threshold.

[0109] On day 1, neuropathic pain was induced by ligating the sciatic nerve with three loose ligatures of Prolene suture (chronic constriction injury). Under anesthesia with isoflurane mixed with oxygen (3:1, 1 L / min), the left hind limb was shaved at mid-thigh level, and the skin was incised using a scalpel. The biceps femoris muscle layer was dissected by making an initial incision with sharp scissors and then spreading it with blunt scissors. The common sciatic nerve was exposed using a pair of forceps, and three loose ligatures of Prolene (7-0, Ethicon) were tied around the sciatic nerve, spaced 1 mm apart. The nerve was then placed back under the muscle layer, and the wound was closed using absorbable sutures (Vicryl).

[0110] Von Frey assessments of mechanical allodynia were performed on days 19 and 22. Animals were then ranked and randomized (based on a Latin square design) into treatment groups according to the percent change in mean mechanical pain threshold (compared to pre-surgery baseline) observed on days 19 and 22. Only animals that demonstrated a PWT percentage change of more than 60% from pre-surgery baseline were included in the study.

[0111] On day 23, mice were dosed at 10 mL / kg with either vehicle, test compound (ip), or pregabalin (15 mg / kg po). Allodynia was then assessed at subsequent intervals, e.g., 2, 4, and 24 hours after dosing. Von Frey measurements were continued until activity disappeared, up to day 27.

[0112] In studies involving repeated dosing, animals were dosed at 10 mL / kg with either vehicle, test compound (ip), or pregabalin (15 mg / kg po) on day 23. Allodynia was then assessed at subsequent intervals, e.g., 4, 24, and 48 hours after dosing. Animals were re-dosed at 68, 116, 164, 212, 260, and 308 hours, with von Frey measurements performed 4 hours after each dose. Thus, allodynia was measured at 72, 120, 168, 216, 264, and 312 hours after the initial dose, equivalent to two, three, four, five, six, and seven repeated doses, respectively.

[0113] Study Evaluation and Statistical Analysis: vF pain threshold (g) was assessed by sequentially applying a series of graded von Frey hairs (0.07 g, 0.16 g, 0.4 g, 0.6 g, and 1 g) to the ipsilateral hind paw, 1 sec on, 1 sec off, repeated 10 times. Each hair was applied perpendicular to the center of the ventral surface of the paw until it bent slightly. The force applied to the animal's ipsilateral hind paw to elicit a response on 5 out of 10 trials was recorded as the paw pain threshold (PWT, g) and expressed as the mean ± SEM. Data were subjected to a two-way repeated measures ANOVA with "treatment" as the between-subject effect and "day" as the within-subject effect (Dixon WJ. Ann Rev Pharmacol Toxicol. 1980 20, 441-62). Post-hoc analyses were performed using planned pairwise comparisons (InVivoStat., Clark et al., 2012 J Psychopharmacology 26(8) 1136-1142.).

[0114] Complete Freund's Adjuvant (CFA)-Induced Inflammatory Model of Chronic Pain Intraplantar injection of complete Freund's adjuvant (CFA) induces hypersensitivity and edema, producing an inflammatory response that mimics some aspects of clinical inflammatory pain. These effects can be investigated using an instrument that measures paw pain thresholds. Naive mice distribute their body weight equally between their two hind paws. When the injected hind paw becomes inflamed and / or painful, body weight is redistributed, resulting in less weight being placed on the affected paw (reduced weight bearing on the injured paw). Weight bearing through each hind paw is measured using an incapacitance tester (Linton Instruments, UK).

[0115] Control group: PBS, 10 mL / kg Reference group: indomethacin 10 mg / kg po Animals: Naive male C57B16 mice were acclimated to the treatment room in home cages with free access to food and water. Environmental conditions: Animals were housed in groups of 2-5 in standard caging and laboratory conditions with a 12 / 12 light / dark cycle and free access to food (5CR4, Purina) and water (except while in the incapacitance box). Acclimation to the incapacitance tester was performed over several days.

[0116] Weight bearing: Mice were placed in an incapacitance tester, and the hind paws were attached to separate sensors. The average force exerted by both hind paws was recorded over a 2-second period. Baseline weight bearing recordings were obtained before the insult was administered. Inflammatory hypersensitivity was induced by intraplantar injection of CFA (20 μl of a 1.5 mg / ml solution) into the left hind paw. Prior to test compound treatment, weight bearing readings were obtained 23 hours after CFA to assess baseline hypersensitivity. Animals were then ranked and randomized into treatment groups according to the CFA response window of a Latin square design. 24 hours after CFA, animals were treated with test compound (usually 0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, or 3 mg / kg ip), vehicle (PBS), or indomethacin (10 mg / kg po). All treatments were normalized to a dose volume of 10 ml / kg. Weight bearing was then assessed 4, 24, 48, and 72 hours after test compound treatment.

[0117] Weight bearing (g) readings were taken for both the right and left hind paws, and the difference was calculated. Data were expressed as the ipsilateral / contralateral % ratio (mean ± SEM). Data were statistically combined by repeated measures ANOVA followed by planned comparison tests using InVivoStat (invivostat.co.uk, Clark et al., 2012).

[0118] Chemotherapy-induced model of chronic pain Oxaliplatin, a platinum-based chemotherapy drug, is commonly used to treat various types of cancer, including colorectal cancer (Ohsawa et al. J Pharmacol Sci, 2014, 125(3):292-9). However, the clinical value of oxaliplatin is diminished by acute and chronic peripheral neuropathy, including mechanical hyperalgesia, which appears as a side effect in both humans (Pasetto et al. Rev Oncol Hematol, 2006, Aug;59(2):159-68) and rodents (Ling et al., Toxicology, 2007 May;234(3):176-84). Oxaliplatin-induced peripheral neuropathy is the most frequent dose-limiting toxicity associated with the therapy, and no treatment is currently available (reference in Ling et al., 2007).

[0119] In rodents, a single intraperitoneal injection of oxaliplatin induces long-lasting cold hypersensitivity (e.g., Ling et al. Pain, 2007, Apr;128(3):225-34; Zhao et al. Mol Pain, 2012, Jul 28;8:55) and can be used to model both thermal and mechanical hyperalgesia, mimicking aspects of clinical oxaliplatin-induced neuropathy (Descoeur et al., EMBO Mol Med. 2011, May;3(5):266-78).

[0120] Control group: PBS, 10 mL / kg Reference group: pregabalin (15 mg / kg po) Animals: Naive male C57B16 mice were 6 weeks old on arrival and were acclimated to the treatment room in home cages with free access to food and water. Environmental conditions: Animals were housed in groups of 2-4 in standard caging and laboratory conditions with a 12 / 12 light / dark cycle and free access to food (5CR4, Purina) and water (except while in the test box). Environmental enrichment was provided from the day of arrival and changed every 3 days to prevent self-injurious behavior.

[0121] Oxaliplatin-induced hypersensitivity: A single dose of oxaliplatin was injected intraperitoneally (ip) at 10 mg / kg in a volume of 10 ml / kg. Oxaliplatin was dissolved in 5% dextrose to 1 mg / ml before use. Animals were injected in the same order as tested.

[0122] Static mechanical tactile allodynia: Static mechanical (tactile) allodynia was assessed by measuring the pain threshold using calibrated (force; g) von-Frey monofilaments (Touch-Test Sensory Evaluator; Scientific Marketing Associates) applied to the plantar surface of the hind paw. Animals were placed in individual Perspex boxes on a raised metal mesh for 30–40 min before testing. A series of graded von Frey hairs (0.07 g, 0.16 g, 0.4 g, 0.6 g, and 1 g) were applied sequentially using a 1-second on, 1-second off protocol repeated 10 times. Each hair was applied perpendicular to the center of the ventral surface of the paw until it bent slightly. The force applied to the animal's hind paw to elicit a response in 5 out of 10 trials was recorded as the paw pain threshold (PWT).

[0123] Baseline PWT was assessed on 3 consecutive days (days -3, -2, and -1). The average of days -2 and -1 was considered the baseline before oxaliplatin administration. Day 0 was the day of oxaliplatin injection.

[0124] Mechanical allodynia was reassessed on days 3 and 4 after administration of a single dose of oxaliplatin to monitor the onset of allodynia. Animals were then ranked and randomized (based on a Latin square design) into treatment groups according to the percent change in mean PWT observed on days 3 and 4 (compared to pre-oxaliplatin baseline). Only animals whose PWT changed by 50% or more compared to pre-oxaliplatin baseline were included in the study. Mechanical pain threshold was used as the primary endpoint.

[0125] Animals were injected ip with FC2-13, vehicle / PBS (10 ml / kg), or pregabalin (15 mg / kg po) on day 5 after oxaliplatin.

[0126] Mechanical allodynia was reassessed on day 5 (4 hours post-dose) and then on days 6 and 7 after administration of oxaliplatin (ie, 24 and 48 hours after FC2-13 treatment).

[0127] Behavioral assessments were performed by operators blinded to the administered test agent or the association of animals with treatment groups to ensure proper and complete blinding of the experiment. Assessments of the effects of pregabalin were performed 60 minutes after dosing on day 5.

[0128] result A galanin-FC fusion was constructed using the human galanin neuropeptide sequence (GenBank: AAB20740.1) and the CH2 / CH3 region of human IgG1. The sequence was designed to include residues from the IgG1 hinge region and a short linker between the galanin sequence (SEQ ID NO: 1, complete galanin underlined) and the FC portion (residues 49-282). Additionally, a 19-amino acid leader sequence (dotted underline) was inserted to facilitate secretion of the protein from cells. This region is then cleaved from the galanin-FC fusion product.

[0129] Various truncations of the galanin peptide were also inserted into the FC fusion constructs by adding a short peptide linker, AEAAAKEAAAKA (SEQ ID NO: 6) (in italics), to the design between the galanin peptide and the FC segment (SEQ ID NO: 5), generating a construct with the sequence set forth in, for example, SEQ ID NO: 2.

[0130] After initial investigation in functional assays, all peptide FC fusions generated consisted of the leader sequence and linker type shown in SEQ ID NO:2.

[0131] In vitro pharmacological profiling of novel hIgG1 FC-peptide fusions Briefly, a panel of functional cell-based microtiter plate assays was developed using recombinant GalR1 and GalR2 receptors stably expressed in CHO cells. Test compounds were typically assayed as 10-point concentration-response curves, using the range that best defined the curve. Nominally, this corresponds to 10 serial (approximately 3-fold) dilutions, with the highest concentration ranging from 10 μM to 1 μM, as appropriate.

[0132] We performed comprehensive pharmacological characterization of both fusion and non-fusion variants of galanin / galanin truncates, along with other peptides described in the literature as galanin receptor agonists. In addition to generating estimates of both potency and efficacy (the latter expressed relative to native galanin), we measured both G protein and non-G protein signaling pathways. In line with well-described secondary signaling messengers of the two galanin receptors, intracellular calcium mobilization was utilized for GalR2 (Figure 1), and cyclic AMP (cAMP) was monitored for GalR1. Additionally, β-arrestin (β-AR) recruitment was measured for both receptor systems.

[0133] Tables 2 and 3 and Figures 1 and 2 show a direct comparison of the potency and relative efficacy of various galanin agonists using these common assay platforms.

[0134] To account for the fact that the observed potency of agonists may be system-dependent and influenced, for example, by receptor expression levels and coupling efficiency, empirical molar ratios (EPMRs) were used where appropriate to highlight receptor selectivity between compounds. These data provided a system-independent measure of activity and allowed for comparison of GalR2 versus GalR1 selectivity between compounds (Tables 2 and 3).

[0135] Despite the effect of curve definition on the maximum asymptote, all FC fusions in the current dataset tended to be full agonists at GalR2 and, in some cases, partial agonists at GalR1. Some compounds appeared to exhibit functional selectivity in signaling, with potency tending to be lower (partial) at GalR1 but not at GalR2 for BA. Gal2-13FC was found to be specific for GalR2 compared to GalR1, in stark contrast to Gal2-30FC, Gal2-18FC, and Gal2-24FC, which all showed a preference for GalR2 over GalR1.

[0136] Ligand binding of FC fusions To further confirm that the responses in the functional assays were mediated by the galanin receptor and to measure affinity rather than potency, radioligand binding was performed with Gal2-12FC, Gal2-13FC, and Gal2-30FC. Briefly, cells overexpressing recombinant GalR2 were used for crude membrane preparation. A competitive-style binding assay was then used to monitor the displacement of [125I]-galanin using various concentrations of Gal-FC fusions. After the incubation period, data were expressed relative to maximum specific galanin binding. Gal2-12FC, Gal2-13FC, and Gal2-30FC all displaced [125I]-galanin in a concentration-dependent manner, with pIC50 values ​​of 7.24, 7.35, and 7.55, respectively (Figure 3). These data confirmed that the positive target engagement and affinity were in good agreement with the potency from the functional studies.

[0137] "Warhead" length effect To assess the effect on activity at GalR2 and selectivity over GalR1, FC fusions were generated using various lengths of galanin warhead linked to the AEAAAKEAAAKA sequence (SEQ ID NO: 6) and the human IgG1 FC region (SEQ ID NO: 5). It is well known that a fragment consisting of residues 2-11 of galanin is the smallest fragment capable of activating the receptor and that this fragment exhibits selectivity over GalR1. However, when fused to the FC region, this chimeric protein did not exhibit measurable activity at GalR2 (Table 2). Chimeric proteins containing residues 2-12, 2-13, 2-14, 2-15, 2-16, 2-17, 2-18, 2-24, and 2-30 were constructed and designated Gal2-12FC, Gal2-13FC, Gal2-14FC, Gal2-15FC, Gal2-16FC, Gal2-17FC, Gal2-18FC, Gal2-24FC, and Gal2-30FC. Equivalent activity at GalR2 was observed for all FC fusions tested (Table 6), indicating that Gal2-12FC is the smallest fragment of galanin that can be used to construct GalR2-activating chimeric proteins. As the size of the galanin fragment increases, FC fusions show increased activity at GalR1 (Table 6). Gal2-14FC and above showed increased GalR1 activity compared to Gal2-12FC and Gal2-13FC. These data confirm that Gal2-12FC and Gal2-13FC are the most selective chimeric proteins.

[0138] Linker effect To assess the in vitro effect of the polypeptide linker region (residues between the galanin 2-13 fragment and the FC fragment), the AEAAAKEAAAKA sequence (SEQ ID NO: 6) was changed to GGS (SEQ ID NO: 11), GGSGGS (SEQ ID NO: 12), GGSGGSGGSGGS (SEQ ID NO: 13), PAPAPAPA (SEQ ID NO: 14), or a linkerless variant in which the galanin fragment was immediately upstream of the fc fragment. These FC fragments were then analyzed for activity at GalR2 or GalR1 in vitro. Gal-FC fusion proteins containing the GGS linker exhibited comparable activity to fusions containing the AEAAAKEAAAKA (SEQ ID NO: 6) linker (Table 7). FC fusions containing the PAPAPAPA (SEQ ID NO: 14) linker, or "no linker," were more active at GalR2. However, this was also associated with increased activity at GalR1, thus resulting in no net gain in selectivity.

[0139] GalR3 activity Three galanin receptors have been identified: GalR1, GalR2, and GalR3. While GalR1 and GalR2 have been well characterized, the precise downstream signaling mechanisms utilized by GalR3 are not fully understood. Therefore, a reporter gene assay was used to measure activity at the GalR3 receptor. This system uses MAP / ERK signaling to measure receptor activity. Activation of the Gqi-coupled receptor leads to downstream activation of ERK1 / 2. Activated ERK translocates to the nucleus, where it phosphorylates and activates transcription factors, leading to binding of serum response elements (SREs). In this assay, SRE activation induces expression of a luciferase reporter gene. Gal2-12FC, Gal2-13FC, and Gal2-30FC showed no measurable activity at GalR3 (Table 5). Galanin 1-30 and Gal2-11 fragments showed the expected activity at GalR3. Similarly, spexin, a described GalR3-specific agonist, was also able to activate GalR3. To ensure that this effect was not an artifact of the reporter gene assay, we also performed GalR1 and GalR2 reporter assays (using the SRE and NFAT-RE, respectively). Gal2-12FC, Gal2-13FC, and Gal2-30FC all exhibited activity at GalR2 but not at GalR1 (Figure 4). Gal2-11 activated GalR2 but not GalR1, whereas galanin 1-30 was active at both (Table 5). These data confirm that the Gal fc fusions do not activate GalR3 and are selective for GalR2.

[0140] Orthologous activity Gal2-12FC, Gal2-13FC, and Gal2-30FC were analyzed for activity in GalR2 from the following species: house mouse (Mus musculus), roof rat (Rattus rattus), and cynomolgus monkey (Macaca fascicularis). These species were chosen because they represent potential downstream in vivo models for use. Because amino acid residues 1-15 of native galanin are conserved across species, we expected that Gal FC fusions containing amino acids 2-13 would retain GalR2 activity in these species. Calcium imaging was used to define receptor activity. For each FC fusion, activity at the orthologous receptor was comparable to that observed with the human receptor (Table 8). These data indicate that Gal2-12FC, Gal2-13FC, and Gal2-30FC exhibit activity at GalR2 orthologs beyond the human receptor, with no species-specific influence from the FC region.

[0141] Ala scanning To investigate the role of specific residues within the galanin fragment and assess whether they could enhance the potency of Gal2-13FC, we generated 13-residue peptides in which natural amino acids were replaced with alanine residues ("ala scanning"). The peptides generated are summarized in Table 9. Replacement of residues 2, 3, 5, 6, 9, 10, 11, and 12 (corresponding to W, T, L, S, Y, L, L, and G) significantly reduced activity at GalR2 (Figure 5). Replacement of residues 4, 8, and 13 (corresponding to N, G, and P) did not result in a reduction in activity. Similarly, Gal2-13ala8 showed reduced GalR1 activity, offering the potential for increased selectivity for GalR2 over GalR1. Notably, none of the substitutions increased agonistic activity at GalR2 or GalR1.

[0142] In vivo profiling of novel human FC fusion peptides Following in vitro optimization of the FC fusions, the compounds were assayed in an "industry standard" preclinical model of NP. Briefly, Gal2-30FC and then Gal2-13FC were first tested in a mouse chronic constriction injury (CCI) model, in which nerve injury induces a phenotype similar to the human NP condition, resulting in static mechanical (tactile) allodynia. Therefore, compounds capable of reversing this phenotype were considered analgesics, at least within this particular model. The CCI model of neuropathic pain involves the loose unilateral placement of three ligatures around the left sciatic nerve, spaced 1 mm apart, at mid-thigh level. This procedure results in the development of hyperalgesia, allodynia, and spontaneous pain (ectopic action potentials), which can be measured using mechanical and thermal behavioral assessments. This model is believed to mimic some of the symptoms and pathogenesis of neuropathic pain observed in the clinic. 64、65 In this study, static mechanical (tactile) allodynia was assessed by measuring pain thresholds using calibrated (force; g) von-Frey (vF) monofilaments applied to the plantar surface of the hind paw. Animals were placed in individual Perspex boxes on a raised metal mesh for 30–40 min before testing. A series of graded von Frey hairs (0.07 g, 0.16 g, 0.4 g, 0.6 g, and 1 g) were applied sequentially using a 1-second on, 1-second off protocol repeated 10 times. Each hair was applied perpendicular to the center of the ventral surface of the paw until it bent slightly. The force applied to the animal's hind paw to elicit a response in 5 out of 10 trials was recorded as the paw pain threshold (PWT).

[0143] Typically, baseline PWT was assessed from the ipsilateral paw for three consecutive days (days -2, -1, and 0), and the average response was recorded as the baseline pain threshold. On day 1, neuropathic pain was induced by ligation of the sciatic nerve. Animals were allowed to recover from surgery, and vF assessments of mechanical allodynia were performed on days 19 and 22. Animals were ranked and randomized (based on a Latin square design) into treatment groups according to the percent change in mean mechanical pain threshold observed on days 19 and 22 (compared to the pre-surgery baseline). Treatment groups and cohort sizes were designed to allow adequate power and post-hoc analysis using, for example, two-way repeated measures ANOVA and planned pairwise comparisons.

[0144] Gal2-30FC was tested at 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg ip, and in line with Study 1, vF assessments were performed at 2, 4, and 24 hours post-dose.

[0145] In this study, Gal2-30FC was effective, reversing CCI-induced mechanical hyperalgesia at 3 mg / kg, 10 mg / kg, and 30 mg / kg ip (Figure 6). The effect was statistically concentration-dependent and statistically significant. The effect was evident after 2 and 4 hours, but not after 24 hours at any of the doses tested. Based on the predicted in vitro stability of human IgG1 FC, the lack of effect of Gal2-30FC at 24 hours was unexpected and suggested that degradation and / or conformational changes of the active peptide warhead were likely the underlying cause.

[0146] Additional studies were commissioned to investigate whether shorter galanin fragments within FC fusions equally shared this tendency, as well as to test molecules that were more likely to be GalR2-specific than GalR1. For reasons of continuity, the study design was kept constant.

[0147] Gal2-13FC was tested at 1 mg / kg, 3 mg / kg, 10 mg / kg, and 30 mg / kg ip, with vF assessments performed 2, 4, and 24 hours after dosing. Gal2-13FC was effective, with statistically significant effects at all doses tested, resulting in a minimally effective dose of 1 mg / kg ip or less (Figures 7, 8, and 10). However, in stark contrast to Gal2-30FC, the effect was maintained and statistically significant beyond 24 hours. In this study, experimenters continued to measure vF as long as the effect persisted or was practically possible. Despite slight, typical variability between cohorts, statistically significant reversal of mechanical hyperalgesia was evident up to 96 hours after dosing and at both the highest and especially the lowest dose tested in these studies (Figures 7, 8, and 10).

[0148] Gal2-13FC was further tested at doses of 0.01 mg / kg, 0.03 mg / kg, and 0.1 mg / kg ip, with vF assessments at 4, 24, and 48 hours. This study demonstrated that Gal2-13FC was effective at low doses. The 0.03 mg / kg ip dose was shown to be effective at 4 hours. The effect of the higher, but relatively low, 0.1 mg / kg dose was also statistically significant at 24 hours (Figure 11).

[0149] Notably, the difference in the minimum effective dose (at 4 hours) between Gal2-13FC (0.03 mg / kg ip) and Gal2-30FC (3 mg / kg ip) was not predicted by their in vitro potencies, which were nearly equivalent (Figures 1 and 4). Although not explicitly shown for these molecules, properties such as recirculation via FCRn and renal excretion are likely to be substantially identical between Gal2-13FC and Gal2-30FC.

[0150] To assess the possibility of effect-desensitization, Gal2-13FC was tested at 0.3 mg / kg and 1 mg / kg ip (as above), with vF assessments repeated after repeated dosing. After vF assessments were performed at 4, 24, and 48 hours following the initial dose, animals were re-dosed and re-assessed by vF on days 3, 5, 7, 9, 11, and 13. Within acceptable experimental variability, there was no loss of effect at either dose over the course of the study (Figure 13).

[0151] In summary, multiple studies have demonstrated that Gal2-13FC is robust, effective (minimum effective dose [MED] ≤ 0.03 mg / kg ip), long-lasting (≥ 96 hours at 1 mg / kg ip), and sustained reversal of CCI-induced mechanical hyperalgesia with repeated administration (≥ 13 days) in a preclinical mouse model of NP (Figures 7, 10, 11, and 13). Notably, the Gal2-13 peptide consistently and repeatedly produces long-lasting effects in vivo. In vitro studies have shown that Gal2-13 is more stable.

[0152] Furthermore, the analgesia conferred by Gal2-13FC was not associated with overt sedation, at least as observed during the study, in stark contrast to clinically relevant drugs such as gabapentin and morphine, where sedation severely limits their usefulness.

[0153] In Gal2-13FC, amino acids 12 and 13 were glycine and proline, respectively. In Gal2-12FC, the proline was absent but was replaced by the first amino acid (alanine) of the linker sequence. To further investigate the importance of glycine and proline at these positions, Gal2-13FC ( ALA-12) was generated. This compound was very similar to Gal2-13FC, e.g., it was unable to activate GalR2 in vitro, despite being identical in amino acid length and containing a proline at position 13. Taken together, these data suggest that glycine and proline at positions 12 and 13, respectively, dramatically affect functional activity, at least when presented as an FC fusion (Table 4).

[0154] The above experiments demonstrated a favorable in vivo profile of Gal2-13FC. In doing so, this study also provided solid confirmation of the involvement and therapeutic potential of galanin and related peptides in interacting with peripheral GalR2 receptors in the pathogenesis of NP. This is also the first positive demonstration of the utility of biopharmaceuticals, specifically FC fusions, for the treatment of NP and appears to preclude the possibility of GalR2 desensitization. The predicted lack of CNS penetrance of the FC fusions and the known receptor selectivity of GalR2 over GalR1 conferred by the peptide warhead design also indicated a role for central and peripheral GalR2 and GalR1 receptors in this phenotype.

[0155] Importantly, Gal2-l3FC demonstrated robust, effective (MED ≤ 1 mg / kg ip) and long-lasting (> 72 h) reversal of CFA-induced mechanical hyperalgesia in a preclinical mouse model of inflammatory pain (Figure 10), demonstrating that Gal2-l3FC is effective at low doses and over a long period of time in treating inflammatory pain and NP.

[0156] Gal2-13FC was also tested at doses of 0.1 mg / kg, 1 mg / kg, and 3 mg / kg i.p. in a chemotherapy-induced model of neuropathic pain, with assessments at 4, 24, and 48 hours post-dose. In this study, Gal2-13FC was effective and was able to maintain the reversal of oxaliplatin-induced mechanical hyperalgesia 4 hours post-dose (day 5 post-oxaliplatin administration) (Figure 12). This study demonstrates the utility of Gal2-13FC for the treatment of neuropathic pain associated with anti-cancer therapy.

[0157] These data clearly validate the role of peripheral activation of the GalR2 receptor in nociception and offer the prospect of therapeutic interventions in which modulation of the galanin / galanin receptor system could circumvent the burden of central CNS side effects from current frontline pharmacotherapies, e.g., sedation, constipation, addiction, nausea, etc. The differences in the in vivo profile of Gal2-30FC compared to Gal2-13FC were not predicted based on their relative in vitro activity (Figure 3).

[0158] table

[0159] TIFF2026502133000001.tif77170

[0160] TIFF2026502133000002.tif132170

[0161] TIFF2026502133000003.tif169170

[0162] TIFF2026502133000004.tif110170

[0163] TIFF2026502133000005.tif52170

[0164] TIFF2026502133000006.tif75170

[0165] TIFF2026502133000007.tif68170

[0166] TIFF2026502133000008.tif52170

[0167] TIFF2026502133000009.tif115170

[0168] array TIFF2026502133000010.tif45170 SEQ ID NO: 1 - Amino acid sequence of Gal1-30FC fusion.

[0169] TIFF2026502133000011.tif45170 SEQ ID NO:2 - Amino acid sequence of Gal2-30FC fusion.

[0170] MEWSGIFLFLVATATDVHS SEQ ID NO:3 - Leader sequence

[0171] WTLNSAGYLLGP SEQ ID NO: 4 - GalR2 binding moiety Gal2-13

[0172] LEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:5 - IgG1 FC region

[0173] AEAAAKEAAAKA SEQ ID NO:6 - Linker

[0174] TIFF2026502133000012.tif22170 SEQ ID NO: 7-Gal2-13FC mature chimeric protein (junctions underlined, linkers in italics)

[0175] TIFF2026502133000013.tif23170 SEQ ID NO: 8 - Gal2-13FC precursor chimeric protein (leader underlined with dotted lines, junctions underlined with solid lines, linkers in italics)

[0176] WTX1NSA X2YLLGX3 (wherein X1, X2 and X3 are independently any amino acid). SEQ ID NO:9

[0177] WTX1NSA X2YLLGX3 (wherein X1 is independently L or A, X2 is independently G or A, and X3 is independently P or A). SEQ ID NO: 10

[0178] GGS SEQ ID NO: 11 - Linker

[0179] GGSGGS SEQ ID NO: 12 - Linker

[0180] GGSGGSGGSGGS SEQ ID NO: 13 - Linker

[0181] PAPAPAPA SEQ ID NO: 14 - Linker

[0182] GWTLNSAGYLLGPHAVGNHRSFSDKNGLTS SEQ ID NO: 15 - Full length human galanin (1-30)

[0183] GWTLNSAGYLLGPHAIDNHRSFHDKYGLA SEQ ID NO: 16 - Full length rat galanin (1-29)

[0184] WTLNSAGYLLGPHAIDNHRSFHDKYGLA SEQ ID NO: 17 - Rat galanin truncates 2-29

[0185] WTLNSAGYLL SEQ ID NO: 18-Gal2-11

[0186] GWTLNSAGYLLGPHA SEQ ID NO: 19-Gal1-15

[0187] GWTLNSAGYLLGPQPPGFSPFR SEQ ID NO:20-M617

[0188] RGRGNWTLNSAGYLLGPVLPPPALALA SEQ ID NO:21-M1145

[0189] WTLNS AGYLLGPKKKKK SEQ ID NO: 22-Nax409-9

[0190] TIFF2026502133000014.tif24170 SEQ ID NO: 23-Gal1-30FC mature chimeric protein (junctions underlined, linkers in italics)

[0191] TIFF2026502133000015.tif24170 SEQ ID NO: 24-Gal2-11FC mature chimeric protein (junctions underlined, linkers in italics)

[0192] TIFF2026502133000016.tif24170 SEQ ID NO: 25-Gal2-12FC mature chimeric protein (junctions underlined, linkers in italics)

[0193] TIFF2026502133000017.tif24170 SEQ ID NO: 26-Gal2-18FC mature chimeric protein (junctions underlined, linkers in italics)

[0194] TIFF2026502133000018.tif24170 SEQ ID NO: 27-Gal2-24FC mature chimeric protein (junctions underlined, linkers in italics)

[0195] TIFF2026502133000019.tif24170 SEQ ID NO: 28-Gal2-30FC mature chimeric protein (junctions underlined, linkers in italics)

[0196] TIFF2026502133000020.tif23170 SEQ ID NO: 29-Gal2-14FC mature chimeric protein (junctions underlined, linkers in italics)

[0197] TIFF2026502133000021.tif24170 SEQ ID NO: 30-Gal2-15FC mature chimeric protein (junctions underlined, linkers in italics)

[0198] TIFF2026502133000022.tif22170 SEQ ID NO: 31 - Gal2-16FC mature chimeric protein (junctions underlined, linkers in italics)

[0199] TIFF2026502133000023.tif24170 SEQ ID NO: 32 - Gal2-17FC mature chimeric protein (junctions underlined, linkers in italics)

[0200] TIFF2026502133000024.tif22170 SEQ ID NO: 33-Gal2-13FC GGS mature chimeric protein (junctions underlined, linkers in italics)

[0201] TIFF2026502133000025.tif22170 SEQ ID NO: 34-Gal2-13FC GGS2 mature chimeric protein (junctions underlined, linkers in italics)

[0202] TIFF2026502133000026.tif22170 SEQ ID NO: 35-Gal2-13FC GGS4 mature chimeric protein (junctions underlined, linkers in italics)

[0203] TIFF2026502133000027.tif22170 SEQ ID NO: 36-Gal2-13FC NL mature chimeric protein (junctions underlined, linkers in italics)

[0204] TIFF2026502133000028.tif22170 SEQ ID NO: 37-Gal2-13FC PA4 mature chimeric protein (junctions underlined, linkers in italics)

[0205] ATLNSAGYLLGP SEQ ID NO: 38-Gal2-13 Ala2

[0206] WALNSAGYLLGP SEQ ID NO: 39-Gal2-13 Ala3

[0207] WTANSAGYLLGP SEQ ID NO: 40-Gal2-13 Ala4

[0208] WTLASAGYLLGP SEQ ID NO: 41-Gal2-13 Ala5

[0209] WTLNAAGYLLGP SEQ ID NO: 42-Gal2-13 Ala6

[0210] WTLNSAAYLLGP SEQ ID NO: 43 - Gal2-13 Ala8

[0211] WTLNSAGALLGP SEQ ID NO: 44-Gal2-13 Ala9

[0212] WTLNSAGYALGP SEQ ID NO: 45-Gal2-13 Ala10

[0213] WTLNSAGYLAGP SEQ ID NO: 46-Gal2-13 Ala11

[0214] WTLNSAGYLLAP SEQ ID NO: 47-Gal2-13 Ala12

[0215] WTLNSAGYLLGA SEQ ID NO: 48-Gal2-13 Ala13

[0216] WTLNSAGYLLGP SEQ ID NO: 49 - GalR2 binding moiety Gal2-12

[0217] MRGSHHHHHH SEQ ID NO:50

[0218] DYKDDDDK SEQ ID NO:51

[0219] KETAAAKFERQHMDS SEQ ID NO:52

[0220] WSHPQFEK SEQ ID NO:53

[0221] EQKLISEEDL SEQ ID NO:54

[0222] MKAEFRRQESDR SEQ ID NO: 55

[0223] MRDALDRLDRLA SEQ ID NO:56

[0224] SEQ ID NO:57 - Human truncated galanin 2-30

[0225] WTLNSAGYLLGPHA SEQ ID NO:58-Gal2-15

[0226] WTLNSAGYLLGPH SEQ ID NO:59-Gal2-14

[0227] TIFF2026502133000029.tif23170 SEQ ID NO: 60 - Gal2-12FC precursor chimeric protein (leader underlined with dotted lines, junctions underlined with solid lines, linkers in italics)

[0228] TIFF2026502133000030.tif23170 SEQ ID NO: 61-Gal2-13FC(Ala12) mature chimeric protein (junctions underlined, linkers in italics)

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Claims

1. A chimeric protein comprising: (i) a galanin fragment consisting of residues 2-13 of galanin (Gal2-13), or residues 2-12 of galanin (Gal2-12), or a variant thereof; (ii) an FC region; and A chimeric protein comprising:

2. The chimeric protein of claim 1, which is a selective agonist of peripheral GalR2.

3. The chimeric protein of claim 1 or 2, wherein the galanin fragment is at the N-terminus of the chimeric protein and the FC region is at the C-terminus.

4. 10. The chimeric protein of any one of the preceding claims, wherein the galanin fragment consists of residues 2 to 13 of galanin (Gal2-13) or a variant thereof.

5. 10. A chimeric protein according to any one of the preceding claims, wherein the galanin fragment consists of the amino acid sequence of SEQ ID NO:

9.

6. 10. A chimeric protein according to any one of the preceding claims, wherein the galanin fragment consists of the amino acid sequence of SEQ ID NO:

10.

7. 10. A chimeric protein according to any one of the preceding claims, wherein the galanin fragment consists of the amino acid sequence of SEQ ID NO:

4.

8. 10. The chimeric protein of claim 1, wherein the Fc region is an IgG1 Fc region.

9. 10. The chimeric protein of any one of the preceding claims, wherein the FC region comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

5.

10. 10. The chimeric protein of claim 1, wherein the FC region comprises the amino acid sequence of SEQ ID NO:

5.

11. 2. The chimeric protein of claim 1, wherein the galanin fragment and the FC region are directly connected.

12. 2. The chimeric protein of claim 1, wherein the galanin fragment and the FC region are connected via a linker.

13. The chimeric protein of claim 12, wherein the linker comprises the amino acid sequence of any one of SEQ ID NO: 6 and SEQ ID NO: 11 to SEQ ID NO:

14.

14. 10. A chimeric protein according to any one of the preceding claims, comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

7.

15. 10. A chimeric protein according to any one of the preceding claims, comprising the amino acid sequence of SEQ ID NO:

7.

16. 10. The chimeric protein according to any one of the preceding claims, wherein the galanin fragment consists of residues 2 to 12 of galanin (Gal2-12) or a variant thereof.

17. The chimeric protein of claim 16, wherein the galanin fragment consists of the amino acid sequence of SEQ ID NO:

49.

18. 18. The chimeric protein of claim 16 or 17, comprising the amino acid sequence of SEQ ID NO:

25.

19. 10. The chimeric protein of any one of the preceding claims, wherein the chimeric protein exhibits analgesic activity in vivo for 48 hours or more.

20. 20. The chimeric protein of claim 19, wherein the chimeric protein reduces paw withdrawal in a CCI mouse model or a CFA-induced hyperalgesia mouse model for 48 hours or more.

21. A nucleic acid encoding a chimeric protein according to any one of the preceding claims.

22. A vector comprising the nucleic acid of claim 21.

23. 23. A recombinant cell comprising the nucleic acid of claim 21 or the vector of claim 22.

24. A pharmaceutical composition comprising the chimeric protein of any one of claims 1 to 20 and a pharmaceutically acceptable excipient.

25. A method for treating pain in an individual, comprising administering to the individual in need thereof the chimeric protein of any one of claims 1 to 20.

26. 26. The method of claim 25, wherein the pain is inflammatory pain or neuropathic pain.

27. A chimeric protein according to any one of claims 1 to 20 for use in a method of treatment of the human or animal body by therapy.

28. A chimeric protein according to any one of claims 1 to 20 for use in a method for the treatment of pain.

29. 29. The chimeric protein for use according to claim 28, wherein the pain is inflammatory pain or neuropathic pain.

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