US 2008.0020978A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2008/0020978 A1 Gegg, JR. et al. (43) Pub. Date: Jan. 24, 2008

(54) CGRP ANTAGONSTS AND Related U.S. Application Data CONUGATES (60) Provisional application No. 60/729,083, filed on Oct. (76) Inventors: Colin V. Gegg JR., Newbury Park, CA 21, 2005. (US); Eileen J. Johnson, Newbury Park, CA (US); Leslie P. Miranda, Publication Classification Thousand Oaks, CA (US); Kenneth W. Walker, Newbury Park, CA (US); (51) Int. Cl. Jerry Ryan Holder, Simi Valley, CA A6II 38/00 (2006.01) (US); Marie E. Wright, Westlake A6IP 43/00 (2006.01) Village, CA (US); Derin C. D'Amico, (52) U.S. Cl...... S14/12 Newbury Park, CA (US) (57) ABSTRACT Correspondence Address: AMGEN INC. Disclosed is a composition of matter that involves a CGRP MAIL STOP 28-2-C peptide antagonist. A pharmaceutical composition is dis ONE AMGEN CENTER DRIVE closed that comprises the composition of matter and a THOUSAND OAKS, CA 91320-1799 (US) pharmaceutically acceptable carrier, which can be config ured for administration to a patient. Also disclosed is a (21) Appl. No.: 111584,177 method of producing the composition of matter. Methods of treating, preventing or mitigating migraine, are also dis (22) Filed: Oct. 19, 2006 closed. Structure of human OCGRP

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CGRP PEPTIDE ANTAGONSTS AND that recognize regulatory such as , CONUGATES and vasoactive intestinal polypeptide (VIP). The best char acterized splice variants of human CT receptor differ 0001) This application claims the benefit of U.S. Provi depending on the presence (formerly CTR or CTR1, now sional Application No. 60/729,083, filed Oct. 21, 2005, known as CTs) or absence (the major splice variant, which is hereby incorporated by reference. formerly CTR, or CTR, now known as CT) of 16 amino 0002 This application incorporates by reference all Sub acids in the first intracellular loop. (Gorn et al., Expression ject matter contained on the compact disc, which is identi of two human skeletal receptor isoforms cloned fied by the name of the file, A-1061.5T25.txt created on from a giant cell tumor of bone: the first intracellular domain October , 2006, the size of which file is KB. modulates ligand binding and signal transduction, J. Clin. Invest., 95:2680-2691 (1995); Hay et al., receptors: 0003. Throughout this application various publications molecular composition and , Biochem. Soc. are referenced within parentheses. The disclosures of these Trans., 32:865-867 (2004); Poyner et al., 2002). The exist publications in their entireties are hereby incorporated by ence of at least two CGRP receptor subtypes had been reference in this application in order to more fully describe proposed from differential antagonist affinities and agonist the state of the art to which this invention pertains. potencies in a variety of in vivo and in vitro bioassays. (Dennis et al., CGRP8-37. A calcitonin gene-related peptide BACKGROUND OF THE INVENTION antagonist revealing calcitonin gene-related peptide receptor heterogeneity in brain and periphery, J. Pharmacol. Exp. 0004) 1. Field of the Invention Ther. 254:123-128 (1990); Dennis et al., Structure-activity 0005 The present invention is related to the biochemical profile of calcitonin gene-related peptide in peripheral and arts, in particular to therapeutic peptide conjugates. brain tissues. Evidence for receptor multiplicity, J. Pharma col. Exp. Ther. 251:718-725 (1989); Dumont et al. A potent 0006 2. Discussion of the Related Art and selective CGRP2 agonist, Cys(Et)2.7hCGRP: com 0007. The calcitonin (CT) superfamily of peptides parison in prototypical CGRP and CGRP2 in vitro assays, includes at least five known members: CT, amylin (AMY), Can. J. Physiol. Pharmacol., 75:671-676 (1997)). adrenomedulin (ADM), and two calcitonin gene-related 0009. The CGRP receptor subtype was found to be peptides, CGRP1 (also known as ctCGRP) and CGRP2 (also sensitive to the antagonist fragment CGRP(8-37). (Chiba et known as BCGRP). Calcitonin is involved in the control of al., Calcitonin gene-related peptide bone metabolism and is also active in the central nervous human CGRP-(8-37), Am. J. Physiol., 256:E331-E335 system (CNS). Amylin also has specific binding sites in the (1989); Dennis et al. (1990); Mimeault et al., Comparative CNS and is thought to regulate gastric emptying and have a affinities and antagonistic potencies of various human cal role in carbohydrate metabolism. ADM is a potent vasodi citonin gene-related peptide fragments on calcitonin gene lator. ADM has specific receptors on astrocytes and its related peptide receptors in brain and periphery, J. Pharma messenger RNA is upregulated in CNS tissues that are col. Exp. Ther. 258:1084-1090 (1991)). By contrast, the Subject to ischaemia. (Zimmermann, et al., Identification of CGRP, receptor was sensitive to linear human CGRP adrenoinedullin receptors in cultured rat astrocytes and in (hCGRP) analogs, in which the cysteine residues at posi neuroblastoinaglioma hybrid cells (NG108-15), Brain Res., tions 2 and 7 were derivatized (e.g., with acetoaminomethyl 724:238-245 (1996); Wang et al., Discovery of adrenoin Cys(ACM) or ethylamide Cys(Et)7)) but CGRP, edullin in rat ischaemic cortex and evidence for its role in receptor was insensitive to fragment CGRP(8-37). (Dennis exacerbating focal brain ischaemic damage, Proc. Natl. et al. (1989); Dennis et al. (1990); Dumont et al. (1997)). In Acad. Sci. USA, 92:11480-11484 (1995)). The biological 1998, the CGRP receptor was identified as a heterodimer activities of CGRP include the regulation of neuromuscular composed of a novel single transmembrane domain acces junctions, of antigen presentation within the immune sys sory , receptor activity-modifying protein 1 tem, of vascular tone and of sensory neurotransinission. (RAMP1), and calcitonin receptor-like receptor (CRLR or (Poyner, D. R., Calcitonin gene-related peptide: multiple “CL”). (McLatchie et al., RAMPs regulate the transport and actions, multiple receptors, Pharmacol. Ther., 56:23-51 ligand specificity of the calcitonin-receptor-like receptor, (1992); Muff et al., Calcitonin, calcitonin gene related peptide, and amylin: homologous peptides, Nature, 393:333-339 (1998)). separate receptors and overlapping biological actions, Eur. J. 0010 CRLR has 55% overall sequence iden Endocrinol., 133: 17-20 (1995)). Three calcitonin receptor tity with CT receptor, although the transmembrane domains stimulating peptides (CRSPs) have also recently been iden are almost 80% identical. (McLatchie et al. (1998); Poyner tified in a number of mammalian species; the CRSPs may et al., International union of pharmacology. XXXII. The form a new subfamily in the CGRP family, however, the mammalian calcitonin gene-related peptides, adrenomedul endogenous molecular forms, receptors, and biological lin, amylin and calcitonin receptors, Pharmacol. Rev., activity of the CRSPs remain unidentified. (Katafuchi, Tand 54:233-246 (2002)). Minamino, N, Structure and biological properties of three 0011 Ligand specificity of CT receptor and CRLR calcitonin receptor-stimulating peptides, novel members of depend on the coexpression of members of a family of the calcitonin gene-related peptide family, Peptides, accessory called the receptor activity modifying 25(11):2039-2045 (2004)). proteins (RAMPs). The RAMP family includes three that act 0008. The CT superfamily peptides act through seven as receptor modulators that determine the ligand specificity transmembrane-domain G-protein-coupled receptors of receptors for the CT family members. RAMPs are type I (GPCRs). The CT receptor and CGRP receptors are type II transmembrane proteins that share about 30% amino acid (“family B') GPCRs, which family includes other GPCRs sequence identity and a common predicted topology, with US 2008/0020978 A1 Jan. 24, 2008

short cytoplasmic C-termini, one trans-membrane domain receptor gene product, Molecular Pharmacology, 56:235 and large extracellular N-termini that are responsible for the 242 (1999); Muff et al. An amylin receptor is revealed specificity. (McLatchie et al. (1998); Fraser et al., The amino following co-transfection of a calcitonin receptor with terminus of receptor activity modifying proteins is a critical receptor activity modifying proteins-1 or -3. Endocrinology, determinant of glycosylation state and ligand binding of 140:2924-2927 (1999); Sexton et al. (2001); Leuthauser et calcitonin receptor-like receptor, Molecular Pharmacology, al., Receptor-activity-modifying protein 1 forms het 55:1054-1059 (1999)). erodimers with two G-protein-coupled receptors to define 0012 CRLR has been shown to form a high affinity ligand recognition, Biochem. J., 351:347-351 (2000); receptor for CGRP, when associated with RAMP1, or, to Tilakaratne et al., Amylin receptor phenotypes derived from preferentially bind ADM when associated with RAMP2 or human calcitonin receptor/RAMP coexpression exhibit RAMP3. (McLatchie et al. (1998); Sexton et al., Receptor pharmacological differences dependent on receptor isoform activity modifying proteins, Cellular Signaling, 13:73-83 and host cell environment, J. Pharmacol. Exp. Ther. 294:61 (2001); Conner et al., Interaction of calcitonin-gene-related 72 (2000): Nakamura et al., Osteociast-like cells express peptide with its receptors, Biochemical Society Transactions receptor activity modifying protein 2: application of laser 30(Part 4): 451-454 (2002)). The glycosylation state of capture microdissection, J. Molec. Endocrinol. 34:257-261 CRLR is associated with its pharmacology. RAMPs 1, 2, and (2005)). 3 transport CRLR to the plasma membrane with similar 0014. The CGRP-sensitive responses mediated via efficiencies, however RAMP1 presents CRLR as a termi CT/RAMPs are blocked by the selective receptor antagonist nally glycosylated, mature glycoprotein and a CGRP recep calcitonin(8-32) but not by CGRP(8-37), which antagonizes tor, whereas RAMPs 2 and 3 present CRLR as an immature, the CRLR/RAMP1 complex (CGRP receptor). (Kuwasako core glycosylated ADM receptor. (Fraser et al. (1999)). et al., Novel calcitonin-(8-32)-sensitive adrenomedullin Characterization of the CRLR/RAMP2 and CRLR/RAMP3 receptors derived from co-expression of calcitonin receptor receptors in HEK293T cells by radioligand binding (I- with receptor activity-modifying proteins, Biochem. Bio ADM as radioligand), functional assay (cAMP measure phys. Res. Commun., 301:460-464 (2003); Leuthauser et al., ment), or biochemical analysis (SDS-polyacrylamide gel 2000). electrophoresis) revealed them to be indistinguishable, even though RAMPs 2 and 3 share only 30% amino acid sequence 0.015 The CCGRP peptide (also known as CGRP1) and identity. (Fraser et al. 1999)). Differences have been (BCGRP peptide (also known as CGRP2) are 37 amino acid observed, however, in the pharmacology for CRLR residues long and differ from each other by three amino expressed with RAMP2 versus RAMP3. Both ccCGRP and acids. These two isoforms have so far proved to be indis CGRP8-37 as well as ADM and ADM 22-52 are active at the tinguishable in their biological activities. (Poyner, D. R. RAMP3 heterodimer, indicating that this complex may act (1992); Muff et al. (1995)). Native human CCGRP and as both a CGRP and an ADM receptor. (Howitt et al., British BCGRP each contain a disulfide bridge between cysteine Journal of Pharmacology, 140:477-486 (2003); Muffet al., residues at amino acid positions 2 and 7 and a carboxy Hypertens. Res., 26:S3-S8 (2003)). Coexpression of human terminal phenylalanine amide, both of which are required for CRLR with rat RAMP1, and vice versa, showed that the biological activity of the native peptides. (Wimalawansa, S RAMP1 species determined the pharmacological character J. Amylin, calcitonin gene-related peptide, calcitonin and istics of the CRLR/RAMP1 complex with respect to several adrenomedullin: a peptide superfamily, Crit. Rev. Neuro small molecule CGRP receptor antagonists tested. (Mallee et biol., 11: 167-239 (1997)). Their binding sites are widely al., Receptor Activity-Modifying Protein 1 determines the distributed among peripheral tissues and in the central species selectivity of non-peptide CGRP receptor antago nervous system, enabling CGRP to exert a wide variety of nists, J. Biol. Chem., 277(16): 14294-14298 (2002)). Unless biological effects, including potent vasodilation. (Van Ros associated with a RAMP, CRLR is not known to bind any Sum et al., Neuroanatomical localization, pharmacological endogenous ligand; it is currently the only GPCR thought to characterization and functions of CGRP related peptides behave this way. (Conner et al. A key role for transmem and their receptors, Neurosci. Biobehav. Rev. 21:649-678 brane prolines in calcitonin receptor-like agonist binding (1997)). and signaling: implications for family B G-protein-coupled 0016 Structural studies investigating the interaction of receptors, Molec. Pharmacol., 67(1):20-31 (2005)). CGRP with its receptor have defined both functional sub 0013 CT receptor has also been demonstrated to form domains and specific residues involved in receptor binding heterodimeric complexes with RAMPs, which are known as and activation. (Conner, A. C., Biochem. Soc. Trans. amylin receptors. Generally, CT/RAMP1 (AMY) receptors 30:451–455 (2002)). As illustrated in FIG. 1, the first seven have high affinity for salmon CT, AMY and CGRP and lower amino acid residues of CGRP, which form a disulphide affinity for mammalian CTs. For CT/RAMP2 (AMY) bonded loop, are thought to interact with the transmembrane receptors and CTVRAMP3 (AMY) receptors, a similar domain of CRLR to cause receptor activation. CGRP(8-37) pattern is principally observed, although the affinity for fragment binds with high affinity to CGRP receptor, but acts CGRP is lower and may not be significant at physiologically as an antagonist. The rest of the CGRP molecule falls into relevant ligand concentrations. The precise receptor pheno three domains or regions: amino acid residues 28-37 and type is dependent on cell type and CT receptor splice variant 8-18 are normally required for high-affinity binding with the (CT or CT), particularly for RAMP2-generated AMY CGRP receptor, while residues 19-27 form a hinge region, receptors. For example, a pure population of osteoclast-like which appears to allow a hairpin-like structure wherein the cells reportedly expressed RAMP2, CT receptor, and CRLR, two sub domains interact with one another at the receptor but not RAMP1 or RAMP3. (Hay et al. (2004); Christopou interface. The 28-37 region is thought to be in direct contact los et al., Multiple amylin receptors arise from receptor with the receptor during binding, while the C-helical region activity-modifying protein interaction with the calcitonin comprising residues 8-18 may make additional receptor US 2008/0020978 A1 Jan. 24, 2008

contacts or may stabilize an appropriate conformation of the hyperresponsiveness, FEBS Journal, 30" FEBS Congress & 28-37 region. It is likely that these binding regions of CGRP 9" IUMB Conference Website, Abstract No. M3-020P interact with CGRP receptor both at the CRLR and at the (2005); Brain, S D et al., Evidence that calcitonin gene extracellular domain of RAMP1. The amidated carboxy related peptide contributes to inflammation in the skin and terminal residue has been shown to be essential for high joint, Ann. NY Acad. Sci., 657(1):412–419 (1992); Cav affinity binding to CGRP receptors by CGRP and CGRP iedes-Bucheli, J. et al., Expression of calcitonin gene-related peptide analogs. Mutation analyses have further implicated peptide(CGRP) in irreversible acute pulpitis, J. Endodontics, select residues R11, R18, T30, V32 S34 and F37 in receptor 30(4):201-204 (2004); Ling, Q D et al., The pattern and binding. (Rist et al. From micromolar to nanomolar affinity: distribution of calcitonin gene-related peptide (CGRP) ter a systematic approach to identify the binding site of CGRP minals in the rat dorsal following neonatal peripheral at the human calcitonin gene-related peptide 1 receptor, J. inflammation, Neuroreport, 14(15):1919-1921 (2003); Li, Y Med. Chem., 41:117-123 (1998); Conner et al., Interaction J et al., CGRP-mediated cardiovascular effect of nitroglyc of calcitonin-gene-related peptide with its receptors, Bio erin, Med Hypotheses, 60(5):693-698 (2003); Beer, Set al., chem. Soc. Trans., 30(4):451–455 (2002); Smith et al., Systemic neuropeptide levels as predictive indicators for Modifications to the N-terminus but not the C-terminus of lethal outcome in patients with postoperative sepsis, Critical calcitonin gene-related peptide(8-37) produce antagonists Care Medicine, 30(8): 1794-1798 (2002)). with increased affinity, J. Med. Chem., 46:2427-2435 0020. Therapeutic administration of CGRP analogs was (2003)). taught by Evans et al. for the lowering of blood pressure and 0017 Moreover, amino-terminal truncations of CGRP gastric acid secretion, and for other effects on, for example, (e.g., CGRP(8-37)) have been identified that are antagonistic ingestion behavior, taste and sensory perception, e.g., noci to the receptor. While further truncations (e.g., CGRP(28 ception. (U.S. Pat. No. 4,530,838; U.S. Pat. No. 4.736,023). 37)) show very little receptor binding, much of the lost 0021. Therapeutic use of CGRP antagonists and CGRP antagonist activity can be restored with three point muta targeting aptamers has been proposed for the treatment of tions (T30D, V32P and G33F) in the CGRP(28-37) region. migraine and other disorders. (E.g., Olesen et al., Calcitonin (Rist, B. et al., J. Med. Chem., 41: 117-123 (1998)). It has gene-related peptide receptor antagonist BIBN 4096 BS for been Suggested that these amino acid Substitutions compen the acute treatment of migraine, New Engl. J. Med., sate for the missing (CGRP(8-27) domain (Carpenter, K. A. 350:1104-1110 (2004); Perspective: CGRP-receptor antago et al., Turn structures in CGRP C-terminal analogues pro nists—a fresh approach to migraine, New Engl. J. Med., mote stable arrangements of key residue side chains, Bio 350:1075 (2004); Vater et al., Short bioactive Spiegelmers to chemistry, 40:8317-8325 (2001)). migraine-associated calitonin gene-related peptide rapidly 0018 CGRP is thought to have a causative role in identified by a novel approach: tailored-SELEX, Nuc. Acids migraine. Migraine pathophysiology involves the activation Res., 31(21 e130):1-7 (2003); WO 96/03993). of the trigeminal ganglia, where CGRP is localized, and CGRP levels significantly increase during a migraine attack. 0022. For example, Noda et al. described the use of This in turn, promotes cranial blood vessel dilation and CGRP or CGRP derivatives for inhibiting platelet aggrega neurogenic inflammation and sensitization. (Doods, H., tion and for the treatment or prevention of arteriosclerosis or Curr. Opin. Investig. , 2:1261-1268 (2001)). CGRP thrombosis. (EP 0385712 B1). has been shown to induce migraine headaches in patients 0023 Liu et al. disclosed therapeutic agents that modu susceptible to migraines. Furthermore, in a recent Phase II late the activity of CT receptor, including vehicle-conju clinical trial, a potent small-molecule CGRP antagonist has gated peptides such as calcitonin and human CCGRP. (WO been shown to alleviate migraine pain. CGRP may also be 01/83526 A2: US 2002/0090646A1). involved in chronic pain syndromes other than migraine. In 0024. Vasoactive CGRP peptide antagonists and their use rodents, intrathecally delivered CGRP induces severe pain, in a method for inhibiting CGRP binding to CGRP receptors and CGRP levels are enhanced in a number of pain models. In addition, CGRP antagonists block neuropathic and cap were disclosed by Smith et al.; such CGRP peptide antago saicin-induced pain in rodents. Together, these observations nists were shown to inhibit CGRP binding to coronary artery imply that a potent and selective CGRP receptor antagonist membranes and to relax capsaicin-treated pig coronary can be an effective therapeutic for treatment of chronic pain, arteries. (U.S. Pat. No. 6,268,474 B1; and U.S. Pat. No. including migraine. 6,756.205 B2). 0025) Rist et al. disclosed peptide analogs with CGRP 0019 CGRP has also been implicated in diabetes mellitus receptor antagonist activity and their use in a for (type II), inflammation, cardiovascular disorders, and in the hemodynamic derangements associated with endotoxemia treatment and prophylaxis of a variety of disorders. (DE and sepsis resulting from postoperative infection and a 19732944 A1). variety of other infectious diseases. (E.g., Khachatryan, Aet 0026. Nevertheless, CGRP peptides have shown a num al., Targeted expression of the neuropeptide calcitonin gene ber of problems as therapeutics. Native CGRP peptides are related peptide to beta cells prevents diabetes in NOD mice, typically nonselective, inactive in oral form, generally have J. Immunol., 158(3):1409-1416 (1997); Ohtori S et al., a short duration of action and can elicit a number of potential Phenotypic inflammation switch in rats shown by calcitonin side effects that can include undesirable effects on blood gene-related peptide immunoreactive dorsal root ganglion pressure. In general, therapeutic peptides and proteins neurons innervating the lumbar facet joints, Spine, exhibit very fast plasma clearance, thus requiring frequent 26(9): 1009-1013 (2001); Qin, X et al. Temporal and spatial injections to ensure steady pharmaceutically relevant blood distribution of Substance P and its receptor regulated by levels of a particular peptide or protein with pharmacologi calcitonin gene-related peptide in the development of airway cal activity. Many pharmaceutically relevant peptides and US 2008/0020978 A1 Jan. 24, 2008

proteins, even those having human primary structure, can be carrier; and to clinical methods involving administration of immunogenic, giving rise to neutralizing antibodies circu the CGRP peptide antagonist to a patient. lating in the bloodstream. This is especially true for intra 0032. The compositions of matter and pharmaceutical venous and Subcutaneous administration, which is of par compositions of the present invention, comprising the CGRP ticular concern for the delivery of most peptide and protein peptide antagonists, can provide Substantially increased in drugs. Vivo pharmacological half-life and/or decreased immunoge 0027. By increasing the molecular volume and by mask nicity, compared to corresponding unconjugated forms or ing potential epitopes, modification of a therapeutic unmodified amino acid sequences, while maintaining rela polypeptide with a vehicle, such as a polyethylene glycol tively high potency. (PEG) polymer, has been shown to be effective in reducing both the rate of clearance as well as the antigenicity of the 0033. The vehicle-conjugated, or unconjugated, CGRP protein. Reduced proteolysis, increased water solubility, peptide antagonist, involved in the inventive composition of reduced renal clearance, and steric hindrance to receptor matter, includes a CGRP peptide that lacks a functional mediated clearance are a number of mechanisms by which CGRP, receptor activation region, but includes from its the attachment of a polymer to the backbone of a polypep C-terminal end to its N-terminal end: tide may prove beneficial in enhancing the pharmacokinetic 0034 the C-terminal carboxy moiety is replaced with a properties of the drug. For example, Davis et al. taught moiety selected from: conjugating PEG or polypropylene glycol to proteins such as and to produce a less immunogenic product 0035 (A) = C(=O)NRR, where R is independently while retaining a Substantial proportion of the biological hydrogen, (C-Cs)alkyl, haloalkyl, aryl or heteroaryl; and activity. (U.S. Pat. No. 4,179,337). 0036 (B) —CHOR where R is H (e.g., representing a 0028. In the actual practice of developing a conjugated peptide alcohol), or (C-C)alkyl, aryl or heteroaryl (e.g., peptide drug, it is not a trivial matter to overcome the representing a peptide ester). For example, in (A) the NRR significantly lower potency that a conjugated form typically can represent an amide moiety (interchangeably designated exhibits relative to the unconjugated form of the peptide. (J. “-amide' or “ NH or “ NH at the C-terminal end of M. Harrist et al., PEGylation: A Novel Process for Modify sequences listed herein), N-mono-substituted or N-di-sub ing Pharmacokinetics, Clin. Pharmacokinet., 40:539-551 stituted alkyl or aryl amide; and (2001); and R. Mehvar, Modulation of the Pharmacokinetics 0037 the CGRP peptide includes a first CGRP1 receptor and Pharmacodynamics of Proteins by Polyethylene Glycol binding region. Conjugation, J. Pharm. Pharmaceut. Sci., 3:125-136 (2000)). 0038. In vehicle-conjugated embodiments, a pharmaceu 0029. It is, therefore, a desideratum to combine the tically acceptable vehicle is conjugated to the CGRP peptide therapeutic benefits of vehicle-conjugated CGRP peptides at a site on the CGRP peptide other than at its carboxy and analogs (particularly, but not limited to, those with terminal amino acid residue. CGRP receptor antagonist activity), such as substantially 0039. Accordingly, the invention is also related to a increased pharmacological half-life and decreased immuno method of producing a composition of matter, which genicity, with little, if any, loss of potency relative to involves obtaining a CGRP peptide that lacks a functional unconjugated forms. These and other benefits are provided CGRP, receptor activation region, which CGRP peptide by the present invention. includes from its C-terminal end to its N-terminal end: an amide moiety at the C-terminus; and a first CGRP1 receptor SUMMARY OF THE INVENTION binding region. The obtained CGRP peptide is conjugated to 0030 The present invention relates to: compositions of a pharmaceutically acceptable vehicle at a site on the matter involving CGRP peptide antagonists (i.e., CGRP obtained peptide other than at the C-terminal amino acid peptides that are CGRP receptor antagonists). In some residue. embodiments, these CGRP peptide antagonists are vehicle conjugated, or alternatively, in other embodiments, they are 0040. The present invention is also directed to a method not conjugated to a vehicle (i.e., “unconjugated' or “free” or of treating migraine, in which a patient in need of Such “naked peptides). Some of the vehicle-conjugated CGRP treatment is administered a therapeutically effective amount peptide antagonists of the invention include those having a of the inventive composition of matter, so that one or more native CGRP peptide sequence. Alternatively, whether in migraine symptoms is alleviated in the patient. vehicle-conjugated or unconjugated embodiments, the 0041. The present invention is also directed to a method CGRP peptide antagonists of the present invention include of preventing or mitigating migraine, which involves admin CGRP peptide analogs containing modifications relative to istering to a patient who has previously experienced a a native CGRP sequence of interest, and can contain amino migraine a prophylactically effective amount of the inven acid residues that are canonical or non-canonical, naturally tive composition of matter, so that at least one symptom of rare, and/or unnatural. The CGRP peptide can possess either a Subsequent migraine is prevented or mitigated. a full length CGRP sequence, or a truncated fragment thereof, as long as it has a first CGRP receptor binding BRIEF DESCRIPTION OF THE FIGURES region proximal to the carboxy terminal end of the peptide. 0042 FIG. 1 shows the native amino acid sequence of 0031. The present invention also relates to methods of full-length human CCGRP (SEQ ID NO:43) with functional producing the compositions of matter, to pharmaceutical domains and secondary structural elements defined. Shaded compositions containing the composition of matter, i.e., the residues (R11, R18, T30, V32, S34 and F35-amide) have CGRP peptide antagonist and a pharmaceutically acceptable been implicated by the prior art in receptor binding, with US 2008/0020978 A1 Jan. 24, 2008 modifications at those positions typically having a positive 0.052 FIG. 11 shows in vitro metabolic identification of or negative effect on binding to CGRP. Source: Conner et Seq ID: No.658 in 100% monkey plasma after 4 hours al., Interaction of calcitonin-gene-related peptide with its incubation at room temperature. receptors, Biochemical Society Transactions, 30(Part 4): 0053 FIG. 12 shows in vivo metabolic identification of 451-454 (2002). Seq ID: No.658 in monkey plasma during 30 minutes of 0.043 FIG. 2 illustrates the proteolytic cleavage frag intravenous infusion with dose of 6 mg/kg. ments M1-M8 of SEQID NO:7 after incubation in 10% rat plasma for 1 hour at room temperature, detected by LC/MS 0054 FIG. 13 illustrates some peptide synthesis tech with the major cleavages occurring after L16, S17, and R18 niques that can be employed in making CGRP peptides in relative to the native human CCGRP sequence. accordance with the present invention. 0044 FIG.3 depicts some embodiments of the positions DETAILED DESCRIPTION OF THE within a CGRP peptide that are separated by 1 or more PREFERRED EMBODIMENTS amino acids that can be joined to form a cyclic lactam. 0055. The present invention relates to a composition of 004.5 FIG. 4 illustrates the effects of some different matter, which involves a vehicle-conjugated CGRP peptide alcohol co-solvents on reaction yields for PEGylation of a antagonist that preferentially binds to a mammalian CGRP relatively soluble CGRP peptide (Table 4, SEQ. ID NO: 7). receptor and antagonizes its biological activity. Mammalian 0046 FIG. 5 shows a comparison of the effects of IPA CGRP receptors can include those of humans or other and TFE on the PEGylation efficiency of a relatively soluble primates, rodents, canines, felines, or any other mammal of CGRP peptide (Table 4, SEQ ID NO:7) and a relatively interest, or of harvested or cultured mammalian cells. insoluble CGRP peptide (Table 7, SEQ ID NO: 31). 0056 “Polypeptide' and “protein’ are used interchange ably herein and include a molecular chain of amino acids 0047 FIG. 6 shows the effect of co-solvent on the PEGy linked through peptide bonds. The terms do not refer to a lation efficiency of a relatively soluble CGRP peptide SEQ. specific length of the product. Thus, “peptides, and “oli ID NO 7 in the presence of gopeptides,” are included within the definition of polypep tide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein frag ps (-O-) or TFE (v) or ments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. HFIPA (E) The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included. 0.048 FIG. 7 shows the effect of co-solvent concentration 0057 “CGRP receptor” means a cell membrane-inte on the PEGylation efficiency of a relatively insoluble CGRP grated CRLR/RAMP1 (or “CRLR-RAMP1’) complex. peptide SEQ ID No.:739 in the presence of 0.058 A“CGRP peptide' is a peptide that preferentially binds the CGRP receptor under physiological conditions of temperature, pH, and ionic strength. For purposes of the present invention, CGRP peptides include those having a full native CGRP peptide sequence and non-native CGRP peptide analogs containing modifications of a native CGRP sequence (e.g., amino acid Substitutions, insertions, dele tions, and/or amino terminal end truncations as further described herein below) relative to a native CGRP sequence 0049 FIG. 8 shows the effect of co-solvent concentration of interest, which can be, e.g., any known mammalian on the PEGylation efficiency of a relatively insoluble CGRP CGRP sequence, such as but not limited to, the native human peptide SEQ ID No.658 in the presence of CCGRP sequence or human BCGRP sequence. In accor dance with the present invention, the CGRP peptide the C-terminal carboxy moiety is replaced with a moiety selected from: (A) —C(=O)NRR, where R is indepen dently hydrogen, (C-C)alkyl, haloalkyl, aryl or heteroaryl; and (B) —CHOR where R is H. (C-C)alkyl, aryl or heteroaryl. In some embodiments, this constitutes a carboxy terminally amidated amino acid sequence. Such as, but not limited to, a sequence having a C-terminal phenylalanina mide residue or tyrosineamide residue. (See, e.g., Smith et 0050 FIG. 9 shows in vitro metabolic identification of al., Modifications to the N-terminus but not the C-terminus Seq ID: No.658 in 100% human plasma after 4 hours of calcitonin gene-related peptide(8-37) produce antagonists incubation at room temperature. with increased affinity, J. Med. Chem., 46:2427-2435 0051 FIG. 10 shows in vitro metabolic identification of (2003)). the Seq ID: No: 144 in 100% human plasma after 4 hours 0059) “Aryl is phenyl or phenyl vicinally-fused with a incubation at room temperature. saturated, partially-Saturated, or unsaturated 3-, 4-, or 5 US 2008/0020978 A1 Jan. 24, 2008

membered carbon bridge, the phenyl or bridge being sub included within the definition of both “lipophilic residue' stituted by 0, 1, 2 or 3 substituents selected from Cs alkyl, and “hydrophilic residue.” The term “nonfunctional amino Chaloalkyl or halo. acid residue' refers to amino acid residues in D- or L-form having side chains that lack acidic, basic, or aromatic 0060) “Heteroaryl” is an unsaturated 5, 6 or 7 membered groups. Exemplary neutral amino acid residues include monocyclic or partially-Saturated or unsaturated 6-, 7-, 8-, methionine, glycine, alanine, Valine, isoleucine, , and 9-, 10- or 11 membered bicyclic ring, wherein at least one ring is unsaturated, the monocyclic and the bicyclic rings norleucine (Nle) residues. containing 1, 2, 3 or 4 atoms selected from N, O and S. 0064.) The CGRP peptide includes a first CGRP receptor wherein the ring is substituted by 0, 1, 2 or 3 substituents binding region, or domain, proximal to its carboxy terminal selected from Cs alkyl, Chaloalkyl and halo. end, which binding region preferentially binds a first binding site on a CRLR-RAMP1 complex. The phrase “proximal to 0061 Although not essential for the practice of the the carboxy terminal end means close to the peptide's present invention, assay methods for the detection of pref C-terminal amino acid residue (regardless of any lack of a erential binding to CGRP receptor are known in the art free carboxyl group thereon) by way of the peptide's pri (e.g., McLatchie et al., Nature, 393:333-339 (1998); Rist et mary structure, i.e., its amino acid sequence (also known as al., J. Med. Chem., 41: 117-123 (1998)), and are further “primary sequence'), not relating to actual spatial distance exemplified herein below. or other secondary or higher order structural considerations, 0062). A “CGRP peptide antagonist” is a CGRP peptide, or, to whether vehicle is conjugated thereto. Typically, the such as, but not limited to, a CGRP peptide analog, that first CGRP receptor binding region involves the ten amino antagonizes, blocks, decreases, reduces, impedes, or inhibits acid residues most proximal to, and including, the carboxy CGRP receptor activation by full length native human terminal residue of the peptide, at amino acid positions CCGRP or f3CGRP under physiological conditions of tem 28-37 relative to the positional order of the native human perature, pH, and ionic strength. CGRP peptide antagonists CCGRP sequence. However, as long as the peptide retains include full and partial antagonists. The present invention detectable specific or preferential binding to CRLR-RAMP1 does not depend on any particular mechanism of antago complex, the first CGRP receptor binding region may be 1, nism. For example, the CGRP peptide antagonist can act as 2, 3, 4, or about 5 amino acid residues longer, or 1, 2, or a competitive antagonist or a noncompetitive antagonist. about 3 amino acid residues shorter, than the CGRP recep Such antagonist activity can be detected by known in vitro tor binding region at amino acid positions 28-37 of the methods or in vivo functional assay methods. (See, e.g., native human CCGRP sequence. Smith et al., Modifications to the N-terminus but not the 0065. In some embodiments, the CGRP peptide also C-terminus of calcitonin gene-related peptide(8-37) produce includes, from the first CGRP receptor binding region to the antagonists with increased affinity, J. Med. Chem., 46:2427 N-terminal end of the peptide: a hinge region; and a second 2435 (2003)). CGRP1 receptor binding region between the hinge region and the N-terminal end of the peptide. Thus, the hinge region 0063) The CGRP peptide is comprised of at least 2 to or domain, is more distal from the carboxy terminal end of about 90 amino acid residues connected in a main chain by the peptide than the first CGRP receptor binding region. peptide bonds. Amino acid residues are commonly catego The phrase “more distal means more distant than a certain rized according to different chemical and/or physical char referent (such as the first CGRP receptor binding region) acteristics. The term “acidic amino acid residue' refers to from the peptide's C-terminal amino acid residue (regardless amino acid residues in D- or L-form having side chains of any lack of a free carboxyl group thereon) by way of the comprising acidic groups. Exemplary acidic residues peptide’s primary structure, i.e., its amino acid sequence (or include aspartate and glutamate residues. The term “aro “primary sequence'), not relating to actual spatial distance matic amino acid residue' refers to amino acid residues in or other secondary or higher order structural considerations, D- or L-form having side chains comprising aromatic or to whether vehicle is conjugated thereto. The hinge region groups. Exemplary aromatic residues include tryptophan, provides a movable joint or axis in the peptide that facili tyrosine, 3-(1-naphthyl)alanine, or phenylalanine residues. tates, participates in, or responds to, CGRP receptor-spe The term “basic amino acid residue' refers to amino acid cific binding by permitting the peptide to bend to better residues in D- or L-form having side chains comprising stabilize a peptide-CRLR-RAMP1 complex. Typically, the basic groups. Exemplary basic amino acid residues include histidine, lysine, homolysine, ornithine, arginine, N-methyl hinge region involves the nine amino acid residues at amino arginine, ()-aminoarginine, ()-methyl-arginine, 1-methyl acid positions 19-27 relative to the positional order of the histidine, 3-methyl-histidine, and homoarginine (hR) resi native human CCGRP sequence. However, a peptide that dues. The term “hydrophilic amino acid residue' refers to retains detectable specific or preferential binding to CRLR amino acid residues in D- or L-form having side chains RAMP1 complex, can have a hinge region 1, 2, 3, 4, 5, 6, comprising polar groups. Exemplary hydrophilic residues 7, 8, 9 or about 10 amino acid residues longer, or 1, 2, 3, 4, include cysteine, serine, threonine, histidine, lysine, aspar 5, or about 6 amino acid residues shorter, than the hinge agine, aspartate, glutamate, glutamine, and citrulline (Cit) region at amino acid positions 19-27 of the native human residues. The terms "lipophilic amino acid residue' refers to CCGRP sequence. amino acid residues in D- or L-form having sidechains 0066 Being between the hinge region and the N-terminal comprising uncharged, aliphatic or aromatic groups. Exem end of the peptide, the second CGRP receptor binding plary lipophilic sidechains include phenylalanine, isoleu region or domain, when present, is located even more cine, leucine, methionine, Valine, tryptophan, and tyrosine. distally from the carboxy terminal end than the hinge region. Alanine (A) is amphiphilic—it is capable of acting as a The phrase "even more distal” means at a greater distance hydrophilic or lipophilic residue. Alanine, therefore, is than a certain referent (such as the hinge region) from the US 2008/0020978 A1 Jan. 24, 2008

CGRP peptide's C-terminal amino acid residue (regardless Springer-Verlag, 1993: “The Practice of Peptide Synthesis, of any lack of a free carboxyl group thereon) by way of the 2nd ed., M. Bodanszky and A. Bodanszky, Eds. Springer peptide's primary structure, i.e., its amino acid sequence Verlag, 1994; “Protecting Groups. P. J. Kocienski, Ed., (also known as "primary sequence'), not relating to actual Georg Thieme Verlag, Stuttgart, Germany, 1994; “Fmoc spatial distance or other secondary or higher order structural Solid Phase Peptide Synthesis, A Practical Approach. W. C. considerations, or to whether vehicle is conjugated thereto. Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields The second CGRP receptor binding region is involved in et al., Synthetic Peptides: A User's Guide, 1990, 77-183, and increasing the binding affinity of the CGRP peptide either elsewhere. A further illustration of peptide synthesis tech through direct binding to the CRLR-RAMP1 complex and/ niques that can be employed in accordance with the present or via interacting with, or otherwise affecting the confor invention is found in FIG. 13. mation of the first CGRP receptor binding region. Typi cally, the second CGRP receptor binding region, if present, 0069. Typically, linear and cyclic CGRP peptides are involves eleven amino acid residues at amino acid positions synthesized using Fmoc Solid-phase peptide synthesis 8-18 relative to the native human CCGRP sequence. How (SPPS) methodologies on a commercially available synthe ever, a peptide that retains detectable specific binding to sizer, such as a Symphony automated synthesizer (Protein CRLR-RAMP1 complex, can have, if present at all, a second Technologies, Inc., Washington, D.C.) or a Liberty micro CGRP receptor binding region 1, 2, 3, 4, 5, 6, 7, 8, 9, or wave assisted automated synthesizer (CEM Corporation, about 10 amino acid residues shorter than the CGRP, Matthews, N.C.). Protected derivatives of conical amino receptor binding region at amino acid positions 8-18 of the acids, Fmoc-Dpr(Mtt)-OH, Fmoc-Dab(Mtt)-OH, and Fmoc native human C.CGRP sequence (SEQ ID NO:43), or can Cit-OH can be purchased from EMD Biosciences, Inc. (La have a second CGRP receptor binding region longer by 1 or Jolla, Calif.). Fmoc-homo Arg(Prmc)-OH can be purchased from Bachem California, Inc. (Torrance, Calif.). All other more amino acid residues up to the maximum length of the non-conical Fmoc-amino acids can be purchased from either CGRP peptide, as described herein. Advanced Chemtech (Louisville, Ky.) or Chem-Impex Inter 0067. The CGRP peptide lacks a “functional CGRP, national, Inc. (Wood Dale, L). The coupling reagents 2-(1- receptor activation” region that is capable of detectably H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluo activating a CGRP receptor (or of activating an amylin rophosphate (HBTU) and receptor, adrenomedulin receptor, or CT receptor) at a 1-Benzotriazoyloxytris(pyrrolidino)phosphonium hexafluo physiologically or pharmacologically relevant concentration rophosphate (PyBOP) can be purchased from Matrix Inno of the peptide. Although not required for the practice of the vation, Inc. (Montreal, Quebec Canada). N-Methyl Pyrroli invention, the skilled artisan is aware of suitable functional done (NMP), dichloromethane (DCM), methanol (MeOH), assays for detecting CGRP receptor activation, or lack acetonitrile (ACN), isopropanol, dimethylsulfoxide thereof. Such as a cAMP-based assay system. In some (DMSO), and anhydrous ethyl ether can be purchased from embodiments the peptide has an amino acid sequence that VWR International (West Chester, Pa.). N,N-dimethylfor includes amino acid positions 1-7, or any portion thereof, mamide (DMF) is purchased from EMD Biosciences. Trif relative to the native CGRP sequence, however modified, luoroacetic acid (TFA), N-ethylmorpholine (NEM), pyri such that the peptide cannot functionally activate the CGRP dine, piperidine, N N-diisopropylethylamine (DIEA), receptor. triisopropylsilane (Tis), phenol, acetic anhydride, and 0.1% 0068. The CGRP peptide (or a peptide portion of the TFA in HO are purchased from Sigma-Aldrich (St. Louis, pharmaceutically acceptable vehicle, when applicable as Mo.). All solvents and reagents are preferably ACS grade or described herein) can be obtained by synthesis employing better and can be used without further purification. Peptides conventional chemical synthetic methods. For example, are assembled on CLEAR-amide-MBHA resin (0.44 meq/g Solid phase peptide synthesis techniques can be used. Such substitution), purchased from Peptides International (Lou techniques are well known in the art and include but are not isville, Ky.). Typically, the syntheses are performed using 16 limited to, those described in Merrifield (1973), Chem. mL polypropylene reaction vessels fitted with course frits Polypeptides, 335-361 (Katsoyannis and Panayotis eds.); (Protein Technologies). Approximately 455 mg resin (0.2 Merrifield (1963), J. Am. Chem. Soc., 85:2149: Davis et al. mmole) is added to each reaction vessel and solvated for 10 (1985), Biochem. Intl., 10:394-414; Stewart and Young min in DMF. The growing peptide chains are assembled on (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941, the amide-resin starting from the C-terminus using the 763; Finn et al. (1976), The Proteins (3rd ed.) 2:105-253; general amino acid cycle as follows: The N'-Fmoc groups and Erickson et al., The Proteins (3rd ed.) 2: 257-527 (1976). are removed by addition of 5 mL 20% piperidine in DMF for The use of protecting groups, linkers, and Solid phase 5 min, followed by a 20 min 5 mL incubation. Amino acids Supports, as well as specific protection and deprotection (3-fold molar excess) are added to the resin (3000 uL of 0.2 reaction conditions, linker cleavage conditions, use of Scav Mamino acid solution in NMP), followed by the addition of engers, and other aspects of solid phase peptide synthesis are 3-fold excess HBTU and 6-fold excess NEM (1.2 mL of 0.5 well known and are also described in “Protecting Groups in M HBTU & 1.0 M NEM in DMF). The mixture is agitated Organic Synthesis,” 3rd Edition, T. W. Greene and P. G. M. by periodic sparging with nitrogen for 45 min, followed by Wuts, Eds. John Wiley & Sons, Inc., 1999; NovaBiochem emptying of the reaction vessel by positive nitrogen pres Catalog, 2000; “Synthetic Peptides, A User’s Guide.” G. A. sure. The resin is washed with 5 mL of DMF (4x30 sec). A Grant, Ed., W.H. Freeman & Company, New York, N.Y., second coupling reaction is repeated for 30 min, the reaction 1992: "Advanced Chemtech Handbook of Combinatorial & vessel emptied, and the N'-Fmoc-protected peptide-resin is Solid Phase Organic Chemistry.” W. D. Bennet, J. W. washed with 5 mL DMF (3x30 sec) and 5 mL DCM (2x30 Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, sec). The amino acid coupling cycle is repeated with and H. H. Saneii, Eds. Advanced Chemtech, 1998; “Prin required amino acids until the desired peptide is assembled. ciples of Peptide Synthesis, 2nd ed., M. Bodanszky, Ed., Following N*-Fmoc deprotection of the final amino acid, US 2008/0020978 A1 Jan. 24, 2008 acetylation of the N'-amine is performed by addition of 2.5 and washed again with DCM following the cyclization mL acetic anhydride/DIEA solution (1.0 M in DMF) to the reactions and thoroughly dried in vacuo prior to cleavage. reaction vessels and mixed for 30 min. If peptides do not Cyclic peptide-resins are cleaved, purified, and analyzed in require cyclization, the acetylated peptide-resin is washed the same manner as described for linear peptides. The with 5 mL DCM (5x30 sec) and dried thoroughly prior to preceding methods are merely illustrative, and the skilled cleavage from the resin and removal of side chain protecting artisan is aware of various other methods and technical groups. Deprotection of the amino acid side chains and variations for synthesizing the inventive CGRP peptides. cleavage of the acetylated-peptide from the resin is per 0070). The CGRP peptide can also be obtained using formed by incubating the peptide-resin with 15 mL cleavage recombinant DNA- and/or RNA-mediated protein expres cocktail (92.5% TFA, 2.5% water, 2.5% T is, 2.5% phenol) sion techniques, or any other methods of preparing peptides for 3 hr. The cleavage product is filtered under positive or, when applicable, fusion proteins. For example, the pep nitrogen gas pressure into tarred 50 mL polypropylene tides can be made in transformed host cells. Briefly, a conical tubes. The resin is washed with 10 mL cleavage recombinant DNA molecule coding for the peptide is pre cocktail for 5 min, filtered, and the filtrates combined. The pared. Methods of preparing such DNA molecules are well cleavage solutions are concentrated to approximately 5 mL known in the art. For instance, sequences encoding the total volume tinder a gentle stream of nitrogen. Cold (-20° peptides can be excised from DNA using suitable restriction C.) anhydrous ethyl ether (up to 50 mL) is added to the enzymes. Alternatively, the DNA molecule can be synthe filtrates. The flocculent peptides are pelleted by centrifuga sized using chemical synthesis techniques, such as the tion (e.g., Eppendorf centrifuge 5702 using a swinging phosphoramidate method. Also, a combination of these bucket rotor) at 3800 rpm for 5 min and the ether is techniques can be used. Any of a large number of available decanted. The peptide pellets are washed with additional and well-known host cells may be used in the practice of this cold anhydrous ethyl ether (up to 50 mL), pelleted by invention. The selection of a particular host is dependent centrifugation, decanted, and dried in vacuo. The crude upon a number of factors recognized by the art. These peptide yields typically range from 60% to 99% of the include, for example, compatibility with the chosen expres theoretical yields. Crude peptides are dissolved in DMSO sion vector, toxicity of the peptides encoded by the DNA and purified by RP-HPLC using, e.g., an Agilent 1100 molecule, rate of transformation, ease of recovery of the preparative chromatography system with a photodiode array peptides, expression characteristics, bio-safety and costs. A detector (Agilent Technologies, Inc., Santa Clara, Calif.) and balance of these factors must be struck with the understand a preparative RP-HPLC bonded silica column (Phenomenex ing that not all hosts may be equally effective for the Jupiter C18(2), 300A, 10 uM, 50x250 mm) and lyophilized expression of a particular DNA sequence. Within these to form amorphous solids. The purified peptides are prefer general guidelines, useful microbial hosts include bacteria ably at least >95% pure as determined by the analytical (such as E. coli sp.), yeast (such as Saccharomyces sp.) and RP-HPLC using a linear gradient of 2-60% B over 60 min other fungi, insects, plants, mammalian (including human) (A=0.1% TFA in HO, B=0.1% TFA in CAN, column= cells in culture, or other hosts known in the art. Modifica Phenomenex Jupiter Proteo, 90 A, 4 M, 2.1x50 mm). tions can be made at the DNA level, as well. The peptide Correct molecular mass can be confirmed by LC-MS meth encoding DNA sequence may be changed to codons more odologies using, e.g., a Waters Acquity HPLC equipped with compatible with the chosen host cell. For E. coli, optimized a LCT Premier XE orthogonal acceleration time-of-flight codons are known in the art. Codons can be substituted to (oa-TOF) benchtop mass spectrometer (Waters Corporation, eliminate restriction sites or to include silent restriction sites, Milford, Mass.). CGRP peptide antagonists cyclized with a which may aid in processing of the DNA in the selected host lactam bridge are prepared by selectively incorporating cell. Next, the transformed host is cultured and purified. residues with nucleophilic side chains that will form the Host cells may be cultured under conventional fermentation lactam protected with 4-methyltrityl (Mtt) and electrophilic conditions so that the desired compounds are expressed. side chains with 2-phenyl-isopropyl (2-PhilPr). Amino acids Such fermentation conditions are well known in the art. involved in the cyclizations are purchased from EMD Bio sciences, Inc. All other amino acids used in the syntheses are 0.071) Obtaining the CGRP peptide, whether the peptide preferably standard t-butyl, pentamethyldihydrobenzofuran is prepared by synthetic or recombinant techniques, can also 5-sulfonyl, or pentamethylchroman-6-sulfonyl side-chain involve suitable protein purification techniques, when appli protected Fmoc amino acids. Mtt and 2-PhilPr groups are cable. In some embodiments of the vehicle-conjugated selectively removed from cyclic CGRP peptide antagonists CGRP peptide antagonists of the invention, the CGRP by addition of 10 mL TFA/T is/DCM (0.3:0.5:9.2) to the peptide portion can be prepared to include a suitable isotopic peptide-resin (5x10 min), followed by washing the peptide label (e.g., 125, 14C, 13C, 35S, H, 2H, 13N, 15N, 18O, 17O, resin with 10 mL of a 2% DIEA solution (2x2 min) and 10 etc.), for ease of quantification or detection. mL DCM (4x1 min). The lactam bridge in cyclic peptides 0072) In accordance with the present invention, are formed by activating electrophilic carboxyl groups with “obtained' or “obtaining a CGRP peptide means that the 5-fold excess PyBOP 7-fold excess DIEA in DMF. The peptide is prepared, synthesized, produced, brought into mixtures are agitated with continuous sparging with nitro existence, purified, isolated, gotten and/or purchased. gen. Reactions are monitored by cleavage of a 1-2 mg aliquot of peptide-resin in 400 u, of cleavage cocktail. 0073). In useful embodiments of the invention, the CGRP followed by filtration, concentration of the filtrate under peptide is modified in one or more ways relative to a native nitrogen, and analyzing by LC-MS methodologies previ CGRP sequence of interest, such as a native human CCGRP ously described for linear peptides. Lactam formation ranges or BCGRP sequence. The one or more useful modifications from 70-99% after approximately 1-4 days at room tem can include amino acid additions or insertions, amino acid perature. The peptide-resin is washed with DMF, acetylated, deletions, peptide truncations, amino acid substitutions, and/ US 2008/0020978 A1 Jan. 24, 2008

or chemical derivatization of amino acid residues, accom a N-terminal region of preferably, 1 to about 50 amino acid plished by known chemical techniques. For example, the residues, more preferably, 1 to about 30 amino acid residues, thusly modified amino acid sequence includes at least one and most preferably, 1 to about 10 amino acid residues (e.g., amino acid residue inserted or substituted therein, relative to a peptide linker, aromatic amino acid residue(s), or one or the amino acid sequence of the native CGRP sequence of more copies of CGRP receptor binding regions, as interest, in which the inserted or substituted amino acid described herein). residue has a side chain comprising a nucleophilic or elec 0077. In further describing the CGRP peptide herein, a trophilic reactive functional group by which the peptide is one-letter abbreviation system is frequently applied to des conjugated to the vehicle. In accordance with the invention, ignate the identities of the twenty "canonical amino acid useful examples of Such a nucleophilic or electrophilic residues generally incorporated into naturally occurring pep reactive functional group include, but are not limited to, a tides and proteins (Table 1). Such one-letter abbreviations thiol, a primary amine, a seleno, a hydrazide, an aldehyde, are entirely interchangeable in meaning with three-letter a carboxylic acid, a ketone, an aminooxy, a masked (pro abbreviations, or non-abbreviated amino acid names. Within tected) aldehyde, or a masked (protected) keto functional the one-letter abbreviation system used herein, an upper case group. Examples of amino acid residues having a side chain letter indicates a L-amino acid, and a lower case letter comprising a nucleophilic reactive functional group include, indicates a D-amino acid. For example, the abbreviation “R” but are not limited to, a lysine residue, a homolysine, an designates L-arginine and the abbreviation “r” designates C.f3-diaminopropionic acid residue, an O.Y-diaminobutyric D-arginine. acid residue, an ornithine residue, a cysteine, a homocys teine, a glutamic acid residue, an aspartic acid residue, or a Selenocysteine residue. TABLE 1. 0074 The useful CGRP peptide according to the present One-letter abbreviations for the canonical amino acids. Three-letter invention can have one or more amino acid additions or abbreviations are in parentheses. insertions, amino acid deletions, peptide truncations, amino Alanine (Ala) acid Substitutions, and/or chemical derivatizations of amino Glutamine (Gln) Leucine (Leu) acid residues, relative to the sequence of a peptide whose Serine (Ser) sequence is described herein, so long as the requisite CGRP Arginine (Arg) receptor antagonist activity is maintained. Examples of the Glutamic Acid (Glu) inventive CGRP peptide antagonists include CGRP peptides Lysine (Lys) Threonine (Thr) having an amino acid primary sequence of any of those set Asparagine (ASn) forth in Table 2A, Table 2B, Table 2C, Table 2D, Table 3A, Glycine (Gly) Table 3B, Table 3 (except SEQID NO: 1), Table 4, or Table Methionine (Met) 7, whether shown in vehicle-conjugated or unconjugated Tryptophan (Trp) form. Aspartic Acid (Asp) Histidine (His) Phenylalanine (Phe) 0075 For example, additional amino acid residues can be Tyrosine (Tyr) included at the N-terminal end of the CGRP peptide, as long Cysteine (Cys) as they do not significantly reduce the potency of CGRP Isoleucine (Ile) receptor binding or otherwise interfere with CGRP receptor Proline (Pro) antagonism. For example, in some embodiments, an aro Valine (Val) matic amino acid residue, such as a tryptophan or tyrosine residue, can be a useful addition at the N-terminal end of the 0078. An amino acid substitution in an amino acid peptide as a chromophore for quantification or detection sequence is typically designated herein with a one-letter purposes, or can, in Some embodiments, actually improve abbreviation for the amino acid residue in a particular potency. Such additions can also be made to N-terminally position, followed by the numerical amino acid position truncated CGRP peptide analogs as described herein. relative to the native CGRP sequence of interest (human 0076. In some embodiments of the present invention, it is CCGRP sequence, unless otherwise specified), which is then useful that the CGRP peptide's sequence include an N-ter followed by the one-letter symbol for the amino acid residue minal truncation from a native CGRP peptide amino acid substituted in. For example, “T30D” symbolizes a substitu sequence length (e.g., deletion of entirety, or part, of the tion of a threonine residue by an aspartate residue at amino activation region or of the second binding region), for acid position 30, relative to the native human C.CGRP example, but not limited to, a peptide having an amino acid sequence (i.e., SEQ ID NO:43), which serves herein as a sequence length of a CGRP fragment, such as CGRP(8-37), reference sequence. By way of further example, “R18hR' or CGRP(9-37), CGRP(10-37), CGRP(11-37), CGRP(12-37), “R18Cit’ indicates a substitution of an arginine residue by CGRP(13-37), CGRP(14-37), CGRP(15-37), CGRP(16 a homoarginine (herein abbreviated “hR' or “harg) residue 37), CGRP(17-37), CGRP(18-37), CGRP(19-37), or a citrulline (herein abbreviated “Cit) residue, respec CGRP(20-37), CGRP(21-37), CGRP(22-37), CGRP(23 tively, atamino acid position 18, relative to the native human 37), CGRP(24-37), CGRP(25-37), CGRP(26-37), CCGRP sequence (i.e., SEQ ID NO:43). An amino acid CGRP(27-37), or CGRP(28-37), regardless of whether the position within the amino acid sequence of any particular fragment has a native amino acid sequence or is a CGRP CGRP peptide (or CGRP peptide analog) described herein analog sequence in other respects. Nevertheless, in accor may differ from its position relative to the native human dance with the invention, such N-terminally truncated CCGRP sequence, i.e., as determined in an alignment of the sequences can, in certain embodiments, have other amino C-terminal end of the peptide's amino acid sequence with acid residues added or attached at the N-terminal end to form the C-terminal end of the native human CCGRP sequence. US 2008/0020978 A1 Jan. 24, 2008

For example, amino acid position 1 of the sequence VTHR (or biphenylalanine; 4Bip), C.-amino-isobutyric acid (Aib), LAGLLSRSGGVVKNNFVPTNVGSKAF (SEQ ID NO:1; beta-alanine, beta-aminopropionic acid, piperidinic acid, human CCGRP(8-37)), thus aligned, corresponds to amino aminocaprioic acid, aminoheptanoic acid, aminopimelic acid position 8 relative to the native human CCGRP refer acid, desmosine, diaminopimelic acid, N-ethylglycine, ence sequence (SEQID NO:43), and amino acid position 30 N-ethylaspargine, hydroxylysine, allo-hydroxylysine, of SEQ ID NO:1 corresponds to amino acid position 37 isodesmosine, allo-isoleucine, N-methylglycine, N-methyli relative to the native human C.CGRP reference sequence soleucine, N-methylvaline, 4-hydroxyproline (Hyp), Y-car (SEQ ID NO:43). As a further example, addition of a boxyglutamate, e-N,N,N-trimethyllysine, e-N-acetyllysine, tryptophan at the N-terminus of a peptide having an amino O-phosphoserine, N-acetylserine, N-formylmethionine, acid sequence SEQ ID NO:1 constitutes a “WO addition.” 3-methylhistidine, 5-hydroxylysine, ()-methylarginine, and but in relation to the sequence WVTHRLAGLLSRSGGV other similar amino acids, and derivatized forms of any of VKNNFVPTNVGSKAF (SEQID NO:4), the tryptophan is these as described herein. Table 1A contains some exem at amino acid position 1, while it corresponds to amino acid plary non-canonical amino acid residues that are useful in position 7 relative to the native human CCGRP sequence accordance with the present invention and associated abbre (SEQ ID NO:43). viations as typically used herein, although the skilled prac titioner will understand that different abbreviations and 0079. In some useful embodiments of the inventive com nomenclatures may be applicable to the same Substance and position of matter, the amino acid sequence of the CGRP my appear interchangeably herein. peptide includes the following three amino acid substitu tions: N31D, S34P, and K35F, relative to the native human TABLE 1A CCGRP sequence (SEQ ID NO:43). These three substitu tions were found to improve the potency of some CGRP Useful non-canonical amino acids for amino acid addition, insertion, or peptide antagonists at the CGRP receptor, as demonstrated Substitution into CGRP peptide sequences in accordance with the herein below. present invention. In the event an abbreviation listed in Table 1A differs from another abbreviation for the same substance disclosed 0080. In certain embodiments of the present invention, elsewhere herein, both abbreviations are understood to be amino acid Substitutions encompass, non-canonical amino applicable. acid residues, which include naturally rare (in peptides or Abbreviation Amino Acid proteins) amino acid residues or unnatural amino acid resi dues. Non-canonical amino acid residues can be incorpo Sar Sarcosine Nle norleucine rated into the peptide by chemical peptide synthesis rather Ile isoleucine than by Synthesis in biological systems, such as recombi 1-Nal 3-(1-naphthyl)alanine nantly expressing cells, or alternatively the skilled artisan 2-Nal 3-(2-naphthyl)alanine can employ known techniques of protein engineering that Bip 4,4'-biphenylalanine use recombinantly expressing cells. (See, e.g., Link et al., Dip 3,3-diphenylalanine Ny norvaline Non-canonical amino acids in protein engineering, Current NMe-Val NC.-methyl valine Opinion in Biotechnology, 14(6):603-609 (2003)). The term NMe-Leu NC.-methyl leucine “non-canonical amino acid residue' refers to amino acid NMe-Nile NC.-methyl norleucine residues in D- or L-form that are not among the 20 canonical Cpg cyclopentylglycine amino acids generally incorporated into naturally occurring Chg cyclohexyl glycine proteins, for example, B-amino acids, homoamino acids, Hyp hydroxy proline Oic Octahydroindole-2-Carboxylic cyclic amino acids and amino acids with derivatized side Acid chains. Examples include (in the L-form or D-form; abbre Ig Indanylglycine viated as in parentheses): citrulline (Cit), homocitrulline Aib aminoisobutyric acid (hCit), N'-methylcitrulline (NMeCit), N'-methylhomoc Aic 2-aminoindane-2-carboxylic itrulline (N'-MeHoCit), ornithine (Orn), N'-Methylomith acid Pi pipecolic acid ine (N'-MeOrn or NMeOrn), sarcosine (Sar), homolysine BhTic B-homo Tic (hLys or hK), homoarginine (harg or hR), homoglutamine BPro 3-homo proline (hO), N'-methylarginine (NMeR), N'-methylieucine (N'- Sar Sarcosine MeL or NMeL), N-methylhomolysine (NMeHoK), N'-me Cpg cyclopentylglycine thylglutamine (NMeO), norleucine (Nle), norvaline (Nva), Tiq ,2,3,4-L-Tetrahydroisoquinoline-1-Carboxylic 1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2- acid Nip Nipecotic Acid carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2- Thz Thiazolidine-4-carboxylic acid naphthyl)alanine (2-Nal), 1.2.3,4-tetrahydroisoquinoline Thi 3-thienylalanine (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pl.- 4GuaPr 4-guanidino proline Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4Pip 4-Amino-1-piperidine-4-carboxylic acid 4-guanidino phenylalanine (Guf), nitrophenylalanine (nitro Idc indoline-2-carboxylic acid ,2,3,4-Tetrahydroisoquinoline-7-hydroxy-3 phe), aminophenylalanine (aminophe or Amino-Phe), ben carboxylic acid Zylphenylalanine (benzylphe), Y-carboxyglutamic acid Bip 4,4'-biphenylalanine (Y-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl Ome-Tyr O-methyl tyrosine phenylalanine (Cpa), C.-aminoadipic acid (Aad), NC.-methyl -Tyr odatyrasine valine (NMeVal), N-O-methyl leucine (NMeLeu), NC.-me Tic ,2,3,4-L-Tetrahydroisoquinoline-3-Carboxylic acid thylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclo gl ndanylglycine hexylglycine (Chg), acetylarginine (acetylarg), C.B-diami BhTic B-homo Tic nopropionoic acid (Dpr), C.Y-diaminobutyric acid (Dab), BhPhe 3-homo phenylalanine diaminopropionic acid (Dap), cyclohexylalanine (Cha), AMeF C-methyl Phenyalanine 4-methyl-phenylalanine (MePhe), B.B-diphenyl-alanine BPle 3-phenylalanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine US 2008/0020978 A1 Jan. 24, 2008 11

0082 In some useful embodiments of the inventive com TABLE 1 A-continued position of matter, a modification of the CGRP peptide is at amino acid positions 3, 4, 9, 10, 11, 12, 14, 15, 16, 17, 18, Useful non-canonical amino acids for amino acid addition, insertion, or 19, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 36, and/or 37, and substitution into CGRP peptide sequences in accordance with the present invention. In the event an abbreviation listed in Table 1A more preferably, at positions 3, 4, 11, 18, 24, 25, 34, 35 differs from another abbreviation for the same substance disclosed and/or 37, or at any combination of these positions, relative elsewhere herein, both abbreviations are understood to be to the human CCGRP reference sequence (SEQID NO:43). applicable. 0083. In some embodiments of the inventive composition Abbreviation Amino Acid of matter, the amino acid sequence of the CGRP peptide is Phg phenylglycine modified, relative to a native CGRP reference sequence Anc 3-amino-2-naphthoic acid (SEQ ID NO:43), such that the modification reduces the Atc 2-aminotetraline-2-carboxylic acid CGRP peptide's susceptibility to enzymatic proteolysis. NMe-Phe NC.-methyl phenylalanine This reduced susceptibility may be due to an effect on a NMe-Lys NC.-methyl lysine binding site or cleavage site for an or Tpi 1,2,3,4-Tetrahydronorharman-3-Carboxylic acid . The native CGRP peptide is degraded in Cpg cyclopentylglycine Dip 3,3-diphenylalanine biological fluid by proteolytic enzymes such as serine pro 4Pal 4-pyridinylalanine teases (trypsin, chymotrypsin, and elastase). The half-life of 3Pal 3-pyridinylalanine CGRP in mammalian plasma is reported to be about 10 min 2Pal 2-pyridinylalanine A. S. Thakor; D. A. Giussani, Circulation. 2005; 112:2510 4Pip 4-Amino-1-piperidine-4-carboxylic acid 2516; Kraenzlin M. E. Ch'ng J. L. Mulderry PK, Ghatei M 4AmP 4-amino-phenylalanine Idc indoline-2-carboxylic acid A, Bloom S R., Regul Pept. 1985 March; 10(2-3): 189-97.). Chg cyclohexyl glycine Proteolytic enzymes have varying Substrate specificities, for hPhe homophenylalanine instance, trypsin, preferentially cleaves at Arg and Lys in BhTrp 3-homotryptophan position P1 with higher rates for Arg. Keil, B. Specificity of pI-Phe 4-iodophenylalanine proteolysis. Springer-Verlag Berlin-Heidelberg-New York, Aic 2-aminoindane-2-carboxylic acid NMe-Lys NC.-methyl lysine pp. 335. (1992). On this basis multiple sites within a given Orn ornithine peptide or protein can be recognized and processed by Dpr 2,3-Diaminopropionic acid different . Dbu 2,4-Diaminobutyric acid homoLys homolysine 0084 With the objective of increasing the stability of N-eMe-K Ne-methyl-lysine CGRP in biological fluid we have investigated the rate and N-et-K Ne-ethyl-lysine site of CGRP breakdown by -linked immunosorbent N-ePr-K Ne-isopropyl-lysine assay (ELISA) and mass spectrometry analysis techniques. bhomok 3-homolysine rLys Lys (CH2NH)-reduced amide bond After the identification of key degradation site in CGRP and rOrn Orin (CH2NH)-reduced amide bond CGRP analogues using this approach, we have replaced Acm acetamidomethyl selected natural amino acids with non-canonical or unnatural Ahx 6-aminohexanoic acid amino acids to reduce protease processing and in turn e Ahx 6-aminohexanoic acid increase the stability of the CGRP peptide in plasma. K(NPeg11) Ne-(O-(aminoethyl)-O'-(2-propanoyl)- undecaethyleneglycol)-Lysine Examples of this approach are the replacement of arginine K(NPeg27) Ne-(O-(aminoethyl)-O'-(2-propanoyl)- (R) with citrulline, and phenylalanine (F) with 3-(1-naph (ethyleneglycol)27-Lysine thyl)alanine. We have also cyclized side-chain to side-chain Cit Citrulline (for example, i, i+4 residues) to form ring structures which hArg homoarginine also increase overall plasma stability (See, e.g., FIG. 3 and hCit homocitrulline NMe-Arq NC.-methyl arginine (NMeR) FIG. 13). The introduction of non-canonical or unnatural Gulf 4-guanidinyl phenylalanine amino acids and cyclic structures has been carried out in an bharg 3-homoarginine iterative manner to identify novel CGRP analogues with 3G-Dpr 2-amino-3-guanidinopropanoic acid significantly improved stability in biological fluid (e.g., 4AmP 4-amino-phenylalanine 100% plasma) with or without PEGylation (20 kDa MeO 4AmPhe 4-amidino-phenylalanine 4AmPig 2-amino-2-(1-cambamimidoylpiperidin-4- PEG), as illustrated in Table 1B below, and as further yl)acetic acid described in Examples 4-6, herein below. 4GuaPr 4-guanidino proline N-Arg NC-(CH2)NHCH(NH)NH2. Substituted glycine TABLE 1B rArg Arg (CH2NH)-reduced amide bond 4PipA 4-Piperidinyl alanine Half-life of some modified CGRP peptides in 100% plasma. NMe-Arg NC.-methyl arginine (or NMeR) NMe-Thr NC.-methyl threonine (or NMeThr) Stability in undiluted plasma at SEQ ID SEQ ID 37° C. as determined by ELISA NO: 172* NO: 173* to in rat plasma 8-9 h 8-9 h 0081. Nomenclature and Symbolism for Amino Acids t2 in Cynomolgus plasma >24 h >24 h and Peptides by the UPAC-IUB Joint Commission on Bio t12 in human plasma >24 h >24 h chemical Nomenclature (JCBN) have been published in the following documents: Biochem. J., 1984, 219, 345-373; Eur. *SEQ ID 172 and SEQ ID 173 are both PEGylated (20 kDa MeO-PEG) at J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2: Lys. Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 0085. Accordingly, in some embodiments of the inven 595-624; Amino Acids and Peptides, 1985, 16, 387-410; tive composition of matter, a modification, such as, but not Biochemical Nomenclature and Related Documents, 2nd limited to, an amino acid substitution of a basic amino acid edition, Portland Press, 1992, pages 39-69. residue (such as a basic amino acid residue described herein US 2008/0020978 A1 Jan. 24, 2008 above, or a functionally equivalent Substitution) is at amino 0111 R11Cit, L12NMeL, R18Cit: acid position positions 3, 4, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 36, and/or 37, and O112 R11Cit, L12Nle, L15N1e, L16Nle, R18Cit: more preferably, at positions 3, 4, 11, 18, 24, 25, 34, 35 0113 R11Cit, L12Nva, L15Nva, L16Nva, R18Cit: and/or 37, or at any combination of these positions, relative to the human CCGRP reference sequence (SEQID NO:43). 0114 R11Cit, S17T, R18Cit: By way of further example, in some embodiments, a Sub 0115 H10Tic, R11Cit, R18Cit; stitution is made at a residue that is adjacent to a proteolytic cleavage site to prevent or reduce enzymatic proteolysis, 0116 R11Cit, G14Sar, R18Cit: e.g., a S17P and/or S19P substitution, relative to the human 0117 R11Cit, L12Nle, L15N1e, L16Nle, S17T, R18Cit, CCGRP sequence (SEQ ID NO:43). In other embodiments, K24h R, G33Sar; Substitution of a charged residue, such as hArg or Gulf, at position 24 relative to the native human CCGRP reference 0118. R11hcit, L12Nle, L15N1e, L16Nle, S17T, sequence (SEQID NO:43) enhances the proteolytic stability R18hCit, K24h R, G33Sar; of the CGRP peptide. 0119). R11NMeR, L12Nle, L15N1e, L16Nle, S17T, 0086. In certain embodiments, the modification is a R18NMeR, K24NMeR, G33Sar; and single amino acid Substitution, e.g.: 0120 R11NMeR, L12Nva, L15Nva, L16Nva, S17T, 0087). R11O; R18NMeR, K24NMeR, G33Sar, or functionally equivalent amino acid substitutions at positions 10, 11, 12, 14, 15, 16, 0088. R11 hR; 17, 18, 19, 24 and/or 33. 0089). R11Cit: 0121 The term “protease' is synonymous with "pepti 0090 R11 hK; dase'. Proteases comprise two groups of enzymes: the which cleave peptide bonds at points within 0.091 R11Orn; the protein, and the , which remove one or 0092). R11Cit: more amino acids from either N- or C-terminus respectively. The term “proteinase' is also used as a synonym for 0093). R11 hCit: endopeptidase. The four mechanistic classes of proteinases 0094). R11NMeR; are: Serine proteinases, cysteine proteinases, aspartic pro teinases, and metallo-proteinases. In addition to these four O095 R11NMeO; mechanistic classes, there is a section of the enzyme nomen 0096). R11NMeHoK; clature which is allocated for proteases of unidentified catalytic mechanism. This indicates that the catalytic mecha 0097. R11NmeOrn; nism has not been identified. 0098. R11NMeCit: 0.122 Cleavage subsite nomenclature is commonly 0099). R11NMeHoCit: adopted from a scheme created by Schechter and Berger (Schechter I. & Berger A. On the size of the active site in 0100 R18Q: proteases. I. Papain, Biochemical and Biophysical Research 0101 R18hR; Communication, 27:157 (1967); Schechter I. & Berger A. On the active site of proteases. 3. Mapping the active site of 0102) R18Cit: papain; specific peptide inhibitors of papain, Biochemical 0103) R18NMeR; and Biophysical Research Communication, 32:898 (1968)). According to this model, amino acid residues in a substrate 0104 R18NmeO; and undergoing cleavage are designated P1, P2, P3, P4 etc. in the 0105 R18NMeCit, or a functionally equivalent amino N-terminal direction from the cleaved bond. Likewise, the acid Substitution, or in Some other embodiments, any com residues in the C-terminal direction are designated P1, P2', bination of these that entails an amino acid substitution at P3', P4'. etc. both position II and position 18, relative to the native human 0123 The skilled artisan is aware of a variety of tools for CCGRP sequence. identifying protease binding or protease cleavage sites of 0106. In certain embodiments, modification can involve interest. For example, the PeptideCutter software tool is double or multiple amino acid Substitutions, including modi available through the ExPASy (Expert Protein Analysis fications at amino acid positions 3, 4, 9, 10, 11, 12, 14, 15. System) proteomics server of the Swiss Institute of Bioin 16, 17, 18, 19, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 36, formatics (SIB: www.expasy.org/tools/peptidecutter). Pep and/or 37, and more preferably, at positions 3, 4, 11, 18, 24, tideCutter searches a protein sequence from the SWISS 25, 34, 35 and/or 37, or at any combination of these PROT and/or TrEMBL databases or a user-entered protein positions, relative to the human C.CGRP reference sequence sequence for protease cleavage sites. Single proteases and (SEQ ID NO:43), e.g.: chemicals, a selection or the whole list of proteases and chemicals can be used. Different forms of output of the 0107 R11hcit, R18hCit: results are available: Tables of cleavage sites either grouped 0108). R11 hK, R18hK; alphabetically according to enzyme names or sequentially according to the amino acid number. A third option for 0109). R11NMeR, R18NMeR; output is a map of cleavage sites. The sequence and the 0110 R11NMeR, R18NMeR, K24NMeR; cleavage sites mapped onto it are grouped in blocks, the size US 2008/0020978 A1 Jan. 24, 2008

of which can be chosen by the user. Other tools are also only moderately affected by residues in position P1". (Keil, known for determining protease cleavage sites. (E.g., Turk, 1992). The Asp-N endopeptidase cleaves specifically bonds B. et al., Determination of protease cleavage site motifs with a D residue in position P1". (Keil, 1992). using mixture-based oriented peptide libraries, Nature Bio 0131 The foregoing is merely exemplary and by no technology, 19:661-667 (2001); Barrett A. et al., Handbook means an exhaustive treatment of knowledge available to the of proteolytic enzymes, Academic Press (1998)). skilled artisan concerning protease binding and/or cleavage 0.124. The serine proteinases include the chymotrypsin sites that the skilled artisan may be interested in eliminating family, which includes mammalian protease enzymes Such in practicing the invention. as chymotrypsin, trypsin or elastase or kallikrein. The serine 0.132. Additional useful embodiments of vehicle-conju proteinases exhibit different substrate specificities, which gated CGRP peptide antagonists can result from conserva are related to amino acid substitutions in the various enzyme tive modifications of the amino acid sequences of the subsites interacting with the substrate residues. Some vehicle-conjugated peptides disclosed herein. Conservative enzymes have an extended interaction site with the substrate modifications will produce vehicle-conjugated peptides hav whereas others have a specificity restricted to the PI sub ing functional, physical, and chemical characteristics similar strate residue. to those of the vehicle-conjugated (e.g., PEG-conjugated) 0125 Trypsin preferentially cleaves at R or K in position peptide from which such modifications are made. Such P1. A statistical study carried out by Keil (1992) described conservatively modified forms of the vehicle- or PEG the negative influences of residues Surrounding the Arg- and conjugated peptides disclosed herein are also contemplated Lys-bonds (i.e. the positions P2 and P1", respectively) during as being an embodiment of the present invention. trypsin cleavage. (Keil, B., Specificity of proteolysis, Springer-Verlag Berlin-Heidelberg-New York, 335 (1992)). 0133. In contrast, substantial modifications in the func A proline residue in position P1’ normally exerts a strong tional and/or chemical characteristics of the CGRP peptides negative influence on trypsin cleavage. Similarly, the posi may be accomplished by selecting Substitutions in the amino tioning of R and K in P1 results in an inhibition, as well as acid sequence that differ significantly in their effect on negatively charged residues in positions P2 and P1". maintaining (a) the structure of the molecular backbone in the region of the Substitution, for example, as an O.-helical 0126 Chymotrypsin preferentially cleaves at a W. Y or F conformation, (b) the charge or hydrophobicity of the mol in position P1 (high specificity) and to a lesser extent at L. ecule at the target site, or (c) the size of the molecule. M or H residue in position P1. (Keil, 1992). Exceptions to these rules are the following: When W is found in position 0.134) For example, a “conservative amino acid substitu P1, the cleavage is blocked when M or P are found in tion may involve a Substitution of a native amino acid position P1' at the same time. Furthermore, a proline residue residue with a normative residue such that there is little or in position P1" nearly fully blocks the cleavage independent no effect on the polarity or charge of the amino acid residue of the amino acids found in position P1. When an M residue at that position. Furthermore, any native residue in the is found in position P1, the cleavage is blocked by the polypeptide may also be substituted with alanine, as has presence of a Y residue in position P1". Finally, when H is been previously described for 'alanine Scanning mutagen located in position P1, the presence of a D. Mor Wresidue esis” (see, for example, MacLennan et al., Acta Physiol. also blocks the cleavage. Scand. Suppl., 643:55-67 (1998); Sasaki et al., 1998, Adv. 0127. Membrane metallo-endopeptidase (NEP: neutral Biophys. 35:1-24 (1998), which discuss alanine scanning endopeptidase, kidney-brush-border neutral proteinase, mutagenesis). enkephalinase, EC 3.4.24.11) cleaves peptides at the amino 0.135) Desired amino acid substitutions (whether conser side of hydrophobic amino acid residues. (Connelly, J C et vative or non-conservative) can be determined by those al., Neutral Endopeptidase 24.11 in Human Neutrophils skilled in the art at the time such substitutions are desired. Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 For example, amino acid Substitutions can be used to iden (1985)). tify important residues of the peptide sequence, or to 0128. Thrombin preferentially cleaves at an R residue in increase or decrease the affinity of the peptide or vehicle position P1. (Keil, 1992). The natural substrate of thrombin conjugated peptide molecules described herein. is fibrinogen. Optimum cleavage sites are when an R residue 0.136 Naturally occurring residues may be divided into is in position P1 and Gly is in position P2 and position P1". classes based on common side chain properties: Likewise, when hydrophobic amino acid residues are found in position P4 and position P3, a proline residue in position 0.137 l) hydrophobic: norleucine (Nor), Met, Ala, Val, P2, an R residue in position P1, and non-acidic amino acid Leu, Ile; residues in position P1" and position P2’. A very important 0.138 2) neutral hydrophilic: Cys, Ser, Thr, Asn., Gln; residue for its natural substrate fibrinogen is a D residue in P10. 0139 3) acidic: Asp, Glu: 0129 Caspases are a family of cysteine proteases bearing 0140 4) basic: H is, Lys, Arg; an active site with a conserved amino acid sequence and 0.141 5) residues that influence chain orientation: Gly, which cleave peptides specifically following D residues. Pro; and (Earnshaw W C et al., Mammalian caspases: Structure, activation, Substrates, and functions during apoptosis, 0.142 6) aromatic: Trp, Tyr, Phe. Annual Review of Biochemistry, 68:383-424 (1999)). 0.143 Conservative amino acid substitutions may involve 0130. The Arg-C proteinase preferentially cleaves at an R exchange of a member of one of these classes with another residue in position P1. The cleavage behavior seems to be member of the same class. Conservative amino acid Substi US 2008/0020978 A1 Jan. 24, 2008

tutions may encompass non-naturally occurring amino acid residue Such as isoleucine, Valine, leucine norleucine, ala residues, which are typically incorporated by chemical pep nine, or methionine for another, the Substitution of one polar tide synthesis rather than by Synthesis in biological systems. (hydrophilic) amino acid residue for another Such as These include peptidomimetics and other reversed or between arginine and lysine, between glutamine and aspar inverted forms of amino acid moieties. agine, between glycine and serine, the Substitution of one basic amino acid residue Such as lysine, arginine or histidine 0144) Non-conservative substitutions may involve the for another, or the Substitution of one acidic residue. Such as exchange of a member of one of these classes for a member aspartic acid or glutamic acid for another. The phrase from another class. Such substituted residues may be intro “conservative amino acid substitution' also includes the use duced into regions of the human antibody that are homolo of a chemically derivatized residue in place of a non gous with non-human antibodies, or into the non-homolo derivatized residue, provided that such polypeptide displays gous regions of the molecule. the requisite CGRP receptor antagonist activity. Other 0145. In making such changes, according to certain exemplary amino acid substitutions that can be useful in embodiments, the hydropathic index of amino acids may be accordance with the present invention are set forth in Table considered. Each amino acid has been assigned a hydro 2. pathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); Valine (+4.2): TABLE 2 leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4): Some Useful Amino Acid Substitutions. threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine Original Exemplary (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); Residues Substitutions glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine Ala Val, Leu, Ile (-3.9); and arginine (-4.5). Arg LyS, Gln, ASn ASn Gln 0146 The importance of the hydropathic amino acid Asp Glu index in conferring interactive biological function on a Cys Ser, Ala Gln ASn protein is understood in the art (see, for example, Kyte et al., Glu Asp 1982, J. Mol. Biol. 157:105-131). It is known that certain Gly Pro, Ala amino acids may be substituted for other amino acids having His ASn, Gln, Lys, Arg a similar hydropathic index or score and still retain a similar Ile Leu, Val, Met, Ala, biological activity. In making changes based upon the hydro Phe, Norleucine Leu Norleucine, Ile, Val, pathic index, in certain embodiments, the Substitution of Met, Ala, Phe amino acids whose hydropathic indices are within t2 is Lys Arg, 1,4-Diamino included. In certain embodiments, those that are within +1 butyric Acid, Gln, are included, and in certain embodiments, those within +0.5 ASn are included. Met Leu, Phe, Ile Phe Leu, Val, Ile, Ala, Tyr 0147. It is also understood in the art that the substitution Pro Ala of like amino acids can be made effectively on the basis of Ser Thr, Ala, Cys hydrophilicity, particularly where the biologically functional Thr Ser protein or peptide thereby created is intended for use in Trp Tyr, Phe Tyr Trp, Phe, Thr, Ser immunological embodiments, as disclosed herein. In certain Wall Ile, Met, Leu, Phe, embodiments, the greatest local average hydrophilicity of a Ala, Norleucine protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and anti genicity, i.e., with a biological property of the protein. 0150. As stated herein above, in accordance with the 0148. The following hydrophilicity values have been present invention, the peptide can also be chemically deriva assigned to these amino acid residues: arginine (+3.0); lysine tized at one or more amino acid residues. Peptides that (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine contain derivatized amino acid residues can be synthesized (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); by known organic chemistry techniques. “Chemical deriva threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine tive' or “chemically derivatized refers to a subject peptide (-0.5); cysteine (-1,0); methionine (-1.3); valine (-1.5); having one or more residues chemically derivatized by leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenyla reaction of a functional side group. Such derivatized mol lanine (-2.5) and tryptophan (-3.4). In making changes ecules include, for example, those molecules in which free based upon similar hydrophilicity values, in certain embodi amino groups have been derivatized to form amine hydro ments, the Substitution of amino acids whose hydrophilicity chlorides, p-toluene Sulfonyl groups, carbobenzoxy groups, values are within t2 is included, in certain embodiments, t-butyloxycarbonyl groups, chloroacetyl groups or formyl those that are within +1 are included, and in certain embodi groups. Free carboxyl groups may be derivatized to form ments, those within +0.5 are included. One may also identify salts, methyl and ethyl esters or other types of esters or epitopes from primary amino acid sequences on the basis of hydrazides. Free hydroxyl groups may be derivatized to hydrophilicity. These regions are also referred to as form O-acyl or O-alkyl derivatives. The imidazole nitrogen "epitopic core regions.” of histidine may be derivatized to form N-im-benzylhisti dine. Also included as chemical derivatives are those pep 0149 Examples of conservative substitutions include the tides which contain one or more naturally occurring amino Substitution of one non-polar (hydrophobic) amino acid acid derivatives of the twenty canonical amino acids, US 2008/0020978 A1 Jan. 24, 2008 whether in L- or D-form. For example, 4-hydroxyproline 0157 Carboxyl sidechain groups (aspartyl or glutamyl) may be substituted for proline; 5-hydroxylysine maybe may be selectively modified by reaction with carbodiimides substituted for lysine: 3-methylhistidine may be substituted (R' N=C=N R") such as 1-cyclohexyl-3-(2-morpholi for histidine; homoserine may be substituted for serine; and nyl-(4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dim ornithine may be substituted for lysine. ethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues may be converted to asparaginyl and 0151. Useful derivatizations include, in some embodi glutaminyl residues by reaction with ammonium ions. ments, those in which the amino terminal of the peptide is chemically blocked so that conjugation with the vehicle will 0158 Glutaminyl and asparaginyl residues may be dea be prevented from taking place at an N-terminal free amino midated to the corresponding glutamyl and aspartyl resi group. There may also be other beneficial effects of such a dues. Alternatively, these residues are deamidated under modification, for example a reduction in the CGRP peptide's mildly acidic conditions. Either form of these residues falls Susceptibility to enzymatic proteolysis. The N-terminus can within the scope of this invention. be acylated or modified to a substituted amine, or derivatized 0159. Cysteinyl residues can be replaced by amino acid with another functional group. Such as an aromatic moiety residues or other moieties either to eliminate disulfide bond (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or ing or, conversely, to stabilize cross-linking. (See, e.g., Bn), or benzyloxycarbonyl (Cbz or Z)), N,N-dimethylgly Bhatnagar et al., J. Med. Chem., 39:3814-3819 (1996)). cine or creatine. For example, in Some embodiments, an acyl moiety, such as, but not limited to, a formyl, acetyl (Ac), 0.160 Derivatization with bifunctional agents is useful propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or for cross-linking the peptides or their functional derivatives nonanoyl, can be covalently linked to the N-terminal end of to a water-insoluble support matrix, if desired, or to other the peptide, which can prevent undesired side reactions macromolecular vehicles. Commonly used cross-linking during conjugation of the vehicle to the peptide. Other agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, exemplary N-terminal derivative groups include —NRR' glutaraldehyde, N-hydroxySuccinimide esters, for example, (other than -NH), NRC(O)R', NRC(O)OR', esters with 4-azidosalicylic acid, homobifunctional imi -NRS(O).R', -NHC(O)NHR', succinimide, or benzy doesters, including disuccinimidyl esters such as 3,3'-dithio loxycarbonyl-NH. (Cbz-NH ), wherein R and R' are bis(Succinimidylpropionate), and bifunctional maleimides each independently hydrogen or lower alkyl and wherein the Such as bis-N-maleimido-1.8-octane. Derivatizing agents phenyl ring may be substituted with 1 to 3 substituents such as methyl-3-(p-azidophenyl)dithiopropioimidate selected from C-C alkyl, C-C alkoxy, chloro, and bromo. yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, 0152. In some embodiments, one or more peptidyl reactive water-insoluble matrices such as cyanogen bro —C(O)NR— linkages (bonds) between amino acid resi mide-activated carbohydrates and the reactive substrates, dues can be replaced by a non-peptidyl linkage. Exemplary e.g., as described in U.S. Pat. Nos. 3.969,287: 3.691,016: non-peptidyl linkages are —CH-carbamate —CH2— 4,195,128; 4,247,642; 4,229,537; and 4,330,440, are OC(O)NR ), phosphonate, —CH2— sulfonamide employed for protein immobilization. CH-S(O)NR ), urea NHC(O)NH ), —CH secondary amine, and alkylated peptide C(O)NR'- 0.161. Other possible modifications include hydroxyla wherein R is lower alkyl). tion of proline and lysine, phosphorylation of hydroxyl groups of Seryl or threonyl residues, oxidation of the Sulfur 0153. In some embodiments, one or more individual atom in Cys, methylation of the alpha-amino groups of amino acid residues can be derivatized. Various derivatizing agents are known to react specifically with selected lysine, arginine, and histidine side chains. Creighton, Pro sidechains or terminal residues, as described in detail below teins: Structure and Molecule Properties (W. H. Freeman & by way of example. Co., San Francisco), 79-86 (1983). 0154 Lysinyl residues and amino terminal residues may 0162 The above examples of derivatizations are not be reacted with succinic or other carboxylic acid anhydrides, intended to be an exhaustive treatment, but merely illustra which reverse the charge of the lysinyl residues. Other tive. Suitable reagents for derivatizing alpha-amino-containing 0.163 Some exemplary embodiments of the inventive residues include imidoesters such as methyl picolinimidate; composition of matter include vehicle-conjugated, or uncon pyridoxal phosphate: pyridoxal; chloroborohydride; trini jugated, CGRP peptide antagonists in which the amino acid trobenzenesulfonic acid; O-methylisourea; 2.4 pentanedi sequence of the CGRP peptide contains (a) any of SEQ ID one; and transaminase-catalyzed reaction with glyoxylate. NOS: 47 through 55 and 82 through 127, herein below: 0155 Arginyl residues may be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1.2-cyclohex (SEQ ID NO: 47) anedione, and ninhydrin. Derivatization of arginyl residues VTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH2, requires that the reaction be performed in alkaline conditions (SEQ ID NO: 48) because of the high pKa of the guanidine functional group. VTHRLAGLLSRSGGVKRNNFVPTDVGPFAF-NH2, Furthermore, these reagents may react with the groups of lysine as well as the arginine epsilon-amino group. (SEQ ID NO: 49) VTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH, 0156 Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing (SEQ ID NO: 50) spectral labels into tyrosyl residues by reaction with aro VTHRLAGLLSRSGGVWRKNFVPTDVGPFAF-NH2, matic diazonium compounds or tetranitromethane. Most (SEQ ID NO:51) commonly, N-acetylimidizole and tetranitromethane are VTHRLAGLLSRSGGVWRNKFVPTDVGPFAF-NH2, used to form O-acetyl tyrosyl species and 3-nitro deriva tives, respectively.

US 2008/0020978 A1 Jan. 24, 2008 17

0167 Xaa is a neutral hydrophobic or neutral polar -continued residue (e.g., Thr, Ser, Ala, Gly, Val, Leu, or Ile); (SEQ ID NO: 122) 0168 Xaa' is a basic amino acid residue (e.g., H is, WTHICitLAGLLSICitSGGVWhArgKNFVPTDWGP1-NalAF N-Methyl-His, Trp, 1-Nal, 2-Nal, Phe, and Tyr); NH; 0169 Xaa' is a basic amino acid residue (e.g., a basic N"-substituted amino acid residue, Arg, N-Methyl-Arg; (SEQ ID NO: 123) homoarginine, Cit, N-Methyl-Cit, Homocitrulline, H is, VTHCitLAGLLSICitSGGVVGuf KNFVPTDVGPFAF-NH; Trp, Guf, and 4-Amino-Phe); (SEQ ID NO: 124) VTHCitLAGLLSICitSGGVWhArgKNFVPTDVGPBipAF 0170 Xaa' is a basic amino acid residue (e.g., a basic N"-substituted amino acid residue, Arg, N-Methyl-Arg; NH; homoarginine, Cit, N-Methyl-Cit, Homocitrulline, H is, Trp, (SEQ ID NO: 125) Gulf, and 4-Amino-Phe): WTHIhArgLAGLLSICitSGGVWhArgkNFVPTDVGP2-NalA 0171 Xaa is a neutral amino acid residue or a basic amino acid residue (e.g., Val, Arg, D-Arg, homoarginine, LyS, D-Lys, homolysine, Orn, Dab, Dpr, homocysteine, and (SEQ ID NO: 126) 4-Amino-Phe); VTHCitLAGLLSICitSGGVWhArgKNFVPTDVGPIglAF 0172 Xaa' is a neutral amino acid residue or a basic NH); amino acid residue (e.g., Arg, D-Arg, homoarginine, LyS, O D-Lys, homolysine, Orn, Dab, Dpr, Homocysteine, and (SEQ ID NO: 127) 4-Amino-Phe); WTHICitLAGLLSICitSGGVWhArgKNIpI-Phe WPTDWGP 0173 Xaa is a neutral amino acid residue or a basic pI-Phe AF-NH2; amino acid residue (e.g., Arg, D-Arg, Lys, D-Lys, homol O ysine, Orn, Dab, Dpr, Cys, homocysteine, and 4-Amino Phe): (b) an N-terminal truncation of any of (a) that preserves the 0174) Xaa is a neutral amino acid residue or a basic hinge region and the first CGRP receptor binding region, amino acid residue (e.g., Arg, D-Arg, ASn, Lys, D-LyS, for example, but not limited to, a sequence having an homolysine, Orn, Dab, Dpr, Cys, homocysteine, and N-terminal truncation of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 4-Amino-Phe); 13, 14, 15, 16, 17, 18, 19, or about 20 contiguous amino acid residues; or 01.75 Xaa' is a neutral polar amino acid residue (e.g., Thr, N'-Methyl-Thr, Ser, N-Methyl-Ser, Sar, Pro, Ala, and (c) any of (a) or (b), in which the amino acid sequence Gly): further contains an aromatic amino acid residue at the 0176) Xaa is a cyclic amino acid residue, a neutral N-terminal end; or hydrophobic amino acid residue, or a neutral polar amino (d) any of (a), (b), or (c), in which the amino acid sequence acid residue (e.g., Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz. contains a modification relative to the native human CCGRP Aib, Sar, and Pip): reference sequence that eliminates a protease binding site or cleavage site. As described herein, some embodiments of the 0177) Xaa is a neutral hydrophobic or aromatic amino peptides described in (a), (b), (c), or (d) are usefully N-ter acid residue (e.g., Phe, D-Phe, Tyr, 1-Nal, 2-Nal, Trp, Bip, minally acylated or otherwise derivatized with another func and His); and tional group as described herein. 0178) Xaa’ is a neutral hydrophobic or aromatic amino acid residue (e.g., Phe, Tyr, 1-Nal, 2-Nal, Trp, His, and Bip). 0164. Some embodiments of the inventive composition of matter involve a CGRP peptide antagonist that comprises 0179. In some preferred embodiments, the composition CGRP peptide that having an amino acid sequence of the of matter includes at its N-terminal an acyl, acetyl (Ac), formula: benzoyl, benzyloxycarbonyl (Cbz or Z), benzyl (Bzl), or dibenzyl (DiBzl or Bn) moiety. In some embodiments, the CGRP peptide is conjugated to a polyethylene glycol (PEG) (SEQ ID NO: 1127) at: Xaa'Xaa’XaaXaaXaaXaaXaavXaaXaaXaa'L'? 0180 (a) 1, 2, 3 or 4 amino functionalized sites of the PEG: 0181 (b) 1, 2, 3 or 4 thiol functionalized sites of the PEG: wherein: 0182 (c) 1, 2, 3 or 4 maleimido functionalized sites of 0165 Xaa, Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa, are the PEG: each independently absent or a hydrophobic amino acid 0183 (d) 1, 2, 3 or 4 N-succinimidyl functionalized residue. sites of the PEG: 0166 Xaa’ is a hydrophobic amino acid residue (e.g., 0.184 (e) 1, 2, 3 or 4 carboxyl functionalized sites of Trp, 1-Nal, 2-Nal, Phe, Tyr, or Bip): the PEG; or US 2008/0020978 A1 Jan. 24, 2008

0185 (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl func 0198 Xaa' is a basic amino acid residue (e.g., Arg, tionalized sites of the PEG. N-Methyl-Arg, homoarginine, Cit, N-Methyl-Cit, 0186. Some other embodiments of the inventive compo Homocitrulline. His, Trp, Guf, Lys, Homolysine, Ornithine, sition of matter incorporate cyclizations to improve stability 4-carboxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, at residues implicated in proteolytic degradation and/or to beta-Homoglutamic acid, homoglutamic acid, or Asp); improve functional activity of the CGRP peptide antagonist. 0199 Xaa' is Ser, Lys, Homolysine, Ornithine, 4-car Some of these contain a CGRP peptide that comprises an boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, amino acid sequence of the formula: beta-Homoglutamic acid, homoglutamic acid, or Asp; 0200) Xaa' is Gly, Lys, Homolysine, Ornithine, 4-car (SEQ ID NO: 1130) boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, Xaa'Xaa’XaaXaaXaaXaaXaavXaaXaa'Xaa' 'Xaa'? beta-Homoglutamic acid, homoglutamic acid, or Asp; XaaXaaXaaXaaXaa17XaaXaaXaa2G2W22 0201 Xaa’ is a neutral amino acid residue or a basic amino acid residue (e.g., Val, Arg, D-Arg, Homoarginine, Xaa2xaa2Xaa2Xaa2F27w 2P2Xaaxaaw? GXaa Lys, D-Lys, Homolysine, Orn, Dab, Dpr, Homocysteine, or XaaXaaXaa 4-Amino-Phe); 0202) Xaa' is a neutral amino acid residue or a basic wherein: amino acid residue (e.g., Arg, D-Arg, Homoarginine, Lys, 0187 Xaa, Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa, are D-Lys, Homolysine, Orn, Dab, Dpr, Homocysteine, or each independently absent or a hydrophobic amino acid 4-Amino-Phe); residue. 0203 Xaa is a neutral amino acid residue or a basic 0188 Xaa’ is a hydrophobic amino acid residue (e.g., amino acid residue (e.g., Lys, D-Lys, Homolysine, Orn, Dab, Trp, 1-Nal, 2-Nal, Phe, Tyr, Bip, Lys, Homolysine, Orni Dpr, Cys, Homocysteine, 4-Amino-Phe, Arg, or D-Arg); thine, 4-carboxy-phenylalanine, 4-Amino-Phe, 4-Amino Phe, beta-glutamic acid, beta-Homoglutamic acid, homo 0204 Xaa’ is a neutral amino acid residue or a basic glutamic acid, or Asp); amino acid residue (e.g., ASn, Lys, D-Lys, Homolysine, Orn, Dab, Dpr, Cys, Homocysteine, 4-Amino-Phe, Arg, or (0189 Xaa is a neutral hydrophobic or neutral polar D-Arg); residue (e.g., Thr, Ser, Ala, Gly, Val, Leu, Ile, Lys, Homol ysine, Ornithine, 4-carboxy-phenylalanine, 4-Amino-Phe, 0205 Xaa' is a neutral polar amino acid residue (e.g., beta-glutamic acid, beta-Homoglutamic acid, homoglutamic Thr, N-Methyl-Thr, Ser, N-Methyl-Ser, Sar, Pro, Ala, Gly, acid, or Asp); LyS, Homolysine, Ornithine, 4-carboxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic acid, 0190. Xaa' is a basic amino acid residue (e.g., H is, homoglutamic acid, or Asp); N-Methyl-His, Trp, 1-Nal, 2-Nal, Phe, Tyr, Lys, Homol ysine, Ornithine, 4-carboxy-phenylalanine, 4-Amino-Phe, 0206 Xaa' is ASn, Lys, Homolysine, Ornithine, 4-car beta-glutamic acid, beta-Homoglutamic acid, homoglutamic boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, acid, or Asp); beta-Homoglutamic acid, homoglutamic acid, or Asp; 0191) Xaa' is a basic amino acid residue (e.g., Arg, 0207 Xaa is Val, Lys, Homolysine, Ornithine, 4-car N-Methyl-Arg, homoarginine, Cit, N-Methyl-Cit, Homoc boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, itrulline. His, Trp, Guf, Lys, Homolysine, Ornithine, 4-car beta-Homoglutamic acid, homoglutamic acid, or Asp; boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, 0208) Xaa is Gly, Lys, Homolysine, Ornithine, 4-car beta-Homoglutamic acid, homoglutamic acid, or Asp); boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, 0192 Xaa' is Leu, Lys, Homolysine. Ornithine, 4-car beta-Homoglutamic acid, homoglutamic acid, or Asp; boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic acid, homoglutamic acid, or Asp; 0209) Xaa is a cyclic amino acid residue, a neutral hydrophobic amino acid residue, or a neutral polar amino 0193 Xaa' is Ala, Lys, Homolysine, Ornithine, 4-car acid residue (e.g., Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz. boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, Aib, Sar, Pip, Lys, Homolysine, Ornithine, 4-carboxy-phe beta-Homoglutamic acid, homoglutamic acid, or Asp; nylalanine, 4-Amino-Phe, beta-glutamic acid, beta-Homo 0194 Xaa' is Gly, Lys, Homolysine, Ornithine, 4-car glutamic acid, homoglutamic acid, or Asp); boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, 0210 Xaa is a neutral hydrophobic or aromatic amino beta-Homoglutamic acid, homoglutamic acid, or Asp; acid residue (e.g., Phe, D-Phe, Tyr, 1-Nal, 2-Nal, Trp, Bip, 0195 Xaa' is Leu, Lys, Homolysine, Ornithine, 4-car His, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic acid, beta-Homoglutamic acid, homoglutamic acid, or Asp; homoglutamic acid, or Asp); 0196) Xaa' is Leu, Lys, Homolysine, Ornithine, 4-car 0211) Xaa is Ala, Lys, Homolysine, Ornithine, 4-car boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic acid, homoglutamic acid, or Asp; beta-Homoglutamic acid, homoglutamic acid, or Asp; 0197) Xaa' is Ser, Lys, Homolysine, Ornithine, 4-car 0212 Xaa is a neutral hydrophobic or aromatic amino boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, acid residue (e.g., Phe, Tyr, 1-Nal, 2-Nal, Trp. His, Bip, Lys, beta-Homoglutamic acid, homoglutamic acid, or Asp; Homolysine, Ornithine, 4-carboxy-phenylalanine, US 2008/0020978 A1 Jan. 24, 2008

4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic acid, to positions on SEQ ID NO:43. Inventive cyclized CGRP homoglutamic acid, or Asp); and wherein a first pair (two) peptide antagonists also include CGRP peptides comprising of amino acid residues selected from Xaa, Xaa, Xaa, the amino acid primary sequence of any of SEQ ID Xaa, Xaa, Xaa' , Xaa, Xaa, Xaa, Xaa, Xaa'7, NOS:155 through 157, 167 through 170, 180 through 228, Xaa, Xaa, Xaa, Xaa, Xaa, Xaa-, Xaa, Xaa, 242 through 280, 348 through 406, 416 through 430, 439 Xaa, Xaa, and Xaa’ are covalently joined to form a first through 498, 512 through 566,580 through 634, 889 through ring and the first pair of amino acid residues forming the first 911, 942 through944, 987,988,994 through996, and 1063 ring are separated by 3 to 7 amino acid residues, more through 1083, whether or not shown in unconjugated or preferably by 3 to 4 amino acid residues, in the CGRP vehicle-conjugated form. peptide's primary sequence (i.e., the chain. For example, in Some embodiments a lactam bridge is formed between 0213. In some preferred embodiments of these inventive residues at positions (relative to the position on the reference compositions containing cyclized CGRP peptides, the com SEQ ID NO:43): 7 and 11, or 8 and 12, or 9 and 13, or 10 position of matter includes at its N-terminal an acyl, acetyl and 14, or 11 and 15, or 12 and 16, or 13 and 17, or 14 and (Ac), benzoyl, benzyloxycarbonyl (Cbz or Z), benzyl (Bzl or 18, or 15 and 19, or 16 and 20, or 17 and 21, or 18 and 22, Bn), or dibenzyl (DiBzl or Bn) moiety. In some embodi or 19 and 23, or 21 and 25, or 22 and 26, or 26 and 31, or ments, the CGRP peptide is conjugated to a polyethylene 27 and 32, or 28 and 33, or 30 and 35, or 31 and 36, or 32 glycol (PEG) at: and 37. Double cyclizations are also useful in accordance 0214 (a) 1, 2, 3 or 4 amino functionalized sites of the with the present invention. Thus in some embodiments of PEG: the composition of matter, a second pair of amino acid residues, both members of which are different from both 0215 (b) 1, 2, 3 or 4 thiol functionalized sites of the members of the first pair of amino acid residues, is selected PEG: from Xaa, Xaa, Xaa, Xaa', Xaa' , Xaa' , Xaa, Xaa, 0216 (c) 1, 2, 3 or 4 maleimido functionalized sites of Xaa, Xaa, Xaa'7, Xaa, Xaa, Xaa, Xaa, Xaa, the PEG: Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa", the second pair of amino acid residues being covalently joined to form 0217 (d) 1, 2, 3 or 4 N-succinimidyl functionalized a second ring. The second pair of amino acid residues sites of the PEG: forming the second ring are also separated by 3 to 7 amino 0218 (e) 1, 2, 3 or 4 carboxyl functionalized sites of acid residues, more preferably by 3 to 4 amino acid residues, the PEG; or in the CGRP peptide's primary sequence, and both members of the second pair of amino acid residues are more proximal 0219 (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl func in the primary sequence to either the C-terminal end or the tionalized sites of the PEG. N-terminal end of the CGRP peptide compared to both 0220 Additional CGRP peptide antagonists that are members of the first pair of amino acid residues. Inventive encompassed within the present invention include any of cyclized CGRP peptide antagonists include, e.g., those those comprising an amino acid primary sequence set forth involving two lactam bridges between residues at positions in the following Table 2A, Table 2B, Table 2C, and Table 9 and 13 (a first ring, for example) and between residues at 2D, whether shown in vehicle-conjugated (e.g., PEGylated positions 15 and 19 (a second ring, for example) in relative form) or unconjugated form.

TABLE 2A Exemplary CGRP peptide sequences. Underlined boldface amino acid residues, if any indicate cyclization between the first underlined boldface residue and the second underlined boldface residue in a sequence. IC 50 values were determined as describe in Example 1 herein: SEQ ID C50 NO CGRP Peptide Sequence (nM) 28Ac-KWVTHCitLAGLLSICitSGGVWRKNFVPTDVGPKAF-NH2 1298 29 Ac-WVTHCitLAGELSICitKGGWh ArgkNFVPTDVGOicFAF-NH2 1.33 30 Ac-WVTHCitLAGLLSICitSGGVWhArgKNFVPTDVGTicFAF-NH2 2.47 31Ac-WVTHCitLAGLLSICitSGGVWhArgKNFVPTDVGPTicAF-NH2 89.91 32Ac-WVTHCitLAGLLSICitSGGVWhArgkNFVOic TDVGOicFA2-Nal-NH2 257 33Ac-WVTHCitLAGLLSICitSGGVWhArgkNFVOic TDWGP1-NalA2-Nal-NH2 2.73 34 Ac-WVTHCitLAGLLSICitSGGVWhArgkNFVOic TDWGPBipAF-NH2 3.85 35Ac-WVTHCitLAGLLSICitSGGVWhArgkNFVOic TDWGPFA2-Nal-NH2 3.22 36 Ac-WVTHCitLAGLLSICitSGGVWhArgkNpI-PhevOic TDWGPpI-PheAF-NH2 2.34

US 2008/0020978 A1 Jan. 24, 2008 45

0226. The term “vehicle' refers to a molecule that pre is one of the hundreds of different proteins dissolved in vents or mitigates in vivo degradation, increases half-life, mammalian blood plasma, including so-called “carrier pro reduces toxicity, reduces immunogenicity, and/or increases teins’ (Such as albumin, transferrin and haptoglobin), biological activity of a therapeutic peptide. In accordance fibrinogen and other blood coagulation factors, complement with the invention, the vehicle is a pharmaceutically accept components, immunoglobulins, enzyme inhibitors, precur able vehicle. The vehicle should be selected such that the sors of Substances Such as angiotensin and and vehicle-conjugated CGRP peptide antagonist achieves a many other types of proteins. The invention encompasses Sufficient hydrodynamic size to prevent clearance by renal the use of any single species of pharmaceutically acceptable filtration in vivo. For example, a vehicle can be selected that vehicle, such as, but not limited to, those described herein, is a polymeric macromolecule, which is Substantially in conjugation with the CGRP receptor-binding peptide, or straight chain, branched-chain, or dendritic in form. Alter the use of a combination of two or more different vehicles. natively, a vehicle can be selected such that, in vivo, the inventive composition of matter will bind to a plasma 0230. In accordance with the present invention, which protein to form a complex. Such that the complex thus also relates to a method of producing a composition of formed avoids Substantial renal clearance. matter, the following steps are included: a CGRP peptide as described herein above is obtained; and the obtained peptide 0227 Exemplary vehicles that can be used, in accordance is conjugated to a pharmaceutically acceptable vehicle at a with the present invention, include a polyalkylene glycol site on the peptide other than at the carboxy terminal amino compound, such as a polyethylene glycolor a polypropylene acid residue. glycol. Other appropriate polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers 0231. In being conjugated, the vehicle, as described of the following types: dextran, colominic acids or other herein, is covalently bound directly to an amino acid residue carbohydrate based polymers, polymers of amino acids, and of the CGRP peptide itself, or optionally, to a peptidyl or biotin derivatives. non-peptidyl linker (including but not limited to aromatic linkers) that is covalently bound to an amino acid residue of 0228. Other examples of the vehicle, in accordance with the CGRP peptide. Any “linker group is optional. When the invention, include a copolymer of ethylene glycol, a present, its chemical structure is not critical, since it serves copolymer of propylene glycol, a carboxymethylcellulose, a primarily as a spacer, which can be useful in optimizing polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1.3.6- pharmacological activity of some embodiments of the inven trioxane, an ethylene/maleic anhydride copolymer, a tive composition. The linker is preferably made up of amino polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrroli acids linked together by peptide bonds. The linker moiety, if done, a poly n-vinyl pyrrolidone, a propylene glycol present, can be independently the same or different from any homopolymer, a propylene oxide polymer, an ethylene oxide other linker, or linkers, that may be present in the inventive polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a composition. linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, 0232. As stated above, the linker, if present, can be an immunoglobulin F, domain (see, e.g., Feige et al., Modi peptidyl in nature (i.e., made up of amino acids linked fied peptides as therapeutic agents, U.S. Pat. No. 6,660,843), together by peptide bonds) and made up in length, prefer an albumin (e.g., human serum albumin; see, e.g., Rosen et ably, of from 1 up to about 40 amino acid residues, more al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US preferably, of from 1 up to about 20 amino acid residues, and 2005/0054051; Bridon et al., Protection of endogenous most preferably of from 1 to about 9 amino acid residues. therapeutic peptides from peptidase activity through conju Preferably, but not necessarily, the amino acid residues in the gation to blood components, U.S. Pat. No. 6,887,470), a linker are from among the twenty canonical amino acids, transthyretin (TTR; see, e.g., Walker et al., Use of tran more preferably, cysteine, glycine, alanine, proline, arginine, sthyretin peptide?protein fusions to increase the serum half asparagine, glutamine, serine and/or lysine. Even more life of pharmacologically active peptides/proteins, US 2003/ preferably, a peptidyl linker is made up of a majority of 0195154 A1: 2003/0191056 A1), or a thyroxine-binding amino acids that are sterically unhindered, such as glycine globulin (TBG). and alanine linked by a . It is also desirable that, if present, a peptidyl linker be selected that avoids rapid 0229. Other embodiments of the vehicle, in accordance proteolytic turnover in circulation in vivo, and/or which with the invention, include peptide ligands or Small includes one or more copies of a CGRP binding region. (organic) molecule ligands that have binding affinity for a Some of these amino acids may be glycosylated, as is well long half-life plasma protein under physiological conditions understood by those in the art. For example, a useful linker of temperature, pH, and ionic strength. Examples include an sequence constituting a sialylation site is XXNXX G albumin-binding peptide or Small molecule ligand, a tran sthyretin-binding peptide or Small molecule ligand, a thy (SEQ ID NO:1104), wherein X, X, X and Xs are each roxine-binding globulin-binding peptide or Small molecule independently any amino acid residue. ligand, an antibody-binding peptide or Small molecule 0233. In other embodiments, the 1 to 40 amino acids are ligand, or another peptide or Small molecule that has an selected from glycine, alanine, proline, asparagine, affinity for a long half-life plasma protein. (See, e.g., Blaney glutamine, and lysine. Preferably, a linker is made up of a et al., Method and compositions for increasing the serum majority of amino acids that are sterically unhindered. Such half-life of pharmacologically active agents by binding to as glycine and alanine. Thus, preferred linkers include transthyretin-selective ligands, U.S. Pat. No. 5,714, 142: polyglycines, poly(Gly-Ala)S, and polyalanines. Some Sato et al., Serum albumin binding moieties, US 2003/ exemplary peptidyl linkers are poly(Gly) is particularly 0069395 A1; Jones et al., Pharmaceutical active conjugates, (Gly), (Gly) (SEQ ID NO:1105), (Gly) (SEQ ID NO:56) U.S. Pat. No. 6,342,225). A “long half-life plasma protein’ and (Gly), (SEQID NO:57), as well as, poly(Gly) Ser (SEQ US 2008/0020978 A1 Jan. 24, 2008 46

ID NO:67), poly(Gly-Ala) and poly(Ala)s. Other spe NO:1126), wherein X, X, X and X are each indepen cific examples of peptidyl linkers include (Gly) Lys (SEQ dently any amino acid residue. ID NO:58), and (Gly) Lys Arg (SEQ ID NO:59). Other 0238. The linkers shown here are exemplary; peptidyl specific examples of linkers are: Other examples of useful linkers within the scope of this invention may be much peptidyl linkers are: longer and may include other residues. A peptidyl linker can contain, e.g., a N-terminal cysteine, another thiol, or nucleo (Gly) Lys (Gly); (SEQ ID NO: 60) phile for conjugation with a vehicle. In another embodiment, the linker contains an N-terminal cysteine or homocysteine (Gly) AsnGlyser (Gly) 2; (SEQ ID NO: 61) residue, or other 2-amino-ethanethiol or 3-amino-pro (Gly) Cys (Gly); (SEQ ID NO: 62) panethiol moiety for conjugation to maleimide, iodoacetaa and mide or thioester, functionalized vehicles. Another useful peptidyl linker is a large, flexible linker comprising a GlyProAsnGlyGly. (SEQ ID NO: 63) random Gly/Ser/Thr sequence, for example: GSG SATGGSGSTASSGSGSATH (SEQ ID NO:64) or HGSG 0234) To explain the above nomenclature, for example, SATGGSGSTASSGSGSAT (SEQ ID NO:66), that is esti (Gly) Lys(Gly), means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly mated to be about the size of a 1 kDa PEG molecule. (SEQ ID NO:60). Other combinations of Gly and Ala are Alternatively, a useful peptidyl linker may be comprised of also useful. amino acid sequences known in the art to form rigid helical 0235) Other preferred linkers are those identified herein structures (e.g., Rigid linker: -AEAAAKEAAAKEAAAK as “L5” (GGGGS: SEQ ID NO:1106), “L10” AGG-) (SEQID NO:65). Additionally, a peptidyl linker can (GGGGSGGGGS: SEQ ID NO:1107), “L25” also comprise a non-peptidyl segment Such as a 6 carbon GGGGSGGGGSGGGGSGGGGSGGGGS: SEQ ID aliphatic molecule of the formula —CH2—CH2—CH2— NO: 1108) and any linkers used in the working examples CH, CH, CH . The peptidyl linkers can be altered to hereinafter. form derivatives as described herein. 0236. In some embodiments of the compositions of this 0239 Optionally, non-peptidyl linkers are also useful for invention, which comprise a peptide linker moiety (L), conjugating the vehicle to the peptide portion of the vehicle acidic residues, for example, glutamate or aspartate residues, conjugated CGRP peptide antagonist. For example, alkyl are placed in the amino acid sequence of the linker moiety linkers such as NH-(CH2), —C(O)—, wherein s=2-20 can be used. These alkyl linkers may further be substituted (L). Examples include the following peptide linker by any non-sterically hindering group Such as lower alkyl Sequences: (e.g., C-C) lower acyl, halogen (e.g., Cl, Br), CN. NH2, phenyl, etc. Exemplary non-peptidyl linkers are PEG linkers GGEGGG; (SEQ ID NO : 1109) (e.g., shown below): GGEEEGGG; (SEQ ID NO : 1110) (I) GEEEG; (SEQ ID NO : 1111) O GEEE; (SEQ ID NO : 1112) GGDGGG; (SEQ ID NO : 1113) ~~r- GGDDDGG; (SEQ ID NO : 1114) GDDDG; (SEQ ID NO : 1115) wherein n is such that the linker has a molecular weight of GDDD; (SEQ ID NO : 1116) about 100 to about 5000 kilodaltons (kDa), preferably about 100 to about 500 kDa. GGGGSDDSDEGSDGEDGGGGS; (SEQ ID NO : 1117) 0240. In one embodiment, the non-peptidyl linker is aryl. WEWEW; (SEQ ID NO : 1118) The linkers may be altered to form derivatives in the same FEFEF; (SEQ ID NO : 1119) manner as described herein. In addition, PEG moieties may be attached to the N-terminal amine or selected side chain EEEWWW; (SEQ ID NO : 1120) amines by either reductive alkylation using PEG aldehydes or acylation using hydroxysuccinimido or carbonate esters EEEFFF; (SEQ ID NO : 1121) of PEG, or by thiol conjugation. WWEEEWW; (SEQ ID NO : 1122) 0241 Non-peptide portions of the inventive composition O of matter, Such as non-peptidyl linkers or non-peptide FFEEEFF (SEQ ID NO : 1123) vehicles can be synthesized by conventional organic chem istry reactions. 0237. In other embodiments, the linker constitutes a 0242. The above is merely illustrative and not an exhaus phosphorylation site, e.g., XXYXXG (SEQ ID tive treatment of the kinds of linkers that can optionally be NO:1124), wherein X, X, X and X are each indepen employed in accordance with the present invention. dently any amino acid residue: XXSXXG (SEQ ID NO:1125), wherein X, X, X and X are each indepen 0243 In one useful embodiment of the inventive com dently any amino acid residue; or XXTXXG (SEQ ID position of matter and/or the method of producing a vehicle US 2008/0020978 A1 Jan. 24, 2008 47 conjugated CGRP peptide antagonist, the peptide is conju concentrations of the reagents can be obtained by relatively gated to the vehicle via covalent linkage at an amino acid simple trial-and-error experiments which are within the residue in the hinge region, Such as, at one of the nine amino ability of an ordinarily skilled artisan with appropriate acid residues at positions 19-27, relative to the native human Scaling-up as necessary. Purification and separation of the CCGRP sequence. In some useful embodiments, the peptide products is similarly achieved by conventional techniques is conjugated to the vehicle, more particularly, at amino acid well known to those skilled in the art. position 22, position 23, position 24, position 25, position 0248. Additionally, physiologically acceptable salts of 26, or position 27, and preferably, at amino acid position 23, the vehicle-conjugated or unconjugated CGRP peptide position 24, or position 25, relative to the native human antagonists of this invention are also encompassed within CCGRP sequence. the present invention. By “physiologically acceptable salts' 0244. In other embodiments of the inventive composition is meant any salts that are known or later discovered to be of matter and/or the method of producing a composition of pharmaceutically acceptable. Some specific examples are: matter, the CGRP peptide is conjugated to the vehicle at an acetate; trifluoroacetate; hydrohalides, such as hydrochlo amino acid residue in the first or the second CGRP receptor ride and hydrobromide; sulfate; citrate; maleate; tartrate; binding region. glycolate; gluconate; succinate; mesylate; besylate; and oxalate salts. 0245. In still another useful embodiment of the inventive composition of matter and/or the method of producing a 0249. As an illustration, in some embodiments of the composition of matter, involving an inventive vehicle-con inventive composition of matter and/or the method of pro jugated CGRP peptide antagonist, the CGRP peptide is ducing a composition of matter, the vehicle is poly(ethylene conjugated at the amino acid residue at the peptide's amino glycol) (PEG). Covalent conjugation of proteins with poly terminal end to the vehicle. (See, e.g., Kinstler et al., (ethylene glycol) (PEG) has been widely recognized as an N-terminally chemically modified protein compositions and approach to significantly extend the in vivo circulating half-lives of therapeutic proteins. PEGylation achieves this methods, U.S. Pat. Nos. 5,985,265, and 5,824,784). effect predominately by retarding renal clearance, since the 0246. It will be appreciated that, since the vehicle PEG moiety adds considerable hydrodynamic radius to the employed for conjugation to the CGRP peptide can be protein. (Zalipsky, S., et al. Use of functionalized poly(eth multivalent (e.g., bivalent, trivalent, tetravalent or a higher ylene glycol)S for modification of polypeptides., in poly order valency), as to the number of residues at which vehicle (ethylene glycol) chemistry: Biotechnical and biomedical can be conjugated, and/or the peptide portion of the inven applications. J. M. Harris, Ed., Plenum Press: New York. tive composition of matter can be multivalent (e.g., bivalent, 347-370 (1992)). Additional benefits often conferred by trivalent, tetravalent or a higher order valency), it is possible PEGylation of proteins include increased solubility, resis by the inventive method of producing a composition of tance to proteolytic degradation, and reduced immunoge matter to produce a variety of conjugated vehicle:peptide nicity of the therapeutic polypeptide. The merits of protein structures. By way of example, a univalent vehicle and a PEGylation are evidenced by the commercialization of univalent peptide will produce a 1:1 conjugate; a bivalent several PEGylated proteins including PEG-Adenosine peptide and a univalent vehicle may form conjugates deaminase (AdagenTM/Enzon Corp.), PEG-L-asparaginase wherein the peptide conjugates bear two vehicle moieties, (OncasparTM/Enzon Corp.), PEG-Interferon C-2b (PEG-In whereas a bivalent vehicle and a univalent peptide may tronTM/Schering/Enzon), PEG-Interferon C-2a (PEGA produce species where two peptide entities are linked to a SYSTM/Roche) and PEG-G-CSF (NeulastaTM/Amgen) as single vehicle moiety; use of higher-valence vehicles can well as many others in clinical trials. lead to the formation of clusters of peptide entities bound to a single vehicle moiety, whereas higher-valence peptides 0250) By "PEGylated peptide' is meant a peptide having may become encrusted with a plurality of vehicle moieties. a polyethylene glycol (PEG) moiety covalently bound to an By way of further example, if the site of conjugation of a amino acid residue of the peptide itself or to a peptidyl or multivalent vehicle to the CGRP peptide is a cysteine or non-peptidyl linker (including but not limited to aromatic other aminothiol the methods disclosed by D'Amico et al. linkers) that is covalently bound to a residue of the peptide. may be employed (U.S. Ser. No. 60/646,685, Method of 0251. By “polyethylene glycol” or “PEG” is meant a conjugating aminothiol containing molecules to water polyalkylene glycol compound or a derivative thereof, with soluble polymers, which application is incorporated herein or without coupling agents or derivatization with coupling or by reference in its entirety). activating moieties (e.g., with aldehyde, hydroxysuccinim idyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, 0247 The peptide moieties may have more than one orthopyridyl disulphide, vinylsulfone, iodoacetamide or a reactive group which will react with the activated vehicle and the possibility of forming complex structures must maleimide moiety). In accordance with the present inven always be considered; when it is desired to form simple tion, useful PEG includes substantially linear, straight chain structures such as 1:1 adducts of vehicle and peptide, or to PEG, branched PEG, or dendritic PEG. (See, e.g., Merrill, use bivalent vehicles to form peptide: vehicle:peptide U.S. Pat. No. 5,171,264; Harris et al., Multiarmed, mono adducts, it will be beneficial to use predetermined ratios of functional, polymer for coupling to molecules and Surfaces, activated vehicle and peptide material, predetermined con U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymer centrations thereof and to conduct the reaction under pre derivatives, U.S. Pat. No. 6,602,498). determined conditions (such as duration, temperature, pH, 0252 PEG is a well-known, water soluble polymer that is etc.) So as to form a proportion of the described product and commercially available or can be prepared by ring-opening then to separate the described product from the other reac polymerization of ethylene glycol according to methods well tion products. The reaction conditions, proportions and known in the art (Sandler and Karo, Polymer Synthesis, US 2008/0020978 A1 Jan. 24, 2008 48

Academic Press, New York, Vol. 3, pages 138-161). In the 5.252.714). Also included within the meaning of “PEG present application, the term “PEG' is used broadly to aldehyde compound are PEG aldehyde hydrates, e.g., PEG encompass any polyethylene glycol molecule, in mono-, bi-, acetaldehyde hydrate and PEG bis aldehyde hydrate, which or poly-functional form, without regard to size or to modi latter yields a bifunctionally activated structure. (See., e.g., fication at an end of the PEG, and can be represented by the Bentley et al., Poly(ethylene glycol) aldehyde hydrates and formula: related polymers and applications in modifying amines, U.S. Pat. No. 5,990.237) (See., e.g., Bentley et al., Poly(ethylene X-O(CH2CH2O), CH2CH2OH, (II) glycol) aldehyde hydrates and related polymers and appli 0253) where n is 20 to 2300 and X is H or a terminal cations in modifying amines, U.S. Pat. No. 5,990.237). An modification, e.g., a C alkyl. activated multi-branched PEG-aldehyde compound can be used (PEG derivatives comprising multiple arms to give 0254. In some useful embodiments, a PEG used in the divalent, trivalent, tetravalent, octavalent constructs). Using invention terminates on one end with hydroxy or methoxy, a 4-arm PEG derivative four (4) CGRP peptides are attached i.e., X is H or CH (“methoxy PEG'). It is noted that the to each PEG molecule. For example, in accordance with the other end of the PEG, which is shown in formula (II) present invention, the CGRP peptide can be conjugated to a terminating in OH, covalently attaches to an activating polyethylene glycol (PEG) at 1, 2, 3 or 4 amino function moiety via an ether oxygen bond, an amine linkage, or amide alized sites of the PEG. linkage. When used in a chemical structure, the term “PEG' includes the formula (II) above without the hydrogen of the 0258. In being conjugated in accordance with the inven hydroxyl group shown, leaving the oxygen available to react tive method, the polyethylene glycol (PEG), as described with a free carbon atom of a linker to form an ether bond. herein, is covalently bound by reductive amination directly More specifically, in order to conjugate PEG to a peptide, the to at least one solvent-exposed free amine moiety of an peptide must be reacted with PEG in an “activated form. amino acid residue of the CGRP peptide itself. In some Activated PEG can be represented by the formula: embodiments of the inventive method, the CGRP peptide is conjugated to a PEG at one or more primary or secondary (PEG)-(A) (III) amines on the CGRP peptide, or to two PEG groups at a where PEG (defined supra) covalently attaches to a carbon single primary amine site on the CGRP peptide (e.g., this can atom of the activation moiety (A) to form an ether bond, an occur when the reductive amination reaction involves the amine linkage, or amide linkage, and (A) contains a reactive presence of excess PEG-aldehyde compound). We have group which can react with an amino, imino, or thiol group observed that when PEGylation by reductive amination is at on an amino acid residue of a peptide or a linker moiety a primary amine on the peptide, it is not uncommon to have covalently attached to the peptide. amounts (1 to 100% range) of reaction product that have two or more PEGs present per molecule, and if the desired 0255 Techniques for the preparation of activated PEG PEGylation product is one with only one PEG per molecule, and its conjugation to biologically active peptides are well then this “over-PEGylation' may be undesirable. When known in the art. (E.g., see U.S. Pat. Nos. 5,643,575, PEGylated product with a single PEG per PEGylation 5,919,455, 5,932,462, and 5.990,237; Thompson et al., product molecule is desired, an embodiment of the inventive PEGylation of polypeptides, EP 0575545 B1; Petit, Site method can be employed that involves PEGylation using specific protein modification, U.S. Pat. Nos. 6,451.986, and secondary amines of the pharmacologically active peptide, 6,548,644; S. Herman et al., Poly(ethylene glycol) with because only one PEG group per molecule will be trans reactive endgroups: 1. Modification of proteins, J. Bioactive ferred in the reductive amination reaction. Compatible Polymers, 10:145-187 (1995): Y. Lu et al., Pegylated peptides III: Solid-phase synthesis with PEGylat 0259 Amino acid residues that can provide a primary ing reagents of varying molecular weight: Synthesis of amine moiety include residues of lysine, homolysine, orni multiply PEGylated peptides, Reactive Polymers, 22:221 thine, C.B-diaminopropionic acid (Dap), C.f3-diaminopropi 229 (1994); A. M. Felix et al., PEGylated Peptides IV: onoic acid (Dpr), and C.Y-diaminobutyric acid (Dab), ami Enhanced biological activity of site-directed PEGylated nobutyric acid (Abu), and C.-amino-isobutyric acid (Aib). GRF analogs, Int. J. Peptide Protein Res., 46:253-264 Amino acid residues that can provide a secondary amine (1995); A. M. Felix, Site-specific poly(ethylene glycol)yla moiety include e-N-alkyl lysine, Cl-N-alkyl lysine, 8-N-alkyl tion of peptides, ACS Symposium Series 680(poly(ethylene ornithine, Cl-N-alkyl ornithine, or an N-terminal proline, glycol)): 218-238 (1997); Y. Ikeda et al., Polyethylene where the alkyl is C to C. glycol derivatives, their modified peptides, methods for 0260. In accordance with the inventive method of pro producing them and use of the modified peptides, EP ducing a composition of matter, the inclusion of an alcohol 0473084 B1; G. E. Means et al., Selected techniques for the co-solvent in a buffer Solution can improve the conjugation modification of protein side chains, in: Chemical modifica efficiency of CGRP peptides to PEG aldehyde compounds tion of proteins, Holden Day, Inc., 219 (1971)). by reductive amination, as is further illustrated in Example 0256 Activated PEG, such as PEG-aldehydes or PEG 8 herein below. The buffer solution for the PEGylation aldehyde hydrates, can be chemically synthesized by known reaction comprises an “alcohol co-solvent, which term means or obtained from commercial sources, e.g., Shearwa encompasses an alcohol solvent, in embodiments in which ter Polymers, (Huntsville, Ala.) or Enzon, Inc. (Piscataway, the (v/v) concentration of the alcohol is 100% as described below. However, in many embodiments, the concentration N.J.). (v/v) of the alcohol co-solvent in an aqueous medium is 0257 An example of a useful activated PEG for purposes about 30% to about 99%, or about 30% to about 90%, or of the present invention is a PEG-aldehyde compound (e.g., about 30% to about 80%, or about 30% to about 70%, with a methoxy PEG-aldehyde), such as PEG-propionaldehyde, about 50% to about 70% being a preferred concentration which is commercially available from Shearwater Polymers range. The skilled artisan will understand how to optimize (Huntsville, Ala.). PEG-propionaldehyde is represented by the alcohol concentration for reductive amination reaction of the formula PEG-CHCHCHO. (See, e.g., U.S. Pat. No. a particular species of CGRP peptide with a particular US 2008/0020978 A1 Jan. 24, 2008 49 species of PEG-aldehyde compound. By way of example, such as 1HF-i-PA or HP-t-BuOH. While this aspect of the for some small CGRP peptides (e.g., about 10-20 amino acid present invention does not depend upon any particular residues in length), an alcohol (e.g., TFE) concentration of mechanism of operation, it is thought that this observation 100% (v/v) is suitable. relates to the increased solubility of aliphatic side chain 0261) Useful aqueous buffer solutions for the reductive residues in aliphatic solvents. In this context, the rank-order amination reaction can be made with any of the buffers of aliphatic character is HP-t-BuOH>HF-i-PA-TFE. The known in the biochemical art that provide buffering from efficiency enhancement afforded by B-fluoro alcohols would about pH 4 to about pH 9, with about pH 5 to about pH 7 be most advantageous to reductive amination reactions. This preferred. These buffers can include, but are not limited to, is hypothetically related to the strong hydrogen bonding acetate, citrate, 2-(N-Morpholino)-ethane sulfonic acid character associated with B-fluoro alcohols such as TFE for (MES), phosphate, N,N-Bis(2hydroxyethyl)glycine two reasons. The first of which is the ability to dissolve (Bicine), or borate buffers. Useful buffer concentrations may polyamide structure by Solvating the amide oxygen. Sec range from 5 mM to 100 mM with 10-50 mM preferred. ondly, the strong H-bonding ability afforded by the B-fluoro Buffers containing a free amine group, such as TRIS, are not alcohol should facilitate imine formation between PEG suitable. aldehyde and an amine present on the peptide in the same 0262 Alcohols that should accelerate the reductive ami manner that acid does. Imine formation is a required step in nation of PEG aldehydes with amine containing peptides the reductive amination process, and is often rate-limiting. include 2.2.2-trifluoroethanol (TFE), 1,1,1,3,3,3- The ability of B-fluoro alcohol to accelerate this step is hexafluoro-2-propanol (HF-i-PA), and 2-trifluoromethyl-1, therefore of particular advantage. 1,1,3,3,3-hexafluoro-2-propanol (HF-t-BuOH), although, in 0264. Another useful activated PEG for generating the principle, any alcohol co-solvent based on structural For PEG-conjugated peptides of the present invention is a PEG mulae IV, V and VI (below) could increase the PEGylation maleimide compound, such as, but not limited to, a methoxy efficiency during reductive amination. The alcohol co-sol PEG-maleimide, such as maleimido monomethoxy PEG, are vent comprises a structure selected from structural Formulae particularly useful for generating the PEG-conjugated pep IV, V and VI: tides of the invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498; C. Delgado et al., The IV uses and properties of PEG-linked proteins. Crit. Rev. R2 Therap. Drug Carrier Systems, 9:249-304 (1992); S. Zalipsky et al., Use of functionalized poly(ethylene glycol)S for modification of polypeptides, in: Poly(ethylene glycol) s chemistry: Biotechnical and biomedical applications (J. M. R3 V Harris, Editor, Plenum Press: New York, 347-370 (1992); S. R3 OH Herman et al., Poly(ethylene glycol) with reactive end groups: 1. Modification of proteins, J. Bioactive Compatible RI Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjuga V - R tion of polymer to colony stimulating factor-1, U.S. Pat. No. y1 4,847.325; G. Shaw et al., Cysteine added variants IL-3 and VI chemical modifications thereof, U.S. Pat. No. 5,166.322 and EP 0469074 B1; G. Shaw et al., Cysteine added variants of EPO and chemical modifications thereof, EP 0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemical modifications thereof, EP 0668354 A1: N. V. Katre et al., Interleukin-2 muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J. Goodson and N. V. Katre, Site wherein R' is independently CFR, n=1 to 3; wherein directed pegylation of recombinant interleukin-2 at its gly Formula V1 comprises at least seven carbon atoms; cosylation site, Biotechnology, 8:343-346 (1990)). R" is a (C. to C.)linear or branched alkyl moiety, a (C. to 0265 A poly(ethylene glycol) vinyl sulfone is another C.)cyclic alkyl moiety, or an aryl or heteroaryl moiety; useful activated PEG for generating the PEG-conjugated peptides of the present invention by conjugation at thiolated R° and R are independently H or R'H: amino acid residues, e.g., at C residues. (E.g., M. Morpurgo et al., Preparation and characterization of poly(ethylene A is (CY)o, wherein o equals 1 to 4, or (CY X—CY), glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996); wherein X is O or NR and Y is independently H or F: see also Harris, Functionalization of polyethylene glycol for R is independently H or a (C. to C.) linear or branched formation of active sulfone-terminated PEG derivatives for alkyl: binding to proteins and biologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208; 5,900,461; 6,610,281 and wherein B is C(R) or N. and 6,894,025; and Harris, Water soluble active sulfones of (B is capable of forming a stable bicyclic structure such as poly(ethylene glycol), WO95/13312 A1). bicyclo2.2.2]octane, or the like.) In some embodiments, if 0266. Another activated form of PEG that is useful in X is O, Y is independently H or F; and if X is NR. Y is H. accordance with the present invention, is a PEG-N-hydrox 0263. In general, the PEGylation efficiency of CGRP ySuccinimide ester compound, for example, methoxy PEG peptide sequences benefits from more hydrophobic alcohols N-hydroxysuccinimidyl (NHS) ester. US 2008/0020978 A1 Jan. 24, 2008 50

0267. Heterobifunctionally activated forms of PEG are preferred combined mass for PEG is from about 20,000 Da also useful. (See, e.g., Thompson et al., PEGylation reagents to 30,000 Da (total n is about 450 to about 680). and biologically active compounds formed therewith, U.S. 0278. The present invention also provides methods of Pat. No. 6,552,170). using the inventive composition of matter and the pharma 0268. In still other embodiments of the inventive method ceutical compositions containing them, e.g., in the inventive of producing a composition of matter, the CGRP peptide is methods of treating migraine, and preventing or mitigating reacted by known chemical techniques with an activated migraine. The inventive methods are of benefit in alleviating PEG compound, such as but not limited to, a thiol-activated and/or mitigating symptoms of migraine. “Alleviated PEG compound, a diol-activated PEG compound, a PEG means to be lessened, lightened, diminished, softened, miti hydrazide compound, a PEG-oxyamine compound, or a gated (i.e., made more mild or gentle), quieted, assuaged, PEG-bromoacetyl compound. (See, e.g., S. Herman, Poly abated, relieved, nullified, or allayed, regardless of whether (ethylene glycol) with Reactive Endgroups: 1. Modification the pain or migraine symptom is entirely erased, eradicated, of Proteins, J. Bioactive and Compatible Polymers, 10: eliminated, or prevented in a particular patient. 145-187 (1995): S. Zalipsky, Chemistry of Polyethylene 0279 If desired, the therapeutic or prophylactic efficacy Glycol Conjugates with Biologically Active Molecules, of the CGRP peptide antagonists may be tested preclinically, Advanced Drug Delivery Reviews, 16:157-182 (1995); R. prior to clinical use in humans, using any appropriate animal Greenwald et al., Poly(ethylene glycol) conjugated drugs model known to those skilled in the art related to a particular and prodrugs: a comprehensive review, Critical Reviews in condition of interest. (See, e.g., Wang and Wang, Animal and Therapeutic Drug Carrier Systems, 17:101-161 (2000)). cellular models of chronic pain, Advanced Drug Delivery 0269. Any molecular mass for a PEG can be used as Reviews 55:949-965 (2003)). An appropriate animal model practically desired, e.g., from about 1,000 Daltons (Da) to for migraine can be selected from numerous methods, as 100,000 Da (n is 20 to 2300). The number of repeating units described, for example, in Bergerot et al., Review Article: “n” in the PEG is approximated for the molecular mass Animal models of migraine: looking at the component parts described in Daltons. It is preferred that the combined of a complex disorder, European Journal of Neuroscience molecular mass of PEG on an activated linker is suitable for 24:1517-1534 (2006); and Akerman, Sand Goadsby PJ, The pharmaceutical use. Thus, the combined molecular mass of role of dopamine in a model of trigeminovascular nocicep the PEG molecule should not exceed about 100,000 Da. tion, Pharmacol. Exp. Ther. 314(1): 162-169 (2005), which 0270. In still other embodiments of the inventive method are both incorporated by reference in there entireties. of producing a composition of matter, the CGRP peptide is 0280 The present invention is directed to a method of reacted by known chemical techniques with an activated treating migraine, and to a method of preventing or miti multi-branched PEG compound (PEG derivatives compris gating migraine. The method of treating migraine includes ing multiple arms to give divalent, trivalent, tetravalent, administering to a patient in need of Such treatment a octavalent constructs). Such as but not limited to, pentaeryth therapeutically effective amount of the inventive composi ritol tetra-polyethyleneglycol ether. Functionalization and tion of matter, Such that one or more migraine symptoms is activated derivatives, such as, but not limited to, N-Succin alleviated in the patient. The inventive method of preventing imidyloxycarbonyl)propyl. p-nitrophenyloxycarbonyl, or mitigating migraine includes administering to a patient (—CO-p-CHNO), 3-(N-maleimido)propanamido, 2-sul who has previously experienced a migraine a prophylacti fanylethyl, and 3-aminopropyl. Using a 4-arm PEG deriva cally effective amount of the inventive composition of tive four CGRP peptide antagonists are attached to each matter Such that at least one symptom of a Subsequent PEG molecule. For example, in accordance with the present migraine is prevented or mitigated. The at least one symp invention, the CGRP peptide can be conjugated to a poly tom is a migraine symptom previously experienced by the ethylene glycol (PEG) at: patient migraineur in a prior migraine attack. 0271 (a) 1, 2, 3 or 4 amino functionalized sites of the 0281. In accordance with these inventive methods, a PEG: patient in need of treatment for migraine, or a patient who has previously experienced a migraine, are well-recogniz 0272 (b) 1, 2, 3 or 4 thiol functionalized sites of the able and/or diagnosed by the skilled practitioner, Such as a PEG: physician, familiar with migraine and its symptoms. 0273 (c) 1, 2, 3 or 4 maleimido functionalized sites of 0282. There are several types of migraine, each with the PEG: unique features or symptoms well known to those of skill in 0274 (d) 1, 2, 3 or 4 N-succinimidyl functionalized the art, but the present invention is not limited to any one sites of the PEG: type and can be useful in treating, alleviating, preventing or mitigating symptoms of any type of migraine. Classic 0275 (e) 1, 2, 3 or 4 carboxyl functionalized sites of migraine and common migraine are the two major varieties. the PEG; or Common migraine (without aura) is the most frequent type, 0276 (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl func accounting for about 80-85% of migraines. Unlike other tionalized sites of the PEG. headaches, migraines usually occur on one side of the head, although the side that is affected can shift with each new 0277 Preferably, the combined or total molecular mass attack. Migraines are also often accompanied by symptoms of PEG used in a PEG-conjugated peptide of the present of abnormal sensitivity to light and/or sound. The pain invention is from about 3,000 Da to 60,000 Da (total n is symptoms of a migraine headache are often described as an from 70 to 1,400), more preferably from about 10,000 Da to intense throbbing or pounding felt in the forehead/temple, 40,000 Da (total n is about 230 to about 910). The most earlaw or around the eyes. Although migraine pain usually US 2008/0020978 A1 Jan. 24, 2008

appears on one side of the head, 30-40% of migraines occur delivery routes and/or forms of administration known in the on both sides. A migraine attack typically lasts about 4 to 72 art. The inventive pharmaceutical compositions may be hours. Migraine symptoms may also include speech diffi prepared in liquid form, or may be in dried powder form, culty, nausea, vomiting, confusion, weakness of an arm or such as lyophilized form. For oral or enteral use, the leg and tingling of face or hands. pharmaceutical compositions can be configured, for example, as tablets, troches, lozenges, aqueous or oily 0283 The basic difference between common and classic Suspensions, dispersible powders or granules, emulsions, types of migraine is the appearance of an "aura.” The aura is the occurrence of neurological symptoms 10-30 minutes hard or Soft capsules, syrups, elixirs or enteral formulas. before the classic migraine attack. During migraine aura, the 0289. In general, the invention encompasses pharmaceu migraineur may see flashing or shimmering lights, ZigZag. tical compositions comprising therapeutically effective lines, geometric shapes, or may temporarily lose vision (e.g., amounts of the vehicle-conjugated or unconjugated CGRP hemianopsia), or experience blind spots called Scotomas, peptide antagonist of the invention (in amounts effective to experience speech disturbances, or experience other sensory prevent, alleviate, mitigate, ameliorate, or abolish chronic phenomena, such as gustatory and/or olfactory hallucina pain, e.g., migraine, or any of the other medical causes, tions. Other symptoms of migraine aura may include numb disorders, or conditions provided herein) together with one ness, tingling, speech difficulties and muscle weakness on or more pharmaceutically acceptable carrier(s). In the prac one side of the body. tice of this invention the “pharmaceutically acceptable car rier is any physiologically tolerated Substance known to 0284 Another type of migraine is basilar migraine, those of ordinary skill in the art useful in formulating which is accompanied by transient brainstem signs thought pharmaceutical compositions, including, any pharmaceuti to be due to vasospastic narrowing of the basilar artery. In cally acceptable diluents, excipients, dispersants, binders, basilar-type migraine, the migraine Sufferer meets the gen fillers, glidants, anti-frictional agents, compression aids, eral criteria for migraine with aura and has two or more of tablet-disintegrating agents (disintegrants), Suspending the following symptoms: dysarthria, Vertigo, tinnitis, hypa agents, lubricants, flavorants, odorants, Sweeteners, perme cusia, double vision (diplopia), bilateral visual symptoms, ation or penetration enhancers, preservatives, Surfactants, ataxia, perioral numbness, decreased level of consciousness, solubilizers, emulsifiers, thickeners, adjuvants, dyes, coat and/or simultaneously bilateral paraesthesias. ings, encapsulating material(s), and/or other additives singly 0285) The above-described symptoms of migraine are or in combination. merely illustrative and are not intended to be an exhaustive 0290 For example, binders can be used to hold the description of all possible migraine symptoms experienced components of the pharmaceutical composition together to by a single patient or by several migraine Sufferers in form a hard tablet, and they include materials from natural composite, and to which the present invention is directed. products Such as acacia, tragacanth, starch and gelatin. Those skilled in the art are aware of various other migraine Others include methyl cellulose (MC), ethyl cellulose (EC) symptoms and constellations of migraine symptoms Suffered and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone by individual patients, and to those migraine symptoms are (PVP) and hydroxypropylmethyl cellulose (HPMC) could also directed the present inventive methods of treating both be used in alcoholic solutions to granulate the thera migraine, or preventing or mitigating migraine. peutic. 0286 The therapeutically effective amount, prophylacti 0291 Disintegrants can be included in the formulation of cally effective amount, and dosage regimen involved in the the pharmaceutical composition into a solid dosage form. inventive methods of treating chronic pain or migraine, or of Materials used as disintegrants include, but are not limited preventing or mitigating migraine, will be determined by the to, starch, including the commercially available disintegrant attending physician, considering various factors which based on Starch, Explotab. Sodium starch glycolate, Amber modify the action of therapeutic agents, such as the age, lite, sodium carboxymethylcellulose, ultramylopectin, condition, body weight, sex and diet of the patient, the Sodium alginate, gelatin, orange peel, acid carboxymethyl severity of the condition being treated, time of administra cellulose, natural sponge and bentonite can also be used. tion, and other clinical factors. Generally, the daily amount Another useful form of the disintegrants are the insoluble or regimen should be in the range of about 1 to about 10,000 cationic exchange resins. Powdered gums may be used as micrograms (ug) of the CGRP peptide per kilogram (kg) of disintegrants and as binders, and these can include powdered body mass, preferably about 1 to about 5000 ug per kilogram gums such as agar, Karaya or tragacanth. Alginic acid and its of body mass, and most preferably about 1 to about 1000 ug Sodium salt are also useful as disintegrants. per kilogram of body mass. 0292 An anti-frictional agent can be included in the formulation of Some embodiments of the pharmaceutical 0287. Accordingly, the present invention also relates to composition to prevent sticking during the formulating the use of one or more of the inventive compositions of process. matter in the manufacture of a medicament for the treatment or prevention of migraine. 0293 Lubricants can be used as a layer between the pharmaceutical composition and the die wall in preparation 0288 Such pharmaceutical compositions can be config of a molded formulation (e.g., a tablet or caplet), and these ured for administration to a patient by a wide variety of can include, but are not limited to: Stearic acid, including its delivery routes, e.g., an intravascular delivery route such as magnesium and calcium salts, polytetrafluoroethylene by injection or infusion, Subcutaneous, intramuscular, intra (PTFE), liquid paraffin, vegetable oils and waxes. Soluble peritoneal, epidural, or intrathecal delivery routes, or for lubricants can also be used, such as Sodium lauryl Sulfate, oral, enteral, pulmonary (e.g., inhalant), intranasal, transmu magnesium lauryl Sulfate, polyethylene glycol of various cosal (e.g., Sublingual administration), transdermal or other molecular weights, Carbowax 4000 and 6000. US 2008/0020978 A1 Jan. 24, 2008 52

0294 Glidants can be included to improve the flow with ethylene oxide) and the like. For enteral administration, properties of the vehicle-conjugated or unconjugated CGRP the carrier can also include an oily ester Such as ethyl oleate peptide antagonists and/or pharmaceutical compositions and isopropyl myristate. during formulation and to aid rearrangement during com 0299 The pharmaceutically acceptable carrier for pres pression. Useful glidants include starch, talc, pyrogenic Surized compositions, such as a composition configured as silica and hydrated silicoaluminate. an inhalant, can be a halogenated hydrocarbon or other pharmaceutically acceptable aerosol propellant. (See, e.g., 0295) To aid dissolution of the compositions of matter Bäckström et al., Aerosol drug formulations containing and/or pharmaceutical compositions of the present invention hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. into an aqueous environment, a surfactant can be included as a wetting agent. Surfactants may include anionic detergents 6,932,962). Such as sodium lauryl Sulfate, dioctyl sodium sulfo Succinate 0300. In another embodiment, the pharmaceutically and dioctyl sodium sulfonate. Cationic detergents might be acceptable carrier is a Solid and the pharmaceutical compo used and could include benzalkonium chloride or benzetho sition is in the form of a powder, granules, microparticles, microspheres, or tablet. The vehicle-conjugated or uncon nium chloride. The list of potential nonionic detergents that jugated CGRP peptide antagonists can be incorporated into could be included in the formulation as surfactants are particulate or microparticle preparations of polymeric com lauromacrogol 400, polyoxyl 40 Stearate, polyoxyethylene pounds such as polylactic acid, polyglycolic acid, etc. or into hydrogenated castor oil 10, 50 and/or 60, glycerol liposomes. Hyaluronic acid may also be used, and this may monostearate, polysorbate 40, 60, 65 and/or 80, sucrose fatty have the effect of promoting Sustained duration in the acid ester, methyl cellulose and/or carboxymethyl cellulose. circulation. Such compositions may influence the physical These surfactants can be present in the formulation of the state, stability, rate of in vivo release, and rate of in vivo vehicle-conjugated or unconjugated CGRP peptide antago clearance of the CGRP peptide antagonists. (See, e.g., nist-containing pharmaceutical compositions, either indi Remington’s Pharmaceutical Sciences, 18th Edition, Mack vidually or as a mixture of surfactants in different ratios. Publishing Co., Easton, Pa., 1435-1712 (1990), which is 0296. Some other additives can also be included in the herein incorporated by reference in its entirety.) A solid formulation of the pharmaceutical composition to enhance carrier can also include one or more Substances that can act uptake of the vehicle-conjugated or unconjugated CGRP as flavoring agents, lubricants, solubilizers, or Suspending peptide antagonists. Additives potentially having this prop agents. erty are for instance the fatty acids oleic acid, linoleic acid 0301 Colorants, odorants, and/or flavoring agents (fla and linolenic acid. Vorants) can also be included as carriers in the pharmaceu 0297. In one embodiment the pharmaceutically accept tical composition formulated (such as by liposome or micro able carrier can be a liquid and the pharmaceutical compo sphere encapsulation) to be further contained within an sition is prepared in the form of a solution, Suspension, edible product, such as a refrigerated beverage containing emulsion, syrup, elixir or pressurized composition. The colorants, odorants and/or flavoring agents. active ingredient(s) (e.g., the inventive composition of mat 0302) In powder forms, the pharmaceutically acceptable ter) can be dissolved, diluted or Suspended in a pharmaceu carrier is a finely divided solid, which is in admixture with tically acceptable liquid carrier Such as water, an organic finely divided active ingredient(s), including the inventive Solvent, a mixture of both, or pharmaceutically acceptable vehicle-conjugated or unconjugated CGRP peptide antago oils or fats. The liquid carrier can contain other suitable nist. For example, in Some embodiments, a powder form is pharmaceutical additives such as detergents and/or solubi useful when the pharmaceutical composition is configured lizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers as an inhalant. (See, e.g., Zeng et al., Method of preparing at appropriate pH (e.g., Tris-HCl, acetate, phosphate), adju dry powder inhalation compositions, WO 2004/017918; vants, anti-oxidants (e.g., ascorbic acid, sodium met Trunk et al., Salts of the CGRP antagonist BIBN4096 and abisulfite), preservatives (e.g., Thimersol, benzyl alcohol), inhalable powdered medicaments containing them, U.S. Pat. Sweeteners, flavoring agents, Suspending agents, thickening No. 6,900,317). agents, bulking Substances (e.g., lactose, mannitol), colors, Viscosity regulators, stabilizers, electrolytes, osmolutes or 0303. In tablet form, the active ingredient(s) are mixed osmo-regulators. with a pharmaceutically acceptable carrier having the nec essary compression properties in Suitable proportions and 0298 Suitable examples of liquid pharmaceutically compacted in the shape and size desired. acceptable carriers for oral and enteral administration of the 0304. The powders and tablets preferably contain up to pharmaceutical composition include water (partially con 99% of the active ingredient(s). Suitable solid carriers taining additives as above, e.g. cellulose derivatives, pref include, for example, calcium phosphate, magnesium Stear erably sodium carboxymethyl cellulose solution), alcohols ate, talc, Sugars, lactose, dextrin, starch, gelatin, cellulose, (including monohydric alcohols and polyhydric alcohols, polyvinylpyrrolidine, low melting waxes and ion exchange e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). The inventive pharmaceutical S1S. compositions can be administered to a patient orally or 0305 One can dilute the vehicle-conjugated unconju enterally in the form of a sterile solution or Suspension gated CGRP peptide antagonists or increase the volume of containing other Solutes or Suspending agents (for example, the pharmaceutical compositions of the invention with an enough saline or glucose to make the solution isotonic), bile inert material. Such diluents can include carbohydrates, salts, acacia, gelatin, Sorbitan monoleate, polysorbate 80 especially, mannitol, C.-lactose, anhydrous lactose, cellulose, (oleate esters of sorbitol and its anhydrides copolymerized Sucrose, modified dextrans and starch. Certain inorganic US 2008/0020978 A1 Jan. 24, 2008 salts may also be used as fillers, including calcium triphos useful for practicing some embodiments of the present phate, magnesium carbonate and Sodium chloride. Some pharmaceutical compositions. (E.g., Chien et al., Transder commercially available diluents are Fast-Flo, Emdex, STA mal /progestin dosage unit, system and process, RX 1500, Emcompress and Avicell. U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., 0306 A variety of conventional thickeners are useful in Diffusion matrix for transdermal drug administration and creams, ointments, Suppository and gel configurations of the transdermal drug delivery devices including same, U.S. Pat. pharmaceutical composition, such as, but not limited to, No. 4,911,916: Teillaud et al., EVA-based transdermal alginate, Xanthan gum, or petrolatum, may also be employed matrix system for the administration of an estrogen and/or a in Such configurations of the pharmaceutical composition of , U.S. Pat. No. 5,605,702; Venkateshwaran et the present invention. A permeation or penetration enhancer, al., Transdermal drug delivery matrix for coadministering Such as polyethylene glycol monolaurate, dimethyl sulfox and another steroid, U.S. Pat. No. 5,783,208: Ebert ide, N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrroli et al., Methods for providing testosterone and optionally done, or 3-hydroxy-N-methyl-2-pyrrolidone can also be estrogen replacement therapy to women, U.S. Pat. No. employed. Useful techniques for producing hydrogel matri 5,460,820). ces are known. (E.g., Feijen, Biodegradable hydrogel matri 0311) A variety of pharmaceutically acceptable systems ces for the controlled release of pharmacologically active for transmucosal delivery of therapeutic agents are also agents, U.S. Pat. No. 4,925,677; Shah et al., Biodegradable known in the art and are compatible with the practice of the pH/thermosensitive hydrogels for sustained delivery of bio present invention. (E.g., Heiber et al., Transmucosal delivery logically active agents, WO 00/38651 A1). Such biodegrad of macromolecular drugs, U.S. Pat. Nos. 5.346,701 and able gel matrices can be formed, for example, by crosslink 5,516.523; Longenecker et al., Transmembrane formulations ing a proteinaceous component and a polysaccharide or for drug administration, U.S. Pat. No. 4,994.439). Transmu mucopolysaccharide component, then loading with the cosal delivery devices may be in free form, Such as a cream, inventive composition of matter to be delivered. gel, or ointment, or may comprise a determinate form Such 0307 Liquid pharmaceutical compositions of the present as a tablet, patch or troche. For example, delivery of the invention that are sterile solutions or Suspensions can be inventive CGRP peptide antagonist can be via a transmu administered to a patient by injection, for example, intra cosal delivery system comprising a laminated composite of muscularly, intrathecally, epidurally, intravascularly (e.g., for example, an adhesive layer, a backing layer, a permeable intravenously or intraarterially), intraperitoneally or Subcu membrane defining a reservoir containing the vehicle-con taneously. (See, e.g., Goldenberg et al., Suspensions for the jugated or unconjugated CGRP peptide antagonist, a peel sustained release of proteins, U.S. Pat. No. 6,245,740 and seal disc underlying the membrane, one or more heat seals, WO 00/38652 A1). Sterile solutions can also be adminis and a removable release liner. (E.g., Ebert et al., Transder tered by intravenous infusion. The vehicle-conjugated or mal delivery system with adhesive overlay and peel seal unconjugated CGRP peptide antagonist can be included in a disc, U.S. Pat. No. 5,662.925; Chang et al., Device for sterile Solid pharmaceutical composition, such as a lyo administering an active agent to the skin or mucosa, U.S. philized powder, which can be dissolved or suspended at a Pat. Nos. 4,849.224 and 4,983,395). convenient time before administration to a patient using 0312 Alternatively, among known tablet or patch sys sterile water, saline, buffered saline or other appropriate tems configured for therapeutic delivery through the oral sterile injectable medium. mucosa (e.g., Sublingual mucosa). Some embodiments can 0308 Implantable sustained release formulations are also comprise an inner layer containing the vehicle-conjugated or contemplated embodiments of the inventive pharmaceutical unconjugated CGRP peptide antagonist, a permeation compositions. For example, the pharmaceutically acceptable enhancer. Such as a bile salt or fusidate, and a hydrophilic carrier, being a biodegradable matrix implanted within the polymer, such as hydroxypropyl cellulose, hydroxypropyl body or under the skin of a human or non-human vertebrate, methylcellulose, hydroxyethyl cellulose, dextran, pectin, can be a hydrogel similar to those described above. Alter polyvinyl pyrrolidone, starch, gelatin, or any number of natively, it may be formed from a poly-alpha-amino acid other polymers known to be useful for this purpose. This component. (Sidman, Biodegradable, implantable drug inner layer can have one surface adapted to contact and adhere to the moist mucosal tissue of the oral cavity and can delivery device, and process for preparing and using same, have an opposing Surface adhering to an overlying non U.S. Pat. No. 4.351,337). Other techniques for making adhesive inert layer. Optionally, such a transmucosal deliv implants for delivery of drugs are also known and useful in ery system can be in the form of a bilayer tablet, in which accordance with the present invention. the inner layer also contains additional binding agents, 0309 Additionally (or alternatively), the present inven flavoring agents, or fillers. Some useful systems employ a tion provides pharmaceutical compositions for use in any of non-ionic detergent along with a permeation enhancer. the various slow or Sustained release formulations or micro These examples are merely illustrative of available trans particle formulations known to the skilled artisan, for mucosal therapeutic delivery technology and are not limiting example, Sustained release microparticle formulations, of the present invention. which can be administered via pulmonary, intranasal, or 0313. In other embodiments, the inventive composition Subcutaneous delivery routes. of matter and/or pharmaceutical composition can be admin 0310. In a further embodiment, the inventive pharmaceu istered to a patient orally. Compositions suitable for oral tical composition is configured as a part of a pharmaceuti administration include Solid forms, such as tablets, pills, cally acceptable transdermal or transmucosal patch or a capsules, caplets, granules, tablets, troches, lozenges, troche. Transdermal patch drug delivery systems, for cachets, pellets and powders, and liquid forms, such as example, matrix type transdermal patches, are known and Solutions, syrups, elixirs, and Suspensions. Forms useful for US 2008/0020978 A1 Jan. 24, 2008 54 enteral administration include sterile solutions, emulsions, U.S. Pat. Nos. 4,193.985; and 4,690,822; 4,572,833 can be and Suspensions. Examples of oral Solid dosage forms are used in the formulation of the inventive pharmaceutical further described generally in Chapter 89 of Remington's compositions. Another form of a controlled release of the Pharmaceutical Sciences, 18th ed., Mack Publishing Co., vehicle-conjugated or unconjugated CGRP peptide antago Easton Pa. 18042, (1990). nists and/or pharmaceutical compositions of this invention is 0314. Also, liposomal or proteinoid encapsulation may by a method based on the Oros therapeutic system (Alza be used to formulate the inventive pharmaceutical compo Corp.), i.e., the inventive CGRP peptide antagonists and/or sitions for oral administration (as, for example, proteinoid pharmaceutical compositions are enclosed in a semi-perme microspheres reported in U.S. Pat. No. 4,925,673; see also able membrane which allows water to enter and push drug Giovagnoli et al., Biodegradable microspheres as carriers for out through a single Small opening due to osmotic effects. native superoxide dismutase and catalase delivery, AAPS Some enteric coatings also have a delayed release effect. PharmSciTech, 5(4), Article 51, (% vww.aapspharm scitech.org) (2004)). Liposomal encapsulation can include 0318. Other coatings may be used for the formulation for derivatization with various polymers. (e.g., U.S. Pat. No. oral administration. These include a variety of Sugars which 5,013,556). A further description of possible solid dosage can be applied in a coating pan. The therapeutic agent could forms suitable for the pharmaceutical composition is given also be given in a film-coated tablet and the materials used in Chapter 10 of Marshall, K., Modern Pharmaceutics, G. S. in this instance are divided into 2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl Banker and C. T. Rhodes, eds. (1979). cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cel 0315) If necessary, the compositions containing the lulose, hydroxypropyl cellulose, hydroxypropyl-methyl cel CGRP peptide antagonists of the invention may be chemi lulose, sodium carboxy-methyl cellulose, providone and the cally modified so that oral delivery is efficacious. Generally, polyethylene glycols. The second group consists of the the chemical modification contemplated is the attachment of enteric materials that are commonly esters of phthalic acid. at least one moiety to the compound molecule itself, wherein A mix of materials might be used to provide the optimum the moiety permits (a) inhibition of proteolysis or acid film coating. Film coating may be carried out in a pan coater hydrolysis in the gastric environment; and (b) uptake into or in a fluidized bed or by compression coating. the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the vehicle-conjugated 0319 Pulmonary administration to a patient of the or unconjugated CGRP peptide antagonists and increase in vehicle-conjugated or unconjugated CGRP peptide antago circulation time in the body. Accordingly, moieties useful as nists and/or pharmaceutical compositions is also useful, in conjugated vehicles in this invention may also be used for accordance with the present invention. The CGRP peptide this purpose. Examples of such moieties include: PEG, antagonists are delivered to the lungs of a patient by inha copolymers of ethylene glycol and propylene glycol, car lation and traverse across the lung epithelial lining to the boxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl blood stream. (See, also, Adjei et al., Pharma. Res., 7:565 pyrrolidone and polyproline. See, for example, Abuchowski 569 (1990); Adjei et al., Intl. J. Pharmaceutics, 63:135-44 and Davis, Soluble Polymer-Enzyme Adducts, Enzymes as (1990), leuprolide acetate; Braquet et al., J. Cardio. Phar Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, macol. 13(5):143-146 (1989), endothelin-1; Hubbard et al., New York, N.Y., 367-383 (1981); Newmark, et al., J. Appl. Annals Int. Med., 3:206-212 (1989), C.1-antitrypsin; Smith Biochem., 4:185-189 (1982). Other polymers that could be et al., J. Clin. Invest. 84:1145-1146 (1989), C.1-proteinase; used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Pre Oswein et al., “Aerosolization of Proteins’. Proc. Symp. ferred for pharmaceutical usage, as indicated above, are Resp. Drug Delivery II, Keystone, Colo., (March 1990), PEG moieties. It is also possible to use a salt of a modified recombinant human ; Debs et al., J. Immu aliphatic amino acid, such as sodium N-(8-2-hydroxyben nol. 140:3482-3488 (1988), interferon-Y and tumor necrosis Zoylamino) caprylate (SNAC), as a carrier to enhance factor C.; and Platz et al., U.S. Pat. No. 5,284.656 (granu absorption of the CGRP peptide antagonists of the invention. locyte colony stimulating factor)). (See, e.g., U.S. Pat. No. 5,792.451, entitled “Oral Drug Delivery Composition and Methods’. 0320 Useful in the practice of the present invention are a wide range of mechanical inhalant dispensing devices (i.e., 0316 The compositions containing the CGRP peptide inhaler devices) designed for pulmonary administration of antagonists of the invention can be included in the formu therapeutic products, including but not limited to nebulizers, lation of the pharmaceutical composition for oral adminis metered dose inhalers, and powder inhalers, all of which are tration as fine multi-particulates in the form of granules or familiar to those skilled in the art. Some specific examples pellets. The formulation of the material for capsule admin of commercially available devices suitable for the practice istration can also be as a powder, as lightly compressed of this invention are the Ultravent nebulizer, manufactured plugs, or even as tablets. by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn 11 nebu lizer, manufactured by Marquest Medical Products, Engle 0317 Controlled release oral formulations of the phar wood, Colo.; the Ventolin metered dose inhaler, manufac maceutical composition may be desirable. The compositions tured by Glaxo Inc., Research Triangle Park, N.C.; and the of matter and/or pharmaceutical compositions of the inven Spinhaler powder inhaler, manufactured by Fisons Corp., tion can be incorporated into an inert matrix which permits Bedford, Mass. (See, e.g., Helgesson et al., Inhalation release by either diffusion or leaching mechanisms e.g., device, U.S. Pat. No. 6,892,728; McDerment et al., Dry gums. Slowly degenerating matrices may also be incorpo powder inhaler, WO 02/11801 A1: Ohki et al., Inhalant rated into the formulation, e.g., alginates, polysaccharides. For example, the pharmaceutical compositions can be indicator, U.S. Pat. No. 6,273,086). coated by the techniques described in U.S. Pat. Nos. 4,256, 0321 All such inhalant dispensing devices require the 108; 4,160,452; and 4.265,874, to form osmotic therapeutic use of formulations suitable for the dispensing of the inven tablets for controlled release. Other techniques for con tive composition of matter. Typically, each formulation is trolled release compositions, such as those described in the specific to the type of inhalant dispensing device employed US 2008/0020978 A1 Jan. 24, 2008

and may involve the use of an appropriate aerosol propellant referents unless the context clearly dictates otherwise. Thus, material, in addition to diluents, adjuvants and/or carriers for example, reference to “a vehicle-conjugated CGRP pep useful in therapy. tide antagonist' or “a vehicle-conjugated CGRP peptide 0322 For pulmonary administration of such inhalant antagonist' includes mixtures of Such conjugates and refer formulations of the pharmaceutical composition, the inven ence to “the method of treatment” includes reference to one tive pharmaceutical composition should most advanta or more methods of treatment of the type which will be geously be prepared in microparticle form with an average known to those skilled in the art or will become known to aerodynamic diameter of about 0.5 to 5 um, for most them upon reading this specification, and so forth. effective delivery to the distal lung. 0329. The invention will be described in greater detail by 0323 Pharmaceutically acceptable carriers suitable for reference to the following examples. These examples are not Such inhalant formulations include carbohydrates such as to be construed in any way as limiting the scope of the trehalose, mannitol. Xylitol. Sucrose, lactose, and Sorbitol. present invention. Other ingredients for use in formulations may include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants may be used. PEG may be used (even apart from EXAMPLES its use as a vehicle to be conjugated to the CGRP peptide antagonist). Dextrans, such as cyclodextran, may be used. Example 1 Bile salts and other related enhancers may be used. Cellulose and cellulose derivatives may be used. Amino acids may be 0330 CGRP peptide synthesis CGRP binding and used. Such as use in a buffer formulation. Also, the use of antagonist activity. A series of CGRP peptide analogs were liposomes, microcapsules or microspheres, inclusion com synthesized by conventional Solid phase peptide synthetic plexes, or other types of carriers is contemplated for some techniques for the purpose of introducing site-selective inhalant formulations. conjugation sites for activated vehicle. 0324 Formulations suitable for use with a nebulizer, 0331. Several conjugation strategies were explored either jet or ultrasonic, will typically comprise the inventive including thiol-mediated alkylation as well as reductive composition of matter dissolved in water. The formulation alkylation and acylation. Also site-selective conjugation may also include a buffer and a simple Sugar (e.g., for sites were engineered at the CGRP peptide N-terminus or peptide stabilization and regulation of osmotic pressure). within the hinge region. The nebulizer formulation may also contain a surfactant, to reduce or prevent Surface induced aggregation of the protein 0332 CGRP peptide synthesis. The following protocol caused by atomization of the Solution in forming the aerosol. was used to generate CGRP analogs as described herein. 0325 Formulations for use with a metered-dose inhaler N'-Fmoc, side-chain protected amino acids and Rink amide device will generally comprise a finely divided powder resin were used. Representative side-chain protection strat containing the inventive compound Suspended in a propel egies were: Asp(OtBu), Arg(Pbf), Cys(Acin), GlucOtBu), lant with the aid of a surfactant. The propellant may be any Glu(O2-PhiPr). His(Trt), Lys(N-Boc), Lys(N-Mtt), conventional material employed for this purpose. Such as a Ser(OtBu), Thr(OtBu), Trp(Boc) and Tyr(OtBu). CGRP chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrof peptide derivatives were synthesized in a stepwise manner luorocarbon, or a hydrocarbon, including trichlorofluo on an ABI433 peptide synthesizer by SPPS using O-Ben romethane, dichlorodifluoromethane, dichlorotetrafluoroet Zotriazole-N,N,N',N'-tetramethyl-uronium-hexafluoro hanol, or 1,1,1,2-tetrafluoroethane, or combinations thereof. phosphate (HBTU)/N,N-diisopropylethylamine (DIEA)/N, Suitable surfactants include sorbitan trioleate and soya leci N-dimethylformamide (DMF) coupling chemistry at 0.2 thin. Oleic acid may also be useful as a Surfactant. (See, e.g., mmol equivalent resin Scale (Fmoc-deprotected Rink amide Backstrom et al., Aerosol drug formulations containing resin). For each coupling cycle, 1 mmol N-Fmoc-amino hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. acid, 4 mmol DIEA and 1 mmol equivalents of HBTU were 6,932,962). used. The concentration of the HBTU-activated Fmoc amino acids was 0.5M in DMF, and the coupling time was 45 min. 0326 Formulations for dispensing from a powder inhaler Fmoc deprotections were carried out with two treatments device will comprise a finely divided dry powder containing using a 30% piperidine in DMF solution first for 2 min and the inventive compound and may also include a bulking then for an additional 20 min. agent, such as lactose, Sorbitol. Sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder 0333 Lactam Formation. In some embodiments of the from the device, e.g., 50 to 90% by weight of the formula CGRP peptides, side-chain to side-chain lactam formation tion. (See, e.g., McDerment et al., Dry powder inhaler, WO was carried out on the assembled N-terminally Fmoc-pro 02/11801 A1). tected peptide resin. The peptide-resin was solvated in DCM 0327 In accordance with the present invention, intranasal for 30 mins, and drained. The Mtt and 2-PhilPr groups delivery of the inventive composition of matter and/or (protecting group at the specified lactam bond forming sites) pharmaceutical compositions is also useful, which allows were removed with 1% TFA in DCM solution containing 5% passage thereof to the blood stream directly after adminis TIS. Treatment of the peptide-resin with the 1% TFA in tration to the inside of the nose, without the necessity for DCM solution was repeated 8 times in 30 min increments, deposition of the product in the lung. Formulations suitable and each treatment was followed by extensive DCM washes. for intranasal administration include those with dextran or The liberate carboxyl and amino groups were then con densed by the addition of 5 equiv of 0.5M benzotriazole-1- cyclodextran, and intranasal delivery devices are known. yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (See, e.g., Freezer, Inhaler, U.S. Pat. No. 4,083,368). (PyBOP) and 10 equiv of DIEA in DMF were added to the 0328. As used in the specification and the appended peptide resin, and left for 24 h. The resin was then wash claims, the singular forms “a”, “an', and “the include plural thoroughly with DMF, DCM, and DCM/MeOH, and dried. US 2008/0020978 A1 Jan. 24, 2008 56

0334 Side Chain Deprotection and Cleavage from Resin. 0336 CGRP binding experiments. CGRP binding Following synthesis and modification, the resin was then assays were set up in 96-well plates at room temperature drained, and washed with DCM, DMF, DCM, and then dried containing: 110 ul binding buffer (20 mM Tris-HCl, pH7.5, in vacuo. The peptide-resin was deprotected and released 5.0 mM MgSO, 0.2% BSA Sigma), 1 tablet of Com from the resin by treatment with a trifluoroacetic acid (TFA)/1.2-ethanedithiol (EDT)/triisopropyl-silane (TIS)/ pleteTM/50 ml buffer protease inhibitor); 20 ul test com H2O (92.5:2.5:2.5:2.5 V/v) solution at room temperature for pound (10x); 20 ul I-ho.CGRP (Amersham Biosciences) 90 min. The volatiles were then removed with a stream of (10x); and 50 ul human neuroblastoma cell (SK-N-MC) nitrogen gas, the crude peptide precipitated twice with membrane suspension (10 ug per well, PerkinElmer). The diethyl ether and collected by centrifugation. plates were incubated at room temperature for 2 hour with 0335) Reversed-Phase HPLC Purification. Reversed shaking at 60 rpm, then the contents of each well were phase high-performance liquid chromatography was per filtered over 0.5% polyethyleneimine (PEI)-treated (at least formed on an analytical (C18, 5 lum, 0.46 cmx25 cm) or a one hour) GF/C 96-well filter plates. The GF/C filter plates preparative (C18, 10 um, 2.2 cmx25 cm) column. Chro were washed 6 times with ice-cold 50 mM Tris, pH 7.5 and matographic separations were achieved using linear gradi dried in an oven at 55C for 1 hour. The bottoms of the GF/C ents of buffer B in A (A=0.1% aqueous TFA: B=90% aq. plates were then sealed, and 40 ul MicroscintTM 20 was ACN containing 0.09% TFA) typically 5-95% over 35 min added to each well, then the tops of the GF/C plates were at a flow rate of 1 mL/min for analytical analysis and 5-65% sealed with TopSealTM-A, a press-on adhesive sealing film, over 90 min at 20 mL/min for preparative separations. and the GF/C plates were counted with TopCount NXT Analytical and preparative HPLC fractions were character (Packard). The data were analyzed by using ActivityBase ized by ESMS and photodiode array (PDA) HPLC, and (IDBS) or Prizm (GraphPad) software. Results of binding selected fractions combined and lyophilized. assays are shown in Table 3A and Table 3B below.

TABLE 3A CGRP binding experiment results:

SEQ ID Ki NO Sequence (nM) 31 Ac-WVTH (Cit)LAGLLS (Cit) SGGVWRKNFVPTDVGPFAF-NH O. 4 6 791 20 K PEG (ald)-Ac-WVTHCitLAGLLSICitSGGVWRKNFVPTDVGPFAF-NH2 1.83 158 Ac-WVTHCitLAGLLSICitSGGVWRK (20 KPEGald)NFVPTDVGPFAF-NH2 2. 61 739Ac-WVTHCitLAGLLSICitSGGVWhArgKNFVPTDVGPFAF-NH2 O53 153 Ac-WVTHCitLAGLLSICitSGGVWhArgK (20 K PEGald)NFVPTDVGPFAF-NH2 1.78 171 Ac-WVTHCitLAGLLSICitSGGVWhArgK (20 KPEGald)NFVPTDVGPFAF-NH2 1.27 658 Ac-WVTHCitLAGLLSICitSGGVWhArgKNFVPTDVGOicFAF-NH2 O3 172 Ac-WVTHCitLAGLLSICitSGGVWhArgK (20 KPEGald)NFVPTDVGOicFAF-NH2 1.27 674 Ac-VWTHCitLAGLLSICitSGGVWhArgkNFVPTDWGP1 -NalAF-NH2 O. 64 173 Ac-WVTHCitLAGLLSICitSGGVWhArgk (20 KPEGald)NFVPTDWGP1-NalAF- 0.84 NH2

0337 TABLE 3B CGRP binding experiment results:

SEQ ID Ki NO CGRP Peptide Sequence (nM) 6Ac-WTHRLAGLLSRSGGVWRKNFWPTDWGPFAF-amide ... 04 229 Ac-VTHRLAGLLSRSGGVWRK P 'NFVPTDWGPFAF-amide ... O 6 230Ac-VTHRLAGLLSRSGGVVRK2 P 'NFVPTDVGPFAF-amide ... 17 231Ac-WVTHRLAGLLSRSGGVVKC (20 kPa PNFVPTDVGPFAF-amide .27 232Ac-WVTHRLAGLLSRSGGVVKC (30 kDa PNFVPTDVGPFAF-amide .27

US 2008/0020978 A1 Jan. 24, 2008 58

TABLE 3B-continued CGRP binding experiment results: SEQ ID NO CGRP Peptide Sequence (nM) >10 OO

>10 OO

1279

>10 OO

30.2

>10 OO

>10 OO

>10 OO

722 a.CDTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 O. O9

723AcDTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 0.26

724ACdTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 1.73

725 ACDtATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 3.25

726ACDTaTCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 O. 12

728ACDTAtCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 7.84

729ACDTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 O-52

73OACDTATCWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 75

731. ACDTATCWThRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2 O 67

732 ACDTATCWtHRLAGLLSRSGGWWKNNFWPTNWGSKAF-NH2

0338 CGRP antagonist activity. The un-conjugated 15 minutes at room temperature. CGRP peptide antagonists CGRP peptide analogs were screened in an in vitro CGRP were then added at varying concentrations in 20 ul buffer receptor mediated cAMP assay to determine intrinsic followed by a 5 minute incubation at 37° C. The CGRP potency prior to vehicle conjugation. agonist, C.CGRP was then added in 20 Jul buffer to each well 0339) Table 3 (herein below) shows the results of such in followed by a 5 minute incubation at 37°C. The medium vitro cAMP-based assays comparing native human was then removed and the cells were lysed with 100 ul lysis ccCGRP(8-37) peptide antagonist (Table 3, SEQ ID NO:1) buffer for 30 minutes at 37° C. for 30 minutes. Using a with a variety of other analogs designed to enhance potency Tropix ELISA kit (Applied Biosystems, Foster City, Calif.), at the CGRP receptor, permit site-selective conjugation a cAMP-AP conjugate was diluted (1:100) and added (30 ul) and/or facilitate handling in vitro. The in vitro cAMP assay to each well of a 96-well ELISA plate. An aliquot of the lysis employed a human neuroblastoma-derived cell line (SK-N- buffer from the cell plate was then added to the ELISA plate MC). SK-N-MC cells express CRLR and RAMP1, which followed by an aliquot of the cAMP antibody. This mixture form CGRP receptor. CGRP receptor is a G-protein was incubated for 1 hour at room temperature with gentle coupled receptor linked to the stimulatory G-protein, G. shaking. The ELISA plate was then washed 6x with wash Upon CGRP binding to the receptor, cAMP levels increase, buffer followed by the additions of a substrate enhancer. mediated by G, activation of adenylyl cyclase and cAMP This mixture was incubated for 30 minutes at room tem dependent protein kinase (PKA). An ELISA assay was perature and then read on a MicroBeta Jet or EnVision plate developed to measure the level of cAMP accumulated in reader. The results were analyzed using Prism (GraphPad). treated SK-N-MC cells as a screening system for the iden 0340 Initial conjugation strategies included engineering tification of CGRPantagonists. Briefly, two days before the an N-terminal cysteine with a hepta-glycine linker to native assay, the cells were plated in 96-well plates in growth CGRP(8-37) antagonist for thiol mediated coupling at the medium. On the day of the assay cells were rinsed with PBS N-terminus. This addition to the N-terminus of the CGRP followed by the addition of 60 ul of plasma free medium for peptide proved deleterious to potency at the CGRP receptor US 2008/0020978 A1 Jan. 24, 2008 59

(Table 3, SEQ ID NO:2 compared to SEQ ID NO:1). Unexpectedly, we discovered that addition of three amino TABLE 3 acid substitutions (T30D, V32P and G33F), used to restore In vitro CGRP receptor-mediated cAMP assays activity in the truncated CGRP peptide antagonist, not only (n = 2-4) comparing native human CCGRP (8-37) restored activity to the N-terminal conjugation analog (Table peptide antagonist (SEQ ID NO: 1) with a 3, SEQ ID NO:3), but improved potency more than 6-fold variety of CGRP peptide analogs. Modifications relative to the native CGRP(8-37) peptide antagonist (Table to the native human CCGRP sequence within each 3, SEQID NO:1). This result was unexpected since the three analog are shown in boldface: amino acid substitutions were described as necessary for SEQ activation of the truncated CGRP(28-37), in which context ID ICso they were proposed to compensate for the missing CGRP NO: Sequence (nM) receptor binding region at CGRP(8-18). (Rist et al. From 1 WTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH 4.94 Micromolar to Nanomolar Affinity: A systematic approach 2 CGGGGGGGWTHRLAGLLSRSGGWWKNNFWPTNWGSKAF- 21. 11 to identify the binding site of CGRP at the human calcitonin NH gene-related peptide 1 receptor, J. Med. Chem., 41:117-123 (1998)). Thus the introduction of three amino acid substi 3 CGGGGGGGWTHRLAGLLSRSGGWWKNNFWPTDWGPFAF- O. F2 tutions (T30D, V32P and G33F) significantly improved the NH potency of full-length CGRP(8-37) peptide antagonist ana 4 WVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH O. 64 logs. 5 Ac-WVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH 0.14 0341 The benefit of the three amino acid substitutions (T30D, V32P and G33F) was again observed when a tryp 6 Ac-VTHRLAGLLSRSGGVWRKNFVPTDVGPFAF-NH2 OTO tophan (W7, relative to the native human C.CGRP sequence) 7 Ac-WVTHRLAGLLSRSGGVVRKNFVPTDVGPFAF-NH 0.13 was added to the N-terminus of CGRP(8-37) (Table 3, SEQ ID NO.4 and SEQID NO:5), which usefully incorporated a 8 Ac-WVTHRLAGLLSRSGGVVRCNFVPTDVGPFAF-NH O5 O chromophore into the CGRP sequence to allow better in 9 Ac-WVTHRLAGLLSRSGGVVCNNFVPTDVGPFAF-NH 2. 15 process monitoring and quantitation using spectroscopic methods. Remarkably, the W7 addition alone (Table 3, SEQ 10 Ac-WVTHRLAGLLSRSGGVCRNNFVPTDVGPFAF-NH O36 ID NO:4) significantly improved the potency of the analog 11 Ac-WVTHRLAGLLSRSGGVKRNNFVPTDVGPFAF-NH 0.51 by greater than 7-fold relative to the native CCGRP(8-37) peptide sequence (Table 3, SEQID NO:1). The combination 96 WFVPTDVGPFAF-NH, 21 68 of all four modifications (Table 3, SEQ ID NO:5) achieved 97 FVPTDVGPFAF-NH, 1739 a greater than 35-fold improved antagonist activity over CCGRP(8-37). 45 CCGGGGGGPTDVGPFAF-NH 5657 0342. With the four amino acid substitutions (W7, T30D, V32P and G33F) and the N-terminus acetylated (Table 3, Example 2 SEQ ID NO:5), there remained only one primary amine in the CGRP hinge region at K24 (relative to the native human 0345 Antagonist activity by vehicle-conjugated CGRP CCGRP sequence) to serve as a reactive nucleophile for peptide antagonists. A variety of CGRP analogs were engi conjugation. Additional analogs (e.g., Table 3, SEQID NO:6 neered for vehicle conjugation through several different and SEQ ID NO:7) were designed in this context by con conjugating chemistries. Three monofunctional 20 kDa verting K24R and N25K to move over the conjugation site methoxy PEG derivatives were conjugated to the appropri one residue in the direction of the C-terminus and to restore ate CGRP peptide analogs resulting in amine, amide or the positive charge of a basic residue to position 24 (relative thioether linkages. The specific conjugation site was also to the native human CCGRP sequence). Unmodified, these analogs showed a similar antagonist activity to the analog varied to include the CGRP peptide N-terminus or amino containing a native hinge region sequence (Table 3, SEQID acid positions 23, 24 or 25 (relative to the native human NO:5). Also, comparison of analogs SEQID NO:6 and SEQ CCGRP sequence) in the CGRP peptide hinge region. ID NO:7 (in Table 3) again demonstrates the unexpected 0346 Table 4 (herein below) shows the results of in vitro beneficial effects of the N-terminal tryptophan. CGRP receptor-mediated cAMP assays comparing a vari 0343. The effect of conjugation at amino acid positions ety of PEG-conjugated CGRP peptide antagonists. The 24 or 25 (relative to the native human C.CGRP sequence) amine-linked PEG-peptides were derived from CGRP pep was also evaluated (see, e.g., Table 4) using cysteine Sub tide antagonists with their N-termini acylated and a single stitutions to introduce a reactive thiol at these positions primary amine engineered into the designated conjugation (Table 3, SEQ ID NO:8 and SEQ ID NO:9). Surprisingly, site (Table 4, SEQID NOS:5, 7 and 11). The activated PEG these analogs were somewhat less active than the equivalent derivative used was a 20 kDa methoxy PEG-aldehyde (in lysine and arginine analogs at the same positions. PEG-ald) and conjugation was achieved by reductive alky 0344 Finally, position 23 (relative to the native human lation in the presence of sodium cyanoborohydride. Briefly, CCGRP sequence) was also evaluated as a potential conju the CGRP peptide was dissolved at 2 mg/ml in an amine-free gation site using V23C, K24R (Table 3, SEQID NO:10) or buffer (20 mM sodium phosphate, pH 6), the mPEG-ald was V23K, K24R (Table 3, SEQ ID NO:11). added in stoichiometric excess (about 5-fold) and sodium US 2008/0020978 A1 Jan. 24, 2008 60 cyanoborohydride was added to a final concentration of 10 compared to the unconjugated N-terminal analog (Table 4. mM. The reaction mixture was stirred at room temperature SEQ ID NO:3). In contrast, PEGylation at an engineered for 24 to 48 hours and was monitored by reverse phase cysteine in the hinge region (Table 4, SEQ ID NO:14) had high-pressure liquid chromatography (RP-HPLC). Upon little effect on the antagonist activity of the parent peptide completion, the reaction was quenched by a 4- to 5-fold (Table 4, SEQ ID NO:9). However, since the thioether dilution into 20 mM sodium acetate buffer, pH 4. Purifica coupling in the hinge region did not result in an especially tion was achieved by preparative cation-exchange chroma potent conjugate, alternative chemistries were explored for tography, eluting with a linear 0-500 mM sodium chloride coupling in the hinge region. gradient. The eluted PEG-peptide was identified by RP HPLC and sodium dodecyl sulfate polyacrylamide gel elec 0350. Using amine (Table 4, SEQ ID NO:12) and amide trophoresis (SDS-PAGE), and was concentrated and dia (Table 4, SEQ ID NO:13) linkages to PEGylate the native lyzed into 10 mM sodium acetate, 5% sorbitol, at pH 4. lysine at position 24 also gave somewhat disappointing Purities of greater than 99% were determined for all the final binding results. Although the parent peptide (Table 4, SEQ pools by RP-HPLC. Peptide mapping and sequencing were ID NO:5) was quite potent, PEGylation through either of used to confirm conjugation of PEG at each of the intended these linkages caused a greater than 15-fold loss in antago conjugation sites. nist activity. Unexpectedly, the CGRP double substitution 0347 The amide-linked PEG-peptides were derived from K24R, N25K (Table 4, SEQID NO:7) tolerated PEGylation the same single, primary amine containing CGRP analogs by either amine or amide linkage very well, Suffering only (Table 4, analogs SEQ ID NOS:5, 7 and 11) used for the about a three-fold loss in antagonist activity (compare SEQ amine-linked conjugates. The activated PEG derivative was ID NO:7 to Table 4, SEQ ID NO:15 and SEQ ID NO:16), a 20 kDa methoxy PEG-N-hydroxysuccinimidyl ester and these conjugates were still about 12-fold more potent (mPEG-NHS), and conjugation was achieved by acylation at than the unPEGylated native sequence CGRP(8-37) peptide pH 8. Briefly, the CGRP peptide was dissolved at 2 mg/ml (Table 4, SEQID NO:1). Both of these conjugates (Table 4, in an amine-free buffer (50 mM Bicine, pH 8); the mPEG SEQ ID NO:15 and SEQ ID NO:16) enjoyed a significant NHS was added in stoichiometric excess (2-10 fold) and was potency benefit from engineering an unreactive basic residue allowed to react for 4- to 24 hours at room temperature. The (e.g., arginine) to replace the native lysine at position 24 reaction was monitored, quenched and purified by the same (relative to the native human C.CGRP sequence) and from method used for the amine-linked PEG-peptides, as moving the conjugation site lysine to position 25 (relative to described above. Although there was a substantial fraction the native human CCGRP sequence). For unknown reasons, of peptide that appeared to be di-PEGylated, it was easily the conjugate Substituting a thioether linkage (Table 4, SEQ separated in purification, and, as described above for the ID NO:17) did not benefit from the same rearrangement at amine-linked PEG-peptide, the mono-PEGylated amide positions 24 and 25 (relative to the native human CCGRP linked peptide was also purified to >99% purity as deter sequence), even though the unPEGylated peptide exhibited mined by RP-HPLC. a more than 4-fold improvement in potency (compare Table 4, SEQID NO:8 to SEQID NO:9). The conjugation site also 0348. Thioether-linked PEG-peptides were derived from could be moved to position 23 (relative to the native human CGRPantagonist analogs with reactive thiols engineered at CCGRP sequence), for example, with the double substitu the desired conjugation site (Table 4, CGRP peptide analogs tion V23C, K24R (Table 4, SEQ ID NO:10 and SEQ ID SEQ ID NOS:3, 8, 9 and 10). The activated PEG derivative NO:11). Remarkably, when PEGylated, this conjugate was a 20 kDa methoxy PEG-maleimide (mPEG-mal) and (Table 4, SEQ ID NO:18) was highly potent, exhibiting an conjugation was achieved by alkylation at pH 6. Briefly, the ICs=0.16 nM in the in vitro cAMP assay. This was nearly CGRP peptide was dissolved at 2 mg/ml in an amine-free 31-fold more potent than the nonPEGylated native sequence buffer (50 mM sodium phosphate, 5 mM EDTA, pH 6), the CGRP(8-37). mPEG-mal was added in a modest stoichiometric excess (1.2-1.5 fold) and was allowed to react 0.5- to 2 hours at 0351. The invention is not limited to the above-exempli room temperature. The reaction was monitored, quenched fied peptide sequence modifications or PEG molecules. For with 5 mM 3-mercaptoethanol, and after quenching was example, efficacious vehicle-peptide conjugates can be allowed to incubate at room temperature another 30 minutes. achieved at other positions in the hinge region or in either of Then, thioether-linked PEG-peptides were purified by the the CGRP receptor binding regions. Further, the introduc same method used for the amine-linked PEG-peptides, as tion of reactive conjugation sites does not rely solely on described above. sequence Substitutions with lysine or cysteine residues, but can include insertions between existing residues, or addition 0349 There were several unexpected results observed of different reactive amino acids. Moreover, since this when these various PEG-CGRP analogs were evaluated invention can utilize synthetic peptides, numerous chemical using the in vitro cAMP assay, as described in Example 1. strategies can be employed to achieve vehicle conjugation at First, there was a very clear preference for coupling in the preferred sites in the CGRP peptide. Finally, the vehicle CGRP hinge region compared to the N-terminus. PEGyla itself is not limited to PEG, but may include any of a broad tion through a thioether linkage at the N-terminus dramati list of biocompatible polymers, polysaccharides, proteins or cally reduced the conjugate's potency by >190-fold, when peptides, as described herein. US 2008/0020978 A1 Jan. 24, 2008 61

TABLE 4 In vitro CGRP receptor-mediated cAMP assays (n = 2-4) comparing a variety of PEG-conjugated peptide antagonists. Conjugates vary in the site of PEGylation (e.g., amino acid positions 23, 24 or 25 relative to the native human CCGRP sequence), and their respective linkage chemistries (amine amide or thioether). Modifications to the native human CCGRP (8-37) peptide antagonist sequence are shown in boldface. Site-specific PEG conjugation is indicated at the underlined residue. SEQ ID 20 k. PEG ICso NO: Linkage Sequence (nM) 1. None VTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-NH, 4.94

5 None Ac-WVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH 0.14

2 Amine Ac-WVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH, 3.05

3 Amide Ac-WVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH 2. 15

9 None Ac-WVTHRLAGLLSRSGGVVCNNFVPTDVGPFAF-NH, 2. 15 4. Thioether Act-WWT LAGLLSRSGGVVCNNFVPTDVGPFAF-NH 2.20

7 None Ac-WVTHRLAGLLSRSGGVVRKNFVPTDVGPFAF-NH, 0.13

5 Amine Ac-WVTHRLAGLLSRSGGVWRKNFVPTDVGPFAF-NH 0.42

6 Amide Ac-WVTHRLAGLLSRSGGVVRKNFVPTDVGPFAF-NH, O 39

8 None Ac-WVTHRLAGLLSRSGGVVRCNFVPTDVGPFAF-NH. O 50 7. Thioether Act-WWT LAGLLSRSGGVVRCNFVPTDVGPFAF-NH, 190

O None Ac-WWT LAGLLSRSGGVCRNNFVPTDVGPFAF-NH O36 8 Thioether Act-WWT LAGLLSRSGGVCRNNFVPTDVGPFAF-NH O. 16

1. None Ac-WVTHRLAGLLSRSGGVKRNNFVPTDVGPFAF-NH 0.51

9 Amine Ac-WVTHRLAGLLSRSGGVKRNNFVPTDVGPFAF-NH O 98

3 None CGGGGGGGVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH2, 20 Thioether cGGGGGGGVTHRLAGLLSRSGGVVKNNFVPTDVGPFAF-NH 138.

Example 3 microplate wells were coated with the capture reagent anti-C-CGRP polyclonal antibody (pAb; 0.050 ug?well incu Increased Circulating Half-Lives. In Vivo Measured bation overnight at 4° C.) raised against the C-terminal by ELISA domain of a modified CGRP peptide C-CGGGGGGGPTD 0352 Typically, injected peptides are rapidly metabo VGPFAF-NH. (SEQID NO:45; designated “cgCGRP27). lized, with circulating half-lives on the order of minutes. The wells were washed and blocked with I-Block buffer (Felix, A., Site-specific Poly(ethylene glycol)ylation of pep (Tropix. Bedford, Mass.). Samples were pretreated in tides, ACS Symposium Series 680 (Polyethylene glycol), I-Block buffer 1:10 to achieve 5% or 10% final (rodent or 218-238 (1997)). In the present example, we demonstrate human) plasma concentration; sample dilutions were carried that peptides of the invention overcome this obstacle by out in I-Block, 5% or 10% matching plasma. Diluted providing an extended pharmacokinetic profile in vivo. samples (50 ul/well) were added to anti-C-CGRP coated wells and were incubated at room temperature for 2 hours 0353. In one experiment C57 Black 6 mice (n=5 per with shaking, after which the wells were washed with timepoint) were each dosed subcutaneously with 5 mg/kg of I-Block buffer (4° C.) to remove unbound CGRP peptide. PEG-CGRP (Table 4, SEQ ID NO: 15) at t=0. Each group Horseradish peroxidase (HRP) labeled Cl-N-CGRP pab con was then sacrificed at t=4, 24, 48 and 72 hrs and the plasma jugate, specific for the N-terminal domain of CGRP was assayed by sandwich ELISA to determine CGRP pep O-CGGGGGGVTHRLAGLLSRSGGVVKNNFVPT DVG tide concentration. PFAF-NH. (SEQ ID NO:46) was diluted in I-Block buffer 0354) The ELISA system employed utilizes antibodies and added to the wells and incubated for 1 hour (at room specific for each of the respective ends of the peptide and temperature with shaking), for detection of captured CGRP accurately quantifies functional CGRP peptide. Briefly, peptide. After another washing step, Pico (Bio FX Labora US 2008/0020978 A1 Jan. 24, 2008 62 tories) Substrate was added to the wells creating a lumines cent signal proportional to the amount of CGRP peptide TABLE 6 bound by the capture reagent in the initial step. The intensity In vitro recovery of unconjugated CGRP peptide (SEQ ID NO: 7) of luminescence was measured with a luminometer. Stan compared to vehicle-conjugated CGRP peptide dard (STD) and quality control (QC) samples were made by SEQ ID NO. 15) in 5% mouse plasma. spiking cgCGRP-27 into 100% matching plasma, were Time (h loaded into wells after pretreatment and in I-Block buffer 1:10 and further dilutions in I-Block buffer, and were O 4 24 48 otherwise assayed in accordance with the ELISA method SEQ ID 112.1 + 1.8 46.1 60.1 2.509 - O.S73 1812 O.754 described above. NO: 7 SEQ ID 82.84 + 8.68 93.93 + 15.52 55.83 + 15.77 26.21 + 3.32 0355 Table 5 shows results demonstrating an increase in NO: 15 pharmacokinetic half-life achieved by Vehicle-conjugated Values are mean (%) it SD. CGRP peptide antagonists of the present invention. PEG CGRP (20 kDa PEG: CGRP peptide sequence at Table 4, SEQID NO:15) was delivered subcutaneously at 5 mg/kg to Example 5 C57 Black 6 mice (n=5), and plasma samples were taken at Substitutions in CGRP Peptide Sequence to Protect time points (t=4, 24, 48 and 72 hours). ELISA was used as from Proteolysis while Preserving Potency described herein above to detect intact CGRP peptide, which persisted in vivo for at least 72 hours. 0357 Despite the significant protection afforded CGRP by this invention from proteolytic degradation (Example 4) TABLE 5 and renal clearance in vivo (Example 3), select fragments of CGRP were still detected in these experiments. To determine In vivo stability of PEG-CGRP peptide (20 kDa PEG: CGRP peptide SEQ ID NO: 15, 5 mg/kg) in C57 Black the most labile proteolytic cleavage sites CGRP analog SEQ 6 mice (n = 5) following Subcutaneous injection. ID NO:7 (Table 3) was spiked into rat plasma at 2.5 mg/ml and incubated for 1 hour at room temperature. The samples Time (h were then diluted 10-fold with water and analyzed by LC/MS. CGRP fragments designated M1-M8 (FIG. 2) were 4 24 48 72 detected, with the major cleavages occurring after R11, L16, Conch CGRP 880 - 248 33 - 12 7 - 1 1 - O S17 and R18 (relative to the native human CCGRP peptide (ng ml) sequence). Combined with literature data (e.g., LeGreves et al., Regulatory Peptides, 25:277-286 (1989); Katayama et Concentration values are mean SD. al., Peptides, 12:563-567 (1991); Tam and Caughey, Deg radation of airway neuropeptides by human lung tryptase, Example 4 Am. J. Resp. Cell Mol. Biol. 3(1):27-32 (1990)), for pro teolytic sites of human CCGRP R11, L15, L16, S17 and R18 Increased Stability of a Vehicle-Conjugated CGRP are potential sites for major proteolytic liabilities. Unfortu nately, the R11, L16 and R18 residues have been previously Peptide Antagonist in Plasma identified as important for receptor binding and antagonist 0356 Although the remarkable increase in the circulating activity. A. C. Conner et al., Interaction of Calcitonin-Gene half-life of a PEG-CGRP peptide antagonist demonstrated in Related Peptide with its Receptors, Biochemical Society Transactions 30:451–455 (2002); Howitt et al., British Jour Example 3 is most likely due to decreased renal clearance, nal of Pharmacology, 138:325-332 (2003)). Nevertheless, it is also possible that the PEG-conjugated peptide enjoys we designed several protease resistant analogs with modi Some additional protection from proteolytic degradation. To fications at R11, L16 and/or R18 with the hope of preserving test this hypothesis, in vitro experiments were performed sufficient activity for efficacious antagonism of the CGRP comparing the stability of a PEG-CGRP peptide antagonist receptor. (SEQ ID NO:15) to its non-PEGylated parent peptide (i.e., 0358 Substitutions for arginine were selected based on SEQ ID NO:7) in 5% mouse plasma at room temperature. preservation of charge and hydrogen bonding potential. The Peptide samples at 2 mg/ml in PBS were spiked with 5% analogs were synthesized as either single or double Substi mouse plasma and analyzed by Sandwich ELISA (as tutions with homoarginine, D-arginine, citrulline, alanine, or described in Example 3 herein above) over 48 hours at room glutamine at positions 11 and/or 18 (relative to the native temperature. Peptide integrity was determined by sandwich human C.CGRP sequence). Each analog was tested before ELISA requiring intact peptide termini. Quality controls and after PEGylation for in vitro activity in the cAMP assay were made at t=0, 4, 24, and 48 hours before preparation of as described in Example 1. the standard curve and quantification; recovery should be 0359 Table 7 shows results of in vitro CGRP receptor 100% if no analyte is lost during analysis; a recovery of mediated cAMP assays comparing a variety CGRP peptide 80-90% at t=0 was observed and represents the assay analogs substituted at positions R11 and/or R18 (relative to variability. The PEG-peptide half-life was more than 9-fold the native human CCGRP sequence). Also shown are the longer than the non-PEGylated parent peptide (see, Table 6). effects of PEGylation and the linkage type on CGRP Clearly, conjugation with vehicle conferred additional sta receptor antagonist activity as measured by the cAMP assay. bility to the CGRP peptide in the presence of plasma. Modifications to the native human CCGRP(8-37) antagonist US 2008/0020978 A1 Jan. 24, 2008

sequence are shown in boldface. Site-specific PEGylation is this position (Table 7, SEQID NO:33) was not well tolerated indicated at the underlined residue. In general, non-PEGy as to in vitro antagonist activity measured by cAMP assay, lated single-substitution homoarginine analogs (Table 7. it was surprising to discover that glutamine (Table 7, SEQID SEQ ID NO:21 and SEQ ID NO:23) were well tolerated, NO:35) and D-arginine (SEQ ID NO:41) were both well retaining their CGRP receptor antagonist activity when tolerated as to antagonist activity measured by CGRP, PEGylated with 20 kDa PEG via an amine linkage (Table 7, receptor-mediated cAMP assay. Unfortunately, when PEGy SEQ ID NO:22 and SEQ ID NO:24). However, a non lated, both the single-substitution glutamine (Table 7, SEQ PEGylated double homoarginine mutant (Table 7, SEQ ID ID NO:36) and D-arginine (Table 7, SEQ ID NO:42) ana NO:25) showed about 5-fold reduced antagonist activity logs Suffered a large loss in in vitro antagonist activity, relative to either of the non-PEGylated peptides with single greater than 12-fold and greater than 32-fold, respectively. substitutions (i.e., SEQ ID NO:21 and SEQ ID NO:23). When this double substitution peptide was PEGylated (Table 0362. Noting that in the context of an amine linkage, the 7, SEQ ID NO:26), the antagonist activity returned to the PEGylated homoarginine-substituted (Table 7, SEQ ID levels of the two peptides with single substitutions. NO:26) and citrulline-substituted (Table 4, SEQID NO:32) analogs seemed to preserve greater potency, a pair of 0360 Citrulline-substituted analogs showed a pattern single—(Table 7, SEQ ID NO:37) and double-glutamine similar to homoarginine-substituted analogs, wherein the (Table 7, SEQ ID NO:39) substitution analogs were pre single substitutions at either positions II or 18 (Table 7, SEQ pared with lysine at position 25 (relative to the native human ID NO:27 and SEQ ID NO:29) were well tolerated, the CCGRP sequence) instead of cysteine. Remarkably, when double substitution (Table 7. SEQ ID NO:31) exhibiting the single-substitution glutamine analog (Table 7, SEQ ID only about 3-fold less potent CGRP receptor antagonism NO:38) was PEGylated in this context, using an amine than either single substitution. However, all the citrulline linkage, the resulting conjugate was more than 42-fold more Substituted analogs Suffered some relatively minor loss of in potent than the equivalent CGRP peptide analog that was vitro antagonist activity when PEGylated (Table 7, SEQ ID PEGylated with a thioether linkage (Table 7, SEQ ID NO:28, SEQ ID NO:30 and SEQ ID NO:32), with the NO:36). Similarly, the double-substituted glutamine analog R18Cit single-substitution analog exhibiting the best ICs of PEGylated through an amine linkage (Table 7, SEQ ID these. NO:40) also showed reasonably good in vitro potency. 0361 Initially, substitutions with natural amino acids, These results reinforce our previous observations that alanine, glutamine and D-arginine were tested only as PEGylation through an amine linkage preserves the largest single-substitutions at position R18 (relative to the native percentage of the analog's potency. Amine coupling of PEG human C.CGRP sequence) and in the context of a cysteine to glutamine-substituted analogs (Table 7, SEQ ID NO:38 PEGylation site at position 25 (relative to the native human and SEQ ID NO:40) resulted in potent vehicle-conjugated CCGRP sequence: Table 4, SEQID NO:8). While alanine at CGRP peptide antagonists.

TABLE 7 In vitro CGRP receptor-mediated cAMP assays comparing a variety of stability analogs substituted at positions R11 and/or R18 (relative to the native human CCGRP sequence). Also shown are the effects of PEGylation and the linkage type on antagonist activity. Modifications to the native human CCGRP (8-37) antagonist sequence are shown in boldface. Site-specific PEGylation is indicated at the underlined residues.

SEQ 20 kDa ID PEG ICso NO: Linkage Sequence (nM) 21 None Ac-WVTH (hR) LAGLLSRSGGVWRKNFVPTDVGPFAF-NH . 44 22 Amine Ac-WVTH (hR) LAGLLSRSGGVVRKNFVPTDVGPFAF-NH, 36 23 None Ac-WVTHRLAGLLS (hR) SGGVWRKNFVPTDVGPFAF-NH 57 24 Amine Ac-WVTHRLAGLLS (hR) SGGVVRKNFVPTDVGPFAF-NH, 33 25 None Ac-WVTH (hR) LAGLLS (hR) SGGVWRKNFVPTDVGPFAF-NH ... 1 26 Amine Ac-WVTH (hR) LAGLLS (hR) SGGVVRKNFVPTDVGPFAF-NH, 37 27 None Ac-WVTH (Cit)LAGLLSRSGGVWRKNFVPTDVGPFAF-NH 57 28 Amine Ac-WVTH (Cit)LAGLLSRSGGVVRKNFVPTDVGPFAF-NH, 08 29 None Ac-WVTHRLAGLLS (Cit) SGGVWRKNFVPTDVGPFAF-NH 58 30 Amine Ac-WVTHRLAGLLS (Cit) SGGVVRKNFVPTDVGPFAF-NH, 96 31. None Ac-WVTH (Cit)LAGLLS (Cit) SGGVWRKNFVPTDVGPFAF-NH ... 8 US 2008/0020978 A1 Jan. 24, 2008 64

TABLE 7-continued In vitro CGRP receptor-mediated cAMP as says comparing a variety of stability analogs substituted at positions R11 and/or R18 (relative to the native human CCGRP sequence). Also shown are the effects of PEGylation and the linkage type on antagonist activity. Modifications to the native human CCGRP (8-37) antagonist sequence are shown in boldface. Site-specific PEGylation is indicated at the underlined residues. SEQ 20 kDa ID PEG ICso NO: Linkage Sequence (nM)

32 Amine Ac-WVTH (Cit)LAGLLS (Cit) SGGVVRKNFVPTDVGPFAF-NH, 3. 74

33None Ac-WVTHRLAGLLSASGGVVRCNFVPTDVGPFAF-NH 2.75 34. Thioether Ac-WVTHRLAGLLSASGGVVRCNFVPTDVGPFAF-NH, 6.2

35 None Ac-WVTHRLAGLLSQSGGVVRCNFVPTDVGPFAF-NH O37 36 Thioether Ac-WVTHRLAGLLSQSGGVVRCNFVPTDVGPFAF-NH, 4 - 69

37 None Ac-WVTHRLAGLLSQSGGVWRKNFVPTDVGPFAF-NH O56

38 Amine Ac-WVTHRLAGLLSQSGGVVRKNFVPTDVGPFAF-NH, O. 11

39 None Ac-WVTHQLAGLLSQSGGVWRKNFVPTDVGPFAF-NH O53

40 Amine Ac-WVTHQLAGLLSQSGGVVRKNFVPTDVGPFAF-NH, 1.33

41 None Ac-WVTHRLAGLLSrSGGVVRCNFVPTDVGPFAF-NH. O59 42 Thioether Ac-WVTHRLAGLLSrSGGVVRCNFVPTDVGPFAF-NH, 1942

Example 6 about 10% of the starting concentration through 2.5 hrs and were 50% depleted at about 45 minutes (Table 8). All three In Vitro Stability of CGRP Peptide Antagonists in di-substituted analogs (R11 hR/R18hR SEQ ID NO:25). Mammalian Plasma R11Cit/R18CitSEQID NO:31), and R11O/R18QSEQID NO:39) were very similar in their degradation profiles, with 0363. In Example 5, several potent CGRP analogs were 10% remaining at about 5 hours and 50% at about 1.5 hours. identified with mutations designed to resist proteolysis at positions R11 and R18 (relative to the native human CCGRP 0367. In contrast, when exposed to 10% human plasma sequence). Although somewhat less active in the in vitro the control peptide (Table 9, SEQID NO:7) and the mono cAMP assay (but with ICs still less than 10 nM), these substituted (R18Q) analog (Table 9, SEQ ID NO:37) both CGRP analogs could prove even more efficacious in vivo, if persisted at about 10% of the starting concentration only relieved of the proteolytic liability associated with the native through about 1 hour and were 50% depleted at <30 minutes R11 and R18 residues. (Table 9). Unlike in the rat plasma experiment (Table 8), in human plasma the (R11 hR/R18hR Table 9, SEQ ID 0364 Consequently, the stability of select, non-PEGy NO:25) analog proved to be less stable than the other lated analogs was evaluated in vitro in the presence of 10% di-Substituted analogs, with 10% remaining at about 2 hours rat or human plasma. Briefly, samples of each peptide were and 50% detectable at about 30 minutes. The other two diluted to 100 ng/ml into PBS containing 10% of rat or di-substitution analogs (R11Cit/R18Cit-SEQ ID NO:31, human plasma. Each sample was then incubated at room and R11O/R18Q-SEQ ID NO:39) were very similar in temperature and analyzed by LC-MS/MS to quantitate the their stability profiles, with 10% remaining at about 4 hours intact peptide remaining at select time points over 6 hours. and 50% at about 1 hour. 0365. The CGRP peptide analogs tested were the di 0368. These two experiments (Table 8 and Table 9) substituted (R11hR/R18hR) analog (Table 7, SEQ ID demonstrated the beneficial effects of adding proteolysis NO:25), the di-substituted (R11Cit/R18Cit) analog (Table 7, resistant substitutions in positions R11 and R18 of CGRP SEQ ID NO:31), the di-substituted (R11O/R18Q) analog peptide antagonists. Even though the peptide degradation (Table 7, SEQ ID NO:39), the mono-substituted (R18Q) profiles were faster in human plasma Table 9) than in rat analog (Table 7, SEQ ID NO:37) and as a control, the plasma (Table 8), the relative stability ranking was the same un-substituted (R11/R18) analog (Table 4, SEQ ID NO:7). in plasma from either species: SEQ ID NO:7 (R11/ 0366 When exposed to 10% rat plasma the control R18)

0369. In some additional experiments, CGRP peptides were incubated at room temperature in 100% plasma TABLE 8 (human or monkey) at initial concentration of 10 g/ml. In vitro stability study of CGRP peptides in 10% rat plasma. Briefly, at time points of 0 hour and 4 hour, 100 ul-plasma Samples of each CGRP peptide analog at 100 ng/ml were exposed to samples were taken, followed immediately by solid phase 10% rat plasma at room temperature and were analyzed by LC-MS/MS extraction. Extracted samples were collected for LC-MS/MS to determine % remaining peptide at each time point. analysis. LC-MS/MS was carried out in a Thermo Finnigan LCQ ion trap equipped with Agilent 1100 LC system. High SEQ ID % Remaining performance liquid chromatographic separation was carried NO Oh O.S 1 h 2h 3h 4h Sh 6 h out on a C18 reverse phase column. Data dependent tandem 37 1OOO 62.7 39.5 1O.S 4.7 2.5 1.3 0.7 mass spectrometric detection was used for peptide sequenc 39 1OOO 83.8 66.2 29.9 20.4 11.8 6.4 4.0 ing. A comparison of in vitro metabolic identification of 7 100.0 SS.4 34.5 8.0 5.2 1.4 1.7 O.6 CGRP peptide SEQ ID NO:658, the non-PEGylated “par 31 1OOO 82.3 59.0 26.1 18.5 10.1 S.2 3.3 ent” peptide of PEGylated CGRP peptide Seq ID: No: 172 25 10O.O 76.9 S8.1 27.5 21.3 16.3 11.O 7.1 (FIG. 9) and CGRP peptide SEQ ID NO: 144, the non PEGylated “parent' peptide of PEGylated CGRP peptide 0371)

TABLE 9

In vitro stability study of CGRP peptides in 10% human plasma. Samples of each CGRP peptide analog at 100 ng/ml were exposed to 10% human plasma at room temperature and was analyzed by LC-MS/MS to determine % remaining peptide at each time point. % Remaining

Peptide Oh O.17 O.S 1 h 2h 3 h 4h Sh 6 h

SEQ ID NO: 1OO.O S9.0 20.6 1O.O 2.3 1.3 0.7 0.7 O.O 37 SEQ ID NO: 1OO.O 74.2 S4.O SO.2 36.7 15.8 11.3 6.3 5.2 39 SEQ ID NO: 1OO.O 60.3 13.8 4.9 O.O O.O O.O O.O O.O 7 SEQ ID NO: 1OO.O 73.0 52.8 44.0 34.9 15.3 11.8 6.1 5.9 31 SEQ ID NO: 1OO.O 88.3 47.3 28.7 11.0 5.4 3.1 3.6 2.8 25

Seq ID No.173 (FIG. 10) in 100% human plasma was made. Example 7 The results in FIG. 9 and FIG. 10 show that the two CGRP analogs (R11Cit/R18Cit/R24h R/P34Oic, SEQID NO:658), Pharmacokinetic (PK) Studies R11Cit/R18Cit/RhR/F35Nal, SEQ ID NO:173) were 0372 Rat pharmacokinetic studies. The results of phar relatively stable after four hours incubation in 100% human macokinetic (PK) studies of some PEG-conjugated CGRP plasma. The major degradation product for peptide SEQ ID peptide antagonists, conducted in male Sprague-Dawley NO:658 was fragment W7-hR24. There were more cleavage rats, are shown in Table 10. There were three rats per group sites for CGRP peptide SEQ ID NO:173; the proteolytic (n=3), each weighing 250-325 g. Each group received either degradation products were fragments W7-P34, W7-hR24 a 2 mg/kg intravenous or Subcutaneous discrete administra tion. The approximate dose Volume was 0.3 mL per rat. and W7-N26. Approximately 0.25 mL of whole blood was taken per time 0370 FIG. 11 shows results of in vitro metabolic iden point at the following post-dose collection times for the tification of CGRP peptide SEQ ID NO:658, the unconju intravenous groups (in hours): 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, gated “parent' peptide of PEGylated CGRP peptide Seq ID 24, 48, 72, 96 and the following time points for the subcu taneous groups (in hours): 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48, 72, No: 172 in 100% Cynomolgus monkey plasma. The results 96. Whole blood samples were collected in microtainer vials indicate that there were differences in in vitro stability in containing EDTA as anticoagulant and were kept on wet ice human and monkey plasma. The major degradation product until processed for plasma. The plasma was obtained by for CGRP peptide SEQ ID NO:658 was fragment W7-K25 centrifuging the whole blood for approximately 5 min at in monkey plasma instead of fragment W7-hR24 in human 11,500 rpm. Plasma was transferred to new tubes and stored plasma. (Also, see Example 7 herein below for comparison frozen at approximately -70° C. until quantitative analysis. with in vivo metabolic identification.) The assay for the quantification of the vehicle-conjugated US 2008/0020978 A1 Jan. 24, 2008 66

CGRP peptides was conducted in accordance with the 0374 Monkey pharmacokinetic studies. Table 11 shows ELISA method described in Example 3 above, using rat the results of pharmacokinetic studies of some PEG-conju plasma. The individual sample concentrations for each time gated, and unconjugated (“naked'), CGRP peptide antago point were averaged (n=3) within a given dose group using nists of the present invention, conducted in male Cynomol Excel 2000 software. The averaged plasma concentration gus monkeys. A complete blood chemistry panel was time data were analyzed by noncompartmental methods performed on each monkey prior to each study day to obtain using WinNonlin v.3.3 software. Nominal sample times baseline values and ensure hematological recovery. A cath were used in the pharmacokinetic analysis. The area under eter was placed in the cephalic vein of three male Cyno molgus monkeys (Macaca fascicularis) for blood sampling the concentration-time curve was calculated using the linear/ during the study. For the intravenous (iv) leg of the study, a log trapezoidal method to determine exposure. catheter was also placed in a saphenous vein for infusion of 0373 The results in Table 10 demonstrate an enhanced test compound. On each study day, the monkeys were placed overall pharmacokinetic profile for the vehicle-conjugated in restraining chairs for the first 4 h of the study to facilitate CGRP peptide antagonist having SEQID NO:32 (including drug administration and blood sampling. For administration R11Cit, R18Cit substitutions in its amino acid sequence of antagonist, each monkey received solubilized peptide relative to the native human C.CGRP sequence), compared to (dose and SEQ ID listed in Table 11 below) either as a a vehicle-conjugated CGRP peptide antagonist having SEQ 30-min intravenous infusion (4 mL/kg/h) or as a single ID NO:15, while the results for SEQID NO:26 and SEQ ID Subcutaneous bolus (approx. 1.0 mL/animal). Blood samples NO:40 were more similar to those for SEQ ID NO:15. were collected from each study animal at various times Following intravenous administration, the mean residence during and after drug administration, plasma samples (100 time (MRT) was 4 times longer for the vehicle-conjugated uL) were isolated by centrifugation and stored at -70° C. CGRP peptide antagonist having SEQID NO:32, compared until analysis. For monkeys receiving intravenous infusion, to the vehicle-conjugated CGRP peptide antagonist having Such a sample was also taken at 20 min during the infusion. SEQ ID NO:15. Following subcutaneous administration, exposure (AUC) was increased by about 15-fold, resulting 0375. As shown in Table 11, SEQ ID NO: 172 peptide in the enhanced bioavailablity (F 96) of 47% versus 12%. displayed low plasma clearance, a small volume of distri Thus, the R11Cit, R18Cit substitutions provided the best bution and a mean residence time (MRT) of 2.5 h. Exposure overall PK profile of the four peptides tested in vivo. following subcutaneous administration of both PEG-conju TABLE 10 Results of pharmacokinetic of vehicle-conjugated CGRP peptide antagonists conducted in male Sprague-Dawley rats. SC Cmax AUCo. AUC of CLF Peptide Ta (h) (ng/mL) tip (h) (ng h/ml) (ng himl) (mL/h/kg) F 9

SEQ ID 2.00 137 22.37 1264 1542 1257 12% NO: 15 SEQ ID 2.00 143 26.17 1188 1204 1660 59% NO: 26 SEQ ID 4.OO 1879 29.98 18933 19046 105 47% NO:32 SEQ ID 4.OO 121 27.15 1212 1223 1634 11% NO: 40 SEQ ID 4.67 662 ND 4349 4375 647 14% NO: 172 SEQ ID 4.OO 643 ND 4385 4396 461 59% NO: 173

IV

AUCo. AUCo.inf CL Vss MRT Peptide t12 (h) (ng himl) (ng himl) (mL/h/kg) (mL/kg) (h) SEQ ID 10.60 12940 12959 154 174 1.10 NO: 15 SEQ ID 7.39 24546 24555 81 83 1.01 NO: 26 SEQ ID 42.78 4O2SO 40431 49 204 4.12 NO:32 SEQ ID 32.57 10651 10666 187 496 2.63 NO: 40 SEQ ID ND 31494 31543 64 117 1.82 NO: 172 SEQ ID ND 80440 80996 27 125 4.97 NO: 173 Injection (dose = 2 mg/kg) was Subcutaneous (SC) or intravenous (IV). The vehicle-con jugated CGRP peptide antagonists were dissolved in aqueous solution (10 mM sodium acetate, 5% sorbitol, pH 4.0). All values represent the mean, n = 3. Listed CGRP peptide sequences are described in detail at Tables 2B, 4, and 7. US 2008/0020978 A1 Jan. 24, 2008 67 gated CGRP peptides evaluated was several fold higher in parent peptides (e.g., compared to relatively soluble parent non-human primates compared to rats (Table 10). The better SEQ. ID NO: 7, disclosed in Table 4) in the aqueous exposure appears to be due to better absorption following conjugation buffers used for coupling vehicle to the peptide Subcutaneous administration, lower plasma clearance and (see, Example 2 herein). In an attempt to better solubilize Smaller Volumes of distribution in the non-human primate these less Soluble peptides during the conjugation reaction, compared to the rat (see Table 10). and thereby improve conjugate yields, a variety of alcohol 0376 Metabolic identification of CGRP peptide SEQ ID co-solvents were tested in the conjugation reaction. NO:658 (sequence disclosed in Table 2B) the “parent” 0378 Briefly, a series of reductive alkylation conjugation peptide of PEGylated CGRP peptide Seq ID: No 172 in reactions using 20 kDa mPEG-ald and the relatively soluble monkeys receiving intravenous infusion thereof (sample CGRP peptide (Table 4, SEQ. ID NO: 7) were prepared as taken at 20 min during the infusion) was analyzed. Samples described in Example 2, with the exception that the alcohols: were taken as described above. After thawing, each 100-11 methanol (MeOH), ethanol (EtOH), isopropanol (IPA) and plasma sample was extracted by Solid phase extraction. tri-fluoroethanol (TFE) were each added at either 25% or Extracted samples were collected for LC-MS/MS analysis. 50% (v/v) ratio. The progress of each reaction was moni LC-MS/MS was carried out in a Thermo Finnigan LCQ ion tored by RP-HPLC after 20 hrs. The mono-PEGylated trap equipped with Agilent 1100 LC system. High perfor peptide product (Table 4, SEQID NO15) was quantitated by mance liquid chromatographic separation was carried out on integration of the RP-HPLC chromatograms and reported as a C18 reverse phase column. Data dependent tandem mass % Product Peak (FIG. 4). These results demonstrate an spectrometric detection was used for peptide sequencing. inhibitory effect by MeOH and EtOH on PEGylation of this The result is shown in FIG. 12, and it indicates that there relatively soluble CGRP peptide whereas IPA showed little were also significant differences between in vivo and in vitro effect and surprisingly TFE showed both positive and nega stability for the same CGRP peptide SEQ ID NO:658 (in tive effects, depending on concentration. vitro metabolic identification conducted as described for 0379 Next, a comparison was made between the rela 100% human plasma in Example 6 herein above; FIG. 11). tively soluble CGRP peptide (sequence disclosed in Table 4. More cleavage sites were observed in the plasma sample SEQ. ID NO: 7) and a relatively insoluble CGRP peptide from the IV infusion study. The degradation products were (sequence disclosed in Table 7, SEQ ID NO: 31). In this fragments W7-T9, T30-A36, W7-N26, T30-F37 and experiment both peptides were PEGylated by reductive W7-hR24. alkylation, as described in Example 2, using 20 kDa mPEG

TABLE 11 Results of pharmacokinetic of vehicle-conjugated CGRP peptide antagonists conducted in male Cynomolgus monkies. MONKEY SC

Dose Tmax Cmax AUC 0-t AUC 0-inf CLF Peptide (mg/kg) (h) (ng/mL) (ng himl) (ng himl) (mL/h/kg) F9o SEQ ID O.8 5.33 3597 24718 24,773 51% NO: 172 (Table 2B) SEQ ID O.8 5.33 1691 11842 12,195 Na NO: 173 (Table 2B) MONKEY IV Ple

Dose T/3 AUC 0-t AUC 0-inf CL Vss MRT Peptide (mg/kg) (h) (ng himl) (ng himl) (mL/h/kg) (mL/kg) (h)

SEQ ID 1.56 2.3 98,843 98,887 16 41 2.54 NO: 172 (Table 2B) SEQ ID 6.0 O.8 16,942 16,964 399 174 O.42 NO: 658 (naked peptide: Table 2A) Administration was by intravenous infusion (4 mL/kg/h) or as a single Subcutaneous bolus (approx. 1.0 mL/animal). All values represent the mean, n = 3.

Example 8 ald in the presence of either 50% IPA or 50% TFE. The progress of each reaction was monitored by RP-HPLC after 0377 Co-solvent mediated conjugation of vehicle to 20 hrs. The mono-PEGylated peptide product (Table 4, SEQ CGRP peptide antagonists. Many of the CGRP peptides that ID NO15 or Table 7, SEQ ID NO:32) was quantitated by were developed to resist proteolysis (see, e.g., Example 4 integration of the RP-HPLC chromatograms and reported as herein) also proved to be considerably less soluble than their % Product Peak (FIG. 5). These results duplicate the modest US 2008/0020978 A1 Jan. 24, 2008 improvement in product (Table 4, SEQID NO: 15) yield for Example 9 the more soluble CGRP peptide also observed in FIG. 4. Surprisingly, both co-solvents provided a much more dra Laser Doppler Blood Flowmetry Assay matic improvement in PEGylation efficiency for the rela tively insoluble CGRP peptide (Table 7, SEQ ID NO: 31), 0385 Methods. An automated laser Doppler blood flow showing increases in product yields of 2.6-fold with IPA and metry device is used (MoorLDIR); Moor Instrument, Devon, 4.1-fold with TFE. UK). The device was turned on prior to the start of experi ments and allowed to warm up for 20 minutes. Blood flow 0380. Over 42 relatively insoluble CGRP peptides have data (in FLUX) were read from software records (see been PEGylated by this method in the presence of 50% TFE with good product yield. At least 34 of these conjugates were below). produced in excess of 60% product yield (data not shown). 0386 For each rat, the experimental procedure was per 0381. In order to determine the optimal concentrations of formed within a single day and was each rat was sacrificed co-solvent for PEGylation of different CGRP peptides titra on the same day immediately after the experiment was over. tion experiments were performed from 30-70% co-solvent On an experiment day, each rat was weighed and adminis using either IPA. TFE or hexafluoro-isopropyl alcohol tered 1 ml/kg (intramuscular injection in right leg) of an (HFIPA or HF-i-PA). The PEGylation reactions were pre anesthetic cocktail consisting of Ketamine, Xylazine and pared as described in Example 2 for reductive alkylation PBS (5:3:2). Each rat was then placed in its home cage for with the three alcohols added at their stated concentrations. 15 min to allow the onset of anesthesia, after which, the The progress of each reaction was monitored by RP-HPLC ventral surface (abdomen) of the body was shaved. A small after 20 hrs. The mono-PEGylated peptide product was dot was placed near the center of the abdomen using a quantitated by integration of the RP-HPLC chromatograms marker. The animal was then ready for the experiment. and reported as % Product Peak. Unfortunately, HFIPA proved partially immiscible in the aqueous reaction buffer 0387 Approximately 20 min after the injection of the rendering the PEGylation results HFIPA cosolvent difficult anesthetic cocktail, baseline blood flow readings were col to use in the existing process. lected for each animal. Each rat was placed under the laser Doppler scanning device, matching the laser with the marker 0382 FIG. 6 shows the effects of IPA. TFE, and HFIPA dot on the abdomen. Using the device's Software package, on the relatively soluble CGRP peptide (sequence disclosed the device was activated and the surface of the abdomen was in Table 4, SEQID NO: 7) examined previously. These data scanned for 1 min with the laser. The scan area was 4 cmx4 show little effect from IPA until the concentration exceeds cm. The software package associated with the device cal 50%. At 70% IPA, a modest 1.1-fold increase in product culates a blood flow flux value with arbitrary units. yield (sequence disclosed in Table 4, SEQ ID NO: 15) was observed. However, 70% TFE shows a more significant 0388. After completion of baseline scanning, the rat was increase in product yield of 1.4-fold. HFIPA showed an removed from the device. The rat then received an injection apparent maximum effect between 50-60%. of CGRP peptide antagonist (PEGylated CGRP peptide or the corresponding unconjugated “parent CGRP peptide) 0383 FIG. 7 shows the effect of IPA, TFE, and HFIPA on a relatively insoluble CGRP peptide Ac-WVTHCitLAG or its vehicle control via intravenous injection through the LLSICitSGGVVDhArg)KNFVPTDVGPFAF-NH, SEQ ID dorsal penis vein or Subcutaneously in the dorsal right flank. NO:739 (sequence disclosed in Table 2B) not previously After a pre-determined interval, based on pharmacokinetic examined. These data show a much more pronounced effect characteristics of the relevant CGRP peptide antagonist, for all three co-solvents. Where IPA produced a maximal agonist (human CCGRP: 0.1 ug. 50 uL: interdermal) was effect at about 60% IPA of 1.8-fold increase in product yield injected such that the point of entry of the needle (30 gauge) for PEGylated CGRP peptide SEQ ID NO:153. With TFE, was remote from the marker dot but the intradermal injec the results were even more dramatic, showing a continued tion bolus was centered under the dot. increase in product yield peaking at about 70% TFE and 3.4-fold. HFIPA also induced a significant increase in prod 0389. At 10 min post-hCCGRP (agonist) injection, the rat uct yield (-3.2-fold) at 50% cosolvent, but yields rapidly was placed under laser again to scan the abdomen at the 10 dropped off at higher HFIPA concentrations. mintime point. At 30 min post-hoCGRP (agonist) injection, the rat was placed under the laser one last time to scan the 0384 FIG. 8 shows the effect of IPA, TFE, and HFIPA on abdomen at the 30 mintime point. The Scanning parameters a relatively insoluble CGRP peptide SEQ ID NO:658 not were the same as those employed at baseline. previously examined. These data show a much more pro nounced effect for all three co-solvents. Where IPA produces 0390 Immediately after the scan at 30 min post-hCGRP, a maximal effect at about 70% IPA of 3.3-fold increase in a blood sample was collected from each rat through via product yield of PEGylated CGRP peptide SEQID NO:172. cardiac puncture using a 21 gauge syringe. Approximately With TFE, the maximum yield was achieved at about 70% 200 uL of plasma was obtained by immediate centrifugation TFE with 4.0-fold increase. Again, the HFIPA gives a of 400 uL whole blood at 14000xg for 10 min at 4°C. The maximal effect of 4.0-fold increased yield at 50% cosolvent, plasma samples were Submitted for analytical determination but then yields quickly drop off at higher HFIPA concen of plasma concentration. trations. Together, these data illustrate a significant and unexpected efficiency benefit to be realized by utilizing 0391. Upon completion of all testing and blood sample co-solvents in PEGylation reactions for CGRP peptides. collection each rat was sacrificed by asphyxiation with CO. US 2008/0020978 A1 Jan. 24, 2008 69

0392 Statistical analysis. Blood flow flux values col hCGRP within a small, defined area of abdominal skin lected at the 10 min and 30 min time points post-hCGRP produced a 30-40% increase in local blood flow. Systemic were converted to % change from baseline values using the injection of several of our CGRP peptide antagonists (either formula: pegylated or non-pegylated analogs; either the i.V. or s.c. route) reduced the effect of hCGRP in a dose-dependent fashion. Thus, this model was able to demonstrate target coverage by our antagonists in vivo in manner that resulted in functional antagonism of an agonist-mediated effect. Differences in in vitro potency against the rat ortholog of the (post-hCGRP flux value) - baseline flux value X = x 100 CGRP1 receptor between pegylated and non-pegylated ver baseline flux sions of peptide antagonists generally resulted in similar differences in potency in vivo. For example, SEQ ID NO:808 and SEQ ID NO:809 (the PEGylated version of SEQ ID NO:808) showed similar in vitro and in vivo potencies (see Table 12). On the other hand, SEQID NO:31 0393 Typically, data were analyzed in two ways: 1) and its pegylated analog SEQID NO:791 showed a marked analysis of variance (ANOVA) followed by Tukey's HSD difference in in vitro functional potency against the rat post hoc tests, and 2) nonlinear least squares regression CGRP1 receptor; the in vitro potency of SEQ ID NO:791 (sigmoidal dose-response) for determination of antagonist was shifted rightward by approximately 9-fold compared ID values and confidence intervals when enough data from with SEQ ID NO:31. This was reflected by an apparently enough doses were collected to allow a full dose-response similar rightward shift in potency in vivo. analysis. In some cases, plasma ICs values were calculated 0395. In summary, the laser Doppler blood flow model by performing least squares linear regression on log plasma demonstrated on-target, functional antagonistic properties of concentration values (nM) vs. '% change in blood flow our peptides in vivo. In vivo potencies generally matched in values calculated for individual rats. vitro potency against the rat ortholog of the CGRP1 receptor.

TABLE 12 Laser Doppler results

Pre in vitro treatinent IDso Efficacy ICso time (plus 95% observed Compound (rat (before confidence Plasma (%) SEQ ID ortholog: Administration agonist; interval) ICso (at highest NO: nM) route minutes) (mg/kg) (nM) dose tested)

O 9 6.4 i.v. 5 O.S2 916 1OO (0.2-1.4) (3 mg/kg)

S.C. 240 1OO 8. b e (10 mg/kg)

5.4 i.v. 5 O.39 1OO (0.2-0.8) (0.6 mg/kg)

14.8 i.v. 5 1OO (5 mg/kg)

130.7 i.v. 5 69 (20 mg/kg) 9 8.9 i.v. 5 O.89 1OO s (0.28-2.8) (6 mg/kg)

49.5 i.v. 5 75.5 (3 mg/kg)

0394 Results and Conclusions. The experimental results 0396 The foregoing being illustrative but not an exhaus are summarized in Table 12 below. In the laser Doppler tive description of the embodiments of the present invention, blood flowmetry experiments, intradermal injection of the following claims are presented. US 2008/0020978 A1 Jan. 24, 2008 70

SEQUENCE LISTING The patent application contains a lengthy “Sequence Listing section. A copy of the “Sequence Listing is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US2008.0020978A1). An electronic copy of the “Sequence Listing will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

We claim: (b) wherein the pharmaceutically acceptable vehicle is 1. A composition of matter comprising: conjugated at one of the additional amino acid residues. 8. The composition of matter of claim 1, wherein the (a) a CGRP peptide antagonist, being a CGRP peptide CGRP peptide has an amino acid sequence that comprises: comprising from its C-terminal end to its N-terminal end: at least one amino acid residue inserted or Substituted therein, relative to reference sequence SEQID NO:43 (i) the C-terminal carboxy moiety is replaced with a or an N-terminally truncated analog: moiety selected from wherein the inserted or substituted amino acid residue has (A) - C(=O)NRR, where R is independently hydrogen, (C-Cs)alkyl, haloalkyl, aryl or het a side chain comprising a nucleophilic or electrophilic eroaryl; and reactive functional group; and (B) —CHOR where R is H. (C-C)alkyl, aryl or wherein the vehicle is conjugated to the CGRP peptide via heteroaryl; the nucleophilic or electrophilic functional group. 9. The composition of matter of claim 8, wherein the at (ii) a first CGRP receptor binding region; and least one amino acid residue inserted or Substituted into the amino acid sequence is a lysine, a homolysine, an O.f3 wherein the CGRP peptide lacks a functional CGRP diaminopropionic acid, an O.Y-diaminobutyric acid, an orni receptor activation region; and thine, a cysteine, a homocysteine, a glutamic acid, an (b) a pharmaceutically acceptable vehicle, wherein the aspartic acid, or a selenocysteine. pharmaceutically acceptable vehicle is conjugated to 10. The composition of matter of claim 8, wherein a basic the CGRP peptide at a site other than at the peptide's amino acid residue, is inserted or Substituted at position 24. C-terminal amino acid residue. relative to reference sequence SEQ ID NO:43. 2. The composition of matter of claim 1, wherein the 11. The composition of matter of claim 10, wherein the CGRP peptide comprises from the first CGRP receptor basic amino acid residue is a histidine residue, a lysine residue, a homolysine, an ornithine residue, an arginine binding region to the N-terminal end of the peptide: residue, a N-methyl-arginine residue, an (D-aminoarginine (iii) a hinge region; and residue, an ()-methyl-arginine residue, a 1-methyl-histidine residue, a 3-methyl-histidine residue, or a homoarginine (iv) a second CGRP receptor binding region between the residue. hinge region and the N-terminal end of the peptide. 12. The composition of matter of claim 1, wherein the 3. The composition of matter of claim 2, wherein the CGRP peptide further comprises at its N-terminal an aro vehicle is conjugated to the CGRP peptide at an amino acid matic amino acid residue. residue in the hinge region. 13. The composition of matter of claim 12, wherein the 4. The composition of matter of claim 2, wherein the aromatic amino acid residue is a tryptophan, tyrosine, 3-(1- vehicle is conjugated to the CGRP peptide at an amino acid naphthyl)alanine, or phenylalanine residue. residue in the first or second CGRP receptor binding region. 14. The composition of matter of claim 12, wherein the 5. The composition of matter of claim 1, wherein the aromatic amino acid residue is a non-canonical amino acid vehicle is conjugated to the CGRP peptide at its N-terminal residue. end. 15. The composition of matter of claim 1, wherein the 6. The composition of matter of claim 3, wherein the amino terminal of the CGRP peptide is chemically blocked vehicle is conjugated to the CGRP peptide at amino acid from conjugation with the vehicle. position 22, position 23, position 24, position 25, position 16. The composition of matter of claim 1, further com 26, or position 27, relative to a native human C.CGRP prising at its N-terminal an acyl moiety, an aromatic moiety, reference sequence (SEQ ID NO:43). or other functional group, covalently linked thereto. 7. The composition of matter of claim 2: 17. The composition of matter of claim 1, wherein the (a) wherein the CGRP peptide comprises an N-terminal CGRP peptide further comprises N31D, S34P and K35F region comprising about one to about fifty additional substitutions relative to reference sequence SEQID NO:43. amino acid residues attached to the N-terminus of the 18. The composition of matter of claim 1, further com second CGRP receptor binding region, and prising a modification relative to reference sequence SEQID US 2008/0020978 A1 Jan. 24, 2008

NO:43, wherein the modification reduces the CGRP pep 27. The composition of matter of claim 26, wherein the tide's Susceptibility to enzymatic proteolysis. vehicle is conjugated to the CGRP peptide at amino acid 19. The composition of matter of claim 18, wherein the position 23, position 24, or position 25, relative to a native Susceptibility to enzymatic proteolysis is proteolysis by a human CCGRP reference sequence (SEQ ID NO:43). serine proteinase, a cysteine protease or a membrane met 28. The composition of matter of claim 26: allo-endopeptidase. 20. The composition of matter of claim 18, wherein the (a) wherein the CGRP peptide comprises an N-terminal modification is at amino acid position 11, 12, 14, 16, 17, 18, region comprising about one to about fifty additional or 19, or at any combination of these positions, relative to amino acid residues attached to the N-terminus of the reference sequence SEQ ID NO:43. second CGRP receptor binding region, and 21. The composition of matter of claim 18, wherein the (b) wherein the pharmaceutically acceptable vehicle is modification is a Substitution of a basic amino acid residue. conjugated at one of the additional amino acid residues. 22. The composition of matter of claim 18, wherein the 29. The composition of matter of claim 26, wherein the modification is an amino acid Substitution selected from CGRP peptide has an amino acid sequence that comprises: R11Q, R11 hR, R11Cit, R11 hK, R11Orn, R11hcit, R11NMeR, R11NMeO, R11NMeHoK, R11NMeOrn, at least one amino acid residue inserted or Substituted R11NMeCit, R11NMeHoCit, R18Q, R18hR, R18Cit, therein, relative to reference sequence SEQID NO:43 R18NMeR, R18NMeO, and R18NMeCit, or any combina or an N-terminally truncated analog: tion of these members wherein there is a substitution at wherein the inserted or substituted amino acid residue has position 11 and position 18 relative to reference sequence a side chain comprising a nucleophilic or electrophilic SEQ ID NO:43. 23. The composition of matter of claim 1, wherein the reactive functional group; and CGRP peptide comprises a C-terminal phenylalaninamide wherein the vehicle is conjugated to the CGRP peptide via residue or tyrosineamide residue. the nucleophilic or electrophilic functional group. 24. The composition of matter of claim 1, wherein the 30. The composition of matter of claim 29, wherein the at pharmaceutically acceptable vehicle is a polyethylene gly least one amino acid residue inserted or substituted into the col, a copolymer of ethylene glycol, a polypropylene glycol, amino acid sequence is a lysine, a homolysine, an O.f3 a copolymer of propylene glycol, a carboxymethylcellulose, diaminopropionic acid, an O.Y-diaminobutyric acid, an orni a polyvinyl pyrrollidone, a poly-1,3-dioxolane, a poly-13, thine, a cysteine, a homocysteine, a glutamic acid, an 6-trioxane, an ethylene/maleic anhydride copolymer, a aspartic acid, or a selenocysteine. polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene 31. The composition of matter of claim 29, wherein a oxide polymer, an ethylene oxide polymer, a polyoxyethy basic amino acid residue, is inserted or Substituted at posi lated polyol, a polyvinyl alcohol, a linear or branched tion 24, relative to reference sequence SEQ ID NO:43. glycosylated chain, a polyacetal, a long chain fatty acid, a 32. The composition of matter of claim 31, wherein the long chain hydrophobic aliphatic group, an immunoglobulin basic amino acid residue is a histidine residue, a lysine Fc domain, an albumin, a transthyretin, a thyroxine-binding residue, a homolysine, an ornithine residue, an arginine globulin, or a ligand that has an affinity for a long half-life residue, a N-methyl-arginine residue, an (D-aminoarginine plasma protein, said ligand being selected from peptide residue, an ()-methyl-arginine residue, a 1-methyl-histidine ligands and Small molecule ligands; or a combination of any residue, a 3-methyl-histidine residue, or a homoarginine of these members. residue. 25. The vehicle-conjugated CGRP peptide antagonist of 33. The composition of matter of claim 26, wherein the claim 1, wherein the peptide further comprises an isotopic CGRP peptide further comprises at its N-terminal an aro label. matic amino acid residue. 26. A composition of matter comprising: 34. The composition of matter of claim 33, wherein the aromatic amino acid residue is a tryptophan, tyrosine, 3-(1- (a) a CGRP peptide antagonist, being a CGRP peptide naphthyl)alanine, or phenylalanine residue. comprising a phenylalaninamide residue or tyrosinea 35. The composition of matter of claim 33, wherein the mide residue at its C-terminal, and from its C-terminal aromatic amino acid residue is a non-canonical amino acid end to its N-terminal end: residue. (i) a first CGRP receptor binding region; 36. The composition of matter of claim 26, wherein the amino terminal of the CGRP peptide is chemically blocked (ii) a hinge region; and from conjugation with the vehicle. (iii) a second CGRP receptor binding region between 37. The composition of matter of claim 26, further com the hinge region and the N-terminal end of the prising at its N-terminal an acyl moiety, an aromatic moiety, peptide; and or other functional group, covalently linked thereto. 38. The composition of matter of claim 26, wherein the wherein the CGRP peptide lacks a functional CGRP CGRP peptide further comprises N31D, S34P and K35F receptor activation region; and substitutions relative to reference sequence SEQID NO:43. (b) a pharmaceutically acceptable vehicle, wherein the 39. The composition of matter of claim 26, further com pharmaceutically acceptable vehicle is conjugated to prising a modification relative to SEQ ID NO:43, wherein the CGRP peptide at an amino acid residue in the hinge the modification reduces the CGRP peptide's susceptibility region. to enzymatic proteolysis. US 2008/0020978 A1 Jan. 24, 2008 72

40. The composition of matter of claim 39, wherein the 49. A pharmaceutical composition, comprising the com Susceptibility to enzymatic proteolysis is proteolysis by a position of matter of claim 26 and a pharmaceutically serine proteinase, a cysteine protease or a membrane met acceptable carrier. allo-endopeptidase. 50. The pharmaceutical composition of claim 49, config 41. The composition of matter of claim 39, wherein the ured for administration to a patient by an intravascular, oral, modification is at amino acid position 3, 4, 9, 10, 11, 12, 14. enteral, transmucosal, transdermal, intranasal, Subcutane 15, 16, 17, 18, 19, 24, 25, 26, 27, 29, 30, 31, 33, 34, 35, 36, ous, or pulmonary delivery route. or 37, or at any combination of these positions, relative to 51. A method of producing a composition of matter, the human CCGRP reference sequence (SEQ ID NO:43). comprising the steps of: 42. The composition of matter of claim 39, wherein the (a) obtaining a CGRP peptide comprising from its C-ter modification is a Substitution of a basic amino acid residue. minal end to its N-terminal end; 43. The composition of matter of claim 39, wherein the modification is an amino acid Substitution selected from (i) the C-terminal carboxy moiety is replaced with a R11Q, R11 hR, R11Cit, R11 hK, R11Orn, R11hcit, moiety selected from R11NMeR, R11NMeO, R11NMeHoK, R11NMeOrn, (A) —C(=O)NRR, wherein R is independently R11NMeCit, R11NMeHoCit, R18Q, R18hR, R18Cit, hydrogen, (C-Cs)alkyl, haloalkyl, aryl or het R18NMeR, R18NMeO, and R18NMeCit, or any combina eroaryl; and tion of these members wherein there is a substitution at position 11 and position 18 relative to reference sequence (B) —CH-OR wherein R is H., (C-C)alkyl, aryl or SEQ ID NO:43. heteroaryl; and 44. The composition of matter of claim 26, wherein the (ii) a first CGRP receptor binding region; and CGRP peptide comprises an amino acid sequence compris 1ng: wherein the CGRP peptide lacks a functional CGRP, receptor activation region; and (a) any one of SEQ ID NOS: 47 through 55, 82 through 127, 1128 and 1129: (b) conjugating the obtained CGRP peptide to a pharma ceutically acceptable vehicle at a site on the obtained (b) an N-terminal truncation of any of (a), of 1 to about peptide other than at the C-terminal amino acid residue, 20 contiguous amino acid residues, that preserves the whereby the composition of matter is produced. hinge region and the first CGRP receptor binding 52. The method of claim 51, wherein the pharmaceuti region; cally acceptable vehicle is a polyethylene glycol, a copoly mer of ethylene glycol, a polypropylene glycol, a copolymer (c) any of (a) or (b), wherein the amino acid sequence of propylene glycol, a carboxymethylcellulose, a polyvinyl further comprises an N-terminal aromatic amino acid pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an residue; or ethylene/maleic anhydride copolymer, a polyaminoacid, a (d) any of (a), (b), or (c), in which the amino acid dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a sequence contains a modification relative to reference propylene glycol homopolymer, a propylene oxide polymer, sequence SEQ ID NO:43 that reduces the CGRP pep an ethylene oxide polymer, a polyoxyethylated polyol, a tide’s Susceptibility to enzymatic proteolysis. polyvinyl alcohol, an immunoglobulin F, domain, an albu 45. The composition of matter of claim 26, wherein the min, a transthyretin, a thyroxine-binding globulin, or a pharmaceutically acceptable vehicle is a polyethylene gly ligand that has an affinity for a long half-life plasma protein, col, a copolymer of ethylene glycol, a polypropylene glycol, said ligand being selected from peptide ligands and Small a copolymer of propylene glycol, a carboxymethylcellulose, molecule ligands; or a combination of any of these members. a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-13, 53. A method of producing a composition of matter, 6-trioxane, an ethylene/maleic anhydride copolymer, a comprising the steps of: polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene (a) obtaining a CGRP peptide comprising a phenylalani oxide polymer, an ethylene oxide polymer, a polyoxyethy namide residue or tyrosineamide residue at its C-ter lated polyol, a polyvinyl alcohol, a linear or branched minal, and from its C-terminal end to its N-terminal glycosylated chain, a polyacetal, a long chain fatty acid, a end: long chain hydrophobic aliphatic group, an immunoglobulin (iii) a first CGRP receptor binding region; Fc domain, an album in, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life (ii) a hinge region; and plasma protein, said ligand being selected from peptide (iii) a second CGRP receptor binding region between ligands and Small molecule ligands; or a combination of any the hinge region and the N-terminal end of the of these members. peptide; and 46. The composition of matter of claim 26, wherein the CGRP peptide further comprises an isotopic label. wherein the CGRP peptide lacks a functional CGRP 47. A pharmaceutical composition, comprising the com receptor activation region; and position of matter of claim 1 and a pharmaceutically accept (b) conjugating the obtained CGRP peptide at an amino able carrier. acid residue in the hinge region to a pharmaceutically 48. The pharmaceutical composition of claim 47, config acceptable vehicle, whereby the composition of matter ured for administration to a patient by an intravascular, oral, is produced. enteral, transmucosal, transdermal, intranasal, Subcutane 54. The method of claim 53, wherein the pharmaceuti ous, or pulmonary delivery route. cally acceptable vehicle is a polyethylene glycol, a copoly US 2008/0020978 A1 Jan. 24, 2008 mer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl -continued pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an VI ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, an immunoglobulin F, domain, an albu min, a transthyretin, a thyroxine-binding globulin, or a ligand that has an affinity for a long half-life plasma protein, said ligand being selected from peptide ligands and Small wherein R' is independently CFR n=1 to 3; wherein molecule ligands; or a combination of any of these members. Formula VI comprises at least seven carbon atoms; 55. The method of claim 51 or 53, wherein the pharma R" is a (C. to C)linear or branched alkyl moiety, a (C. to ceutically acceptable vehicle is a polyethylene glycol (PEG). C.)cyclic alkyl moiety, or an aryl or heteroaryl moiety; 56. The method of claim 51 or 53, wherein the step of conjugating further comprises reacting the obtained CGRP R° and R are independently H or R'H: peptide with a PEG-aldehyde compound. A is (CY)o, whereino equals 1 to 4, or (CY X—CY), 57. The method of claim 51 or 53, wherein the step of wherein X is O or NR and Y is independently H or F: conjugating further comprises reacting the obtained CGRP R is independently H or a (C. to C) linear or branched peptide with a PEG-maleimide compound. alkyl: 58. The method of claim 51 or 53, wherein the step of conjugating further comprises reacting the obtained CGRP and wherein B is C(R) or N. peptide with a PEG-N-hydroxysuccinimide ester compound. 67. The composition of matter of claim 1, wherein the 59. The method of claim 51 or 53, wherein the step of CGRP peptide comprises an amino acid sequence of the conjugating further comprises reacting the obtained CGRP formula: peptide with a thiol-activated PEG compound. 60. The method of claim 51 or 53, wherein the step of (SEQ ID NO: 1127) conjugating further comprises reacting the obtained CGRP Xaa'XaaXaa-XaaXaaXaaXaa'vXaaXaa'Xaa'L'A' peptide with a diol-activated PEG compound. 61. The method of claim 51 or 53, wherein the step of conjugating further comprises reacting the obtained CGRP peptide with a PEG-hydrazide compound. 62. The method of claim 51 or 53, wherein the step of conjugating further comprises reacting the obtained CGRP Xaa', Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa", are each peptide with a PEG-aminooxy compound. independently absent or a hydrophobic amino acid 63. The method of claim 51 or 53, wherein the step of residue; conjugating further comprises reacting the obtained CGRP peptide with a PEG-bromoacetyl compound. Xaa’ is a hydrophobic amino acid residue, 64. The method of claim 56, wherein reacting the obtained Xaa is a neutral hydrophobic or neutral polar residue: CGRP peptide with a PEG-aldehyde compound is accom plished at a free amine moiety on the CGRP peptide in a Xaa" is a basic amino acid residue, buffer Solution comprising an alcohol co-solvent. Xaa' is a basic amino acid residue; 65. The method of claim 64, wherein the alcohol co solvent is selected from 2.2.2-trifluoroethanol (TFE), 1,1,1, Xaa' is a basic amino acid residue, 3,3,3-hexafluoro-2-propanol (HF-i-PA), and 2-trifluorom ethyl-1,1,1,3,3,3-hexafluoro-2-propanol (HF-t-BuOH). Xaa’ is a neutral amino acid residue or a basic amino acid residue; 66. The method of claim 64, wherein the alcohol co solvent comprises a structure selected from structural For Xaa' is a neutral amino acid residue or a basic amino acid mulae IV, V and VI: residue; Xaaf is a neutral amino acid residue or a basic amino acid IV residue; R2 Xaa is a neutral amino acid residue or a basic amino acid residue; sR3 Xaa' is a neutral polar amino acid residue. V R3 OH Xaa is a cyclic amino acid residue, a neutral hydropho bic amino acid residue, or a neutral polar amino acid RI residue; \y1 - R' Xaa is a neutral hydrophobic or aromatic amino acid residue; and US 2008/0020978 A1 Jan. 24, 2008 74

Xaa is a neutral hydrophobic or aromatic amino acid (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized residue. sites of the PEG. 68. The composition of matter of claim 67, wherein the 82. The composition of matter of claim 1, wherein the CGRP peptide comprises at its N-terminal an acyl, acetyl, CGRP peptide comprises an amino acid sequence of the benzoyl, benzyloxycarbonyl, benzyl, or dibenzyl moiety. formula: 69. The composition of matter of claim 67, wherein: Xaa’ is Trp, 1-Nal, 2-Nal, Phe, Tyr, or Bip. (SEQ ID NO: 1130) 70. The composition of matter of claim 67, wherein: Xaa'XaaXaaXaaXaaXaaXaavXaaXaa'Xaa' 'Xaa'? Xaa is Thr, Ser, Ala, Gly, Val, Leu, or Ile. XaaXaaXaaXaaXaa17XaaXaaXaa2G2W22 71. The composition of matter of claim 67, wherein: Xaa' is selected from His, N-Methyl-His, Trp, 1-Nal, Xaa2xaa2Xaa2xaa2F27w 2P2Xaaxaaw?GXaa 2-Nal, Phe, and Tyr. XaaXaaXaa 72. The composition of matter of claim 67, wherein: wherein: Xaa' and Xaa' are each independently a basic amino acid residue or basic N-substituted amino acid residue Xaa', Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa", are each selected from Arg, N' Methyl-Arg; homoarginine, Cit, independently absent or a hydrophobic amino acid N-Methyl-Cit, Homocitrulline. His, Trp, Guf, and residue; 4-Amino-Phe. Xaa’ is a hydrophobic amino acid residue, 73. The composition of matter of claim 67, wherein: Xaa is a neutral hydrophobic or neutral polar residue; Xaa’ is selected from the group selected from Val, Arg, D-Arg, homoarginine, Lys, D-Lys, homolysine, Orn, Xaa' is a basic amino acid residue. Dab, Dpr, homocysteine, and 4-Amino-Phe. Xaa' is a basic amino acid residue: 74. The composition of matter of claim 67, wherein: Xaa' is Leu, Lys, Homolysine, Ornithine, 4-carboxy Xaa' is selected from Arg, D-Arg, homoarginine, Lys, phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta D-Lys, homolysine, Orn, Dab, Dpr, Homocysteine, and Homoglutamic acid, homoglutamic acid, or Asp; 4-Amino-Phe. Xaa' is Ala, Lys, Homolysine, Ornithine, 4-carboxy 75. The composition of matter of claim 67, wherein: phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta Xaa is selected from Arg, D-Arg, Lys, D-Lys, homol Homoglutamic acid, homoglutamic acid, or Asp; ysine, Orn, Dab, Dpr, Cys, homocysteine, and Xaa' is Gly, Lys, Homolysine, Ornithine, 4-carboxy 4-Amino-Phe. phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta 76. The composition of matter of claim 67, wherein: Homoglutamic acid, homoglutamic acid, or Asp; Xaa is selected from Arg, D-Arg, ASn, Lys, D-Lys, homolysine, Orn, Dab, Dpr, Cys, homocysteine, and Xaa' is Leu, Lys, Homolysine, Ornithine, 4-carboxy 4-Amino-Phe. phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta 77. The composition of matter of claim 67, wherein: Homoglutamic acid, homoglutamic acid, or Asp; Xaa' is selected from Thr, N-Methyl-Thr, Ser, N-Me Xaa' is Leu, Lys, Homolysine, Ornithine, 4-carboxy thyl-Ser, Sar, Pro, Ala, and Gly. phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta 78. The composition of matter of claim 67, wherein: Homoglutamic acid, homoglutamic acid, or Asp; Xaa is selected from Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Xaa' is Ser, Lys, Homolysine, Ornithine, 4-carboxy Thz, Aib, Sar, and Pip. phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta 79. The composition of matter of claim 67, wherein: Homoglutamic acid, homoglutamic acid, or Asp; Xaa is selected from Phe, D-Phe, Tyr, 1-Nal, 2-Nal, Trp, Xaa' is a basic amino acid residue. Bip, and His. Xaa' is Ser, Lys, Homolysine, Ornithine, 4-carboxy 80. The composition of matter of claim 67, wherein: phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta Xaa-7 is selected from Phe, Tyr, 1-Nal, 2-Nal, Trp. His, Homoglutamic acid, homoglutamic acid, or Asp; and Bip. Xaa' is Gly, Lys, Homolysine, Ornithine, 4-carboxy 81. The composition of matter of claim 67, wherein the phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta CGRP peptide is conjugated to a polyethylene glycol (PEG) Homoglutamic acid, homoglutamic acid, or Asp; at Xaa’ is a neutral amino acid residue or a basic amino acid (a) 1, 2, 3 or 4 amino functionalized sites of the PEG, residue; (b) 1, 2, 3 or 4 thiol functionalized sites of the PEG, Xaa' is a neutral amino acid residue or a basic amino acid (c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG, residue; (d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the Xaa is a neutral amino acid residue or a basic amino acid PEG: residue; (e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG, Xaa' is a neutral amino acid residue or a basic amino acid O residue; US 2008/0020978 A1 Jan. 24, 2008

Xaa' is a neutral polar amino acid residue. 89. The composition of matter of claim 82, wherein: Xaa' is Asn. Lys, Homolysine, Ornithine, 4-carboxy Xaa' is Arg, D-Arg, Homoarginine, Lys, D-Lys, Homol phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta ysine, Orn, Dab, Dpr, Homocysteine, or 4-Amino-Phe. Homoglutamic acid, homoglutamic acid, or Asp; 90. The composition of matter of claim 82, wherein: Xaa is Val, Lys, Homolysine, Ornithine, 4-carboxy Xaa is Lys, D-Lys, Homolysine, Orn, Dab, Dpr, Cys, phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta Homocysteine, 4-Amino-Phe, Arg, or D-Arg. Homoglutamic acid, homoglutamic acid, or Asp; 91. The composition of matter of claim 82, wherein: Xaa is Gly, Lys, Homolysine, Ornithine, 4-carboxy Xaa’ is Asn. Lys, D-Lys, Homolysine, Orn, Dab, Dpr. phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta Cys, Homocysteine, 4-Amino-Phe, Arg, or D-Arg. Homoglutamic acid, homoglutamic acid, or Asp; 92. The composition of matter of claim 82, wherein: Xaa' is a cyclic amino acid residue, a neutral hydropho Xaa' is Thr, N-Methyl-Thr, Ser, N-Methyl-Ser, Sar, bic amino acid residue, or a neutral polar amino acid Pro, Ala, Gly, Lys, Homolysine, Ornithine, 4-carboxy residue; phenylalanine, 4-Amino-Phe, beta-glutamic acid, beta Homoglutamic acid, homoglutamic acid, or Asp. Xaa is a neutral hydrophobic or aromatic amino acid 93. The composition of matter of claim 82, wherein: residue; Xaa is a Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Xaa is Ala, Lys, Homolysine, Ornithine, 4-carboxy Pip, Lys, Homolysine, Ornithine, 4-carboxy-phenyla phenylalanine. 4-Amino-Phe, beta-glutamic acid, beta lanine, 4-Amino-Phe, beta-glutamic acid, beta-Homo Homoglutamic acid, homoglutamic acid, or Asp; glutamic acid, homoglutamic acid, or Asp. Xaa is a neutral hydrophobic or aromatic amino acid 94. The composition of matter of claim 82, wherein: residue; and Xaa is a Phe, D-Phe, Tyr, 1-Nal, 2-Nal, Trp, Bip, His, wherein a first pair of amino acid residues selected from LyS, Homolysine, Ornithine, 4-carboxy-phenylalanine, Xaa, Xaa, Xaa, Xaa', Xaa'', Xaa' , Xaa, Xaa, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic Xaa, Xaa, Xaa'7, Xaa, Xaa, Xaa, Xaa, acid, homoglutamic acid, or Asp. Xaa, Xaa, Xaa, Xaa, Xaa, Xaa, and Xaa-7 95. The composition of matter of claim 82, wherein: are covalently joined to form a first ring and the first Xaa-7 is a Phe, Tyr, 1-Nal, 2-Nal, Trp. His, Bip, Lys, pair of amino acid residues forming the first ring are Homolysine, Ornithine, 4-carboxy-phenylalanine, separated by 3 to 7 amino acid residues in the CGRP 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic peptide’s primary sequence. acid, homoglutamic acid, or Asp. 83. The composition of matter of claim 82, wherein the 96. The composition of matter of claim 82, wherein the CGRP peptide comprises at its N-terminal an acyl, acetyl, CGRP peptide is conjugated to a polyethylene glycol (PEG) benzoyl, benzyloxycarbonyl, benzyl, or dibenzyl moiety. at: 84. The composition of matter of claim 82, wherein: (a) 1, 2, 3 or 4 amino functionalized sites of the PEG, Xaa’ is selected from Trp, 1-Nal, 2-Nal, Phe, Tyr, Bip, LyS, Homolysine, Ornithine, 4-carboxy-phenylalanine, (b) 1, 2, 3 or 4 thiol functionalized sites of the PEG, 4-Amino-Phe, 4-Amino-Phe, beta-glutamic acid, beta (c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG, Homoglutamic acid, homoglutamic acid, and Asp. 85. The composition of matter of claim 82, wherein: (d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG: Xaa is selected from Thr, Ser, Ala, Gly, Val, Leu, Ile, Lys, Homolysine, Ornithine, 4-carboxy-phenylalanine, (e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG, 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic O acid, homoglutamic acid, and Asp. (f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized 86. The composition of matter of claim 82, wherein: sites of the PEG. 97. The composition of matter of claim 1, comprising a Xaa' is selected from His, N-Methyl-His, Trp, 1-Nal, CGRP peptide antagonist comprising the amino acid pri 2-Nal, Phe, Tyr, Lys, Homolysine, Ornithine, 4-car mary sequence of any of SEQID NOS:155 through 157, 167 boxy-phenylalanine, 4-Amino-Phe, beta-glutamic acid, through 170, 180 through 228, 242 through 280, 348 through beta-Homoglutamic acid, homoglutamic acid, and Asp. 406, 416 through 430, 439 through 498, 512 through 566, 87. The composition of matter of claim 82, wherein: 580 through 634, 889 through 911, 942 through 944, 987, Xaa' is selected from Arg, N'-Methyl-Arg, homoargin 988, 994 through 996, and 1063 through 1083. ine, Cit, N-Methyl-Cit, Homocitrulline. His, Trp, Guf, 98. The composition of matter of claim 1, comprising a LyS, Homolysine, Ornithine, 4-carboxy-phenylalanine, CGRP peptide antagonist that comprises an amino acid 4-Amino-Phe, beta-glutamic acid, beta-Homoglutamic primary sequence of any of those set forth in Table 2A, Table acid, homoglutamic acid, and Asp. 2B, Table 2C, Table 2D, Table 3A, Table 3B, Table 3, Table 4, or Table 7, except SEQ ID NO:1. 88. The composition of matter of claim 82, wherein: 99. A composition of matter, comprising a CGRP peptide Xaaf is Val, Arg, D-Arg, Homoarginine, Lys, D-Lys, antagonist comprising the amino acid primary sequence of Homolysine, Orn, Dab, Dpr, Homocysteine, or any of SEQID NOS: 155 through 157, 167 through 170, 180 4-Amino-Phe. through 228, 242 through 280, 348 through 406, 416 through US 2008/0020978 A1 Jan. 24, 2008 76

430, 439 through 498, 512 through 566, 580 through 634, (b) any of (a), wherein the amino acid sequence further 889 through 911, 942 through 944, 987, 988, 994 through comprises an N-terminal aromatic amino acid residue. 996, and 1063 through 1083. 107. A composition of matter comprising a CGRP peptide 100. A composition of matter, comprising a CGRP peptide antagonist that has an amino acid sequence comprising: antagonist having an amino acid primary sequence of any of those set forth in Table 2A, Table 2B, Table 2C, Table 2D, (a) any one of SEQ ID NOS: 47 through 55, 82 through Table 3A, Table 3B, Table 3, Table 4, or Table 7, except SEQ 127, 1128 and 1129; or ID NO:1. 101. A method of treating migraine, comprising admin (b) any of (a), wherein the amino acid sequence further istering to a patient in need of Such treatment a therapeuti comprises an N-terminal aromatic amino acid residue. cally effective amount of the composition of matter of any 108. The composition of matter of claim 82, wherein a of claims 1, 26, 99, or 100, such that one or more migraine second pair of amino acid residues, both members of which symptoms is alleviated in the patient. are different from both members of the first pair of amino 102. A method of preventing or mitigating migraine, acid residues, is selected from Xaa’. Xaa, Xaa, Xaa". comprising administering to a patient who has previously Xaa, Xaa' , Xaa, Xaa, Xaa, Xaa, Xaa'7, Xaa, experienced a migraine a prophylactically effective amount Xaa, Xaa, Xaa, Xaa, Xaa’ Xaa, Xaa, Xaa, of the composition of matter of any of claims 1, 26, 99, or Xaa and Xaa, the second pair of amino acid residues 100. Such that at least one symptom of a Subsequent being covalently joined to form a second ring, wherein the migraine is prevented or mitigated. second pair of amino acid residues forming the second ring 103. The composition of matter of claim 1, 26.99, or 100, are separated by 3 to 7 amino acid residues in the CGRP wherein the pharmaceutically acceptable vehicle is a poly peptide’s primary sequence, and wherein both members of alkylene glycol. the second pair of amino acid residues are more proximal in 104. A pharmaceutical composition, comprising the com the primary sequence to either the C-terminal end or the position of matter of claim 98 or 99, and a pharmaceutically N-terminal end of the CGRP peptide compared to both acceptable carrier. members of the first pair of amino acid residues. 105. The method of claim 51 or 53, wherein the pharma 109. The method of claim 64, wherein the concentration ceutically acceptable vehicle is a polyalkylene glycol. of the alcohol co-solvent in the buffer solution is about 30% 106. The composition of matter of claim 1 or claim 26, to about 100% (v/v). comprising the CGRP peptide antagonist having an amino 110. The method of claim 66, wherein if X is O, Y is acid sequence comprising: independently H or F; and if X is NR. Y is H. (a) any one of SEQ ID NOS: 47 through 55, 82 through 127, 1128 and 1129; or