Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/57527—Calcitonin gene related peptide
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to peptide agonists of the calcitonin/calcitonin gene -related peptide (CT/CGRP) family of peptide hormones and therapeutic uses thereof.
• CT/CGRP calcitonin/calcitonin gene -related peptide
• the CT/CGRP peptide family includes calcitonin gene-related peptide (CGRP), adrenomedullin (ADM), intermedin (IM), calcitonin (CT) and amylin.
• CGRP calcitonin gene-related peptide
• ADM adrenomedullin
• IM intermedin
• CT calcitonin
• amylin amylin.
• CTR calcitonin receptor
• CTR calcitonin receptor
• CRLR calcitonin receptor-like receptor
• calcitonin receptor is the main mediator for calcitonin action, it preferentially binds amylin, when the receptor is associated with a receptor activity modifying protein (RAMP) (see, e.g., Tilikaratne, et al. 2000, J. Pharmacol. Exp. Ther. 294(l):61-72).
• RAMP receptor activity modifying protein
• Cloning and functional studies have shown that CGRP, ADM, IM and, to a lesser extent, amylin likewise interact with different combinations of CRLIl and the three receptor activity modifying proteins (RAMP-1, RAMP-2 and R.AMP-3); see, e.g., McLatchie et al 1998, Nature 393:333-339 and Roh et al.
• calcitonin receptor-like receptor CRLR
• RAMPs receptor activity-modifying proteins
• CGRP calcitonin gene-related peptide
• ADM adrenomeduUin
• IM intermedin
• IM has been shown to be a nonselective agonist for all three RAMP/CRLR co-receptors.
• the physiological functions of the hormone peptides in the CT/CGRP family are determined by receptor-binding specificity and the tissue expression profiles of individual ligands and their respective receptors and have been shown to be involved in cardiovascular morphogenesis, sensor ⁇ ' neurotransmission, inflammatory reactions, nociceptive behavior and glucose homeostasis (see, e.g., Hay, et al. 2001, Trends Pharmacol Sci. 22:57-59; Shindo, et al 2001, Circulation 104: 1964-1971; Zhang et al. 2001, Pain 89:265-273; Salmon et al (1999) Neuroreport 10:849-854; Salmon, et al. 2001 , Nat. Neurosci. 4: 357-358; and Mulder, et al 2000, Am. Physiol. 278:E684-E691).
• Calcitonin gene-related peptide is a peptide which, in several species, exists in two forms, designated CGR -alpha and CGRP-beta (or CGRP-I and CGRP-II, respectively).
• CGR -alpha and CGRP-beta or CGRP-I and CGRP-II, respectively.
• CGRP peptides are highly conserved across species, for example, human and rat CGRP-alpha peptides share 89% amino acid homology (the mature peptides differing by four amino acids) compared to 92% amino acid homology between human alpha- and beta-CGRP (which differ by 3 amino acids).
• CGRP is a 37 amino acid vasoactive neuropeptide that is released from sensor ⁇ ', motor and enteric nerves comprising an amphiphilic a-helical secondary structure in the amino acid sequence between residues 8-25.
• CGRP has potent vasodilator ⁇ ' and cardiotonic action, as described, for example, in U.S. Pat. No. 4,530,838 to Evans, et al CGRP is present in both the central and peripheral nervous systems and is concentrated in those areas of the body receiving sensory input from the dorsal horn with limited amounts associated with autonomic input.
• the peptide is present in the nuclei of sensory and motor cranial nerves and in cell bodies in the hypothalamus, preoptic area, ventromedial thalamus, hippocampus, and the like (Poyner, D. 1992, Pharmac. Ther. 56:23-51).
• CGRP is known to be involved in various pharmacological effects, such as: 1 ) vasodilation, 2) muscle and liver AMP kinase (AMPK) activation and lipolysis and/or fat oxidation, 3) reduction in food intake, 4) inhibition of gastric emptying and modification of gut function and 5) increased glycolysis and inhibition of glycogen synthesis, The net physiological significance of these effects is not completely understood; however, evidence of CGRP' s role in fatty acid oxidation and regulation of lipid availability and utilization has been demonstrated (Daiiaher, et al, 2008 Endocrinology 149(1): 154-160).
• AMPK muscle and liver AMP kinase
• Metabolic syndrome is a disease state manifested by obesity, insulin resistance, dyslipidemia and hypertension. Today, these four manifestations are treated by selective treatment paradigms. Native CGRP has a half-life of less than 30 minutes, and a short duration of pharmacological actions after CGRP infusions is evident. Due to vasodilatory effects of administered CGRP, in vivo pharmacological studies of native CGRP can be difficult due to the secondary effects of vasodilation and compensatory' vasoconstrictive actions.
• CGRP pharmacological usefulness of CGRP, particularly with respect to long-term or chronic use thereof requires the generation of CGRP analogues with prolonged action and some effects may even only be obtained with longer-acting analogues.
• analogues having prolonged action are desirable.
• the present invention provides calcitonin gene -related peptide (CGRP) agonists comprising the structure of Formula I:
• X 1 is an N-terminal fragment comprising at least about five to about seven amino acid residues having the general formula:
• X t is Ala or Cys or is not present
• X 2 is Cys, Ser or Giy, provided however that at least one of Xi and X 2 is Cys and only one of Xi or X 2 is Cys
• X 3 is Asp or Asn
• X.i is Ala or Ser and wherein the terminal Cys of X 1 is capable of forming a disulfide bridge with the Cys residue in Xj or X 2
• Y 1 is a central core comprising at least about 12 to about 24 and preferably about 15 amino acid residues wherein at least some of the residues of the central core are capable of forming an a-helix under physiological conditions, said central core having the general formula:
• X 5 Lys Gly Arg X 6 X 7 Gin X s X 9 X 10 Arg X n X i2 Thr X 13 (SEQ ID NO: 7) wherein X 5 is Val or Met, X is Lys or Tyr, X 7 is Ser or Thr, Xs is Asp or Glu, X9 is Phe or Lys, X 10 is His or Asn, Xn is Phe or Lys, X 12 is His or Gin, X i3 is Phe or Tyr;
• Z 1 is a C-terminal fragment comprising at least about six to about twelve amino acid residues, preferably about ten amino acid residues, said C-terminal fragment comprising a C-terminal amide, said fragment having the general formula:
• CGRP agonist has a higher binding affinity for human CGRP receptor than does human CGRP
• X 1 comprises 6 or 7 amino acids. In some embodiments, Y comprises 15 amino acids. In some embodiments, Z comprises 10 amino acids. In some embodiments, X comprises 6 or 7 amino acids, Y comprises 15 amino acids andl r ) comprises 10 amino acids.
• X is Cys, and X " is Ser or Gly.
• X * is Cys, and X 1 is Ala.
• X 1 comprises SEQ ID NO: 9.
• Z comprises SEQ ID NO: 10.
• a calcitonin gene-related peptide (CGRP) agonist is provided.
• the calcitonin gene-related peptide (CGRP) agonist selected from the group comprising SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 or a pharmaceutically acceptable salt thereof.
• the calcitonin gene-related peptide (CGRP) agonist comprises at least 90% sequence identity to the amino acid sequence of SEQ ID NO:l , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5 or a pharmaceutically acceptable salt thereof.
• the calcitonin gene-related peptide (CGRP) agonist comprises at least 80% sequence identity to the amino acid sequence of SEQ ID N(): l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID N():5 or a pharmaceutically acceptable salt thereof
• the calcitonin gene-related peptide (CGRP) agonist comprises an N-terminal addition of an XTENS polypeptide.
• the calcitonin gene-related peptide (CGRP) agonist comprises an amino terminus modified with a derivative group, In some embodiments, the derivative group is selected from acetyl, aryl, aralkyl, acyl, epoxysuccinyl and cholesteryl groups.
• the calcitonin gene-related peptide (CGRP) agonist comprises a carboxy-terminus modified with a derivative group.
• the derivative group is selected from alcohol, aldehyde, epoxysuccinate, acid halide, carbonyl, halomethane, diazomethane groups and carboxamide.
• the calcitonin gene-related peptide (CGRP) agonist comprises a C-terminal carboxy group replaced by an amide group, In some embodiments, the amide group comprises the structure --C(0)NH 2 .
• a pharmaceutical composition comprising a calcitonin gene -related peptide (CGRP) agonist of the present invention.
• the pharmaceutical composition comprises an acceptable excipient and any of the embodiments described above.
• a method of treating a metabolic disorder comprises administering to a subject in need thereof an effective amount of the calcitonin gene-related peptide (CGRP) agonist of the present invention.
• the subject is human.
• a method of treating a metabolic disorder selected from the group comprising metabolic syndrome, diabetes and obesity comprises administering to a subject in need thereof an effective amount of the calcitonin gene-related peptide (CGRP) agonist.
• the subject is human.
• a method of increasing glycolysis in a subject comprises administering an effective amount of the calcitonin gene-related peptide(CGRP) agonist of the present invention.
• a method of increasing AMPK activation in a subject is provided.
• the method comprises administering an effective amount of the calcitomn gene -related peptide (CGRP) agonist of the present invention.
• the subject is suffering from insulin resistance. In some embodiments, the subject is suffering from type-2 diabetes mellitus. In some embodiments, the subject is suffering from hypertension. In some embodiments, the subject is suffering from dysiipidaemia. In some embodiments, the subject is suffering from atherosclerosis, In some embodiments, the subject is suffering from thrombosis. BRIEF DESCRIPTION OF THE DRAWINGS
• FIG, 1 provides results of the CALCRL:RAMP1 - CRE ⁇ bia Freestyle 293F agonist screen described in Example 1 below,
• the graph of concentration of ACX017 (SEQ ID NO: 1) versus percent activation of the CGRP receptor demonstrates that a CGRP agonist of the present invention activated the CGRP receptor with an EC50 concentration of about 105nM.
• FIG. 2 provides results of the CALCRL:RAMP3 - CRE-bla Freestyle 293F agonist screen described in Example 1 below.
• the graph of concentration of ACX017 (SEQ ID NO: 1) versus percent activation of the adrenomedullin 2 (AM 2) receptor demonstrates that a CGRP agonist of the present invention did not acti vate the adrenomedullin 2 ( AM2) receptor.
• FIGS. 3-6 provide results of the four-day food intake study, described in Example 2 herein. The effect on cumulative food intake for each of Days 1 -4 are shown in FIGS. 3-6, respectively. Graphed is cumulative food intake of each of the test group (single dose of ACX017 (SEQ ID NO: l) and placebo group over the course of eight hours.
• the present invention provides calcitonin gene-related peptide agonist having the structure of Formula I:
• X 1 is an N-termina! fragment comprising at least about five to about seven amino acid residues having the general formula:
• X ⁇ is Ala or Cys or is not present
• X 2 is Cys, Ser or Gly, provided however that at least one of Xj and X 2 is Cys and only one of Xj or X 2 is Cys
• X 3 is Asp or Asn and 3 ⁇ 4 is Ala or Ser and wherein the terminal Cys of X 1 is capable of forming a disulfide bridge with the Cys residue in X ⁇ or X 2 ;
• Y 1 is a central core comprising at least about 12 to about 24 and preferably about 15 amino acid residues wherein at least some of the residues of the central core are capable of forming an a-helix under physiological conditions, said central core having the general formula:
• X 5 is Val or Met
• Xg is Lys or Tyr
• X 7 is Ser or Thr
• X 8 is Asp or Glu
• X9 is Phe or Lys
• X 10 is His or Asn
• Xn is Phe or Lys
• X 12 is His or Gin
• X 13 is Phe or Tyr;
• Z 1 is a C -terminal fragment comprising at least about six to about twelve amino acid residues, preferably about ten amino acid residues, said C-terminal fragment comprising a C-terminal amide, said fragment having the general formula:
• X 14 is Arg or Gin and X 15 is Asn or Ala;
• CGRP agonist has a higher binding affinity for human CGRP receptor than does human CGRP.
• X 1 comprises the sequence, SEQ ID NO: 9 (ACDTATC), [0030]
• Z 1 comprises the sequence, SEQ ID NO: 10 (PRTNVGSKAF).
• Some CGRP agonists comprise: an amino acid sequence having at least 80%, preferably 90%, sequence identity to the amino acid sequence of SEQ ID NOS: 1, 2, 3, 4 or 5 wherein said peptide retains agonist activity.
• Some embodiments provide a pharmaceutical composition comprising a pharmaceutically acceptable excipient and a calcitonin gene-related peptide agonist as disclosed and described herein.
• Some embodiments provide a method of treating a condition associated with insufficient levels of CGRP or that are ameliorated by increased CGRP said method comprising the administration of a calcitonin gene -related peptide agonist as disclosed and described herein, to an individual, the method comprising administering to the individual an effective amount of a calcitonin gene-related peptide agonist as disclosed and described herein.
• Some embodiments provide a calcitonin gene-related peptide agonist having the structure selected from the following peptide sequences, listed in Table 1.
• the amino acid sequence of the calcitonin gene-related peptide agonist can be modified, relative to the sequence of SEQ ID NOS: 1, 2, 3, 4 or 5 such that the modification reduces the calcitonin gene -related peptide agonist's susceptibility to enzymatic proteolysis, in some embodiments this modification may comprise the -terminal addition of a sequence comprising all or part of the 864 residue XTENS polypeptide, a polypeptide that has been shown to increase protein stability after administration to a subject. See, for example, Schellenberger, et al, 2009, Nature Biotechnology 27(12): 1186-1192, which is hereby incorporated by reference in its entirety.
• agonist refers to a biologically active ligand which binds to its complementary biologically active receptor and activates the latter either to cause a biological response in the receptor or to enhance preexisting biological activity of the receptor.
• amides are typically formed from the corresponding carboxylic acid and an amine.
• amide formation can be accomplished via conventional synthetic techniques. See, for example, March, 1992 Advanced Organic Chemistry, 4th Ed., John Wiley & Sons, New York, p. 393 and Mark, et al, 1980 Encyclopedia of Chemical Technology, John Wiley & Sons, New York.
• a-helix means a structural component that forms an a-helical protein structure or any other structural analogue which results in a similar positioning of the X and Z domains on a receptor
• pharmaceutically acceptable carrier refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
• pharmaceutically acceptable salt refers to the non-toxic alkali metal, alkaline earth metal, and ammonium salts commonly used in the pharmaceutical industry including the sodium, potassium, lithium, calcium, magnesium, barium, ammonium, and protamine zinc salts, which are prepared by methods well known in the art.
• non-toxic acid addition salts which are generally prepared by reacting the modified calcitonin gene-related peptide agonists disclosed herein with a suitable organic or inorganic acid.
• Representative salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, oxalate, valerate, oleate, laurate, borate, benzoate, lactate, phosphate, tosyiate, citrate, maleate, fumarate, succinate, tartrate, napsylate, and the like.
• the term refers to those salts which retain the biological effectiveness and properties of the free bases and which are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycoiic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, menthanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
• organic acids such as acetic acid, propionic acid, glycoiic acid, pyruvic acid,
• terapéuticaally- or pharmaceutically-effective amount refers to the amount of composition sufficient to induce a desired biological result. That result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
• amino- and/or earboxy-terminus of the peptide compounds disclosed herein can be unmodified hydrogen.
• the amino- and/or carboxy-terrninus of the peptide compound can be modiiied with a derivative group.
• ⁇ -derivative groups which can be present at the N-temiinus of a peptide compound include acetyl, aryl, araikyl, acyl, epoxysuccinyi and cholesteryl groups.
• Carboxy-derivatrve groups which can be present at the C- terminus of a peptide compound include alcohol, aldehyde, epoxysuccinate, acid halide, carbonyl, halomethane, diazomethane groups and carboxamide.
• the amino terminus is unmodified hydrogen and the carboxy-terrninus is modified with a carboxamide derivative group.
• Amino acids are sometimes referred to herein by the usual three letter codes and/or one letter codes. If, due to typing errors, there are deviations from the commonly used codes, the commonly used codes apply.
• Percent (%) amino acid sequence identity with respect to the polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST ' , BLAST-2, ALIGN, ClustalW ' 2, Kalign, MA FFT, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• calcitonin gene-related peptide agonists described herein can be prepared by classical methods known in the art, for example, by using standard solid phase techniques. See, for example, Merrifield, 1963 J. Am. Chem. Soc. 85:2149, incorporated herein by reference in their entirety.
• CGRP agonists of the present invention preferably comprise a C-terminal amide, wherein the C-terminal carboxyl group is replaced by an amide ⁇ C(0)NR'R 4 , wherein R' and R 4 are preferably hydrogen (H) but may either or both be a functional group selected, for example, to protect the C-terminal amide from enzymatic degradation and/or to prolong the half- life of the peptide compound in vivo, provided however such functional groups do not interfere with, or are modified or removed in vivo to permit, binding of the peptide compound to the CGRP receptor.
• Such functional groups are well known to those of skill in the art; see for example, ahns, A. and Bundgaard, H. 1991 Pharmaceutical Research 8(12):1533-1538. Techniques for synthesis of such C-terminal amides are also well known to those of skill in the art.
• the calcitonin gene-related peptide agonists as disclosed and described herein may also be prepared by recombinant DMA techniques well known in the art.
• Such recombinantly produced CGRP peptide agonists may be further modified, for example to replace the C-terminal carboxyl group with an amide, after recombinant production using techniques well known in the art, including for example, enzymatic conversion of the acid to the amide.
• compositions containing the CGRP peptide agonists of the present invention can be administered for prophylactic and/or therapeutic treatments.
• compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest or ameliorate the symptoms of the disease and/or its complications.
• An amount adequate to accomplish this is defined as a "therapeutically effective amount” or “therapeutically effective dose”. Amounts effective for this use will depend, for example, on the severity of the disease and the weight and general state of the patient, and can be readily determined by one of ordinary skill in the art.
• compositions containing the compounds disclosed herein are administered to a patient susceptible to or otherwise at risk of a particular disease and are sufficient to prevent, delay or lessen the severity of the disease.
• Such an amount is defined to be a "prophylactically effective amount” or “prophylactically effective dose.”
• the precise amounts again depend, for example, on the patient's state of health and weight, and can be readily determined by one of ordinary skill in the art.
• a therapeutically effective amount of a compound of the present invention is administered to a subject (for example, patient or animal) suffering from insulin resistance, type-2 diabetes mellitus, hypertension, obesity, dyslipidaemia, atherosclerosis and/or thrombosis.
• the dosage ranges for the administration of a compound of this invention are those large enough to produce a therapeutic effect.
• compositions comprising, as an active ingredient, at least one of the peptides disclosed herein in association with a pharmaceutical carrier or diluent.
• These pharmaceutical compositions can be administered by any means, as known to those of ski ll in the art, and include, without limitation, oral, pulmonary, parenteral (intramuscular, intraperitoneal, intravenous, or subcutaneous injection), inhalational (via a fine powder formulation, or aerosol), transdermal, intranasal or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration.
• parenteral intramuscular, intraperitoneal, intravenous, or subcutaneous injection
• inhalational via a fine powder formulation, or aerosol
• transdermal intranasal or sublingual routes of administration and can be formulated in dosage forms appropriate for each route of administration.
• Suitable routes of administration are likewise known to those of skill in the art and may include, for example and without limitation, oral, transmucosal or topical; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, intraperitoneal, intranasal, or intraocular injections, as well as needle-free subcutaneous delivery.
• parenteral delivery including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, intraperitoneal, intranasal, or intraocular injections, as well as needle-free subcutaneous delivery.
• the compounds can also be administered in sustained or controlled release dosage forms, including without limitation, depot injections, osmotic pumps, transdermal (including electrotransport) patches, and the like, for prolonged and/or timed, pulsed administration at a predetermined rate.
• compositions of the present embodiments may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tab letting processes.
• compositions for use in accordance with the present embodiments thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically and which are appropriate for the intended route of administration.
• physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically and which are appropriate for the intended route of administration.
• excipients Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences, above.
• the amount of a compound of this invention administered to an animal to achieve a desired level or concentration of the compound of this invention will depend on a number of factors well known to practitioners, such as compound half-life (for example, serum half-life), and the frequency and mode of administration.
• compound half-life for example, serum half-life
• the dose of a compound of the present invention may be administered in the range from 20 picograms to 1 gram, more often between 3 nanograms and 500 micrograms per dose.
• the unit dosage (in some cases daily dosage) is less than about 100 micrograms, less than about 10 micrograms, less than about 1 microgram, less than about 100 nanograms, less than about 10 nanograms, less than about 1 nanogram, less than about 100 picograms, or less than about 10 picograms.
• compositions are administered 1 to 4 times per day or as a single acute dose.
• peptides will be administered for a period of continuous therapy, for example for a week or more, or for months or years,
• Dosage amount and interval may be adjusted individually to provide plasma levels of the active moiety which are sufficient to maintain the modulating effects, or minimal effective concentration (MEC).
• MEC minimal effective concentration
• the MEC will vary for each compound but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations.
• Dosage intervals can also be determined using MEC value.
• Compositions should be administered using a regimen which maintains plasma levels above the MEC for 10- 90% of the time, preferably between 30-90% and most preferably between 50-90%.
• a compound of the present invention can be formulated or co-administered with other active agents that, for example, also treat metabolic or cardiovascular diseases/disorders.
• the compounds described herein are useful in vitro as unique tools for understanding the biological role of CORP.
• the present compounds are also useful in the development of other compounds that bind to and activate CGRP receptors, because the present compounds provide important information on the relationship between structure and activity to facilitate such development.
• C ALCRL RAMP 1 - CRE-bla Freestyle 293F - Agonist Screen (CGRP Receptor) [0068]
• CALCRL RAMP 1 -CRE-bla Freestyle 293F cells were thawed and re-suspended In Assay Media (DMEM, 10% dialyzed FBS, 25 mM HEPES pH 7,3, 0, 1 mM
• NEAA 100 U/mL/IOO ⁇ ' ⁇ Pen Strep
• 32 ⁇ of ceil suspension 10,000 cells
• Cells in Assay Media were incubated for 16-24 hours in the plate at 37°C/5% C02 in a humidified incubator.
• 4 p.L of a 10X serial dilution of CGRP or ACX017 was added to appropriate wells of the plate.
• 4 ⁇ of Assay Media was added to all wells to bring the final assay volume to 40 ⁇ ,.
• the plate was incubated for 5 hours at 37°C/5% C02 in a humidified incubator, 8 ⁇ of 1 ⁇
• Substrate + Solution D Loading Solution was added to each well and the plate was incubated for
• CALCRL:RAMP3 CRE-bla Freestyle 293F - Agonist Screen (Adrenomedullin 2 Receptor)
• CALCRL:RAMP3 -CRE-bla Freestyle 293F cells were thawed and resuspended In Assay Media (DMEM, 10% dialyzed FBS, 25 mM HEPES pH 7,3, 0, 1 mM
• NEAA 100 U/mL/100 ⁇ ⁇ Pen/Strep
• 32 ⁇ of cell suspension 10,000 cells was added to each well of a 384-well Poly-D-Lysine assay plate.
• Cells in Assay Media were incubated for 16-24 hours in the plate at 37°C/5% C02 in a humidified incubator.
• 4 ⁇ iL of a 10X serial dilution of Adrenomedullin or ACX017 were added to appropriate wells of the plate.
• 4 ⁇ . of Assay Media was added to all wells to bring the final assay volume to 40 ⁇ ,
• the plate was incubated for 5 hours at 37°C/5% C02 in a humidified incubator.
• 8 ⁇ , of 1 ⁇ Substrate + Solution D Loading Solution was added to each well and the plate was incubated for 2 hours at room temperature. The plate was read on a fluorescence plate reader,
• Full Agonist control The full agonist control contained 0, 1% DMSO, cells and a maximum concentration of the known agonist, The full agonist control was used to determine the upper end of the assay or 100%) activation.
• the no agonist control contained 0.1 % DMSO, cells and assay media in place of the agonist, The no agonist control was used to determine the lower end of the assay or 0% ac tivation,
• Cell-free Control The cell-free control contained 0, 1% DMSO and assay media, It was used to determine the background fluorescence for both coumarin and fluorescein wavelengths. This value was used for background subtraction.
• Known Agonist A known agonist titration was run on every plate for each cell-line to ensure the cell line was activated within an expected EC50/IC50 range as previously determined.
• FIG. 1 shows that ACX017 demonstrated agonist activity against the CGRP receptor with an approximate EC50 concentration of 105nM.
• FIG. 2 shows that ACX017 did not activate the adrenoniedullin 2 (AM2) receptor. As such ACX017 was shown to exhibit binding and functional specificity to the CGRP receptor in a dose dependent response.
• AM2 adrenoniedullin 2
• Food consumption was measured pre-dose and at 2, 4, 6 and 8 hours post dose. Food intake was measured by weighing food hoppers on Days 1 , 2, 3 and four in the hour before end of the light phase and at 2, 4, 6 and 8 hours post dosing.

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