US00725.6253B2

(12) United States Patent (10) Patent No.: US 7,256.253 B2 Bridon et al. (45) Date of Patent: Aug. 14, 2007

(54) PROTECTION OF ENDOGENOUS 6,500,918 B2 12/2002 Ezrin et al. THERAPEUTIC FROM 6,514,500 B1 2/2003 Bridon et al. PEPTIDASE ACTIVITY THROUGH 6,593,295 B2 7/2003 Bridon et al. CONUGATION TO BLOOD COMPONENTS 6,602,981 B2 8/2003 Ezrin et al. 6,610,825 B2 8, 2003 Ezrin et al. (75)75 Inventors: Dominique P. Bridon, San Francisco, 6,706,892 B1 3/2004 Ezrin et al. CA (US); Alan M. Ezrin, Moraga, CA (US); Peter G. Milner, Los Altos, CA 6,849,714 B1 2/2005 Bridon et al. (US); Darren L. Holmes, Anaheim, CA 2002fOO18751 A1 2/2002 Bridon et al. (US); Karen Thibaudeau, Rosemere 2003, OO73630 A1 4/2003 Bridon et al. (CA) 2003/O105867 A1 6/2003 Colrain et al. 2003. O108568 A1 6/2003 Bridon et al. (73) Assignee: Conjuchem Biotechnologies Inc., 2003/0170250 A1 9, 2003 EZrin et al. Montreal (CA) 2004/O127398 A1 7, 2004 Bridon et al. (*) Notice: Subject to any disclaimer, the term of this 2004/O138100 A1 7/2004 Bridon et al. patent is extended or adjusted under 35 2004/O156859 A1 8, 2004 Ezrin et al. U.S.C. 154(b) by 0 days. 2004/0248782 A1 12/2004 Bridon et al. 2004/0266673 Al 12/2004 Bakis et al. (21) Appl. No.: 11/066,697 2005, 0037974 A1 2/2005 Krantz et al. 2005, OO65075 A1 3, 2005 Erickson et al. (22) Filed: Feb. 25, 2005 2005, 0070475 A1 3, 2005 Bridon et al. (65) Prior Publication Data US 2005/O187159 A1 Aug. 25, 2005 FOREIGN PATENT DOCUMENTS Related U.S. Application Data DE 252539 12/1987 (63) Continuation of application No. 09/657.276, filed on Sep. 7, 2000, now Pat. No. 6,887,470. (51) Int. Cl. (Continued) A6 IK 38/00 (2006.01) C07K I4/00 (2006.01) OTHER PUBLICATIONS (52) U.S. Cl...... r r 530/300: 514/2; 424/185.1 Gunnes et al., “Haemodynamic effects of pharmacological doses of (58) Field of Classification Search ...... None in patients with impaired left ventricular function'. Euro See application file for complete search history. pean Journal 7: 146-149 (1986). DO.* (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner Robert A. Wax 4,206,199 A 6/1980 Fujino et al. Assistant Examiner—Anand Desai 4,251,631 A 2f1981 Simon (74) Attorney, Agent, or Firm Jones Day 4,423,034 A 12/1983 Nakagawa et al. 4.462,941. A 7/1984 Lee et al. (57) ABSTRACT 4,859,604 A 8, 1989 Gould et al. 5,493,007 A 2f1996 Burnier et al. is: 121: i.e., al. A secretin O secretin derivative protected against peptidase 5,654,276 A 8, 1997 Barrett et al. activity. The secretin or derivative comprises a peptidic 5,837.247 A 1 1/1998 Oppenhelmet al. sequence and a reactive group selected from the group 5,840,733. A 11/1998 Krantz et al. consisting of Succinimidyl and maleimido groups capable of 5,843.440 A 12/1998 Pouletty et al. reacting with an amino group, hydroxyl group or thiol group 5,874,408 A 2/1999 Nayar on a blood component to form a stable covalent bond. The 5,877,151 A 3, 1999 Pereira reactive group is attached at a position along the peptidic 5,942,620 A 8, 1999 Krantz et al. sequence that provides, when conjugated to a blood com 6,087,375 A 7/2000 Bridon et al. ponent, a higher stability against peptidase degradation than 6,103,233 A 8/2000 Pouletty et al. the unconjugated secretin or derivative, and therefore an 6,107,489 A 8, 2000 Krantz et al. increased maintenance of the therapeutic activity compared 6, 197,813 B1 3/2001 Hegenauer to the unconjugated secretin or derivative. Such a compound 6,277,583 B1 8, 2001 Krantz et al. is thus effective to provide a source of secretin having a high 6,277,863 B1 8, 2001 Krantz et al. stability against peptidases. A method for synthesizing Such 6,329,336 B1 12/2001 Bridon et al. 6,403,324 B1 6, 2002 Krantz et al. a compound is also described. 6,437,092 B1 8, 2002 Ezrin et al. 6,440,417 B1 8/2002 Thibaudeau et al. 2 Claims, No Drawings US 7,256.253 B2 Page 2

FOREIGN PATENT DOCUMENTS Smith, Francine et al. (1989) “Atrial Natriuretic Factor During Fetal and Postnatal Life: A Review,” J. Dev Physiol., 12:55-62. DE 1993678O 2, 2001 EP O602290 6, 1994 Stehle, Gerd et al. (1997) “The Loading Rate Determines Tumor WO WO-93,25217 12/1993 Targeting Properties of Methotrexate-Albumin Conjugates in Rats.” WO WO-95.00534 1, 1995 Anti-Cancer Drugs, 8:677-685. WO WO-95/10302 4f1995 Oren, Ziv et al. (1998) “Mode of Action of Linear Amphipathic WO WO-96,06626 3, 1996 O-Helical Antimicrobial Peptides.” Biopolymers ( Science), WO WO-96,28544 9, 1996 47:451-463. WO WO-99/24075 5, 1999 Akil. Huda et al. (1984) “Endogenous Opioids: Biology and Func WO WO-99.244.62 5, 1999 WO WO-99/48536 9, 1999 tion.” Ann. Rev. Neurosci., 7:223-255. WO WOOOf 69900 11 2000 Hirai, Yuko et al. (1979) “A New Mast Cell Degranulating Peptide WO WO-OOf 76550 12/2000 Mastoparan in the Venom of Vespula Lewisii.” Chem. Pharm. WO WO-OOf 76551 12/2000 Bull., 27(8): 1942-1944. Mumby, Susanne et al. (Jan. 1986) “Antisera of Designed Speci OTHER PUBLICATIONS ficity for Subunits of Guanine Nucleotide-Binding Regulatory Pro Paige et al., “Prolonged circulation of recombinant human granu teins.” Proc. Natl. Acad. Sci. 83:265-269. locyte-colony stimulating factor by covalent linkage to albumin Otteson, Kenneth M. (1983) “Comparisons of Peptide Sequences through a heterobifunctional polyethylene glycol. Pharmaceutical with Deprotection Problems in Fmoc Solid Phase Peptide Synthe Research 12(12): 1883-1888 (1995).* sis.” Proceedings of the 8" American Peptide Symposium, pp. U.S. Appl. No. 09/424,571, Ezrin et al. 409-412. U.S. Appl. No. 09/530,891, Blanchard et al. Selkoe, Dennis J. “Physiological Production of the B-Amyloid U.S. Appl. No. 09/623.533, filed Sep. 5, 2000, Boudjellab et al. Protein and the Mechanism of Alzheimer's Disease.” TINS, U.S. Appl. No. 09/623.543, filed Sep. 5, 2000, Beliveau et al. 16(10):403-409. U.S. Appl. No. 09/657,336, filed Sep. 7, 2000, Boudjellab et al. Ito, K. et al. (Dec. 1995) “Preparation of Specific Antiserum of U.S. Appl. No. 09/657.431, filed Sep. 7, 2000, Beliveau et al. Secretin and its Use in Time-Resolved Fluoroimmunoassay,” Jour U.S. Appl. No. 11/040,810, filed Jan. 21, 2005, Bakis et al. nal of the Pharmaceutical Society of Japan, abstract. U.S. Appl. No. 1 1/067.556, filed Feb. 25, 2005, Bidon et al. Singer, M. V. et al. (1981) “Effect of Adding Albumin to Solutions Breton, J. et al. (Aug. 1995) “Prolonged Half-life in the Circulation of Secretin on Pancreatic Volume and Bicarbonate Response.” of a Chemical Conjugate Between a Pro-urokinase Derivative and Scandinavian Journal of Gastroenterology, abstract. Human Serum Albumin.” Eur: J. Biochem. 231(3):563-9. “Trimethylaminuria (Fish-Malodor Syndrome) and the Flavin Davis et al., 1991, in Peptide and Protein Drug Delivery, V.H.L. Lee Monooxygenases.” Biothech Report, Abstract, 1994/1995 pp. 106 (ed.), Chap. 21, Marcel Dekker, Inc., pp. 831-864. 107. Poznansky M.J. "Enzyme-protein conjugates: new possibilities for Thim. Lars et al. (1998) “Molecules in Focus: CART, a new enzyme therapy.” Pharmacol Ther. 1983:21(1):53-76. Anorectic Peptide.” Int. J. Biochem. Cell. Biol. 30: 1281-1284. Poznansky et al. “Growth -albumin conjugates. Reduced Endocrinology, 1982, 110(3):1049-1051. renal toxicity and altered plasma clearance.” FEBS Lett. Oct. 24. Kapas, Supriya et al. (Oct. 27, 1995) “Cloning and Expression of 1988:239(1):18-22. cDNA Encoding a Rat Adrenomedullin Receptor,” Journal of Biological Chemistry, 270(4):25344-25347. * cited by examiner US 7,256.253 B2 1. 2 PROTECTION OF ENDOGENOUS sive optimization programs to attempt to develop non THERAPEUTIC PEPTIDES FROM peptide, organic compounds which mimic the activity seen PEPTIDASE ACTIVITY THROUGH with therapeutic peptides without incurring an unacceptable CONUGATION TO BLOOD COMPONENTS side effect profile. For example, cyclic peptides, peptidomi metics and Small molecules coming from expensive SAR RELATED APPLICATIONS (Structure Activity Relationship) and molecular modeling studies have led to the development of an incredible amount This application is a Continuation Application of U.S. of peptide mimics. However, these peptide mimics in no patent application Ser. No. 09/657.276 filed Sep. 7, 2000 way reflect the exact original biological nature of the thera now U.S. Pat No. 6,887,470, which is expressly incorpo 10 peutic peptide, and thus are inferior to the endogenous rated by reference in its entirety. therapeutic peptide as therapeutic agents. An alternative to creating peptide mimics is to block the FIELD OF THE INVENTION action of peptidases to prevent degradation of the therapeu tic peptide or to modify the therapeutic peptides in Such a This invention relates to modified therapeutic peptides. In 15 way that their degradation is slowed down while still main particular, this invention relates to protection of endogenous taining biological activity. Such methods include conjuga therapeutic peptides from peptidase activity through a modi tion with polymeric materials such as dextrans, polyvinyl fication that enables the peptide to selectively conjugate to pyrrolidones, glycopeptides, polyethylene glycol and blood components, thus protecting the peptide from pepti polyamino acids, conjugation with adroitin Sulfates, as well dase activity and increasing the duration of action of the as conjugation with polysaccharides, low molecular weight therapeutic peptide for the treatment of various disorders. compounds such as aminolethicin, fatty acids, vitamin B, and glycosides. These conjugates, however, are still often BACKGROUND OF THE INVENTION Susceptible to protease activity. In addition, the therapeutic activity of these peptides is often reduced by the addition of Many endogenous peptides have been described as key 25 the polymeric material. Finally, there is the risk of the components of biological processes. Some of these peptides conjugates generating an immune response when the mate have been identified as key therapeutic agents for the rial is injected in vivo. Several methods include ex vivo management of various disorders. In general, endogenous conjugation with carrier proteins, resulting in the production peptides are more desirable as therapeutic agents than Syn of randomized conjugates. Since conjugates are difficult to thetic peptides with non-native sequences, because they do 30 manufacture, and their interest is limited by commercial not produce an immune response due to their endogenous availability of the carriers, as well as by their poor pharmaco character. In addition, endogenous peptides are highly spe economics. cific for their target receptors and are easy to synthesize and There is thus a need for novel methods to modify thera manufacture. However, a major difficulty with the delivery peutic peptides to protect them from peptidase activity and of such therapeutic peptides is their short plasma half-life, 35 to provide longer duration of action in vivo, while main mainly due to rapid serum clearance and proteolytic degra taining low toxicity yet retaining the therapeutic advantages dation via the action of peptidases. of the modified peptides. Peptidases break a peptide bond in peptides by inserting a water molecule across the bond. Generally, most peptides SUMMARY OF THE INVENTION are broken down by peptidases in the body in a manner of 40 a few minutes or less. In addition, Some peptidases are This invention is directed to overcoming the problem of specific for certain types of peptides, making their degrada peptide degradation in the body by modifying the therapeu tion even more rapid. Thus, if a peptide is used as a tic peptide of interest and attaching it to protein carriers, therapeutic agent, its activity is generally reduced as the Such that the action of peptidases is prevented, or slowed peptide quickly degrades in the body due to the action of 45 down. More specifically, this invention relates to novel peptidases. chemically reactive derivatives of therapeutic peptides that One way to overcome this disadvantage is to administer can react with available functionalities on blood proteins to large dosages of the therapeutic peptide of interest to the form covalent linkages, specifically a therapeutic peptide patient so that even if some of the peptide is degraded, maleimide derivative. The invention also relates to novel enough remains to be therapeutically effective. However, 50 chemically reactive derivatives or analogs of Such therapeu this method is quite uncomfortable for the patient. Since tic peptides. The invention additionally pertains to the most therapeutic peptides cannot be administered orally, the therapeutic uses of Such compounds. therapeutic peptide would have to be either constantly The present invention is directed to modifying and attach infused, frequently administered by intravenous injections, ing therapeutic peptides to protein carriers, preferentially or administered frequently by the inconvenient route of 55 albumin, through in vivo or ex vivo technology to prevent or Subcutaneous injections. The need for frequent administra reduce the action of peptidases by virtue of a synthetic tion also results in many potential peptide therapeutics modification on the first residue to be cleaved. Therapeutic having an unacceptably high projected cost per treatment peptides are usually active at the N-terminus portion, at the course. The presence of large amounts of degraded peptide C-terminus portion, or in an interior portion of the peptide may also generate undesired side effects. 60 chain. Using the technology of this invention, a site other Discomfort in administration and high costs are two than the active portion of a therapeutic peptide is modified reasons why most therapeutic peptides with attractive bio with certain reactive groups. These reactive groups are activity profiles are not developed as drug candidates. capable of forming covalent bonds with functionalities Instead, these therapeutic peptides are used as templates for present on blood components. The reactive group is placed the development of peptidomimetic compounds to Substitute 65 at a site such that when the therapeutic peptide is bonded to for the therapeutic peptide. Biotechnology and large phar the blood component, the peptide retains a Substantial pro maceutical firms frequently undertake lengthy and expen portion of the parent compounds activity. US 7,256.253 B2 3 4 The modification of the therapeutic peptide through the The present invention is also directed to a method for chemical modification used in the invention is done in Such protecting a therapeutic peptide from peptidase activity in a way that all or most of the peptide specificity is conserved vivo, the peptide being composed of between 3 and 50 despite attachment to a blood component. This therapeutic amino acids and having a carboxy terminus and an amino peptide-blood component complex is now capable of trav terminus and a carboxy terminal amino acid amino acid and eling to various body regions without and being degraded by an amino terminal amino acid. The method comprises the peptidases, with the peptide still retaining its therapeutic following steps: activity. The invention is applicable to all known therapeutic (a) modifying the peptide by attaching a reactive group to peptides and is easily tested under physiological conditions the carboxy terminal amino acid, to the amino terminal by the direct comparison of the pharmacokinetic parameters 10 amino acid, or to an amino acid located between the amino for the free and the modified therapeutic peptide. terminal amino acid and the carboxy terminal amino acid, The present invention is directed to a modified therapeutic Such that the modified peptide is capable of forming a peptide capable of forming a peptidase stablilized therapeu covalent bond in vivo with a reactive functionality on a tic peptide composed of between 3 and 50 amino acids. The blood component; peptide has a carboxy terminal amino acid, an amino ter 15 (b) forming a covalent bond between the reactive group minal amino acid, a therapeutically active region of amino and a reactive functionality on a blood component to form acids and a less therapeutically active region of amino acids. a peptide-blood component conjugate, thereby protecting The peptide comprises a reactive group which reacts with the peptide from peptidase activity; and amino groups, hydroxyl groups, or thiol groups on blood (c) analyzing the stability of the peptide-blood component components to form a stable covalent bond and thereby conjugate to assess the protection of the peptide from forms the peptidase stabilized therapeutic peptide. In the peptidase activity. peptide of the invention the reactive group is selected from the group consisting of Succinimidyl and maleimido groups These steps may be performed either in vivo or ex vivo. and the reactive group is attached to an amino acid posi The present invention is also directed to a method for tioned in the less therapeutically active region of amino 25 protecting a therapeutic peptide from peptidase activity in acids. vivo, the peptide being composed of between 3 and 50 In one embodiment, the therapeutically active region of amino acids and having a therapeutically active region of the peptide includes the carboxy terminal amino acid and the amino acids and a less therapeutically active region of amino reactive group is attached to said amino terminal amino acid. acids. The method comprises the following steps: In another embodiment, the therapeutically active region 30 (a) determining the therapeutically active region of amino of the peptide includes the amino terminal amino acid and acids; the reactive group is attached to the carboxy terminal amino (b) modifying the peptide at an amino acid included in the acid. less therapeutically active region of amino acids by attach In another embodiment, the therapeutically active region ing a reactive group to the amino acid to form a modified of the peptide includes the carboxy terminal amino acid and 35 peptide. Such that the modified peptide has therapeutic the reactive group is attached to an amino acid positioned activity and is capable of forming a covalent bond in vivo between the amino terminal amino acid and the carboxy with a reactive functionality on a blood component; terminal amino acid. (c) forming a covalent bond between the reactive entity In yet another embodiment, the therapeutically active and a reactive functionality on a blood component to form region includes the amino terminal amino acid and the 40 a peptide-blood component conjugate, thereby protecting reactive group is attached to an amino acid positioned the peptide from peptidase activity; and between the amino terminal amino acid and the carboxy (d) analyzing the stability of the peptide-blood component terminal amino acid. conjugate to assess the protection of the peptide from The present invention is also directed to a method of peptidase activity. synthesizing the modified therapeutic peptide. The method 45 comprises the following steps. In the first step, if the These steps may be performed either in vivo or ex vivo. therapeutic peptide does not contain a cysteine, then the The peptides useful in the compositions and methods of peptide is synthesized from the carboxy terminal amino acid the present invention include, but are not limited to, the and the reactive group is added to the carboxy terminal peptides presented in SEQ ID NO:1 to SEQ ID NO:1617. amino acid. Alternatively, a terminal lysine is added to the 50 carboxy terminal amino acid and the reactive group is added DETAILED DESCRIPTION OF THE to the terminal lysine. In the second step, if the therapeutic INVENTION peptide contains only one cysteine, then the cysteine is reacted with a protective group prior to addition of the Definitions reactive group to an amino acid in the less therapeutically 55 active region of the peptide. In the third step, if the thera To ensure a complete understanding of the invention, the peutic peptide containe two cysteines as a disulfide bridge, following definitions are provided: then the two cysteines are oxidized and the reactive group is Reactive Groups: Reactive groups are entities capable of added to the amino terminal amino acid, or to the carboxy forming a covalent bond. Such reactive groups are coupled terminal amino acid, or to an amino acid positioned between 60 or bonded to a therapeutic peptide of interest. Reactive the carboxy terminal amino acid and the amino terminal groups will generally be stable in an aqueous environment amino acid of the therapeutic peptide. In the fourth step, if and will usually be carboxy, phosphoryl, or convenient acyl the therapeutic peptide contains more than two cysteines as group, either as an ester or a mixed anhydride, or an imidate, disulfide bridges, the cysteines are sequentially oxidized in thereby capable of forming a covalent bond with function the disulfide bridges and the peptide is purified prior to the 65 alities such as an amino group, a hydroxy or a thiol at the addition of the reactive groups to the carboxy terminal target site on mobile blood components. For the most part, amino acid. the esters will involve phenolic compounds, or be thiol US 7,256.253 B2 5 6 esters, alkyl esters, phosphate esters, or the like. Reactive and the resulting peptidase Stabalized peptide (as defined groups include Succimidyl and maleimido groups. below) administered in vivo. The terms “modified therapeu Functionalities: Functionalities are groups on blood com tic peptide' and “modified peptide' may be used inter ponents, including mobile and fixed proteins, to which changeably in this application. reactive groups on modified therapeutic peptides react to Peptidase Stabalized Therapeutic Peptides—A peptidase form covalent bonds. Functionalities usually include stabalized therapeutic peptide is a modified peptide that has hydroxyl groups for bonding to ester reactive groups, thiol been conjugated to a blood component via a covalent bond groups for bonding to maleimides, imidates and thioester formed between the reactive group of the modified peptide groups; amino groups for bonding to activated carboxyl, and the functionalities of the blood component, with or phosphoryl or any other acyl groups on reactive groups. 10 without a linking group. Peptidase stabalized peptides are Blood Components: Blood components may be either more stable in the presence of peptidases in vivo than a fixed or mobile. Fixed blood components are non-mobile non-stabalized peptide. A peptidase stabalized therapeutic blood components and include tissues, membrane receptors, peptide generally has an increased half life of at least interstitial proteins, fibrin proteins, collagens, platelets, 10-50% as compared to a non-stabalized peptide of identical endothelial cells, epithelial cells and their associated mem 15 sequence. Peptidase stability is determined by comparing brane and membraneous receptors, somatic body cells, skel the half life of the unmodified therapeutic peptide in serum etal and Smooth muscle cells, neuronal components, osteo or blood to the halflife of a modified counterpart therapeutic cytes and osteoclasts and all body tissues especially those peptide in serum or blood. Half life is determined by associated with the circulatory and lymphatic systems. sampling the serum or blood after administration of the Mobile blood components are blood components that do not modified and non-modified peptides and determining the have a fixed situs for any extended period of time, generally activity of the peptide. In addition to determining the activ not exceeding 5, more usually one minute. These blood ity, the length of the therapeutic peptide may also be components are not membrane-associated and are present in measured. the blood for extended periods of time and are present in a Therapeutic Peptides—As used in this invention, thera minimum concentration of at least 0.1 ug/ml. Mobile blood 25 peutic peptides are amino acid chains of between 2-50 components include serum albumin, transferrin, ferritin and amino acids with therapeutic activity, as defined below. Each immunoglobulins such as IgM and IgG. The half-life of therapeutic peptide has an amino terminus (also referred to mobile blood components is at least about 12 hours. as N-terminus or amino terminal amino acid), a carboxyl Protective Groups: Protective groups are chemical moi terminus (also referred to as C-terminus terminal carboxyl eties utilized to protect peptide derivatives from reacting 30 terminal amino acid) and internal amino acids located with themselves. Various protective groups are disclosed between the amino terminus and the carboxyl terminus. The herein and in U.S. Pat. No. 5,493,007 which is hereby amino terminus is defined by the only amino acid in the incorporated by reference. Such protective groups include therapeutic peptide chain with a free C-amino group. The acetyl, fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycar carboxyl terminus is defined by the only amino acid in the bonyl (Boc), benzyloxycarbonyl (Cbz), and the like. The 35 therapeutic peptide chain with a free C-carboxyl group. specific protected amino acids are depicted in Table 1. Therapeutic peptides used in the present invention contain Linking Groups: Linking groups are chemical moieties atherapeutically active region generally located at the amino that link or connect reactive groups to therapeutic peptides. terminus, at the carboxyl terminus, or at an internal amino Linking groups may comprise one or more alkyl groups, acid. The therapeutically active region may be identified alkoxy group, alkenyl group, alkynyl group or amino group 40 using blind or structure activity relationship (SAR) driven Substituted by alkyl groups, cycloalkyl group, polycyclic Substitution, as defined in more detail in this application. group, aryl groups, polyaryl groups, Substituted aryl groups, SAR is an analysis which defines the relationship between heterocyclic groups, and Substituted heterocyclic groups. the structure of a molecule and its pharmacological activity Linking groups may also comprise poly ethoxy aminoacids for a series of compounds. Alternatively, where the thera Such as AEA ((2-amino)ethoxyacetic acid) or a preferred 45 peutically active region has previously been defined and is linking group AEEA (2-(2-amino)ethoxy)ethoxy acetic available in the literature, it may be obtained by referring to acid). A preferred linking group is aminoethoxyethoxyacetic references such as scientific journals. Knowledge of the acid (AEEA). location of the therapeutically active region of the peptide is Sensitive Functional Groups—A sensitive functional important for modifying the therapeutic peptide, as defined group is a group of atoms that represents a potential reaction 50 in more detail below. site on a therapeutic peptide. If present, a sensitive func Therapeutic peptides used in this invention also contain a tional group may be chosen as the attachment point for the less therapeutically active region generally located at the linker-reactive group modification. Sensitive functional amino terminus, at or near the carboxyl terminus, or at or groups include but are not limited to carboxyl, amino, thiol, near an internal amino acid. The less therapeutically active and hydroxyl groups. 55 region is a region of amino acids that does not coincide with Modified Therapeutic Peptides—A modified therapeutic the therapeutically active region of the therapeutic peptide. peptide peptide is a therapeutic peptide that has been modi The less therapeutically active region is generally located fied by attaching a reactive group, and is capable of forming away from the therapeutically active region, Such that modi a peptidase stabalized peptide through conjugation to blood fication at the less therapeutically active region does not components. The reactive group may be attached to the 60 substantially affect the therapeutic activity of the therapeutic therapeutic peptide either via a linking group, or optionally peptide. For example, if the therapeutically active region is without using a linking group. It is also contemplated that located at the amino terminus, the therapeutic peptide will be one or more additional amino acids may be added to the modified at either the carboxyl terminus or at an internal therapeutic peptide to facilitage the attachment of the reac amino acid. Alternatively, if the therapeutically active region tive group. Modified peptides may be administered in vivo 65 is located at the carboxyl terminus, the therapeutic peptide Such that conjugation with blood components occurs in Vivo, will be modified at either the amino terminus or at an or they may be first conjugated to blood components in vitro internal amino acid. Finally, if the therapeutically active US 7,256.253 B2 7 8 region is located at an internal region, the therapeutic 1. Therapeutic Peptides Used in the Present Invention peptide will be modified at either the amino terminus or the Peptide fragments chosen from the determined amino acid carboxyl terminus. sequence of a therapeutic peptide as provided in the attached “Therapeutic activity” is any activity directed toward SEQUENCE LISTING constitute the starting point in the healing or curing a biological disorder in a patient. Examples 5 development comprising the present invention. The peptides of said therapeutic peptides include pituitary Such range from 2 to 50 amino acids in length. The interchange as , , melanocyte stimulating hormones, able terms “peptide fragment” and "peptide moiety are adrenocorticotropic hormones, growth hormones; hypotha meant to include both synthetic and naturally occurring lamic hormones such as releasing factor, amino acid sequences derivable from a naturally occurring corticotropin releasing factor, releasing peptides, 10 amino acid sequence. In one embodiment, peptide and peptide fragments are releasing hormone and its associated peptides, synthesized by conventional means, either by bench-top release hormones, thyrotropin releasing methods or by automated peptide synthesis machines as hormone, , and ; hormones Such discussed in detail below. However, it is also possible to as calcitonins, precursors, and calcitonin gene 15 obtain fragments of the peptides by fragmenting the natu related peptides; parathyroid hormones and their related rally occurring amino acid sequence, using, for example, a proteins; pancreatic hormones such as and insulin proteolytic enzyme. Further, it is possible to obtain the like peptides, , Somatostatin, pancreatic polypep desired fragments of the therapeutic peptide through the use tides, , peptide YY. and Y: digestive of recombinant DNA technology, as disclosed by Maniatis, hormones such as , gastrin releasing peptides, gastrin T. et al., Molecular Biology: A Laboratory Manual, Cold inhibitory peptides, , secretin, , and Spring Harbor, N.Y. (1982), which is hereby incorporated by vasoactive intestinal peptide; natriuretic peptides such as reference. The use of other new modifications to existing atrial natriuretic peptides, brain natriuretic peptides, and methodologies is also contemplated. C-type natriuretic peptides; neurokinins such as neurokinin The present invention includes peptides which are deriv A, , and , related peptides 25 able from the naturally occuring sequence of the therapeutic Such as renin Substrates and inhibitors and ; peptide. A peptide is said to be "derivable from a naturally , including big , occurring amino acid sequence' if it can be obtained by antagonists, and peptides; and other peptides fragmenting a naturally occurring sequence, or if it can be Such as adrenomedullin peptides, allatostatin peptides, amy synthesized based upon a knowledge of the sequence of the loid beta protein fragments, antibiotic and antimicrobial 30 naturally occurring amino acid sequence or of the genetic peptides, apoptosis related peptides, bag cell peptides, material (DNA or RNA) which encodes this sequence. , bone Gla protein peptides, CART peptides, Included within the scope of the present invention are those chemotactic peptides, cortistatin peptides, fibronectin frag molecules which are said to be "derivatives' of a peptide. ments and fibrin related peptides, FMRF and analog pep 35 Such a "derivative has the following characteristics: (1) it tides and related peptides, growth factors and related shares Substantial homology with the therapeutic peptide or peptides, Githerapeutic peptide-binding protein fragments, a similarly sized fragment of the peptide and (2) it is capable guanylin and uroguanylin, inhibin peptides, interleukin and of functioning with the same therapeutic activity as the interleukin receptor proteins, laminin fragments, frag peptide. ment peptides, leucokinins, mast cell degranulating pep 40 A derivative of a peptide is said to share “substantial tides, pituitary adenylate cyclase activating polypeptides, homology' with the peptide if the amino acid sequences of the derivative is at least 80%, and more preferably at least pancreastatin, peptide T. polypeptides, virus related pep 90%, and most preferably at least 95%, the same as that of tides, signal transduction reagents, toxins, and miscella either the peptide or a fragment of the peptide having the neous peptides Such as adjuvant peptide analogs, alpha same number of amino acid residues as the derivative. 45 mating factor, antiarrhythmic peptide, antifreeze polypep The derivatives of the present invention include fragments tide, anorexigenic peptide, bovine pineal antireproductive which, in addition to containing a sequence that is Substan peptide, bursin, C3 peptide P16, tumor necrosis factor, tially homologous to that of a naturally occurring therapeutic cadherin peptide, chromogranin. A fragment, contraceptive peptide may contain one or more additional amino acids at , conantokin G, conantokin T. crustacean cardio 50 their amino and/or their carboxy termini as discussed in active peptide, C-telopeptide, cytochrome b588 peptide, detail below. Thus, the invention pertains to polypeptide decorsin, delicioius peptide, delta-sleep-inducing peptide, fragments of the therapeutic peptide that may contain one or diazempam-binding inhibitor fragment, nitric oxide Syn more amino acids that may not be present in a naturally thase blocking peptide, OVA peptide, platelet calpain inhibi occurring therapeutic peptide sequence provided that Such tor (P1), plasminogen activator inhibitor 1, rigin, Schizo 55 fragments have a therapeutic activity which exceeds that of phrenia related peptide, serum thymic factor, sodium the therapeutic peptide. potassium Atherapeutic peptidease inhibiro-1, speract, Similarly, the invention includes polypeptide fragments sperm activating peptide, systemin, thrombin receptor ago which, although containing a sequence that is Substantially nist, thymic humoral gamma2 factor, , thy homologous to that of a naturally occurring therapeutic mosin alpha 1, factor, tuftsin, adipokinetic hormone, 60 peptide, may lack one or more additional amino acids at uremic pentapeptide and other therapeutic peptides. their amino and/or their carboxy termini that are naturally Taking into account these definitions, the focus of this found on the therapeutic peptide. Thus, the invention per invention is to modify therapeutic peptides to protect them tains to polypeptide fragments of the therapeutic peptide that from peptidase activity in vivo and thereby extend the may lack one or more amino acids that are normally present effective therapeutic life of the therapeutic peptide in ques 65 in the naturally occurring peptide sequence provided that tion as compared to administration of the peptide per se to Such polypeptides have a therapeutic activity which exceeds a patient. that of the therapeutic peptide. US 7,256.253 B2 10 The invention also encompasses the obvious or trivial growth-promoting and lactogenic activity. Mature GH (22. variants of the above-described fragments which have 000 daltons) is synthesized in acidophilic pituitary somato inconsequential amino acid Substitutions (and thus have tropes as a single polypeptide chain. Because of alternate amino acid sequences which differ from that of the natural RNA splicing, a small amount of a somewhat Smaller sequence) provided that such variants have an activity which 5 molecular form is also secreted. is substantially identical to that of the above-described derivatives. Examples of obvious or trivial substitutions There are a number of genetic deficiencies associated with include the substitution of one basic residue for another (i.e. GH. GH-deficient dwarfs lack the ability to synthesize or Arg for Lys), the substitution of one hydrophobic residue for secrete GH, and these short-statured individuals respond another (i.e. Leu for Ile), or the substitution of one aromatic 10 well to GH therapy. Pygmies lack the IGF-1 response to GH residue for another (i.e. Phe for Tyr), etc. but not its metabolic effects; thus in pygmies the deficiency As is known in the art, the amino acid residues may be in is post-receptor in nature. Finally, Laron dwarfs have normal their protected or unprotected form, using appropriate amino or excess plasma GH, but lack GH receptors and have or carboxyl protecting groups as discussed in detail below. low levels of circulating IGF-1. The defect in these indi The variable length peptides may be in the form of the free 15 viduals is clearly related to an inability to respond to GH by amines (on the N-terminus), or acid-addition salts thereof. the production of IGF-1. The production of excessive Common acid addition salts are hydrohalic acid salts, i.e., amounts of GH before epiphyseal closure of the long bones HBr, H1, or, more preferably, HC1. Useful cations are alkali leads to gigantism, and when GH becomes excessive after or alkaline earth metallic cations (i.e., Na, K. Li, Ca, Ba, epiphyseal closure, acral bone growth leads to the charac etc.) or amine cations (i.e., tetraalkylammonium, trialkylam- 20 teristic features of acromegaly. Using GH as a therapeutic monium, where alkyl can be C-C). agent would aid in treating these disorders, and potentially Any peptide having a therapeutic activity may be used in stimulate growth in other cases of short stature with low or this invention. The following list of peptides provides normal GH levels. examples of peptides that may be used in this invention, but Melanocyte Stimulating Hormones (MSH) (SEQ ID is not exhaustive and in no way limits the number or type of 25 peptides that may be used in this invention. These thera NOS: 25-39)—Melanocyte stimulating hormone (MSH) is peutic peptides and fragments produced from these peptides generated in the intermediary pituitary under the control of may be modified according to the present invention, and . MSH may have important physiological roles in used therapeutically in the body. the control of vertebrate pigment cell melanogenesis, neural 30 functioning related to learning and behavior, and fetal devel A. Pituitary Hormones (SEQ ID NOS: 1-72) opment. See Sawyer, T. K. et al., Proc. Nat. Acad. Sci USA, Adrenocortiocotropic Hormones (ACTH, aka corticotro 79, 1751 (1982). pin) (SEQ ID NOS: 1-22) The endocrine functions of the adrenal cortex are regulated by an hor Oxytocin (SEQ ID NOS: 40-44). Oxytocin is involved mone, ACTH, ACTH, a 39-amino acid peptide is generated as in the enhancement of lactation, contraction of the uterus, in the corticotrophic cells of the anterior pituitary under the and relaxation of the pelvis prior to childbirth. Oxytocin control of corticotropin releasing factor. ACTH is derived by secretion in nursing women is stimulated by direct neural post-translational modification from a 241-amino acid pre feedback obtained by stimulation of the nipple during suck cursor known as pro-opiomelanocortin (POMC). ling. Its physiological effects include the contraction of The biological role of ACTH is to maintain the bulk and 40 mammary gland myoepithelial cells, which induces the the viability of the adrenal cortex and to stimulate the ejection of milk from mammary glands, and the stimulation production of adrenal cortex Steroids, principally of uterine Smooth muscle contraction leading to childbirth. and costicosterone. The mechanism of action of ACTH Oxytocin causes myoepithelial cells Surrounding secretory involves binding to the ACTH receptor followed by activa acini of mammary glands to contract, pushing milk through tion of adenylate cyclase, elevation of cyclic AMP (cAMP), 4s ducts. In addition, it stimulates the release of prolactin, and and increased protein kinase A (PKA) activity of adrenal prolactin is trophic on the breast and stimulates acinar cortex tissue. The main effect of these events is to increase formation of milk. A conjugated oxytocin could thus be used the activity of a side chain-cleaving enzyme, which converts to aid lactation and help relax the pelvis prior to birth. It cholesterol to pregnenolone. Because of the distribution of could also be used to prevent post partum uterine hemorrage. enzymes in the various adrenal cortex Subdivisions, the so Vasopressin (ADH) (SEQ ID NOS: 46-72) Vasopressin principal physiological effect of ACTH is production of the is also known as antidiuretic hormone (ADH), because it is glucocorticosteroids. the main regulator of body fluid osmolarity, causing antidi Aside from its function controlling adrenal cortical activ uresis and increase in blood pressure. Vasopressin binds ity, ACTH appears to have diverse biological roles including plasma membrane receptors and acts through G-proteins to modulation of endocrine and exocrine glands, temperature 55 activate the cyclic AMP/protein kinase A (cAMP/PKA) regulation and influences on nerve regeneration and devel regulatory system. The secretion of vasopressin is regulated opment. In addition, ACTH and its fragments affect moti in the by osmoreceptors, which sense water vation, learning, and behavior. The use of ACTH as a concentration and stimulate increased vasopressin Secretion therapeutic agent may thus help the control of these func when plasma osmolarity increases. The secreted vasopressin tions. ACTH release from the anterior pituitary is mediated 60 increases the reabsorption rate of water in tubule by corticotropin releasing factor (CRF). cells, causing the excretion of urine that is concentrated in Growth Hormone Peptides (SEQ ID NOS: 23-24.45)– Na" and thus yielding a net drop in osmolarity of body Human (hPL), growth hormones, and fluids. Vasopressin deficiency leads to watery urine and prolactin (Pri) comprise the growth hormone family. All polydipsia, a condition known as diabetes insipidus. Using have about 200 amino acids, 2 disulfide bonds, and no 65 conjugated vasopressin or vasopressin fragments would thus glycosylation. Although each has special receptors and prevent these disorders and allow the regular maintenance of unique characteristics to their activity, they all possess the body’s osmolarity. US 7,256.253 B2 11 12 B. Hypothalamic Hormones (Releasing Factors) is believed to synchronize growth throughout the body, Corticotropin Releasing Factor (CRF) & related peptides resulting in a homeostatic balance of tissue size and mass. (SEQ ID NOS: 73-102) Corticotrophin-releasing factor IGF-1 secreted by peripheral tissues is generally considered (CRF), a 41 amino acid peptide, plays a significant role in to be autocrine or paracrine in its biological action. The use coordinating the overall response to stress through actions of a conjugated GRF as a therapeutic agent to increase GH both in the brain and the periphery. In the brain, CRF is release, would then help treat disorders involving growth produced and secreted primarily from parvocellular neurons functions regulated by GRF. of the paraventricular hypothalamic nucleus. From there, the Lutenizing Hormone Release Hormones (LH-RH) (SEQ CRF-containing neurons project to the portal capillary Zone ID NOS: 135-161)—Luteinizing hormone releasing hor of the median eminence and act to stimulate the Secretion of 10 mone is the key mediator in the neuroregulation of the adrenocorticotrophic hormone (ACTH), beta-endorphin, secretion of , luteinizing hormone (LH) and and other (POMC)-derived peptides follicle stimulating hormone (FSH). LH-RH can modify from the pituitary gland. The subsequent ACTH-induced sexual behavior by regulating plasma gonadotropin and sex release of adrenal glucocorticoids represents the final stage steroid levels. See Vale, W. W. et al., Peptides, Structure and in the hypothalamic-pituitary-adrenal axis (HPA), which 15 Biological Function, Proceedings of the Sixth American mediates the endocrine response to stress. Besides its neu Peptide Symposium, Gross, E. and Meienhofer, M., eds., roendocrine role, CRF also functions as a neurotransmitter 781 (1979). A conjugated LH-RH agent could be used to and neuromodulator to elicit a wide spectrum of autonomic, stimulate ovulation in humans or animals as an aid to behavioral and immune effects to physiological, pharmaco fertility. logical, and pathological stimuli. (SEQ ID NOS: 162-164) Orexins are a family Clinical studies indicated that CRF hypersecretion is of from the hypothalamus that have been associated with various diseases, such as major depression, recently discovered and characterized. Orexins stimulate anxiety-related illness, eating disorder, as well as inflam appetite and food consumption. Their genes are expressed matory disorder. Low levels of CRF have been found in bilaterally and symmetrically in the lateral hypothalamus, Alzheimer's disease, dementias, obesity, and many endo 25 which was earlier determined to be the “feeding center of crine diseases. Therefore, the use of CRF as a therapeutic the hypothalamus. In contrast, the so-called satiety center is agent to counter the effects associated with high levels or expressed in the ventromedial hypothalamus and is domi low levels of CRF will provide a basis for the treatment of nated by the leptin-regulated neuropeptide network. diseases that are associated with abnormal CRF levels. Prolactin Releasing Peptides (SEQ ID NOS: 65-170)– Several peptide antagonists and nonpeptide antagonists have 30 Prolactin is produced by acidophilic pituitary lactotropes. been discovered and widely studied, including a-helical Prolactin releasing peptides act on lactotrope to release CRF(9-41), Astressin, D-Phe(CRF(12-41) (peptide antago prolactin. PRL initiates and maintains lactation in mammals, nist) and CP-154526 (nonpeptide antagonist). These CRF but normally only in mammary tissue that has been primed antagonists may provide a novel agent for treatment of with estrogenic sex hormones. A conjugated PRP could be depression, anxiety and other CRF related illnesses. Conju 35 used to increase lactation in humans or animals. gated CRF peptides could thus be used to maintain adrenal Somatostatin (SEQ ID NOS: 171-201)—Also known as health and viability during long term steroid use or as Growth Hormone Release Inhibiting Factors (GIF), soma anti-inflamatory agents. tostatin is a 14 amino acid peptide is secreted by both the Gonadotropin Releasing Hormone Associated peptides hypothatamus and by d cells of the (its pancreatic (GAP) (SEQ ID NOS: 103-110) GAP is contained in the 40 version is discussed below). Somatostatin has been reported precursor molecule to gonadotropin-releasing hormone to modulate physiological functions at various sites includ (GnRH). GAP has prolactin inhibiting properties. Gn-RH is ing pituitary, pancreas, gut and brain. It inhibits the release a hormone secreted by the hypothalamus that stimulate the of growth hormone, insulin, and glucagon. It has many release of gonadotrophic hormones follicle stimulating hor biological roles, including: inhibition of basal and stimu mone (FSH) and luteinizing hormone (LH). Low levels of 45 lated hormone secretion from endocrine and exocrine cells, circulating sex hormone reduce feedback inhibition on an effect on locomotor activity and cognitive function, and GnRH synthesis, leading to elevated levels of FSH and LH. possible therapeutic value in Small cell lung cancer. See The latter peptide hormones, bind to gonadal tissue, result Reubi, J. C. et al. Endocrinology, 110, 1049 (1982). A ing in sex hormone production via cyclic AMP (cAMP) and conjugated Somatostatin could be used to treat giantism in protein kinase A (PKA) mediated pathways. A conjugated 50 children or acromegaly in the adult. GnRM could be used to aid fertility, or as a contraceptive in Thyrotropin Releasing Hormone (THR) and Analogs either males or females. This agent would have use in (SEQ ID NOS: 202-214) THR stimulates the production animals as well as humans. of thyroid stimulating hormone (TSH, also known as thy Growth Hormone Releasing Factor (GRF) (SEQID NOS: rotropin) and prolactin Secretion. In adults, TSH is respon 111-134)—GRF is a hypothalamic peptide that plays a 55 sible for up-regulating general protein synthesis and induc critical role in controlling the synthesis and secretion of ing a state of positive nitrogen balance. In the embryo, it is growth hormone in the anterior pituitary. Some structurally necessary for normal development. Hypothyroidism in the unrelated short peptides have also been reported to elicit embryo is responsible for cretinism, which is characterized growth hormone secretion by a different mechanism. by multiple congenital defects and mental retardation. A Under the influence of GRF, growth hormone is released 60 conjugated THR could then be used as a therapeutic agent in into the systemic circulation, causing the target tissue to the treatment of these disorders. It could also be used to treat secrete IGF-1. Growth hormone also has other more direct pituitary causes of thyroid insufficiency or in the diagnosis metabolic effects; it is both hyperglycemic and lipolytic. The of human tumors of the thyroid. principal source of systemic IGF-1 is the liver, although most other tissues secrete and contribute to systemic IGF-1. 65 C. Liver IGF-1 is considered to be the principal regulator of Calcitonins (CT) & Caltitonins Precursor Peptides (SEQ tissue growth. In particular, the IGF-1 secreted by the liver ID NOS: 215-224)—Calcitonin (CT) is a 32-amino acid US 7,256.253 B2 13 14 peptide secreted by C cells of the thyroid gland. Calcitonin tor) that recognizes the N-terminal (1-34) region of these is employed therapeutically to relieve the symptoms of peptides. Hence, when tumor-derived PTHrP enters the osteoporosis, although details of its mechanism of action circulation, it activates receptors in classic PTH target remain unclear. However, it has been observed that CT organs such as bone and kidney and elicits PTH-like bio induces the synthesis of (PTH) in 5 activity. By promoting bone resorption and inhibiting cal isolated cells, which leads in vivo to increased plasma Ca" cium excretion, circulating PTHrP gives rise to the common levels. In addition, CT has been shown to reduce the paraneoplastic syndrome of malignancy-associated humoral synthesis of osteoporin (Opn), a protein made by osteoclasts hypercalcemia. and responsible for attaching osteoclasts to bone. Thus, Although initially discovered in tumors, PTHrP was sub using conjugated CT as a therapeutic peptide would elevate 10 sequently shown to be expressed in a remarkable variety of plasma Ca" via PTH induction and reduce bone reabsorp normal tissues including the fetal and adult skeleton, where tion by decreasing osteoclast binding to bone. acting in concert with its amino terminal PTH-1 receptor, it Calcitonins Gene Related Peptide (CGRP) (SEQID NOS: serves to regulate cellular growth and differentiation. The 225-253)—CGRP is a 37 amino acid peptide that results anabolic effects of intermittent PTH administration on bone from alternative splicing of calcitonin gene transcripts. It 15 and its therapeutic potential in osteoporosis have been exists in at least two forms: alpha-CGRP (or CGRP-I) and extensively explored. With the recognition that PTHrP is the beta-CGRP (or CGRP-II). CGRP has considerable homol endogenous ligand for the PTH/PTHrP receptor in osteo ogy with amylin and adrenomedullin, and is widely distrib blasts, its use as an anabolic agent has also been investi uted both centrally and peripherally in organs including the gated. Modified PTHrP peptides could be used for similar skin, the heart, the pancreas, the lungs, and the kidneys. indications as PTH. CGRP has many biological roles, affecting the nervous and E. Pancreatic Hormones—The principal role of the pancre cardiovascular systems, inflammation and metabolism. atic hormones is the regulation of whole-body energy D. Parathyroid Hormones and Related Proteins metabolism, principally by regulating the concentration and Parathyroid Hormones (PTH) (SEQID NOS: 254-293)– 25 activity of numerous enzymes involved in catabolism and Parathyroid hormone (PTH) is synthesized and secreted by anabolism of the major cell energy Supplies. chief cells of the parathyroid in response to systemic Ca" Amylin (SEQ ID NOS: 310–335) Pancreatic beta-cell levels. It plays a major role in the modulation of serum hormone amylin is a 37-amino-acid peptide related to CGRP calcium concentration and thereby affect the physiology of and calcitonin. It is co-secreted with insulin from pancreatic mineral and bone metabolism. The Ca" receptor of the 30 beta-cells. Amylin is deficient with type 1 diabetes mellitus. responds to Ca" by increasing intracel Amylin appears to work with insulin to regulate plasma lular levels of PKC, Ca" and IP; this stage is followed, glucose concentrations in the bloodstream, Suppressing the after a period of protein synthesis, by PTH secretion. The postherapeutic peptiderandial secretion of glucagon and synthesis and secretion of PTH in chief cells is constitutive, restraining the rate of gastric emptying. People with diabetes but Ca" regulates the level of PTH in chief cells (and thus 35 have a deficiency in the secretion of amylin that parallels the its secretion) by increasing the rate of PTH proteolysis when deficiency in insulin secretion, resulting in an excessive plasma Ca" levels rise and by decreasing the proteolysis of inflow of glucose into the bloodstream during the posthera PTH when Ca" levels fall. The role of PTH is to regulate peutic peptiderandial period. Ca" concentration in extracellular fluids. The feedback loop While insulin replacement therapy is a cornerstone of that regulates PTH secretion therefore involves the parathy 40 diabetes treatment, replacement of the function of both roids, Ca", and the target tissues described below. amylin and insulin may allow a more complete restoration of PTH acts by binding to cAMP-coupled plasma membrane the normal physiology of glucose control. Type 2 diabetes is receptors, initiating a cascade of reactions that culminates in characterized by islet amyloid deposits, which are primarily the biological response. The body's response to PTH is composed of the amyloidogenic human form of islet amy complex but is aimed in all tissues at increasing Ca" levels 45 loid polypeptide. A conjugated amylin could be used in the in extracellular fluids. PTH induces the dissolution of bone management of diabetes to limit post prandial hyperglyse by stimulating osteoclast activity, which leads to elevated 18 plasma Ca" and phosphate. In the kidney, PTH reduces Glucagon (SEQ ID NOS: 336-376) Glucagon is a renal Ca" clearance by stimulating its reabsorption; at the 29-amino acid hormone synthesized by the a cells of the same time, PTH reduces the reabsorption of phosphate and 50 islets of Langerhans as a very much larger thereby increases its clearance. Finally, PTH acts on the molecule. Like insulin, glucagon lacks a plasma carrier liver, kidney, and intestine to stimulate the production of the protein, and like insulin its circulating half life is also about 1,25-dihydroxycholecalciferol (), 5 minutes. As a consequence of the latter trait, the principal which is responsible for Ca" absorption in the intestine. A effect of glucagon is on the liver, which is the first tissue conjugated PTH could be used to regulate calcium homeo 55 perfused by blood containing pancreatic secretions. Gluca Stasis in patients with parathyroid hormone deficiency states. gon binds to plasma membrane receptors and is coupled Inhibitor analogues could be used to block PTH action in through G-proteins to adenylate cyclase. The resultant renal failure or other patients with excessive PTH levels. increases in cAMP and PKA reverse all of the effects Parathyroid Hormone Related Proteins (PTHrP) (SEQ ID described above that insulin has on liver. The increases also NOS: 294-309) Parathyroid hormone-related protein 60 lead to a marked elevation of circulating glucose, with the (PTHrP) has received attention as a physiological regulator glucose being derived from liver gluconeogenesis and liver attenuating chondrocytic differentiation and preventing apo glycogenolysis. A conjugated glucagon construct could be ptotic cell death. PTHrP was initially identified as a tumor used to manage brittle diabetes with recurrent hypoglycemia derived, secretory protein with structural similarity to par or to prevent or treat iatrogenic hypoglycemia. athyroid hormone (PTH), the major regulator of calcium 65 Insulin and Insulin-Like peptides (SEQ ID NOS: 377 homeostasis. PTH and PTHrP bind to a common G protein 382). The earliest of these hormones recognized was insu coupled cell surface receptor (PTH/PTHrP or PTH-1 recep lin, a disulfide bonded of 21 and 30 amino acids US 7,256.253 B2 15 16 produced by the pancreas, whose major function is to Pancreatic Polypeptides (PP) (SEQ ID NOS: 390-396)– counter the concerted action of a number of hyperglycemia (PP) is a 36-amino acid hormone generating hormones and to maintain low blood glucose produced by F cells within the pancreatic islets and the levels. Insulin is a member of a family of structurally and exocrine pancreas. It is a member of the PP fold family of functionally similar molecules that include IGF-1, IGF-2, regulatory peptides, and increases glycogenolysis and regu and . The tertiary structure of all 4 molecules is lates gastrointestinal activity. A conjugated pancreatic similar, and all have growth-promoting activities, but the polypeptide could thus be used to alter absorption and dominant role of insulin is metabolic while the dominant metabolism of foods. roles of the IGFs and relaxin are in the regulation of cell Peptide YY (SEQID NOS: 397-400) PYY is a thirty six growth and differentiation. 10 amino acid long peptide, first isolated from porcine intesti Insulin is synthesized as a preprohormone in the b cells of nal tissue and mainly localized in intestinal endocrine cells. the islets of Langerhans. Its is removed in the It has many biological activities, including a range of cisternae of the endoplasmic reticulum and it is packaged activities within the digestive system and potent inhibition into secretory vesicles in the Golgi, folded to its native of intestinal electrolyte and fluid secretion. structure, and locked in this conformation by the formation 15 Somatostatin (SEQ ID NOS: 171-201) The somatosta of 2 disulfide bonds. Specific protease activity cleaves the tin secreted by d cells of the pancreas is a 14-amino acid center third of the molecule, which dissociates as C peptide, peptide identical to somatostatin secreted by the hypothala leaving the amino terminal B peptide disulfide bonded to the mus. In neural tissue Somatostatin inhibits GH secretion and carboxy terminal A peptide. thus has systemic effects. In the pancreas, somatostatin acts Insulin generates its intracellular effects by binding to a a paracrine inhibitor of other pancreatic hormones and thus plasma membrane receptor, which is the same in all cells. also has systemic effects. It has been speculated that Soma The receptor is a disulfide-bonded glycoprotein. One func to statin Secretion responds principally to blood glucose tion of insulin (aside from its role in signal transduction) is levels, increasing as blood glucose levels rise and thus to increase glucose transport in extrahepatic tissue is by leading to down-regulation of glucagon secretion. A conju increasing the number of glucose transport molecules in the 25 gated Somatostatin could then be used to aid in the man plasma membrane. Glucose transporters are in a continuous agement of diabetes. state of turnover. Increases in the plasma membrane content F. Digestive Hormones of transporters stem from an increase in the rate of recruit Cholecystokinin (CCK) & related peptides (SEQ ID ment of new transporters into the plasma membrane, deriv 30 NOS: 401-416) CCK is a polypeptide of 33 amino acids ing from a special pool of preformed transporters localized originally isolated from pig Small intestine that stimulates in the cytoplasm. gallbladder contraction and bile flow and increases secretion In addition to its role in regulating glucose metabolism of digestive enzymes from pancreas. It exists in multiple (and its therapeutic use in treating diabetes), insulin stimu forms, including CCK4 and CCK-8, with the octapeptide lates lipogenesis, diminishes lipolysis, and increases amino 35 representing the dominant molecular species showing the acid transport into cells. Insulin also modulates transcrip greatest activity. It belongs to the CCK/gastrin peptide tion, altering the cell content of numerous mRNAs. It family and is distributed centrally in the nervous system and stimulates growth, DNA synthesis, and cell replication, peripherally in the gastrointestinal system. It has many effects that it holds in common with the IGFs and relaxin. A biological roles, including stimulation of pancreatic secre conjugated insulin could thus be used to manage diabetes. 40 tion, gall bladder contraction and intestinal mobility in the (SEQID NOS: 383-389) Neuropeptide GI tract as well as the possible mediation of satiety and Y (NPY), a peptide with 36 amino acid residues, is one of painful stimuli. A conjugated CCK could be used in diag the most abundant neuropeptides in both the peripheral and nostic studies of the gallbladder or in chronic cholecystisis. the central nervous systems. It belongs to the pancreatic Gastrin Releasing Peptide (GRP) (SEQ ID NOS: 417 polypeptide family of peptides. Like its relatives, peptide 45 429)—GRP is a 27-amino acid peptide originally isolated YY (PYY) and pancreatic polypeptide (PP), NPY is bent from porcine non-antral gastric tissue, and is the homolog of into hairpin configuration that is important in bringing the the frog skin peptide named bombesin growth. It is widely free ends of the molecule together for binding to the recep distributed both centrally and peripherally in tissues includ tOrS. ing brain, lung and gastrointestinal tract It regulates a variety NPY exerts a wide range of effects in the central nervous 50 of cell physiological processes including secretion, Smooth system (CNS) and the periphery. Its CNS actions include muscle contraction, neurotransmission and cell growth. A major effects on feeding and energy expenditure, and alter conjugated GRP could be used in the treatment of adynamic ations in heart rate, blood pressure, arousal and mood. In the illeus or constipation in the elderly. periphery, NPY causes vasoconstriction and hypertension; it Gastrin & related peptides (SEQ ID NOS: 417-429)— is also found in the gastrointestinal and urogenital tract, 55 Gastrin is a polypeptide of 17 amino acids produced by implicating its functions by action upon gastrointestinal and stomachantrum, which stimulates acid and pepsin Secretion. renal targets. In recent studies, hypothalamic NPY has been Gastrin also stimulates pancreatic secretions. Multiple active found to play a fundamental role in developing the features products are generated from the gastrin precursor, and there of obesity, it is a major transducer in the pathways signalling are multiple control points in gastrin biosynthesis. Biosyn body fat to the hypothalamus, and in regulating body fat 60 thetic precursors and intermediates (progastrin and Gly content. Leptin, an obese gene product, has been found to ) are putative growth factors; their products, the decrease NPY gene expression in obese (ob?ob) mice. Insu amidated gastrins, regulate epithelial cell proliferation, the lin and corticosteroids are also involved in the regulation of differentiation of acid-producing parietal cells and hista hypothalamic NPY synthesis, with insulin decreasing and mine-secreting enterochromaffin-like (ECL) cells, and the corticosteroids increasing NPY expression. A conjugated 65 expression of genes associated with histamine synthesis and NPY could be used to treat obesity and MODM (Type II storage in ECL cells, as well as acutely stimulating acid DM) in obese patients. secretion. Gastrin also stimulates the production of members US 7,256.253 B2 17 18 of the (EGF) family, which in turn Atrial-Natriuretic Peptides (ANP) (SEQ ID NOS: 465 inhibit parietal cell function but stimulate the growth of 507)—ANP is a 28-amino acid containing Surface epithelial cells. Plasma gastrin concentrations are a disulfide bond. It exerts natriuretic, diuretic, and vasore elevated in subjects with Helicobacter pylori, who are laxant effects and play an important role in the body's blood known to have increased risk of duodenal ulcer disease and 5 volume and blood pressure homeostasis. See Smith, F. G. et gastric cancer. The use of gastrin or gastrin antagonists as a al., J. Dev. Physiol. 12, 55 (1989). The mechanisms con therapeutic agent may therefore contribute to treating major trolling ANP release have been the subject of intense upper gastrointestinal tract disease. research, and are now fairly well understood. The major Gastrin Inhibitory peptides (SEQ ID NOS: 417-429)— determinant of ANP secretion is myocyte stretch. Although 10 much less is known about the factors regulating BNP release Gastrin inhibitory peptide is a polypeptide of 43 amino acids from the heart, myocyte stretch has also been reported to that inhibits secretion of gastrin. A conjugated GIP could be stimulate BNP release from both atria and ventricles. How used to treat severe peptic ulcer disease. ever, whether wall stretch acts directly or via factors such as Motilin (SEQ ID NOS: 430-433) Motilin is a polypep endothelin-1, nitric oxide, or II liberated in tide of 22 amino acids that controls gastrointestinal muscles. 15 response to distension has not been established. Recent Motilin-producing cells are distributed in the , studies show that by stimulating endothelin type A receptors upper jejunum, and colorectal adenocarinomas and in mid endothelin plays an important physiological role as a media gut carcinoids. Motilin stimulates gut motility. tor of acute-volume load-induced ANP secretion from atrial Secretin (SEQID NOS: 434–441) Secretin is a polypep myocytes in conscious animals. In fact, endogenous para tide of 27 amino acids secreted from duodenum at pH values crine/autocrine factors liberated in response to atrial wall below 4.5, stimulates pancreatic acinar cells to release stretch rather than direct stretch appears to be responsible for bicarbonate and H2O. Secretin is a neurotransmitter (a activation of ANP secretion in response to volume load, as chemical messenger) in the neuropeptide group. It is one of evidenced by almost complete blockade of ANP secretion the hormones that controls (gastrin and cholecys during combined inhibition of endothelin type A/B and tokinin are the others). It is a polypeptide composed of 27 25 angiotensin II receptors. Furthermore, under certain experi amino acids and is secreted by cells in the digestive system mental conditions angiotensin II and nitric oxide may also when the empties. Secretin stimulates the pancreas exert a significant modulatory effect on stretch-activated to emit digestive fluids that are rich in bicarbonate which ANP secretion. The molecular mechanisms by which endot neutralizes the acidity of the intestines, the stomach to helin-1, angiotensin II, and nitric oxide synergistically regu produce pepsin (an enzyme that aids digestion of protein), 30 late stretch-activated ANP release are yet unclear. Abstract and the liver to produce bile. Volume 75 Issue 11/12 (1997) pp 876-885, Journal of Secretin may be useful in treating autism. In one study, Molecular Medicine. A conjugated would be useful in the children with autistic spectrum disorders underwent upper management of malignant hypertension or severe hyperten gastrointestinal endoscopy and intravenous administration sion and renal failure. of Secretin to stimulate pancreaticobiliary secretion. All 35 Brain Natriuretic Peptides (BNP) (SEQ ID NOS: 507 three had an increased pancreaticobiliary secretory response 516)—Brain (BNP), a member of the when compared with nonautistic patients (7.5 to 10 mL/min natriuretic peptide family, is produced and released from versus 1 to 2 mL/min). Within 5 weeks of the secretin cardiac ventricles. BNP regulates the body fluid volume, infusion, a significant amelioration of the children's gas blood pressure, and vascular tones through the A-type gua trointestinal symptoms was observed, as was a dramatic 40 nylate cyclase-coupled receptor. The BNP plays a role in improvement in their behavior, manifested by improved eye electrolyte-fluid homeostasis such as atrial natriuretic pep contact, alertness, and expansion of expressive language. tides (ANP). A conjugated BNP could be useful in the These clinical observations suggest an association between management of heart failure. gastrointestinal and brain function in patients with autistic C-Type Natriuretic Peptides (CNP) (SEQ ID NOS: 517 behavior. 45 524)—C-type matriuretic peptide (CNP), the third member Vasoactive Intestinal Peptide (VIP) and related peptides of the natriuretic peptide family, is produced in vascular (SEQ ID NOS: 442-464) VIP is a polypeptide of 28 endothelial cells (ECs) and acts as an endothelium-derived residues produced by hypothalamus and GI tract. It relaxes relaxing peptide. Although atrial and brain natriuretic pep the GI, inhibits acid and pepsin secretion, acts as a neu tides are well known to be involved in the regulation of rotransmitter in peripheral autonomic nervous system, and 50 cardiovascular and endocrine functions as circulating hor increases secretion of H2O and electrolytes from pancreas mones, the roles of the C-type natriuretic peptide (CNP) and gut. It was originally discovered in lung and intestine remain unknown. and is also found in tissues including brain, liver, pancreas, CNP is found principally in the Smooth muscle and lymphocytes. It is structurally related to and vascular endothelial cells while ANP and BNP are a family of peptides which include PACAP, PHI, secretin 55 cardiac hormones. ANP is synthesized mainly in the atria of and glucagon. It has a diverse range of biological actions the normal adult heart, while BNP is produced by both the including vasodilation, electrolyte secretion, modulation of atria and Ventricles. immune function and neurotransmission. A conjugated VIP may be useful in the treatment of achlorhydria, ischemic H. Tachykinins (SEQ ID NOS: 525-627) A family of 60 peptides, including and Substance P, that share colitis and irritable bowel syndrome (IBS). a common C terminal sequence (F-X-GLM-NH2) which is G. Natriuretic Peptides There are three members in the required for full biological activity including Neurokinin A, natriuretic peptide hormone family, atrial natriuretic peptide B, and Substance P. (ANP), B-type matriuretic peptide (BNP. brain natriuretic Neurokinin A Neurokinin A is a decapeptide, previously peptide), and C-type natriuretic peptide (CNP), that are 65 known as Substance K. It is is a member of the tachykinin involved in the regulation of blood pressure and fluid family of neuropeptides which includes substance P and homeostasis. neurokinin B. It exhibits a variety of activities related to US 7,256.253 B2 19 20 Smooth muscle contraction, pain transmission, bronchocon J. Endothelins and Related Peptides (SEQ ID NOS: 685 striction, vasodilation and modulation of the immune sys 744)—The endothelin peptide family consists of the 21 tem. amino acid isoforms endothelin-1, endothelin-2, endothelin Neuromedin-Neuromedins, Smooth-muscle-stimulating 3, Sarafotoxin (a Snake venom) and Scorpion toxin. peptides, are commonly divided into four groups: bombesin Endothelins (ET) and Big Endothelins—Endothelins are like, -like, -like and neuromedins U. found on endothelial cells in a wide variety of organ These neuropeptides and their receptors are localized to all systems. Examples of pathologies and physiological pro components of the HPA hypophyseal pituitary axis, the only cesses associated with changes in endothelin levels and exemption seems to be neurokinin B, which is not detected synthetics include: atherosclerosis and hypertension, coro 10 nary vasospasm, acute renal failure, changes in intracellular in the adenohypophysis. Neuromedins exert a manifold Ca2" levels, and effects on the renin-angiotensin system. effect on HPA axis, and their action on the adrenal Suggests Endothelins are released in reponse to variations in angio their involvement in the regulation of growth, structure and tensin II, vasopressin, and (e.g. TGF-Bm TNF-C. function of the adrenal cortex. Neuromedins may exert both IL-B-) levels as well as other physiological events including direct and indirect effects on the adrenal cortex. Direct effect 15 increase blood flow. is proven by the stimulation of mineralo- and glucocorticoid The endothelin family of peptides consists of highly therapeutic peptides by isolated or cultured adrenocortical potent endogenous vasoconstrictor agents first isolated from cells and by mobilisation of intracellular Ca2+. Indirect endothelial cell supernatant. They regulate blood flow to effects, on the other hand, may be mediated by ACTH, organs by exporting a vasoconstrictive effect on arteries. arginine-vasopressin, angiotensin II, catecholamines or by Endothelins are derived from big-endothelin, which is other regulatory Substances of medullary origin. cleaved by a unique membrane-bound metalloprotease, Substance P and Related Peptides—Substance P is an endothelin-converting enzyme, into the 21-amino-acid bio eleven amino acid peptide, first isolated from brain and active forms (ET-1, ET-2 and ET-3). intestine. It has been proposed as a neuromodulator involved Of the 3 isoforms (ET-1, ET-2, ET-3), endothelin-1 is the in pain transmission in the spinal cord. It also affects 25 major isoform and plays an important role for regulation of contraction of Smooth muscle, reduction of blood pressure, vascular function. Endogenous endothelin peptides and their stimulation of secretory tissue, and release of histimine from receptors are differentially distributed throughout the many mast cells. Smooth muscle tissues including blood vessels, uterus, blad 30 der and intestine. Through this widespread distribution and I. Renin Related Peptides localization, they exert biological functions in regulating Angiotensins (SEQID NOS: 628-677)—Angiotensin is a Vascular tone and causing mitogenesis. ETS and their recep 10 amino acid peptide derived from enzymatic cleavage of tor Subtypes are also present in various endocrine organs. It a2-globin by the kidney enzyme renin. The C-terminal 2 appears to act as a modulator of Secretion of prolactin, amino acids are then released to yield angiotensin I, which 35 gonadotropins GH and TSH. Endothelin may also be the is responsible for essential hypertension through stimulated disease marker or an etiologic factor in ischemic heart synthesis and release of from adrenal cells. It is disease, atherosclerosis, congestive heart failure, renal fail a multifunctional hormone regulating blood pressure, ure, systemic hypertension, pulmonary hypertension, cere plasma Volume, neuronal function thirst, and water intake. bral vasospasm. Angiotensin II is an octapeptide derived from angiotensin 40 Exogenously administered endothelin-1 has been demon I by angiotensin converting enzyme, and is widely distrib strated to increase peripheral resistance and blood pressure uted both centrally and peripherally in organs such as the in a dose-dependent manner. However, during the first heart, the kidneys, and the liver. Angiotensin IV is the minutes of intravenous administration endothelins also terminal hexapeptide fragment of angiotensin II formed decrease peripheral resistance and blood pressure, presum metabolically by proteolytic cleavage from either angio 45 ably due to the release of vasodilatory compounds Such as tensin I or angiotensin II. It plays a role in vascular control, nitric oxide, prostacyclin, and atrial natriuretic peptide. cardiac growth, renal blood flow and memory function. ET(A) Receptor Antagonists—Endothelin receptors exist Angiotensin II is the key peptide hormone that regulates as two types: A (ET-A) and B (ET-B1 and ET-B2). ETA vascular Smooth muscle tone, blood pressure, free water receptors are responsible for while ET-B1 and ET-B2 medi intake and sodium retension. It controls vascular homeosta 50 ate vasorelaxation and vasoconstriction respectively. sis compensating for loss of intravascular volume by stimu Sarafotoxin Peptides—As already described, endothelin lating increased vasospastic tone, increase Sodium retention (ET) peptides are potent growth factors binding to G pro and increased free water intake. tein-coupled receptors. (S6) isolated from Renin substrates and Inhibitors (SEQ ID NOS: 678 Atractaspis engaddensis are highly homologous to endot 684)—Renin is a very specific aspartic protease, which is 55 helins. Sarafotoxin peptides have marked vasoconstrictive synthesized and released by differentiated smooth muscle activity and are responsible for the ischemic limb loss that cells in the vasculature of the kidney called granular epi follows snake or scorpion bites. They could be used thera thelial cells. Renin is specific for its Substrate, angiotensi peutically as a peptidase Stabalized peptide as a vasopressive nogen, which it cleaves specifically at the Leu'-Val' bond agent in shock and sepsis. to form the decapeptide, angiotensin I (Al). The renin 60 angiotensin System is involved in the control of fluid and K. Opioid Peptides (SEQ ID NOS: 745-927) Opioids are mineral balance throughout the vertebrates. Renin can be a large class of drugs, used clinically as painkillers, that found in mammals, birds, reptiles, amphibians, bony fishes, include both plant-derived and synthetic alkaloids and pep cartilaginous fishes, and agnathans. Specific renin inhibitors tides found endogenously in the mammalian brain. While can also be designed, with therapeutic applications for 65 the plant-derived alkaloids have been known and used for treatment of for example hypertension and congestive heart thousands of years, the endogenous opioid peptides were failure (Blundell et al., 1987). discovered only in the mid-1970s. US 7,256.253 B2 21 22 Opioids include casomorphin peptides, demorphins, assist development of immature, precursor cells into fully , , deltorphins, , and ana competent T cells. Thymic peptides seem to regulate the logs and derivatives of these. expression of various and monokine receptors on T Casomorphin Peptides—Casomorphin peptides are novel cells and induce Secretion of IL-2, interferon alpha, and opioid peptides derived from casein(S-casomorphins). Beta (disease-fighting Substances) when the casomorphins are the more exStensive studied opioid pep immune system is challenged. There are reports that the use tides arising from food proteins (beta-caseins). They were of thymic hormones in children with immuno-deficiencies originally isolated from bovine beta-casein, the same caused by chemotherapy has resulted in an increase in sequences occur in ovine and buffalo beta-caseins. circulating T cells, normalization of Subsets, and Dermorphins—Demorphin is a is a seven amino acid 10 restoration of delayed hypersensitivity reactions. peptide, originally isolated from Phylomedusa sauvagei frog Thymopoietin is the largest of the known skin. It is a ligand which binds with high affinity to the L thymic hormones and consists of 49 amino acids. opioid receptor, and has many biological roles including Thymulin Previously known as thymic serum factor, analgesia, endocrine modulation, immunomodulation, thymulin is the smallest of the chemically characterized increased K' conductance and inhibition of action poten 15 thymic hormones and consists of 9 amino acids. Thymulin tials. is the hormone responsible for stimulating the production of /New-Endorphin Precursor Related Peptides— immune-system T cells Dynorphins are a class of endogenous opioids that exist in Thymopentin Thymopentin is a Small, synthesized thy multiple forms in the central nervous system. Dynorphins mic peptide drug, also known as therapeutic peptide-5 or are derived from the precursor ( Timunox. In the U.S. it is being developed as an AIDS B). Dynorphin, also known as Dynorphin A1-17, is a well therapy by the Immunobiology Research Institute. Thymo known opioid which has the sequence Tyr-Gly-Gly-Phe pentin has been studied more extensively than most other Leu-Arg-Arg-Ile-Arg-Pro'-Lys-Leu-Lys-Trp-Asp-Asn thymic peptide drugs. At least one study has claimed a Gln. SEQID NO:1. A number of derivatives and analogs of significant rise in T cells and slight clinical improvement in dynorphin are known including Dyn A1-13, SEQID NO: 2 25 those patients who received thymopentin three times a week, Dyn A2-13, SEQID NO:3, Dyn A1-12, Dyn A2-12 and Dyn compared to untreated control participants. Compared to the A2-17 as well as amide analogs such as those mentioned in 14 untreated control participants, those taking the drug U.S. Pat. No. 4.462,941 of Lee et al., N-terminus truncated showed greater “immunologic stability” and some clinical dynorphin analogs such as those described in International improvement. Patent Application WO 96/06626 of Lee et al. and des-Tyr 30 Thymosin is a mixture of 15 or more pro or des-Tyr-Gly analogs such as those disclosed in Interna teins. One of these proteins is thymosin alpha-1 which tional Patent Application WO 93/25217 also of Lee et al. consists of 28 amino acids. Thymosin has therapeutic use for The dynorphis are highly potent opioids, and demonstrate the treatment of primary immunodeficiencies and as a selective affinity for the kappa receptor. See Goldstein, A., booster for influenza vaccine in renal dialysis patients. It is Peptides, Structure and Function, Proceedings of the 8th 35 also being tested in ongoing clinical trials for activity against American Peptides Symposium, Hruby, V. J. and Rich, D. chronic hepatitis B and C, HIV infection, and certain forms H., eds., 409 (1983). of cancer. Endorphins—The endorphis are derived from the precur Thymic Humoral Factor (THF) THF is a thymic peptide sor protein-. They have been found to elicit several currently being examined as an anti-HIV treatment. In biological reactions such as analgesia, behavioral changes 40 preclinical studies in rats with CMV-related immunosup and growth hormone release. See Akil. H. et al., Ann. Rev. pression, THF restored immune competence through modu Neurosci., 7, 223 (1984). lation of T cells. In addition, it may have therapeutic use in Enkepalins & Related Peptides—Enkephalins and endor the treatment of herpes, causing (at least in one study) the phins are that inhibit transmission of pain viral infection’s rapid regression and increase of T-cell impulses. The activity of neurons in both the central and 45 populations. peripheral nervous systems is affected by a large number of neurohormones that act on cells quite distant from their site L. Other Peptides of release. Neurohormones can modify the ability of nerve Adrenomedullin Peptides (AM) (SEQ ID NOS: 935 cells to respond to synaptic neurotransmitters. Several Small 945)—Adrenomedullin is a potent vasodilator peptide that peptides with profound effects on the nervous system have 50 exerts major effects on cardiovascular functions. Its sys been discovered recently, for example enkephalins (e.g. temic administration causes a rapid and profound fall in Met- and Leu-enkephalin) and endorphins (e.g. B blood pressure and an increase in pulmonary blood flow. Its endorphin). These three contain a common tetrapeptide other actions are bronchodilatation, being an inhibitor of sequence (Tyr-Gly-Gly-Phe) that is essential to their func drinking behavior and an inhibitor of angiotensin-induced tions. Enkephalins and endorphins function as natural pain 55 aldosterone secretion. See The Journal of Biological Chem killers or opiates and decrease the pain responses in the istry, Vol. 270, No. 43, pp 25344-25347, 1995 and in the central nervous system. See also Akil. H. etl al., Ann. Rev. references cited therein Allatostatin Peptides (SEQID NOS: 946-949) Allatost Neurosci., 7, 223 (1984). atins are 6-18 amino acid peptides synthesized by insects to L. Thymic peptides (SEQ ID NOS: 928-934) The thymus 60 control production of juvenile hormones, which in turn is thought to be responsible for the development and regu regulate functions including metamorphosis and egg pro lation of T cell immunity in both infants and adults. The duction. While neuropeptides of the allatostatin family thymus seems to exert its regulatory functions through the inhibit in vitro production of juvenile hormone, which secretion of various noncellular, hormonelike products via modulates aspects of development and reproduction in the its epithelial cells, called thymic peptides. 65 cockroach, Diploptera punctata, they are Susceptible to Thymic peptides are reported to have many effects on T inactivation by peptidases in the hemolymph, gut, and bound cells. Several studies have reported that thymic peptides can to internal tissues. US 7,256.253 B2 23 24 Amyloid Beta-Protein Fragments (AB fragments) (SEQ the alveolar fluid, and thus impairs the bactericidal activity ID NOS: 950-1010) These are the principle component of of B-defensins, which are salt sensitive. Andreu D. (Ed.) the amyloid plaques that accumulate intracellularly and (1998) “Antimicrobial peptides’ Biopolymers (Peptide Sci extracellularly in the neuritic plaques in the brain in Alzhe ence) vol 47, N, pp. 413-491. A. Andreu, L. Rivas (1998) imer's Disease. A? is a 4.5 kD protein, about 40-42 amino 5 Animal Antimicrobial Peptides. An Overview, Biopolymers acids long, that is derived from the C-terminus of amyloid (Pep. Sci.) 47: pp. 415-433. precursor protein (APP). APP is a membrane-spanning gly Antioxidant Peptides (SEQ ID NOS: 1048-1050) Anti coprotein that, in the normal processing pathway, is cleaved oxidants are agents that prevents oxidative damage to tissue. inside the AB protein to produce C-SAPP, a secreted form of Mammalian cells are continuously exposed to activated APP. Formation of C-SAPP precludes formation of AB. It has 10 oxygen species such as Superoxide, hydrogen peroxide, been proposed that AB accumulates by virtue of abnormal hydroxyl radical, and singlet oxygen. These reactive oxygen processing of APP, so that compounds that inhibit the intermediates are generated in vivo by cells in response to activity of enzymes responsible for AB production are being aerobic metabolism, catabolism of drugs and other Xenobi sought. See, e.g., Wagner et al. Biotech. Report (1994/1995), otics, ultraviolet and X-ray radiation, and the respiratory pp. 106-107; and Selkoe (1993) TINS 16:403409. Under 15 burst of phagocytic cells (such as white blood cells) to kill certain conditions AB peptides first aggregate and then are invading bacteria Such as those introduced through wounds. deposited as a folded B-sheet structure that is characteristic Hydrogen peroxide, for example, is produced during respi of amyloid fibrils. B-amyloid (1-42) forms aggregates at a ration of most living organisms especially by stressed and significantly greater rate and to a greater extent than B-amy injured cells. loid (1-40). One example of antioxidant peptides is natural killer Antimicrobial Peptides (SEQ ID NOS: 1011-1047)— enhancing factor B (NKEF-B), which belongs to a highly Antimicrobial peptides are a key component of the innate conserved family of recently discovered antioxidants. Natu immune systems of most multicellular organisms, being ral killer-enhancing factor (NKEF) was identified and active against one or more microorganisms such as bacteria, cloned on the basis of its ability to increase NK cytotoxicity. fungi, protozoa, yeast, and mycobacteria. Examples of Such 25 Two genes, NKEF-A and -B, encode NKEF proteins and peptides include defensin, cecropin, buforin, and magainin. sequence analysis presented Suggests that each belongs to a Despite broad divergences in sequence and taxonomy, most highly conserved family of antioxidants. The role of antimicrobial peptides share a common mechanism of NKEF-B as an antioxidant has been demonstrated by its action, i.e. membrane permeabilization of the pathogen. protection of transfected cells to oxidative damage by hydro They are classified in two broad groups: linear and cyclic. In 30 gen peroxide. NKEF-B has antioxidant activities toward the linear antimicrobial peptides, there are two subgroups: prooxidants such as alkyl hydroperoxide and MeHg. linear peptides tending to adopt C-helical amphipathic con Together with its antioxidant activity, the induction of formation and linear peptides of unusual composition, rich NKEF-B by HP indicates that NKEF-B is an important in amino acids such as Pro, Arg, or Trp. The cyclic group oxidative stress protein providing protection against a vari encompasses all cysteine-containing peptides, and can be 35 further divided into two subgroups corresponding to single ety of Xenobiotic toxic agents. or multiple disulfide structures. Apoptosis Related Peptides (SEQID NOS: 1051-1075)– Most antimicrobial peptides provoke an increase in Animal cells can self-destruct via an intrinsic program of plasma membrane permeability. There is also evidence of cell death (Steller, 1995). Apoptosis is a form of pro other mechanisms, such as inhibition of specific membrane 40 grammed cell death that is characterized by specific mor proteins, synthesis of stress proteins, arrest of DNA synthe phologic and biochemical properties (Wyllie et al., 1980). sis, breakage of single-strand DNA by defensins, interaction Morphologically, apoptosis is characterized by a series of with DNA (without arrest of synthesis) by buforins, or structural changes in dying cells: blebbing (i.e. blistering) of production of hydrogen peroxide. Antimicrobial peptides the plasma membrane, condensation of the cytoplasm and can also act by triggering self-destructive mechanisms such 45 nucleus, and cellular fragmentation into membrane apop as apoptosis in eukaryotic cells or autolysis in bacterial totic bodies (Steller, 1995; Wyllie et al., 1980). targets. Antimicrobial peptides are also known to act as Biochemically, apoptosis is characterized by the degra inhibitors of enzymes produced by pathogenic organisms, dation of chromatin, initially into large fragments of 50-300 either by serving as pseudo-Substrates or by tight binding to kilobases and Subsequently into Smaller fragments that are the active sight that disturbs the access of the substrate. 50 monomers and multimers of 200 bases (Oberhammer et al., Increased levels of antimicrobial peptides have been 1993; Wylie, 1980). Other biochemical indicators of apo reported for several animal and human infections for ptosis are induced or increased levels of the protein clusterin example for C-defensins in Mycobacterium, Pasteurella, or (Pearse et al., 1992), also known as TRPM-2 or SGP-2, and Cryptopondium infections and for a variety of peptides in activation of the enzyme typell transglutaminase, which blisters and wound fluid. Inflammatory situations or stimuli 55 crosslinks proteins to the envelope of apoptotic bodies are also associated with induction of antibiotic peptides. (Fesus et al., 1991). Apoptosis is a complex phenomenon of Depleted levels of antimicrobial peptides are associated to related morphological and biochemical processes that can several pathologies. Thus, patients of specific granule-defi vary with tissue and cell type (Zakeri et al., 1995). ciency syndrome, completely lacking in C-defensins, Suffer The execution of apoptosis minimizes the leakage of from frequent and severe bacterial infections. Low levels of 60 cellular constituents from dying cells (apoptosis causes the histatins from saliva in HIV patients has been correlated cell to involute). For example, proteases could damage with a higher incidence of oral candidiasis and fungal adjacent cells or stimulate an inflammatory response. This infections. Perhaps the most compelling illustration of the cardinal feature of apoptosis distinguishes it from necrosis, implication of antimicrobial peptides in human pathology which usually results from trauma that causes injured cells comes from cystic fibrosis, a genetic disease associated with 65 to Swell and lyse, releasing the cytoplasmic material that recurrent bacterial infections of the airways. The defective stimulates an inflammatory response (Steller, 1995; Wyllie chloride channel causing the disease increases the Salinity of et al., 1980) US 7,256.253 B2 25 26 Bag Cell Peptides (BCP) (SEQ ID NOS: 1076-1080) between soluble mediators and membrane-bound cellular The bag cells of the marine mollusc Aply adhesion molecules. Soluble cellular chemotactic factors, sia californica are involved in the egg-laying behavior of the which are produced in the damaged tissue by a variety of animal. These neurosecretory cells synthesize an egg-laying resident cells, set up a chemical concentration gradient out hormone (ELH) precursor protein, yielding multiple bioac to the plasma compartment. Interaction of these factors with tive peptides, including ELH, several bag cell peptides their receptors on leukocytes leads to a directional migration (BCP) and acidic peptide (AP). The bag cells of the marine of the leukocytes toward increasing concentrations of the mollusc Aplysia Californica are well-characterized neuroen chemotactic factor. Simultaneously, various adhesion pep docrine cells that initiate egg laying. During sexual matu tides are upregulated on the leukocyte which mediate the ration, these cells (bag cell neurons), develop the capability 10 initial rolling on the endothelial tissue, binding to a specific of storing hormones that are released during periods of ligand on the activated endothelial tissue, and finally migra nervous system stimulation. The hormones are important to tion between endothelial cells into the tissue. The steps in the process of egg laying, and so must not be released before this cascade of events are mediated by the interaction of the animal is sexually mature. Alpha-bag cell peptide belong specific cell surface proteins, termed “cell adhesion mol to a small family of structurally related peptides that can 15 ecules such as. E-selectin (ELAM-1, endothelial leukocyte elicit bag-cell activity in vitro. adhesion molecule-1), ICAM-1 (intercellular adhesion mol Bombesin (SEQ ID NOS: 1081-1090) Bombesin is a ecule-1), and VCAM-1 (vascular cell adhesion molecule-1). bioactive tetradecapeptide neuropeptide that belongs to a Chemotactic Peptides (SEQ ID NOS: 1102-1113)– family of peptides sharing a common C terminal sequence, Chemotactic peptides are peptides that stimulate the migra Trp-Ala-X-Gly-His-Met-NH2, and the N terminal region. It tion of white cells, leukocytes and macrophages into tissues has a modulatory role found in nerves of the brain and gut at the site of infection or injury or alternatively the prevent that prevents gastric injury by release of endogenous gastrin. the migration of these same cells away from these sites. The mammalian homologue of bombesin is gastrin-releas Complement Inhibitors (SEQ ID NOS: 1114-1120)– ing peptide (GRP). Inhibition of complement attack on Xenotransplants may be Bone Gla Protein Peptides (SEQ ID NOS: 1091-1097)— 25 accomplished by the use of complement inhibitors. The (bone Gla-protein, or BGP) is produced and rejection of transplanted organs may involve both an secreted by osteoblasts in the process of bone formation. As extremely rapid hyperacute rejection (HAR) phase and a with collagen, this protein is a component of bone matrix. slower cellular rejection phase. HAR of xenotransplants is Serum osteocalcin rises when bone formation rates increase. initiated by preformed “natural antibodies that bind to Levels are high during puberty when bone growth is most 30 donor organ endothelium and activate complement attack by rapid. Often levels are also high in diseases having high the recipient immune system. Activation of complement bone turnover, such as hyperparathyroidism and hyperthy leads to the generation of fluid phase (C3a, C5a) and roidism. In postmenopausal osteoporosis, osteocalcin levels membrane bound (C3b and C5b-9, i.e., C5b, C6, C7, C8, and are sometimes increased, reflecting the increased turnover of C9) proteins with chemotactic, procoagulant, proinflamma bone secondary to rapid bone resorption. In senile 35 tory, adhesive, and cytolytic properties. Complement inhibi osteoporosis, occurring in more elderly Subjects, osteocalcin tors inhibit this process. levels are more likely to be low, reflecting reduced rates of Cortistatin Peptides (SEQ ID NOS: 1121-1124) Cor both bone turnover and bone formation. A treatment regimen tistatin, whose mRNA accumulates during sleep deprivation, that increases bone formation also raises the serum osteo apparently acts by antagonizing the effects of acetylcholine calcin levels. 40 on cortical excitability, thereby causing synchronization CART Peptides (SEQ ID NOS: 1098-1100) Cocaine brain slow waves. Cortistatin-14 (CST-14) shares 11 of its and amphetamine regulated transcript peptide (CART), is a 14 residues with somatostatin-14 (SRIF-14), yet its effects recently discovered hypothalamic peptide with a potent on sleep physiology, locomotor behavior and hippocampal appetite Suppressing activity. In the rat the CART gene function are quite different from those of Somatostatin. encodes a peptide of either 129 or 116 amino acid residues 45 Fibronectin Fragments & Fibrin Related Peptides (SEQ whereas only the short form exists in humans. The predicted ID NOS: 1125-1174)—Fibronectin is a large glycoprotein signal sequence is 27 amino acid residues resulting in a that is composed of blocks of three types of repeating, prohormone of 102 or 89 residues. The C-terminal end of homologous peptide sequences. Several of the homologous CART, consisting of 48 amino acid residues and 3 disul blocks form functional domains that are organized in a linear phide bonds, is thought to constitute a biologically active 50 array on two nearly identical Subunit arms. Each arm can be part of the molecule. divided into functional domains, which are often referred to In the central nervous system CART is highly expressed by one of the substances which bind in that region, for in many hypothalamic nuclei. Some of which are involved in example the heparin-binding fragment, the fibrin binding regulating feeding behavior. The CART mRNA is regulated fragment, and the cell-binding fragment. In several cell by leptin, and the expressed CART is a potent inhibitor of 55 types, the Arg-Gly-Asp (RGD) sequence in the cell-binding feeding that even overrides the feeding response induced by domain of fibronectin interacts with a cell-surface glyco neuropeptide Y. The putative CART receptor is therefore a protein designated lib/IIIa. Fibronectin also binds to extra potential therapeutic target for an anti-obesity drug. See cellular and basement-membrane components, to the enve CART, a new anorectic peptide Thim L. Kristensen P; lope glycoprotein of viruses, to a variety of bacteria Larsen PJ: Wulff BS, Int J Biochem Cell Biol, 30(12): 60 including Staphylococci and streptococci, and to parasites 1281-4 1998 Dec. Such as Trypanosoma Cruzi and Leishmania species. Cell Adhesion Peptides (SEQ ID NO: 1101) Cellular Fibronectin has several adhesive functions, for example adhesion peptides are directly involved in the cellular cell-to-cell adhesion, cell-to-basement-membrane attach response to external stimuli. For example, during an inflam ment, and clot stabilization. In addition, fibronectin pro matory response, leukocytes must leave the plasma com 65 motes embryogenesis, nerve regeneration, fibroblast migra partment and migrate to the point of antigenic insult. The tion, macrophage function, and pathogen (virus, fungus, mechanism of this migratory event is a complex interplay bacteria, and protozoa) binding to mammalian cells and US 7,256.253 B2 27 28 extracellular matrix. Thus, fibronectin is involved in the and 116 amino acids). Its role is inhibition of FSH secretion. pathogenesis of infections from the initiation of the infection The two inhibin isoforms, inhibin A and inhibin B, are through the final stages of wound healing. See Proctor, R. A., produced by the gonads in the course of gamete maturation Rev. Infect. Dis., 9, 317 (1987). and have different patterns of secretion during the menstrual FMRF and Analog peptides (SEQID NOS: 1175-1187)– 5 cycle. Inhibins are also produced by the and fetal FMRF are neuropeptides encoded in the FMRFamide gene membranes and may be involved in physiological adaptation and have a common C-terminal FMRFamide but different of pregnancy. Clinically, inhibins may serve as sensitive N-terminal extensions. FMRFamide-related peptides tumor markers in postmenopausal women, or as useful tools (FaRPs) are present throughout the animal kingdom and for evaluating ovarian reserve in infertile women; they may affect both neural and gastrointestinal functions. Organisms 10 also be used in the diagnosis of materno-fetal disorders and have several genes encoding numerous FaRPs with a com to prevent maturation of the ovum or to inhibit ovulation. mon C-terminal structure but different N-terminal amino Interleukin (IL) and Interleukin Receptor Proteins (SEQ acid extensions. ID NOS: 1256-1263)—Interleukins are growth factors tar Galanin & related peptides (SEQ ID NOS: 1188-1208)— geted to cells of hematopoietic origin. A variety of biological Galanin is a 29-30 amino acid peptide originally isolated 15 activities associated with immune and inflammatory from pig Small intestine. It is found in two biologically responses have been ascribed to interleukins. These active forms: GAL (1-19), and GAL (1-30), a non-amidated responses include fever, cartilage breakdown, bone resorp form. It has many biological roles including: the inhibition tion, thymocyte proliferation, activation of T and B lym of the release of biogenic amines in the hypothalamus, the phocytes, induction of acute-phase protein synthesis from pre- and post-synaptic inhibition of cholinergic function, the 20 hepatocytes, fibroblast proliferation, and differentiation and maintenance of gastrointestinal homeostasis, and the regu proliferation of bone marrow cells. lation of insulin and glucagon Secretion. Laminin Fragments (SEQ ID NOS: 1264-1284)—Lami Growth Factors & related peptides (SEQ ID NOS: 1209 nin, the major noncollagenous glycoprotein of basement 1240)—Growth factors are a family of proteins that regulate membranes, has been shown to promote the adhesion, cell division. Some growth factors are cell type specific, 25 spreading, and migration of a variety of tumor cell types in stimulating division of only those cells with appropriate vitro. In particular, the major current studies in the labora receptors. Other growth factors are more general in their tory utilize intact laminin, purified proteolytic fragments of effects. There are also extracellular factors that antagonize laminin, and synthetic peptides of laminin to identify func the effects of growth factors, slowing or preventing division tionally active sites on this large protein. Components of (for example transforming growth factor beta and tumor 30 Such basement membranes are important modulators of necrosis factor). These extracellular signals act through cell growth, development, and differentiation for various cell surface receptors very similar to those for hormones, and by types. A conjugated laminin could be used to prevent infla similar mechanisms: the production of intracellular second mation or fibrosis in tissues. messangers, protein phosphorylation, and ultimately, alter This category also includes the peptide kringle-5 (or K-5). ation of gene expression. 35 As used herein, the term "kringle 5” refers to the region of Gtherapeutic peptide-Binding protein fragments (SEQID mammalian plasminogen having three disulfide bonds NOS: 1241-1246)—Members of a family of Gtherapeutic which contribute to the specific three-dimensional confir peptide-binding regulatory proteins (G-proteins) transduce mation defined by the fifth kringle region of the mammalian signals from membrane-bound receptors to intracellular plasminogen molecule. One Such disulfide bond links the effectors. The family includes G and G, which are respon- 40 cysteine residues located at amino acid positions 462 and sible for stimulation and inhibition, respectively, of adeny 541, a second links the cysteine residues located at amino late cyclase. Transducin (T), localized in the disc mem acid positions 483 and 524 and a third links the cysteine branes of retinal rod outer segments, couples activation of residues located at amino acid positions 512 and 536. The rhodopsin by light to increased cyclinc GMP phosphodi term "kringle 5 peptide peptides' refers to peptides with esterase activity. Go, found originally in bovine brain, is a 45 anti-angiogenic activity of between 4 and 104 amino acids fourth member of the family. (inclusive) with a substantial sequence homology to the Purified G proteins have similar physical properties. They corresponding peptide fragment of mammalian plasmino are heterodimers composed of C, B, and Y subunits. The C. gen. subunits bind and hydrolyze Gtherapeutic peptide. See S. M. Leptin Fragment Peptides (SEQ ID NOS: 1285-1288)— Mumby et al., PNAS 83,265 (1986) and Lehningerp. 764. 50 Leptin, the protein product of the obesity gene, is secreted by Guanylin and Uroguanvlin (SEQID NOS: 1247-1249)– fat cells. Leptin is involved in the regulation of bodyweight Guanylin and uroguanylin are peptides isolated from intes and metabolism in man and might also be involved in the tinal mucosa, and urine, which regulate cyclic GMP pro pathophysiology of the insulin resistance syndrome, which duction in enterocytes bind to and activate guanylate cyclase is associated with the development of cardiovascular dis C and control salt and water transport in many epithelia in 55 CaSS vertebrates, mimicking the action of several heat-stable Leucokinins (SEQID NOS: 1289-98) Leucokinins are a bacteria enterotoxins. In the kidney, both of them have group of widespread insect hormones that stimulate gut well-documented natriuretic and kaliuretic effects. motility and tubule fluid secretion rates. In tubules, their Chloride secretion in the intestine is regulated by these major action is to raise chloride permeability by binding to hormones via activation of guanylate cyclase C (GC-C). 60 a receptor on the basolateral membrane. Both peptides are expressed in a variety of tissues and Pituitary Adenylate Cyclase Activating Polypeptide organs, including the kidney. In the isolated perfused kidney (PACAP) (SEQ ID NOS: 1299-1311). It is a thirty-eight and in vivo these hormones induce natriuresis and diuresis, amino acid peptide first isolated from ovine hypothalamus, however, localisation and cellular mechanisms of their which also occurs in a 27 amino acid form called PACAP action in the kidney are still unknown. 65 27. PACAP has been localized in the hypothalamus, else Inhibin Peptides (SEQ ID NOS: 1250-1255) Inhibin is where in the brain, respiratory tract and gastrointestinal composed of two subunits (C. is 134 amino acids; B is 115 system. It has many biological actions, including neurotrans US 7,256.253 B2 29 30 mitter and hormonal functions, involvement in regulation of of 0.5 ug/ml and releases histamine from the cells in the energy metabolism, and neuronal cytoprotective activity. same concentration. See IY. Hirai et al., Chem. Pharm. Bull. Pancreastatin (SEQID NOS: 1312-1324) Pancreastatin 27, 1942 (1979). is a 49 amino acid peptide first isolated, purified and Other examples of Such toxins include omega-agatoxin characterized from porcine pancreas. Its biological activity 5 TK, agelenin, apamin, calcicudine, calciseptine, charbdot in different tissues can be assigned to the C-terminal part of oxin, chlorotoxin, conotoxins, endotoxin inhibitors, gegra the molecule. Pancreastatin has a prohormonal precursor, phutoxins, iberiotoxin, kaliotoxin, mast cell degranulating chromogranin A, which is a glycoprotein present in neu peptides, margatoxin, neurotoxin NSTX-3, PLTX-II, scyl roendocrine cells, including the endocrine pancreas latoxin, Stichodactyla toxin, and derivatives and fragments Polypeptides (SEQ ID NOS: 1325-1326) these are 10 thereof. repetitive chains. Two examples are provided: (pro-Hyp Trypsin Inhibitors (SEQ ID NOS: 1416-1418) Trypsin Gly) 10*20H20 and Poly-L-Lysine Hydrochloride. inhibitors functions as an inhibitors of trypsin, as well as Signal Transduction Reagents (SEQ ID NOS: 1327 other serine proteases. Useful for treatment of lung inflam 1367) Signal transduction is the process by which an mation, pancreatitis, myocardial infarction, cerebrovascular extracellular signal (for example chemical, mechanical, or 15 ischemia electrical) is amplified and converted to a cellular response. Virus Related Peptides (SEQ ID NOS: 1419-1529)— Many reagents are involved in this process, for example Virus related peptides are proteins related to viruses, for achatin-1, glycogen synthase, autocamtide 2, calcineurin example virus receptors, virus inhibitors, and envelope autoinhibitory peptide, calmodulin dependent protein kinase proteins. Examples include but are not limited to peptide II, calmodulin dependent protein kinase Substrate, calmodu inhibitors of human immunodeficiency virus (HIV), respi lin dependent protein kinase Substrate analog, CKS-17, ratory syncytial virus (RSV), human parainfluenza virus Cys-Kemptide, autocamtide 2, malantide, melittin, phos (HPV), measles virus (MeV), and simian immunodeficiency phate acceptor peptide, protein kinase C fragments, P34cd2 virus (SIV), fluorogenic Human CMV Protease Substrate, kinase fragment, P60c-Src Substrate II, protein kinase A HCV Core Protein, HCV NS4A Protein, Hepatitis B Virus 25 Receptor Binding Fragment, Hepatitis B Virus Pre-S fragments, tyrosine protein kinase Substrate, syntide 2, S6 Region, Herpes Virus Inhibitor 2, HIV Envelope Protein kinase Substrate peptide 32, tyrosine specific protein kinase Fragment, HIV gag fragment, HIV substrate, HIV-1 Inhibi inhibitor, and their derivatives and fragments. tory Peptide, peptide T, T21, V3 decapeptide, Virus Repli Thrombin Inhibitors (SEQ ID NOS: 1368-1377) cation Inhibitor Peptide, and their fragments and derivatives. Thrombin is a key regulatory enzyme in the coagulation 30 These peptides can be administered therapeutically. For cascade; it serves a pluralistic role as both a positive and example, peptide T is a chain of 8 amino acids from the V2 negative feedback regulator. In addition to its direct effect on region of HIV-1 gp120. These amino acids look like a hemostasis, thrombin exerts direct effects on diverse cell portion of HIV’s outer envelope. It is under investigation as types that Support and amplify pathogenesis of arterial a treatment for HIV-related neurological and constitutional thrombus disease. The enzyme is the strongest activator of 35 symptoms, as peptide T may be able to alleviate symptoms platelets causing them to aggregate and release Substances like fevers, night Sweats, weight loss, and fatigue. It has also (eg. ADPTXA.sub.2 NE) that further propagate the throm been shown to resolve psoriatic lesions. botic cycle. Platelets in a fibrin mesh comprise the principal Miscellaneous peptides (SEQ ID NOS: 1529-1617)– framework of a white thrombus. Thrombin also exerts direct Including adjuvant peptide analogs, alpha mating factor, effects on endothelial cells causing release of vasoconstric 40 antiarrhythmic peptide, anorexigenic peptide, alpha-1 antit tor Substances and translocation of adhesion molecules that rypsin, bovine pineal antireproductive peptide, bursin, C3 become sites for attachment of immune cells. In addition, peptide P16, cadherin peptide, chromogranin. A fragment, the enzyme causes mitogenesis of Smooth muscle cells and contraceptive tetrapeptide, conantokin G, conantokin T. proliferation of fibroblasts. From this analysis, it is apparent crustacean cardioactive peptide, C-telopeptide, cytochrome that inhibition of thrombin activity by thrombin inhibitors 45 b588 peptide, decorsin, delicious peptide, delta-sleep-induc constitutes a viable therapeutic approach towards the attenu ing peptide, diazempam-binding inhibitor fragment, nitric ation of proliferative events associated with thrombosis. oxide synthase blocking peptide, OVA peptide, platelet Toxins (SEQ ID NOS: 1378-1415) A toxin can be calpain inhibitor (P1), plasminogen activator inhibitor 1, conjugated using the present invention to target cancer cells, rigin, Schizophrenia related peptide, Sodium potassium receptors, viruses, or blood cells. Once the toxin binds to the 50 Atherapeutic peptidease inhibitor-1, speract, sperm activat target cells the toxin is allowed to internalize and cause cell ing peptide, Systemin, thrombin receptor agonist (three toxicity and eventually cell death. Toxins have been widely peptides), tuftsin, adipokinetic hormone, uremic pentapep used as cancer therapeutics. tide, Antifreeze Polypeptide, tumor necrosis factor, leech One example of a class of toxins is the mast cell degranu Des Asp10 Decorsin, L-Ornithyltaurine Hydrochloride, lating peptide, a cationic 22-amino acid residue peptide with 55 p-Aminophenylacetyl Tuftsin, Ac-Glu-Glu-Val-Val-Ala two disulfide bridges isolated from bee venom, causes mast Cys-pNA, Ac-Ser-Asp-Lys-Pro, Ac-rfwink-NH2, Cys-Gly cell degranulation and histamine release at low concentra Tyr-Gly-Pro-Lys-Lys-Lys-Arg-Lys-Val-Gly-Gly, DAla-Leu, tions and has anti-inflammatory activity at higher concen D-D-D-D-D, D-D-D-D-D-D, N-P-N-A-N-P-N-A, V-A-I-T- trations. It is a powerful anti-inflammatory, more than 100 V-L-V-K, V-G-V-R-V-R, V-I-H-S, V-P-D-P-R, Val-Thr-Cys times more effective than hydrocortisone in reducing inflam 60 Gly, R-S-R, Sea Urchin Sperm Activating Peptide, SHU mation. Because of these unique immunologic properties, 9119 MC3-R & MC4-R Antagonist, glaspimod MCD peptide may serve as a useful tool for studying (immunostimulant, useful against bacterial infections, fun secretory mechanisms of inflammatory cells such as mast gal infections, immune deficiency immune disorder, leuko cells, basophils, and leukocytes, leading to the design of penia), HP-228 (, useful against chemotherapy compounds with therapeutic potential. An example of a mast 65 induced emesis, toxicity, pain, diabetes mellitus, inflamma cell degranulating peptide is mastoparans, originating from tion, rheumatoid arthritis, obesity), alpha 2-plasmin inhibitor wasp venom. It degranulates mast cells in the concentration (plasmin inhibitor), APC tumor Suppressor (tumor Suppres US 7,256.253 B2 31 32 Sor, useful against neoplasm), early pregnancy factor (im reaction of maleimido groups with sulfhydryls is 1000-fold munosuppressor), endozepine diazepam binding inhibitor faster than with amines. A stable thioether linkage between (receptor peptide), gamma interferon (useful against leuke the maleimido group and the sulfhydryl is formed which mia), glandular kallikrei n-1 (immunostimulant), placental cannot be cleaved under physiological conditions. ribonuclease inhibitor, sarcolecin binding protein, Surfactant protein D. Wilms tumor Suppressor, wilm's tumor Suppres sor, GABAB 1b receptor peptide, prion related peptide (iPrP13), choline binding protein fragment (bacterial related O peptide), telomerase inhibitor, cardiostatin peptide, endosta tin derived peptide (angiogenesis inhibitor), prion inhibiting 10 peptide, N-methyl D-aspartate receptor antagonist, C-pep tide analog (useful against diabetic complications). O—sh C- -- 2. Modified Therapeutic Peptides O This invention relates to modified therapeutic peptides 15 O and their derivatives. The modified therapeutic peptides of the invention include reactive groups which can react with H available reactive functionalities on blood components to N-R form covalent bonds. The invention also relates to such modifications. Such combinations with blood components O-s and methods for their use. These methods include extending O the effective therapeutic in vivo half life of the modified O therapeutic peptides. O O HO | H. H. H To form covalent bonds with functionalities on a protein, N-R He- HO-C-C-C-C-N-R one may use as a reactive group a wide variety of active 25 carboxyl groups, particularly esters, where the hydroxyl moiety is physiologically acceptable at the levels required to modify the therapeutic peptide. While a number of different O hydroxyl groups may be employed in these linking agents, the most convenient would be N-hydroxysuccinimide 30 The therapeutic peptides and peptide derivatives of the (NHS), and N-hydroxy-sulfosuccinimide (sulfo-NHS). invention may be modified for specific labeling and non Primary amines are the principal targets for NHS esters as specific labeling of blood components. diagramed in schematic 1A below. Accessible C-amine A. Specific Labeling groups present on the N-termini of proteins react with NHS Preferably, the therapeutic peptides of this invention are esters. However, C.-amino groups on a protein may not be 35 designed to specifically react with thiol groups on mobile desirable or available for the NHS coupling. While five blood proteins. Such reaction is preferably established by amino acids have nitrogen in their side chains, only the covalent bonding of a therapeutic peptide modified with a e-amine of lysine reacts significantly with NHS esters. An maleimide link (e.g. prepared from GMBS, MPA or other amide bond is formed when the NHS ester conjugation maleimides) to a thiol group on a mobile blood protein Such reaction reacts with primary amines releasing N-hydrox 40 as serum albumin or IgG. ySuccinimide as demonstrated in schematic 1A below. Under certain circumstances, specific labeling with male imides offers several advantages over non-specific labeling of mobile proteins with groups such as NHS and sulfo-NHS. Schematic 1A Thiol groups are less abundant in vivo than amino groups. NHS - Ester Reaction Scheme 45 Therefore, the maleimide derivatives of this invention will covalently bond to fewer proteins. For example, in albumin O (the most abundant blood protein) there is only a single thiol group. Thus, therapeutic peptide-maleimide-albumin conju R-C-O-N + R-NH, P H7-9-- gates will tend to comprise approximately a 1:1 molar ratio of therapeutic peptide to albumin. In addition to albumin, IgG molecules (class II) also have free thiols. Since IgG O molecules and serum albumin make up the majority of the O soluble protein in blood they also make up the majority of the free thiol groups in blood that are available to covalently bond to maleimide-modified therapeutic peptides. R-C-N-R + HO-N Further, even among free thiol-containing blood proteins, specific labeling with maleimides leads to the preferential formation of therapeutic peptide-maleimide-albumin conju O gates, due to the unique characteristics of albumin itself. The 60 single free thiol group of albumin, highly conserved among In the preferred embodiments of this invention, the func species, is located at amino acid residue 34 (Cys). It has tionality on the protein will be a thiol group and the reactive been demonstrated recently that the Cys of albumin has group will be a maleimido-containing group Such as gamma increased reactivity relative to free thiols on other free maleimide-butyralamide (GMBA) or MPA. The maleimido thiol-containing proteins. This is due in part to the very low group is most selective for Sulfhydryl groups on peptides 65 pK value of 5.5 for the Cys of albumin. This is much lower when the pH of the reaction mixture is kept between 6.5 and than typical pK values for cysteines residues in general, 7.4 as shown in schematic 1 B below. At pH 7.0, the rate of which are typically about 8. Due to this low pK, under US 7,256.253 B2 33 34 normal physiological conditions Cys of albumin is pre Solutions and in the presence of free amines. Since male dominantly in the ionized form, which dramatically imide-therapeutic peptides will only react with free thiols, increases its reactivity. In addition to the low pK value of protective groups are generally not necessary to prevent the Cys, another factor which enhances the reactivity of Cys' maleimide-therapeutic peptides from reacting with itself. In is its location, which is in a crevice close to the Surface of 5 addition, the increased stability of the peptide permits the one loop of region V of albumin. This location makes Cys' use of further purification steps such as HPLC to prepare very available to ligands of all kinds, and is an important highly purified products suitable for in vivo use. Lastly, the factor in Cys's biological role as free radical trap and free increased chemical stability provides a product with a longer thiol scavenger. These properties make Cys' highly reactive shelf life. with therapeutic peptide-maleimides, and the reaction rate 10 B. Non-Specific Labeling acceleration can be as much as 1000-fold relative to rates of The therapeutic peptides of the invention may also be reaction of therapeutic peptide-maleimides with other free modified for non-specific labeling of blood components. thiol containing proteins. Bonds to amino groups will generally be employed, particu Another advantage of therapeutic peptide-maleimide-al larly with the formation of amide bonds for non-specific bumin conjugates is the reproducibility associated with the 15 labeling. To form Such bonds, one may use as a chemically 1:1 loading of peptide to albumin specifically at Cys. reactive group coupled to the therapeutic peptide a wide Other techniques, such as glutaraldehyde, DCC, EDC and variety of active carboxyl groups, particularly esters, where other chemical activations of for example, free amines lack the hydroxyl moiety is physiologically acceptable at the this selectivity. For example, albumin contains 52 lysine levels required. While a number of different hydroxyl groups residues, 25-30 of which are located on the surface of may be employed in these linking agents, the most conve albumin and accessible for conjugation. Activating these nient would be N-hydroxysuccinimide (NHS) and N-hy lysine residues, or alternatively modifying peptides to droxy-sulfosuccinimide (sulfo-NHS). couple through these lysine residues, results in a heterog Other linking agents which may be utilized are described enous population of conjugates. Even if 1:1 molar ratios of in U.S. Pat. No. 5,612,034, which is hereby incorporated peptide to albumin are employed, the yield will consist of 25 multiple conjugation products, some containing 0, 1, 2 or herein. The various sites with which the chemically reactive more peptides per albumin, and each having peptides ran groups of the non-specific therapeutic peptides may react in domly coupled at any one of the 25-30 available lysine sites. vivo include cells, particularly red blood cells (erythrocytes) Given the numerous combinations possible, characterization and platelets, and proteins, such as immunoglobulins, of the exact composition and nature of each batch becomes 30 difficult, and batch-to-batch reproducibility is all but impos including IgG and IgM, serum albumin, ferritin, steroid sible, making such conjugates less desirable as a therapeutic. binding proteins, transferrin, thyroxin binding protein, C-2- Additionally, while it would seem that conjugation through macroglobulin, and the like. Those receptors with which the lysine residues of albumin would at least have the advantage derivatized therapeutic peptides react, which are not long of delivering more therapeutic agent per albumin molecule, 35 lived, will generally be eliminated from the human host studies have shown that a 1:1 ratio of therapeutic agent to within about three days. The proteins indicated above (in albumin is preferred. In an article by Stehle, et al., “The cluding the proteins of the cells) will remain in the blood Loading Rate Determines Tumor Targeting Properties of stream at least three days, and may remain five days or more Methotrexate-Albumin Conjugates in Rats.” Anti-Cancer (usually not exceeding 60 days, more usually not exceeding Drugs, Vol. 8, pp. 677-685 (1997), incorporated herein in its 40 30 days) particularly as to the half life, based on the entirety, the authors report that a 1:1 ratio of the anti-cancer concentration in the blood. methotrexate to albumin conjugated via glutaraldehyde gave For the most part, reaction will be with mobile compo the most promising results. These conjugates were taken up nents in the blood, particularly blood proteins and cells, by tumor celis, whereas conjugates bearing 5:1 to 20:1 more particularly blood proteins and erythrocytes. By methotrexate molecules had altered HPLC profiles and were 45 “mobile' is intended that the component does not have a quickly taken up by the liver in vivo. It is postulated that at fixed situs for any extended period of time, generally not these higher ratios, conformational changes to albumin exceeding 5 minutes, more usually one minute, although diminish its effectiveness as a therapeutic carrier. Some of the blood components may be relatively stationary Through controlled administration of maleimide-thera for extended periods of time. Initially, there will be a peutic peptides in Vivo, one can control the specific labeling 50 relatively heterogeneous population of labeled proteins and of albumin and IgG in vivo. In typical administrations, cells. However, for the most part, the population within a 80-90% of the administered maleimide-therapeutic peptides few days after administration will vary substantially from will label albumin and less than 5% will label IgG. Trace the initial population, depending upon the half-life of the labeling of free thiols such as glutathione will also occur. labeled proteins in the blood stream. Therefore, usually Such specific labeling is preferred for in vivo use as it 55 within about three days or more, IgG will become the permits an accurate calculation of the estimated half-life of predominant labeled protein in the blood stream. the administered agent. Usually, by day 5 post-administration, IgG, serum albu In addition to providing controlled specific in vivo label min and erythrocytes will be at least about 60 mole %, ing, maleimide-therapeutic peptides can provide specific usually at least about 75 mole %, of the conjugated com labeling of serum albumin and IgG ex vivo. Such ex vivo 60 ponents in blood, with IgG, IgM (to a substantially lesser labeling involves the addition of maleimide-therapeutic pep extent) and serum albumin being at least about 50 mole %, tides to blood, serum or saline solution containing serum usually at least about 75 mole%, more usually at least about albumin and/or IgG. Once modified ex vivo with maleimide 80 mole %, of the non-cellular conjugated components. therapeutic peptides, the blood, serum or saline solution can The desired conjugates of non-specific therapeutic pep be readministered to the blood for in vivo treatment. 65 tides to blood components may be prepared in vivo by In contrast to NHS-peptides, maleimide-therapeutic pep administration of the therapeutic peptides directly to the tides are generally quite stable in the presence of aqueous patient, which may be a human or other mammal. The US 7,256.253 B2 35 36 administration may be done in the form of a bolus or ization of any of the chiral centers contained therein. Suit introduced slowly over time by infusion using metered flow able protecting groups are 9-fluorenylmethyloxycarbonyl or the like. (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl If desired, the Subject conjugates may also be prepared ex (CbZ), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, vivo by combining blood with modified therapeutic peptides isobornyloxycarbonyl, C. C.-dimethyl-3,5-dimethoxybenzy of the present invention, allowing covalent bonding of the loxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxy modified therapeutic peptides to reactive functionalities on carbonyl, and the like. The 9-fluorenyl-methyloxycarbonyl blood components and then returning or administering the (Fmoc) protecting group is particularly preferred for the conjugated blood to the host. Moreover, the above may also synthesis of Itherapeutic peptide fragments. Other preferred be accomplished by first purifying an individual blood 10 side chain protecting groups are, for side chain amino groups component or limited number of components, such as red like lysine and arginine, 2.2.5.7.8-pentamethylchroman-6- blood cells, immunoglobulins, serum albumin, or the like, Sulfonyl (pmc), nitro, p-toluenesulfonyl, 4-methoxyben and combining the component or components ex vivo with Zene-Sulfonyl, Cbz, Boc, and adamantyloxycarbonyl; for the chemically reactive Itherapeutic peptides. The labeled tyrosine, benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlo blood or blood component may then be returned to the host 15 robenzyl, isopropyl, t-butyl (t-Bu), cyclohexyl, cyclopenyl to provide in vivo the subject therapeutically effective and acetyl (Ac); for serine, t-butyl, benzyl and tetrahydro conjugates. The blood also may be treated to prevent coagu pyranyl; for histidine, trity1, benzyl, Cbz, p-toluenesulfonyl lation during handling ex vivo. and 2,4-dinitrophenyl; for tryptophan, formyl; for asparti cacid and glutamic acid, benzyl and t-butyl and for cysteine, 3. Synthesis of Therapeutic Peptides Used in the Present triphenylmethyl (trityl). Invention In the Solid phase peptide synthesis method, the C-C- Peptide fragments may be synthesized by standard meth terminal amino acid is attached to a Suitable solid Support or ods of Solid phase peptide chemistry known to those of resin. Suitable solid supports useful for the above synthesis ordinary skill in the art. For example, peptide fragments may are those materials which are inert to the reagents and be synthesized by solid phase chemistry techniques follow 25 reaction conditions of the stepwise condensation-deprotec ing the procedures described by Steward and Young (Stew tion reactions, as well as being insoluble in the media used. ard, J. M. and Young, J. D., Solid Phase Peptide Synthesis, The preferred solid support for synthesis of C-C-terminal 2nd Ed., Pierce Chemical Company, Rockford, Ill., (1984) carboxy peptides is 4-hydroxymethylphenoxymethyl-co using an Applied BioSystem synthesizer. Similarly, multiple poly(styrene-1% divinylbenzene). The preferred solid sup fragments may be synthesized then linked together to form 30 port for C-C-terminal amide peptides is the 4-(2',4'- larger fragments. These synthetic peptide fragments can also dimethoxyphenyl-Fmoc-aminomethyl) be made with amino acid substitutions at specific locations. phenoxyacetamidoethyl resin available from Applied For solid phase peptide synthesis, a Summary of the many Biosystems (Foster City, Calif.). The C-C-terminal amino techniques may be found in J. M. Stewart and J. D. Young, acid is coupled to the resin by means of N,N'-dicyclohexy Solid Phase Peptide Synthesis, W. H. Freeman Co. (San 35 lcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC) Francisco), 1963 and J. Meienhofer, Hormonal Proteins and O O-benzotriazol-1-yl-N,N,N',N'-tetramethyluronium Peptides, vol. 2, p. 46, Academic Press (New York), 1973. hexafluorophosphate (HBTU), with or without 4-dimethy For classical solution synthesis see G. Schroder and K. laminopyridine (DMAP), 1-hydroxybenzotriazole (HOBT), Lupke. The Peptides,Vol. 1, Acacemic Press (New York). In benzotriazol-1-yloxy-tris(dimethylamino)phosphonium general, these methods comprise the sequential addition of 40 hexafluorophosphate (BOP) or bis(2-oxo-3-oxazolidinyl) one or more amino acids or Suitably protected amino acids phosphine chloride (BOPCI), mediated coupling for from to a growing peptide chain. Normally, either the amino or about 1 to about 24 hours at a temperature of between 10° carboxyl group of the first amino acid is protected by a and 50° C. in a solvent such as dichloromethane or DMF. Suitable protecting group. The protected or derivatized When the solid support is 4-(2',4'-dimethoxyphenyl amino acid is then either attached to an inert Solid Support or 45 Fmoc-aminomethyl)phenoxy-acetamidoethyl resin, the utilized in Solution by adding the next amino acid in the Fmoc group is cleaved with a secondary amine, preferably sequence having the complimentary (amino or carboxyl) piperidine, prior to coupling with the C-C-terminal amino group Suitably protected and under conditions suitable for acid as described above. The preferred method for coupling forming the amide linkage. The protecting group is then to the deprotected 4-(2',4'-dimethoxyphenyl-Fmoc-aminom removed from this newly added amino acid residue and the 50 ethyl)phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl next amino acid (Suitably protected) is added, and so forth. N.N.N',N'-tetramethyluroniumhexafluoro-phosphate After all the desired amino acids have been linked in the (HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 proper sequence, any remaining protecting groups (and any equiv.) in DMF. The coupling of Successive protected amino Solid Support) are removed sequentially or concurrently to acids can be carried out in an automatic polypeptide Syn afford the final polypeptide. By simple modification of this 55 thesizer as is well known in the art. In a preferred embodi general procedure, it is possible to add more than one amino ment, the Cl-N-terminal amino acids of the growing peptide acid at a time to a growing chain, for example, by coupling chain are protected with Fmoc. The removal of the Fmoc (under conditions which do not racemize chiral centers) a protecting group from the Cl-N-terminal side of the growing protected tripeptide with a properly protected dipeptide to peptide is accomplished by treatment with a secondary form, after deprotection, a pentapeptide. 60 amine, preferably piperidine. Each protected amino acid is A particularly preferred method of preparing compounds then introduced in about 3-fold molar excess, and the of the present invention involves Solid phase peptide Syn coupling is preferably carried out in DMF. The coupling thesis wherein the amino acid C.-N-terminal is protected by agent is normally O-benzotriazol-1-yl-N,N,N',N'-tetram an acid or base sensitive group. Such protecting groups ethyluronium hexafluorophosphate (HBTU, 1 equiv.) and should have the properties of being stable to the conditions 65 1-hydroxybenzotriazole (HOBT, 1 equiv.). of peptide linkage formation while being readily removable At the end of the solid phase synthesis, the polypeptide is without destruction of the growing peptide chain or racem removed from the resin and deprotected, either in Succes US 7,256.253 B2 37 38 sively or in a single operation. Removal of the polypeptide loxymethyl, 9-fluorenylmethyloxycarbonyl (Fmoc) and the and deprotection can be accomplished in a single operation like and silyl groups such as trimethylsilyl and the like. by treating the resin-bound polypeptide with a cleavage (2) Carboxy Protective Groups reagent comprising thianisole, water, ethanedithiol and tri As discussed above, the term "carboxy protecting group' fluoroacetic acid. In cases wherein the C-C-terminal of the refers to a carboxylic acid protecting ester or amide group polypeptide is an alkylamide, the resin is cleaved by ami employed to block or protect the carboxylic acid function nolysis with an alkylamine. Alternatively, the peptide may ality while the reactions involving other functional sites of be removed by transesterification, e.g. with methanol, fol the compound are performed. Carboxy protecting groups are lowed by aminolysis or by direct transamidation. The pro disclosed in Greene, “Protective Groups in Organic Synthe tected peptide may be purified at this point or taken to the 10 sis' pp. 152-186 (1981), which is hereby incorporated by next step directly. The removal of the side chain protecting reference. Additionally, a carboxy protecting group can be groups is accomplished using the cleavage cocktail used as a pro-drug whereby the carboxy protecting group described above. The fully deprotected peptide is purified by can be readily cleaved in vivo, for example by enzymatic a sequence of chromatographic steps employing any or all of hydrolysis, to release the biologically active parent. Such the following types: ion exchange on a weakly basic resin 15 carboxy protecting groups are well known to those skilled in (acetate form); hydrophobic adsorption chromatography on the art, having been extensively used in the protection of underivitized polystyrene-divinylbenzene (for example, carboxyl groups in the penicillin and cephalosporin fields as Amberlite XAD); silica gel adsorption chromatography; ion described in U.S. Pat. Nos. 3,840,556 and 3,719,667, the exchange chromatography on carboxymethylcellulose; par disclosures of which are hereby incorporated herein by tition chromatography, e.g. on Sephadex G-25, LH-20 or reference. Representative carboxy protecting groups are countercurrent distribution; high performance liquid chro C-C loweralkyl (e.g., methyl ethyl or t-butyl and the like): matography (HPLC), especially reverse-phase HPLC on arylalkyl such as phenethyl or benzyl and substituted deriva octyl- or octadecylsilyl-silicabonded phase column packing. tives thereof such as alkoxybenzyl or nitrobenzyl groups and Molecular weights of these therapeutic peptides are deter the like: arylalkenyl such as phenylethenyl and the like: aryl 25 and substituted derivatives thereof such as 5-indanyl and the mined using Fast Atom Bombardment (FAB) Mass Spec like; dialkylaminoalkyl Such as dimethylaminoethyl and the troscopy. like); alkanoyloxyalkyl groups such as acetoxymethyl, The therapeutic peptides of the invention may be synthe butyryloxymethyl, Valeryloxymethyl, isobutyryloxymethyl, sized with N- and C-terminal protecting groups for use as isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloy pro-drugs. 30 loxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl, pivaloy (1) N-Terminal Protective Groups loxymethyl, propionyloxymethyl and the like; cycloal As discussed above, the term “N-protecting group’ refers kanoyloxyalkyl groups such aS to those groups intended to protect the Cl-N-terminal of an cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxym amino acid or peptide or to otherwise protect the amino ethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbony group of an amino acid or peptide against undesirable 35 loxymethyl and the like; aroyloxyalkyl Such as benzoy reactions during synthetic procedures. Commonly used loxymethyl, benzoyloxyethyl and the like: N-protecting groups are disclosed in Greene, “Protective arylalkylcarbonyloxyalkyl Such as benzylcarbonyloxym Groups In Organic Synthesis,” (John Wiley & Sons, New ethyl, 2-benzylcarbonyloxyethyl and the like: alkoxycarbo York (1981)), which is hereby incorporated by reference. nylalkyl or cycloalkyloxycarbonylalkyl Such as methoxy Additionally, protecting groups can be used as pro-drugs 40 carbonylmethyl, cyclohexyloxycarbonylmethyl, which are readily cleaved in vivo, for example, by enzy 1-methoxycarbonyl-1-ethyl and the like: alkoxycarbony matic hydrolysis, to release the biologically active parent. loxyalkyl or cycloalkyloxycarbonyloxyalkyl such as meth Cl-N-protecting groups comprise loweralkanoyl groups such oxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl, as formyl, acetyl (Ac), propionyl, pivaloyl, t-butylacetyl 1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbony and the like; other acyl groups include 2-chloroacetyl, 45 loxy-1-ethyl and the like: aryloxycarbonyloxyalkyl Such as 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, 2-(phenoxycarbonyloxy)ethyl, 2-(5-indanyloxycarbony o-nitrophenoxyacetyl, -chlorobutyryl, benzoyl, 4-chlo loxy)ethyl and the like; alkoxyalkylcarbonyloxyalkyl such robenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like: as 2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like; Sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbo and the like; carbamate forming groups such as benzyloxy 50 nyloxy)ethyl and the like: arylalkenyloxycarbonyloxyalkyl carbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzy Such as 2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and loxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxy the like; alkoxycarbonylaminoalkyl Such as t-butyloxycar carbonyl, p-bromobenzyloxycarbonyl, 3,4- bonylaminomethyl and the like: alkylaminocarbonylami dimethoxybenzyloxycarbonyl, 3,5- noalkyl Such as methylaminocarbonylaminomethyl and the dimethoxybenzyloxycarbonyl, 2,4- 55 like: alkanoylaminoalkyl Such as acetylaminomethyl and the dimethoxybenzyloxycarbonyl, 4-ethoxybenzyloxycarbonyl, like; heterocycliccarbonyloxyalkyl Such as 4-methylpiper 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4, 5-tri azinylcarbonyloxymethyl and the like; dialkylaminocarbo methoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methyl nylalkyl Such as dimethylaminocarbonylmethyl, diethylami ethoxycarbonyl, C.C.-dimethyl-3,5-dimethoxybenzyloxycar nocarbonylmethyl and the like: (5-(loweralkyl)-2-oxo-1,3- bonyl, benzhydryloxycarbonyl, t-butyloxycarbonyl, 60 dioxolen4-yl)alkyl such as (5-t-butyl-2-oxo-1,3-dioxolen4 diisopropylmethoxycarbonyl, isopropyloxycarbonyl, yl)methyl and the like; and (5-phenyl-2-oxo-1,3-dioxolen4 ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2.2.2.- yl)alkyl such as (5-phenyl-2-oxo-1,3-dioxolen4-yl)methyl trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophe and the like. noxycarbonyl, fluorenyl-9-methoxycarbonyl, cyclopenty Representative amide carboxy protecting groups are ami loxycarbonyl, adamantyloxycarbonyl, 65 nocarbonyl and loweralkylaminocarbonyl groups. cyclohexyloxycarbonyl, phenylthiocarbonyl and the like: Preferred carboxy-protected compounds of the invention arylalkyl groups such as benzyl, triphenylmethyl, benzy are compounds wherein the protected carboxy group is a US 7,256.253 B2 39 40 loweralkyl, cycloalkyl or arylalkyl ester, for example, involved in pharmacological activity, or if no carboxylic methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl acids are available, then any other sensitive functional group ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl not critical for the retention of pharmacological activity will ester, octyl ester, cyclohexyl ester, phenylethyl ester and the be selected as the attachment point for the linker-reactive like or an alkanoyloxyalkyl, cycloalkanoyloxyalkyl, aroy 5 group modification. If several sensitive functional groups loxyalkyl or an arylalkylcarbonyloxyalkyl ester. Preferred are available on a therapeutic peptide, a combination of amide carboxy protecting groups are loweralkylaminocar protecting groups will be used in Such a way that after bonyl groups. For example, aspartic acid may be protected addition of the linker/reactive group and deprotection of all at the C-C-terminal by an acid-labile group (e.g. t-butyl) and the protected sensitive functional groups, retention of phar protected at the B-C-terminal by a hydrogenation labile 10 macological activity is still obtained. If no sensitive func group (e.g. benzyl) then deprotected selectively during Syn tional groups are available on the therapeutic peptide, or if thesis. a simpler modification route is desired, synthetic efforts will Alternatively, it is also possible to obtain fragments of the allow for a modification of the original peptide in Such a way peptides by fragmenting the naturally occurring amino acid that retention of biological activity and retention of receptor sequence, using, for example, a proteolytic enzyme accord 15 or target specificity is obtained. In this case the modification ing to methods well known in the art. Further, it is possible will occur at the opposite end of the peptide. to obtain the desired fragments of the therapeutic peptide An NHS derivative may be synthesized from a carboxylic through the use of recombinant DNA technology using acid in absence of other sensitive functional groups in the methods well known in the art. therapeutic peptide. Specifically, Such a therapeutic peptide 4. Modification of Therapeutic Peptides is reacted with N-hydroxysuccinimide in anhydrous CH, Cl The manner of producing the modified therapeutic pep and EDC, and the product is purified by chromatography or tides of the present invention will vary widely, depending recrystallized from the appropriate solvent system to give upon the nature of the various elements comprising the the NHS derivative. molecule. The synthetic procedures will be selected so as to 25 Alternatively, an NHS derivative may be synthesized be simple, provide for high yields, and allow for a highly from a therapeutic peptide that contains an amino and/or purified stable product. Normally, the reactive group will be thiol group and a carboxylic acid. When a free amino or thiol created as the last stage, for example, with a carboxyl group, group is present in the molecule, it is preferable to protect esterification to form an active ester will be the last step of these sensitive functional groups prior to perform the addi the synthesis. Specific methods for the production of modi 30 tion of the NHS derivative. For instance, if the molecule fied therapeutic peptides of the present invention are contains a free amino group, a transformation of the amine described below. into a Fmoc or preferably into a tRoc protected amine is Generally, the modified therapeutic peptides of the necessary prior to perform the chemistry described above. present invention may be made using blind or structure The amine functionality will not be deprotected after prepa activity relationship (SAR) driven substitution. SAR is an 35 ration of the NHS derivative. Therefore this method applies analysis which defines the relationship between the structure only to a compound whose amine group is not required to be of a molecule and its pharmacological activity for a series of freed to induce a pharmacological desired effect. If the compounds. Various studies relative to individual therapeu amino group needs to be freed to retain the original biologi tic peptides show how the activity of the peptide varies cal properties of the molecule, then another type of chem according to the variation of chemical structure or chemical 40 istry described in example 3-6 has to be performed. properties. More specifically, first the therapeutic activity of In addition, an NHS derivative may be synthesized from the free peptide is assayed. Next, the peptide is modified a therapeutic peptide containing an amino or a thiol group according to the invention, either at the N-terminus, at the and no carboxylic acid. When the selected molecule contains C-terminus, or in the interior of the peptide with the linking no carboxylic acid, an array of bifunctional linkers can be group only. The linking group will include the reactive 45 used to convert the molecule into a reactive NHS derivative. group as discussed above. The therapeutic activity of this For instance, ethylene glycol-bis(Succinimydylsuccinate) modified peptide-linking group is assayed next, and based (EGS) and triethylamine dissolved in DMF and added to the on the detected activity a decision is made regarding the free amino containing molecule (with a ratio of 10:1 in favor modification site. Next, the peptide conjugate is prepared of EGS) will produce the mono NHS derivative. To produce and its therapeutic activity is determined. If the therapeutic 50 an NHS derivative from a thiol derivatized molecule, one activity of the peptide after conjugation is not substantially can use N-I-maleimidobutyryloxysuccinimide ester reduced (i.e. if the therapeutic activity is reduced by less (GMBS) and triethylamine in DMF. The maleimido group than 10 fold), then the stability of the peptide is measured as will react with the free thiol and the NHS derivative will be indicated by its in vivo lifetime. If the stability is not purified from the reaction mixture by chromatography on improved to a desired level, then the peptide is modified at 55 silica or by HPLC. an alternative site, and the procedure is repeated until a An NHS derivative may also be synthesized from a desired level of therapeutic activity and a desired stability therapeutic peptide containing multiple sensitive functional are achieved. groups. Each case will have to be analyzed and solved in a More specifically, each therapeutic peptide selected to different manner. However, thanks to the large array of undergo the derivatization with a linker and a reactive group 60 protecting groups and bifunctional linkers that are commer will be modified according to the following criteria: if a cially available, this invention is applicable to any thera terminal carboxylic group is available on the therapeutic peutic peptide with preferably one chemical step only to peptide and is not critical for the retention of pharmacologi derivatize the therapeutic peptide (as described in example cal activity, and no other sensitive functional group is 1 or 3) or two steps (as described in example 2 and involving present on the therapeutic peptide, then the carboxylic acid 65 prior protection of a sensitive group) or three steps (protec will be chosen as attachment point for the linker-reactive tion, activation and deprotection). Under exceptional cir group modification. If the terminal carboxylic group is cumstances only, would we require to use multiple steps US 7,256.253 B2 41 42 (beyond three steps) synthesis to transform a therapeutic azidobenzoyl hydrazide, N-4-(p-azidosalicylamino)butyl peptide into an active NHS or maleimide derivative. 3'-2'-pyridyldithio)propionamide), bis-sulfosuccinimidyl A maleimide derivative may also be synthesized from a Suberate, dimethyl adipimidate, disuccinimidyl tartrate, therapeutic peptide containing a free amino group and a free N-y-maleimidobutyryloxysuccinimide ester, N-hydroxy sul carboxylic acid. To produce a maleimide derivative from a 5 foSuccinimidyl-4-azidobenzoate, N-succinimidyl 4-azi amino derivatized molecule, one can use N-y-m a leimi dophenyl-1,3'-dithiopropionate, N-succinimidyl 4-io dobutyryloxysuccinimide ester (GMBS) and triethylamine doacetylaminobenzoate, glutaraldehyde, and Succinimidyl in DMF. The succinimide ester group will react with the free 4-N-maleimidomethylcyclohexane-1-carboxylate. amino and the maleimide derivative will be purified from the reaction mixture by crystallization or by chromatography on 10 Even more specifically, the peptides are preferably modi silica or by HPLC. fied according to the nature of their substituants and the Finally, a maleimide derivative may be synthesized from presence or absence of free cysteines. Most peptides can be a therapeutic peptide containing multiple other sensitive gathered into three distinct categories: (1) peptides that functional groups and no free carboxylic acids. When the 15 contain no cysteines; (2) peptides that contain one cysteine, selected molecule contains no carboxylic acid, an array of (3) peptides that contain two cysteines as a disulfide bridge bifunctional crosslinking reagents can be used to convert the (cystine); and (4) peptides that contain multiple cysteines. molecule into a reactive NHS derivative. For instance male A. Peptides that Contain No Cysteines imidopropionic acid (MPA) can be coupled to the free amine Where the peptide contains no cysteine, addition from the to produce a maleimide derivative through reaction of the C terminus is performed with all residues cleaved from the free amine with the carboxylic group of MPA using HBTU/ Support resin and fully protected. Solution phase activation HOBt/DIEA activation in DMF. Alternatively, a lysine resi of C-terminus with EDC and NHS can be reacted with an due can be added on the C-terminus end of the peptide to amino-alkyl-maleimide in one pot. The peptide is then fully allow for conjugation onto the -amino group of the lysine as 25 deprotected. Alternatively, a lysine residue can be added on described in the examples below. This added lysine allows the C-terminus of the peptide to allow modification at the for simple and efficient modification at the C-terminus of the epsilon amino group of the lysine while keeping the carboxy peptide while keeping the terminal end capped by an amide terminus capped with an amide group. Such an addition of function as designed by the initial choice of the resin a lysine residue is preferably performed only where the Many other commercially available heterobifunctional 30 addition does not substantially affect the therapeutic activity crosslinking reagents can alternatively be used when of the peptide. The generalized reaction scheme for C-ter needed. A large number of bifunctional compounds are minus modification of peptides that contain no cysteines is available for linking to entities. Illustrative reagents include: illustrated in the schematic diagram below.

NHP NH2 1946 TFA 596, TIS in CHCl2 O He Pd(0), HOAc NMM in CHCI A(aa)n A(aa)m RH A(aa)n N N Resin RH1 n N n Resin H H O

R = Boc or Ac R = Ac or H R" = NH2 or H P = the protecting group Mtt or Aloc Aaa = an amino acid including Lys protected as Boc

O COH 1N-1 2 Coupling Agent N US 7,256.253 B2

-continued O O i-N- N

O Cleavage O a

A(aa)n A(aa)m A(aa)n A(aa)m RH Y n N n Resin RH1 n N YCOR" H O

If an N-terminus modification is favored, and again for a peptide containing no cysteine, addition on the N terminus -continued is performed with all residues still on the support resin and fully protected. Addition of activated NHS-Malbifunctional O linker could be performed on deprotected N-terminus with A(aa)n -- N 1n-CO2H peptide still on resin. The peptide is then fully deprotected. HN Resin N Aaa = an amino acid O Examples of therapeutic peptides that contain no cystein and 25 undergo a C-terminus modification are described in examples 7-26. Examples of therapeutic peptides that con tain no cystein and undergo a N-terminus modification are opin Reagent described in examples 27-38. The generalized reaction scheme for N-terminus modification of peptides that contain 30 no cysteines is illustrated in the schematic diagrams below, using hetero NHS maleimide (GMBS like) and 3-MPA, respectively. 35 O O N ~us N1 A(aa)n Resin O O N H X O 40 A(aa)n n -- N1 n N HN COR N O O O levue R = NH2 or H X = alkyl or aromatic Aaa = an amino acid 45

50 O

1 A(aa)n O N ~us N YCO.R H 55 N O R = NH2 or H 3-MPA O 7 O 60 N Yx l N1 A(aa) YCOR Alternatively, the peptide may be modified at an internal H amino acid (i.e. neither at the C-terminus nor at the N-ter O minus). The generalized reaction scheme for modification at Hetero NHS Maleimide (GMBS like) 65 an internal amino acid of a peptide that contains no free cysteines is illustrated in the schematic diagrams below, using homo bis NHS and hetero NHS maleimide.

US 7,256.253 B2 47 48 Peptides that contain no cysteine and can be modified as described above include fragments of the Kringle 5 peptide, -continued of the GLP-1 peptide, of , human growth hormone releasing factor, the 1-24 fragment of human neuropeptide Y, and human secretin. Full description of the 5 chemistry for each of these peptides is reported in the Example section. B. Peptides that Contain One Cysteine Where the peptide contains one cysteine, the cysteine 10 must stay capped after addition of the maleimide. If the cysteine is involved in binding site, assessment has to be A(aa) made of how much potency is lost is cysteine is capped by a YCOR O a protecting group. If the cysteine can stay capped, then the 15 synthetic path is similar to that described in section A above for either a C or an N terminus modification. Hetero NHS Maleimide (GMBS like) Alternatively, the peptide may be modified at an internal Examples of therapeutic peptides that contain one cys amino acid (i.e. neither at the C-terminus nor at the N-ter teine include G. (the alpha subunit of Gtherapeutic peptide minus). The generalized reaction scheme for modification at binding protein), the 724-739 fragment of rat brain nitric an internal amino acid of a peptide that contains no cysteines oxide synthase blocking peptide, the alpha Subunit 1-32 is illustrated in the schematic diagram below, using homobis fragment of human Tyro inhibin, the 254-274 fragment of maleimide and hetero NHS maleimide (GMBS like). HIV envelope protein, and P34cdc2 kinase fragment. 25 C. Peptides that Contain Two Cysteines as a Disulfide Bridge (Cystine) Where the peptide contains two cysteines as a disulfide bridge, the peptide is cleaved from the support resin before SH O X O addition of the maleimide. For a modification of the peptide -- N1 NN 30 from the C terminus end, all protecting groups are present RN1.A(aa) "N N A(aa)"COR N A except at the carboxy terminus (which stays unprotected due O O O to cleavage from the Support resin) and at the two cysteines, which need to be deprotected when peptide is cleaved from R = Ac or H X = alkyl or aromatic R = NH2 or H resin. Mild air oxidation yields the disulfide bridge, and the Aaa = an amino acid 35 peptide can be purified at that stage. Solution phase chem istry is then required to activate the C-terminus in presence of the disulfide bridge and add the maleimide (through an amino-alkyl-maleimide) to the C-terminus. The peptide is then fully deprotected. 40 For a modification of the peptide at the N-terminus, the O O peptide can remain on the Support resin. The two cysteines X are selectively deprotected before addition of the maleimide. 1 N. Air oxidation, potentially helped by a catalyst (heteroge neous) can yield the disulfide with the peptide still on the O O 45 resin. Maleimide is then added on the N-terminus and S peptide cleaved from resin and fully deprotected. The gen eralized reaction scheme for modification at an internal amino acid of a peptide that contains two cysteines in a A(aa) A(aa)m RHN1 NN YCOR disulfide bridge is illustrated in the schematic diagram H 50 below. O Homobis Maleimide

SH O O 55 -X --O A(aa) A(aa)m A(aa)o A(aa)88 A(aa)88 “r RHN1 "N "COR N O O O A Fmoc1 Cys1 o, Resin TI(CFCO)3 O \m Acm A(aa)n A(aa)m A(aa)o R = Ac or H X = alkyl or aromatic 60 Fmoc Cys1 Oys Resin R = NH or H V V Aaa = an amino acid SS

1.20% Piperidine 2. 3-Maleimidopropionic 65 acid US 7,256.253 B2 49 50

-continued -continued 7 N A(aa)n A(aa)m A(aa)o / O n- Cys-V OysV YCOR O O S-S N A(aa)n A(aa)m. A(aa)o Cys Oys Resin R = NH2 or H O O y \ 10 Alternatively, the peptide may be modified at an internal tes amino acid (i.e. neither at the C-terminus nor at the N-ter minus). The generalized reaction scheme for modification at is an internal amino acid of a peptide that contains two cysteines in a disulfide bridge is illustrated in the schematic diagram below.

Peptide Scheme 10 NHP

TI(CFCO) ---

Aaa) (Aaa) (Aaa) (Aaa) RH 1. ( cys 1. Cys 1. na N p Ya Resin \ V H Acm Acm O

R = Boc or Ac R = Ac or H R" = NH or H P = the protecting group Mtt or Aloc Aaa = an amino acid including Lys protected as Boc NHP

RH1 (Aaa) cys 1 (Aaa)m Cys 1 (Aaa)o a. N (Aaa) a. Resin. V V H S-S O

1946 TFA 59%, TIS in CHCl2 O Pd(0), HOAc NMM in CHCI

NH2

1.3- Maleimidopropionic acid 2. Cleavage prop 1. (Aaa)n 1. (Aaa)m a 1 (Aaa) n (Aaa)p a. RH Cys Cys N Resin V V H S-S O US 7,256.253 B2 51 52

-continued O

HN --> N O

O

RH1 (Aaa) cys 1. (Aaa)m Cys 1. (Aaa) NN (Aaa) YCOR" V V H S S

Examples of therapeutic peptides that contain two cys physiologically acceptable stabilizers, and the like. The teines as a disulfide bridge include human osteocalcin 149. compositions may be lyophilized for convenient storage and human diabetes associated peptide, the 5-28 fragment of transport. human/canine atrial natriuretic peptide, bovine bactenecin, The modified Itherapeutic peptides will for the most part and human Tyro-cortistatin 29. be administered orally, parenterally, Such as intravascularly D. Peptides Containing Multiple Cysteines (IV), intraarterially (IA), intramuscularly (IM), subcutane Where the peptide contains multiple cysteines as disulfide ously (SC), or the like. Administration may in appropriate situations be by transfusion. In some instances, where reac bridges, the peptide is cleaved from the support resin before 25 tion of the functional group is relatively slow, administration addition of the maleimide. For a modification of the peptide may be oral, nasal, rectal, transdermal or aerosol, where the from the C terminus end, all protecting groups are present nature of the conjugate allows for transfer to the vascular except at the carboxy terminus (which stays unprotected due system. Usually a single injection will be employed to cleavage from the Support resin) and at the two cysteines although more than one injection may be used, if desired. that are supposed to build a disulfide bridge. Cysteines that 30 The modified therapeutic peptides may be administered by are involved in other disulfide bridges are deprotected any convenient means, including syringe, trocar, catheter, or sequencially in pairs using a choice of protecting groups. It the like. The particular manner of administration will vary is recommended to build and purify each bridge one at a depending upon the amount to be administered, whether a time prior to moving on to the next bridge. Mild air single bolus or continuous administration, or the like. Pref oxidation yields the disulfide bridge, and the peptide should 35 erably, the administration will be intravascularly, where the be purified at each stage. Solution phase chemistry is then site of introduction is not critical to this invention, preferably required to activate the C-terminus in presence of the at a site where there is rapid blood flow, e.g., intravenously, disulfide bridge and add the maleimide (through an amino peripheral or central vein. Other routes may find use where alkyl-maleimide) to the C-terminus. The peptide is then fully the administration is coupled with slow release techniques or deprotected. 40 a protective matrix. The intent is that the Itherapeutic For a modification of the peptide from the N terminus end, peptides be effectively distributed in the blood, so as to be one can leave the peptide on the Support resin and selectively able to react with the blood components. The concentration deprotect the first two cysteines to build the disulfide under of the conjugate will vary widely, generally ranging from mild air oxidation. Subsequent deprotection will offer the 45 about 1 pg/ml to 50 mg/ml. The total administered intra other disulfides before addition of the maleimide. Air oxi vascularly will generally be in the range of about 0.1 mg/ml dation, potentially helped by a catalyst (heterogeneous) can to about 10 mg/ml, more usually about 1 mg/ml to about 5 yield the disulfides with the peptide still on the resin. mg/ml. Maleimide is then added on the N-terminus and peptide By bonding to long-lived components of the blood, such cleaved from resin and fully deprotected. 50 as immunoglobulin, serum albumin, red blood cells and Alternatively, the peptide may be modified at an internal platelets, a number of advantages ensue. The activity of the amino acid (i.e. neither at the C-terminus nor at the N-ter modified therapeutic peptides compound is extended for minus). days to weeks. Only one administration need be given Peptides containing multiple cysteines include human during this period of time. Greater specificity can be endothelins and Lys4 Sarafotoxin S6c. 55 achieved, since the active compound will be primarily bound to large molecules, where it is less likely to be taken 5. Administration of the Modified Therapeutic Peptides up intracellularly to interfere with other physiological pro The modified therapeutic peptide will be administered in CCSSCS. a physiologically acceptable medium, e.g. deionized water, The formation of the covalent bond between the blood phosphate buffered saline (PBS), saline, aqueous ethanol or 60 component may occur in vivo or ex vivo. For ex vivo other alcohol, plasma, proteinaceous solutions, mannitol, covalent bond formation, the modified Itherapeutic peptide aqueous glucose, alcohol, vegetable oil, or the like. Other is added to blood, serum or saline solution containing human additives which may be included include buffers, where the serum albumin or IgG to permit covalent bond formation media are generally buffered at a pH in the range of about between the modified therapeutic peptide and the blood 5 to 10, where the buffer will generally range in concentra 65 component. In a preferred format, the therapeutic peptide is tion from about 50 to 250 mM, salt, where the concentration modified with maleimide and it is reacted with human serum of salt will generally range from about 5 to 500 mM, albumin in saline solution. Once the modified therapeutic US 7,256.253 B2 53 54 peptide has reacted with the blood component, to form a use antibodies specific to the therapeutic peptides or thera therapeutic peptide-protein conjugate, the conjugate may be peutic peptide analogs or their derivatives and conjugates, administered to the patient. and to use such antibodies as a treatment for toxicity Alternatively, the modified therapeutic peptide may be potentially associated with Such therapeutic peptides, ana administered to the patient directly so that the covalent bond logs, and/or their derivatives or conjugates. This is advan forms between the modified Itherapeutic peptide and the tageous because the increased stability and life of the blood component in vivo. therapeutic peptides in vivo in the patient might lead to In addition, where localized delivery of therapeutic pep novel problems during treatment, including increased pos tides is desired, several methods of delivery may be used: sibility for toxicity. It should be mentioned, however, that in A. Open Surgical Field Lavage 10 Some cases, the traditional antibody assay may not recognize There are a number of indications for local therapeutic the difference between cleaved and uncleaved therapeutic compounds which would entail administration of the thera peptides. In Such cases, other assay techniques may be peutic compound as an adjunct to open Surgery. In these employed, for example LC/MS (Liquid Chromatography/ cases, the therapeutic compound would either be lavaged in Mass Spectrometry). the Surgical site (or a portion of that site) prior to closure, or 15 The use of antibodies, either monoclonal or polyclonal, the therapeutic compound would be incubated for a short having specificity for a particular therapeutic peptide, analog time in a confined space (e.g., the interior of a section of an or derivative thereof, can assist in mediating any Such artery following an endarterectomy procedure or a portion of problem. The antibody may be generated or derived from a GI tract during resection) and the excess fluid Subsequently host immunized with the particular therapeutic peptide, evacuated. analog or derivative thereof, or with an immunogenic frag B. Incubation of Tissue Grafts ment of the agent, or a synthesized immunogen correspond Tissue grafts Such as autologous and Xenobiotic vein/ ing to an antigenic determinant of the agent. Preferred artery and valve grafts as well as organ grafts can be antibodies will have high specificity and affinity for native, pretreated with therapeutic compounds that have been modi derivatized and conjugated forms of the therapeutic peptide fied to permit covalent bond formation by either incubating 25 or therapeutic peptide analog. Such antibodies can also be them in a therapeutic solution and/or perfusing them with labeled with enzymes, fluorochromes, or radiolabels. Such a solution. Antibodies specific for derivatized therapeutic peptides C. Catheter Delivery may be produced by using purified therapeutic peptides for A catheter is used to deliver the therapeutic compound the induction of derivatized therapeutic peptide-specific either as part of an endoscopic procedure into the interior of 30 antibodies. By induction of antibodies, it is intended not an organ (e.g., bladder, GI tract, vaginafuterus) or adjunctive only the stimulation of an immune response by injection into to a cardiovascular catheter procedure Such as a balloon animals, but analogous steps in the production of synthetic angioplasty. Standard catheters as well as newer drug deliv antibodies or other specific binding molecules such as ery and iontophoretic catheters can be utilized. screening of recombinant immunoglobulin libraries. Both D. Direct Injection 35 monoclonal and polyclonal antibodies can be produced by For certain poorly vascularized spaces such as intra procedures well known in the art. In some cases, the use of articular joint spaces, a direct injection of a therapeutic monoclonal antibodies may be preferred over polyclonal compound may be able to bioconjugate to Surface tissues antibodies, such as when degradation occurs over an area not and achieve a desirable duration of drug effect. Other covered by epitope/antibody recognition. applications could include intra medullary, intratumor, intra 40 The antibodies may be used to treat toxicity induced by vaginal, intrauterine, intra intestinal, intra eustachian tube, administration of the therapeutic peptide, analog or deriva intrathecal, Subcutaneous, intrarticular, intraperitoneal or tive thereof, and may be used ex vivo or in vivo. Ex vivo intraocular injections as weel as via bronchoscope, via methods would include immuno-dialysis treatment for tox nasogastiric tube and via nophrostomy. icity employing antibodies fixed to Solid Supports. In vivo 45 methods include administration of antibodies in amounts 6. Monitoring the Presence of Modified Therapeutic Peptide effective to induce clearance of antibody-agent complexes. Derivatives The antibodies may be used to remove the therapeutic Another aspect of this invention relates to methods for peptides, analogs or derivatives thereof, and conjugates determining the concentration of the therapeutic peptides thereof, from a patient’s blood ex vivo by contacting the and/or analogs, or their derivatives and conjugates in bio 50 blood with the antibodies under sterile conditions. For logical samples (such as blood) and determining the pepti example, the antibodies can be fixed or otherwise immobi dase stability of the modified peptides. The blood of the lized on a column matrix and the patient’s blood can be mammalian host may be monitored for the presence of the removed from the patient and passed over the matrix. The modified therapeutic peptide compounds one or more times. therapeutic peptide analogs, derivatives or conjugates, will By taking a portion or sample of the blood of the host, one 55 bind to the antibodies and the blood containing a low may determine whether the therapeutic peptide has become concentration of the therapeutic peptide, analog, derivative bound to the long-lived blood components in sufficient or conjugate, then may be returned to the patient’s circula amount to be therapeutically active and, thereafter, the level tory system. The amount of therapeutic peptide compound of therapeutic peptide compound in the blood. If desired, removed can be controlled by adjusting the pressure and one may also determine to which of the blood components 60 flow rate. Preferential removal of the therapeutic peptides, the therapeutic peptide derivative molecule is bound. This is analogs, derivatives and conjugates from the plasma com particularly important when using non-specific therapeutic ponent of a patient’s blood can be affected, for example, by peptides. For specific maleimide-therapeutic peptides, it is the use of a semipermeable membrane, or by otherwise first much simpler to calculate the half life of serum albumin and separating the plasma component from the cellular compo IgG. 65 nent by ways known in the art prior to passing the plasma One method for determining the concentration of the component over a matrix containing the anti-therapeutic therapeutic peptide, analogs, derivatives and conjugates is to antibodies. Alternatively the preferential removal of thera US 7,256.253 B2 55 56 peutic peptide-conjugated blood cells, including red blood Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, cells, can be effected by collecting and concentrating the Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf-OH, Fmoc-Pro-OH. blood cells in the patient’s blood and contacting those cells Deblocking of the Fmoc group the the N-terminal of the with fixed anti-therapeutic antibodies to the exclusion of the resin-bound amino acid was performed with 20% piperidine serum component of the patient’s blood. in DMF for about 15-20 minutes. Coupling of the acetic acid The antibodies can be administered in vivo, parenterally, was performed under conditions similar to amino acid to a patient that has received the therapeutic peptide, ana coupling. Final cleavage from the resin was performed using logs, derivatives or conjugates for treatment. The antibodies cleavage mixture as described above. The product was will bind the therapeutic peptide compounds and conjugates. isolated by precipitation and purified by preparative HPLC Once bound the therapeutic peptide, activity will be hin 10 to afford the desired product as a white solid upon lyo dered if not completely blocked thereby reducing the bio philization. logically effective concentration of therapeutic peptide com pound in the patient’s bloodstream and minimizing harmful Example 2 side effects. In addition, the bound antibody-therapeutic peptide complex will facilitate clearance of the therapeutic 15 Addition of Lys at C-Terminus of Kringle-5 peptide compounds and conjugates from the patient’s blood Stream. Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys The invention having been fully described is now exem NH2TFA.3TFA plified by the following non-limiting examples. Using automated peptide synthesis, the following pro EXAMPLES tected amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, A. General Method of Synthesis of a Modified Therapeutic Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Peptide Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH. Deblocking of Solid phase peptide synthesis of the modified peptide on 25 the Fmoc group the the N-terminal of the resin-bound amino a 100LL mole scale was performed on a Symphony Peptide acid was performed with 20% piperidine in DMF for about Synthesizer using Fmoc protected Rink Amide MBHA resin, 15-20 minutes. Coupling of the acetic acid was performed Fmoc protected amino acids, O-benzotriazol-1-yl-N,N,N', under conditions similar to amino acid coupling. Final N-tetramethyl-uronium hexafluorophosphate (HBTU) in cleavage from the resin was performed using cleavage N,N-dimethylformamide (DMF) solution and activation 30 mixture as described above. The product was isolated by with N-methyl morpholine (NMM), and piperidine depro precipitation and purified by preparative HPLC to afford the tection of Fmoc groups (Step 1). The deprotection of the desired product as a white solid upon lyophilization. terminal Fmoc group is accomplished using 20% piperidine (Step 2) followed by either the coupling of 3-maleimidopro Example 3 pionic acid (3-MPA), the coupling of acetic acid or the 35 coupling of one or multiple Fmoc-AEEA followed by the Addition of Lys at N-Terminus of Kringle-5 coupling of 3-MPA (Step 3). Resin cleavage and products isolation are performed using 86% TFA/5% TIS/5% HO/ Preparation of NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys 2% thioanisole and 2% phenol, followed by precipitation by Leu-Tyr-Asp-Tyr-Lys-NH2.3TFA dry-ice cold EtO (Step 4). The products are purified by 40 preparative reverse phase HPLC using a Varian (Rainin) Using automated peptide synthesis, the following pro preparative binary HPLC system using a Dynamax Cs, 60 tected amino acids were sequentially added to Rink Amide A, 8 um, 21 mmx25 cm column equipped with a Dynamax MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, Cls, 60 A, 8 um guard module, 21 mmx25 cm column and Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, UV detector (Varian Dynamax UVD II) at 214 and 254 45 Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, nm. The product should have >95% purity as determined by Fmoc-ASn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tEu)- RP-HPLC mass spectrometry using a Hewlett Packard OH, Fmoc-TyrctBu)OH. Deblocking of the Fmoc group the LCMS-1 100 series spectrometer equipped with a diode the N-terminal of the resin-bound amino acid was performed array detector and using electro-spray ionization. with 20% piperidine in DMF for about 15-20 minutes. 50 Coupling of the acetic acid was performed under conditions B. Alteration of the Native Peptide Chain similar to amino acid coupling. Final cleavage from the resin To facilitate modification of the peptide, one or more was performed using cleavage mixture as described above. amino acid residues may be added to the peptide as The product was isolated by precipitation and purified by described in examples 1 to 5, and/or one or more amino acid preparative HPLC to afford the desired product as a white residues may be replaced with other amino acid residues. 55 Solid upon lyophilization. This alteration aids attachment of the reactive group. Example 4 Example 1 Addition of Lys at N-Terminus of Kringle-5, Addition of Lys at C-Terminus of Kringle-5 60 Substitution of Cys with Ala at Position 524 Preparation of Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-N H.3TFA Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg Lys-Leu-Tyr-Asp-Tyr-Lys-NH2.4TFA Using automated peptide synthesis, the following pro 65 tected amino acids were sequentially added to Rink Amide Using automated peptide synthesis, the following pro MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, tected amino acids were sequentially added to Rink Amide US 7,256.253 B2 57 58 MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Rink Amide MBHA Resin Fmoc-ASn(Trt)-OH, Fmoc-Thr(tEu)-OH, Fmoc-Thr(tEu)- OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Step 1 |rs Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val OH, Fmoc-Asp(OtBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc HN-HaEGTFTSDVSSYLEGQAAKEFIAWLVKGR-PS Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH. Step 2 s TFA/5% TIS/5% thioanisole/5% phenol Deblocking of the Fmoc group the the N-terminal of the 10 resin-bound amino acid was performed with 20% piperidine TFA TFA TFA in DMF for about 15-20 minutes. Coupling of the acetic acid was performed under conditions similar to amino acid HN-HaEGTFTSDVSSYLEGQAAKEFLAWLVKGR-NH coupling. Final cleavage from the resin was performed using TFA cleavage mixture as described above. The product was D-Ala-GLP-1 (7-36)-NH2 isolated by precipitation and purified by preparative HPLC 15 to afford the desired product as a white solid upon lyo philization. C. Preparation of Modified Peptides From Peptides Con taining No Cysteines Example 5 Preparation of maleimido peptides from therapeutic pep tides containing multiple protected functional groups and no Addition of Lys at N-Terminus of Kringle-5 Cysteine is exemplified by the synthesis of peptides as described below. The peptide may be modified at the N-ter Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val minus, the C-terminus, or at an amino acid located between Gly-Gly-Pro-Trp-Lys-NH.2TFA 25 the N-terminus and the C-terminus. The modified peptide is synthesized by linking off the N-terminus of the natural Using automated peptide synthesis, the following pro peptide sequence or by linking off the modified C-terminus tected amino acids were sequentially added to Rink Amide of the natural peptide sequence. One or more additional MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc amino acids may be added to the therapeutic peptide to 30 facilitate attachment of the reactive group. Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)- 1. Modification of the Therapeutic Peptide at the C-Ter OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, minus Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Pbf)-OH. Deblocking of the Fmoc group the the N-terminal of the Example 7 resin-bound amino acid was performed with 20% piperidine 35 in DMF for about 15-20 minutes. Coupling of the acetic acid Modification of RSV Peptide at the e-Amino Group was performed under conditions similar to amino acid of the Added C-terminus Lysine Residue coupling. Final cleavage from the resin was performed using cleavage mixture as described above. The product was Preparation of Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile isolated by precipitation and purified by preparative HPLC 40 Lys-Glu-Asn-Lys-Met-Asn-Gly-Ala-Lys-Val-Lys to afford the desired product as a white solid upon lyo Leu-Ile-Lys-Gln-Glu-Leu-Asp-Lys-Tyr-Lys-Asn philization. Ala-Val-Lys-(Ne-MPA) Example 6 Solid phase peptide synthesis of the DAC analog on a 100 45 umole Scale is performed using manual Solid-phase synthe Preparation of D-Ala2 GLP-1 (7-36) Amide sis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following protected amino acids Solid phase peptide synthesis of the GLP-1 analog on a are sequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc 100 umole scale is performed using manual Solid-phase Val-OH, Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Lys synthesis and a Symphony Peptide Synthesizer using Fmoc 50 (Boc)-OH, Fmoc-Tyr?tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc protected Rink Amide MBHA resin, Fmoc protected amino Asp(tBu)-OH, Fmoc-Leu-OH, Fmoc-GluctBu)-OH, Fmoc acids, O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc hexafluorophosphate (HBTU) in N,N-dimethylformamide Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Lys (DMF) solution and activation with N-methyl morpholine (Boc)-OH, Fmoc-Ala-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)- (NMM), and piperidine deprotection of Fmoc groups (Step 55 OH, Fmoc-Met-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)- 1). Resin cleavage and product isolation is performed using OH, Fmoc-GluctBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu by precipitation by dry-ice cold EtO (Step 2). The product OH, Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)- is purified by preparative reversed phased HPLC using a OH, Fmoc-Ile-OH, Fmoc-Val-OH. They are dissolved in Varian (Rainin) preparative binary HPLC system: gradient 60 N,N-dimethylformamide (DMF) and, according to the elution of 30-55% B (0.045% TFA in HO (A) and 0.045% sequence, activated using O-benzotriazol-1-yl-N,N,N',N'- TFA in CHCN (B)) over 180 min at 9.5 mL/min using a tetramethyl-uronium hexafluorophosphate (HBTU) and Phenomenex Luna 10L phenyl-hexyl, 21 mmx25 cm column Diisopropylethylamine (DIEA). Removal of the Fmoc pro and UV detector (Varian Dynamax UVD II) at 214 and 254 tecting group is achieved using a solution of 20% (V/V) nm to afford the desired peptide in >95% purity, as deter 65 piperidine in N,N-dimethylformamide (DMF) for 20 min mined by RP-HPLC. These steps are illustrated in the utes (step 1).The selective deprotection of the Lys (Aloc) schematic diagram below. group is performed manually and accomplished by treating US 7,256.253 B2 59 60 the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in (DMF) and 3 times with isopropanol. The peptide is cleaved 5 mL of CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). from the resin using 85% TFA/5% TIS/5% thioanisole and The resin is then washed with CHC13 (6x5 mL), 20% HOAc 5% phenol, followed by precipitation by dry-ice cold EtO in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). (Step 4). The product is purified by preparative reverse The synthesis is then re-automated for the addition of the 5 phase HPLC using a Varian (Rainin) preparative binary 3-maleimidopropionic acid (Step 3). Between every cou HPLC system: gradient elution of 30-55% B (0.045% TFA pling, the resin is washed 3 times with N,N-dimethylforma in HO (A) and 0.045% TFA in CHCN (B)) over 180 min mide (DMF) and 3 times with isopropanol. The peptide is at 9.5 mL/min using a Phenomenex Luna 10L phenyl-hexyl, cleaved from the resin using 85% TFA/5% TIS/5% thioani 21 mmx25 cm column and UV detector (Varian Dynamax sole and 5% phenol, followed by precipitation by dry-ice 10 UVD II) at 214 and 254 nm to afford the desired DAC in cold Et2O (Step 4). The product is purified by preparative >95% purity, as determined by RP-HPLC. reverse phase HPLC using a Varian (Rainin) preparative The structure of this product is

HN --> N O 7

TFA TFATFA TFA TFA HN -Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys -NH2 binary HPLC system: gradient elution of 30-55% B (0.045% Example 9 TFA in H20 (A) and 0.045% TFA in CH3CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10L phenyl 30 Modification of Dyn A 2-13 at the e-Amino Group hexyl, 21 mmx25 cm column and UV detector (Varian of the Added C-terminus Lysine Dynamax UVD II) at 214 and 254 nm to afford the desired Residue Synthesis of Dyn A 2-13(Ne-MPA)-NH DAC in >95% purity, as determined by RP-HPLC. Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu Example 8 35 Lys-(Ne-MPA)-NH Modification of Dyn A 1-13 at the e-Amino Group Using automated peptide synthesis, the following pro of the Added C-terminus Lysine tected amino acids were sequentially added to Rink Amide Residue Synthesis of Dyn A 1-13(Ne-MPA)-NH MBHA resin: Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc 40 Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc Leu-Lys-(Ne-MPA)-NH Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Boc-Gly-OH. Manual synthesis was employed for the remaining steps: Solid phase peptide synthesis of a modified Dyn A 1-13 on selective removal of the Mtt group and coupling of MPA a 100 umole Scale was performed using manual Solid-phase 45 using HBTU/HOBt/DIEA activation in DMF. The target synthesis, a Symphony Peptide Synthesizer and Fmoc pro dynorphin analog was removed from the resin; the product tected Rink Amide MBHA. The following protected amino was isolated by precipitation and purified by preparative acids were sequentially added to resin: Fmoc-Lys(Aloc)-OH, HPLC to afford the desired product as a white solid upon Fmoc-Leu-OH, Fmo -Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc lyophilization in a 35% yield. Anal. HPLC indicated product Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc 50 Arg(Pbf-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Gly to be >95% pure with R. 30.42 min. ESI-MS m/z for OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH. They were C.H.I.N.O.s (MH), calcd 1590.0, found MH 531.3. dissolved in N,N-dimethylformamide (DMF) and, according Example 10 to the sequence, activated using O-benzotriazol-1-yl-N,N. N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) 55 Modification of Dyn A 1-13 at the e-Amino Group and Diisopropylethylamine (DIEA). Removal of the Fmoc of the Added C-terminus Lysine protecting group is achieved using a solution of 20% (V/V) Residue Synthesis of Dyn A piperidine in N,N-dimethylformamide (DMF) for 20 min 1-13(AEA-MPA)-NH utes (Step 1).The selective deprotection of the Lys (Aloc) group is performed manually and accomplished by treating 60 Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys the resin with a solution of 3 eq of Pd(PPh) dissolved in 5 Leu-Lys-(AEA-MPA)-NH mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl (6x5 mL), 20% HOAc in Using automated peptide synthesis, the following pro DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The tected amino acids were sequentially added to Rink Amide synthesis is then re-automated for the addition of the 3-ma 65 MBHA resin: Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc leimidopropionic acid (Step 3). Between every coupling, the Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc resin is washed 3 times with N,N-dimethylformamide Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc US 7,256.253 B2 61 62 Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, and Boc-TyrBoc)-OH. Manual synthesis was employed for the remaining steps: selective removal of the Mitt group, the coupling of three-Fmoc-AEA-OH groups (AEA-aminoethoxyacetic acid) with Fmoc removal in-be tween each coupling, and MPA acid using HBTU/HOBt/ DIEA activation in DMF. The target dynorphin analog was removed from the resin; the product was isolated by pre cipitation and purified by preparative HPLC to afford the desired product as a white solid upon lyophilization in a 29% 10 yield. Anal. HPLC indicated product to be >95% pure with R. 33.06 min. ESI-MS m/z for CHNO (MH"), calcd 2057.2, found MH 515.4, MH 686.9, MH." 1029.7.

TFA TFATFA TFA TFA HN Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys -NH2

Example 11 Ile-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc 35 Leu-OH, Fmoc-Phe-OH, Fmoc-Gly-OH, and Fmoc-Gly Modification of Dyn A 2-13 at the e-Amino Group OH. Manual synthesis was employed for the remaining of the Added C-terminus Lysine steps: selective removal of the Mitt group, the coupling of Residue Synthesis of Dyn A three-Fmoc-AEA-OH groups, with Fmoc removal in-be 2-13(AEA-MPA)-NH tween each coupling, and MPA using HBTU/HOBt/DIEA 40 activation in DMF. The target dynorphin analog was Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu removed from the resin; the product was isolated by pre Lys-(AEA-MPA)-NH. cipitation and purified by preparative HPLC to afford the desired product as a white solid upon lyophilization in a 29% Using automated peptide synthesis, the following pro yield. Anal. HPLC indicated product to be >95% pure with tected amino acids were sequentially added to Rink Amide 45 R. 31.88 min. ESI-MS m/z for Css Has NO (MH), MBHA resin: Fmoc-Lys(Mtt)-OH, Fmoc-Leu-OH, Fmoc calcd 1894.3, found MH 474.6, MH 632.4 MH' Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc 948.10.

TFA TFATFA TFA TFA H2N Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys -NH2 US 7,256.253 B2 63 64 Example 12 Example 13 Modification of Neuropeptide Y at the e-Amino Modification of GLP-1 (7-36) at the C-Terminus Group of the Added C-terminus Lysine Residue Arginine Preparation of Tyr-Pro-Ser-Lys-Pro-Asp-Asn-Pro Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg Preparation of GLP-1 (7-36)-EDA-MPA Tyr-Tyr-Ser-Ala-Leu-Lys-(N-eMPA)-NH Solid phase peptide synthesis of a modified GLP-1 analog Solid phase peptide synthesis of a modified neuropeptide 10 on a 100 umole scale is performed manually and on a Yanalog on a 100 umole Scale is performed using manual Symphony Peptide Synthesizer SASRIN (super acid sensi solid-phase synthesis, a Symphony Peptide Synthesizer and tive resin). The following protected amino acids are sequen Fmoc protected Rink Amide MBHA. The following pro tially added to the resin: Fmoc-Arg(Pbf)-OH, Fmoc-Gly tected amino acids were sequentially added to resin: Fmoc OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Lys(Aloc)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser 15 (thBu)-OH, Fmoc-Tyr(tEu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Asp Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, (thBu)-OH, Fmoc-Glu(tEu)-OH, Fmoc-Ala-OH, Fmoc-Pro Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-GluctBu)- Gly-OH, Fmco-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr OH, Fmoc-Gly-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Asp(tEu)-OH, Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, (thBu)-OH, Fmoc-Ser(tPu)-OH, Fmoc-Ser(tBu)-OH, Fmoc Fmoc-Ser(thBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Val-OH, Fmoc-Asp(tEu)-OH, Fmoc-Ser(tBu)-OH, Fmoc They are dissolved in N,N-dimethylformamide (DMF) and, Thr(tEu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tEu)-OH, Fmoc according to the sequence, activated using O-benzotriazol Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Boc-His 1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate 25 (Trt)-OH. They are dissolved in N,N-dimethylformamide (HBTU) and Diisopropylethylamine (DIEA). Removal of (DMF) and, according to the sequence, activated using the Fmoc protecting group is achieved using a solution of O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium 20% (V/V) piperidine in N,N-dimethylformamide (DMF) hexafluorophosphate (HBTU) and Diisopropylethylamine for 20 minutes (Step 1).The selective deprotection of the Lys (DIEA). Removal of the Fmoc protecting group is achieved (Aloc) group is performed manually and accomplished by 30 treating the resin with a solution of 3 eq of Pd(PPh.) using a solution of 20% (V/V) piperidine in N,N-dimethyl dissolved in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h formamide (DMF) for 20 minutes (Step 1). The fully pro (Step 2). The resin is then washed with CHCl (6x5 mL), tected peptide is cleaved from the resin by treatment with 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF 1% TFA /DCM (Step 2). Ethylenediamine and 3-maleimi (6x5 mL). The synthesis is then re-automated for the addi 35 dopropionic acid are then sequentially added to the free tion of the 3-maleimidopropionic acid (Step 3). Between C-terminus (Step 3). The protecting groups are then cleaved every coupling, the resin is washed 3 times with N.N- dimethylformamide (DMF) and 3 times with isopropanol. and the product isolated using 86% TFA/5% TIS/5% HO/ The peptide is cleaved from the resin using 85% TFA/5% 2% thioanisole and 2% phenol, followed by precipitation by TIS/5% thioanisole and 5% phenol, followed by precipita 40 dry-ice cold EtO (Step 4). The product is purified by tion by dry-ice cold EtO (Step 4). The product is purified by preparative reverse phase HPLC using a Varian (Rainin) preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system using a Dynamax Cs, 60 preparative binary HPLC system: gradient elution of A, 8 um, 21 mmx25 cm column equipped with a Dynamax 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Cls, 60 A, 8 um guard module, 21 mmx25 cm column and CHCN (B)) over 180 min at 9.5 mL/min using a Phenom 45 enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV UV detector (Varian Dynamax UVD II) at 214 and 254 nm detector (Varian Dynamax UVD II) at 214 and 254 nm to to afford the desired DAC in >95% purity, as determined by afford the desired DAC in >95% purity, as determined by RP-HPLC. These steps are illustrated in the schematic RP-HPLC. diagram below.

SASRIN Resin Step 1 |rs Boc-HAEGTFTSDVSSYLEGQAAKEFLAWLVKGR-Resin Step 2 t TFADCM

Boc-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-OH

Step 3 1. ethylenediamine P U2. 3-maleimidopropionic acid US 7,256.253 B2 65 66

-continued O Boc-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-O NN 1N1 N CN O O

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

O HN-HAEGTFTSDVSSYLEGQAAKEFLAWLVKGR-O n N 1N1 N CN O O GLP-1 (7-36)-EDA-MPA

2O Example 14 Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc His(Trt)-OH. They are dissolved in N,N-dimethylforma Modification of Exendin-4 at the C-terminus Serine mide (DMF) and, according to the sequence, activated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium Preparation of Exendin-4 (1-39)-EDA-MPA 25 hexafluorophosphate (H BTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protecting group is achieved Solid phase peptide synthesis of a modified Exendin-4 using a solution of 20% (VIV) piperidine in N,N-dimethyl analog on a 100 umole Scale is performed manually and on formamide (DMF) for 20 minutes (Step 1). The fully pro a Symphony Peptide Synthesizer SASRIN (super acid sen tected peptide is cleaved from the resin by treatment with sitive resin). The following protected amino acids are 30 1% TFA/DCM (Step 2). Ethylenediamine and 3-maleimi sequentially added to the resin: Fmoc-Ser(tBu)-OH, Fmoc dopropionic acid are then sequentially added to the free Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, C-terminus (Step 3). The protecting groups are then cleaved Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, and the product isolated using 86% TFA/5% TIS/5% HO/ Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn 2% thioanisole and 2% phenol, followed by precipitation by (Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp 35 dry-ice cold EtO (Step 4). The product is purified by (Boc)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc preparative reverse phase HPLC using a Varian (Rainin) Phe-OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val preparative binary HPLC system using a Dynamax Cs, 60 OH, Fmoc-Ala-OH, Fmoc-GlucCtBu)-OH, Fmoc-Glu A, 8 um, 21 mmx25 cm column equipped with a Dynamax (OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc Cls, 60 A, 8 um guard module, 21 mmx25 cm column and Gln(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, 40 UV detector (Varian Dynamax UVD II) at 214 and 254 nm Fmoc-Leu-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, to afford the desired DAC in >95% purity, as determined by Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, RP-HPLC.

SASRIN Resin Step 1 es

Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Resin Step 2 TFADCM

Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-OH

Step 3 1. ethylenediamine P U2. 3-maleimidopropionic acid US 7,256.253 B2 67 68

-continued O Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-O NN 1N1 N CN O O

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

O HN-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-O NN 1N1 N - N ? O O Exendin-4 (1-39)-EDA-MPA

Example 15 TFA in CHCN (B)) over 180 mm at 9.5 mL/min using a Phenomenex Luna 10L phenyl-hexyl, 21 mmx25 cm column Modification of Secretin Peptide at the e-Amino and UV detector (Varian Dynamax UVD II) at 214 and 254 Group of the Added C-terminus Lysine Residue nm to afford the desired DAC in >95% purity, as determined 25 by RP-HPLC. Preparation of His-Ser-Asp-Gly-Thr-Phe-Thr-Ser Glu-Leu-Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu Example 16 Gln-Arg-Leu-Leu-Gln-Gly-Leu-Val-Lys-(Ne-MPA)- Modification of Kringle-5 at the e-Amino Group of NH, 30 the Added C-terminus Lysine Residue Solid phase peptide synthesis of a modified secretin Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr peptide analog on a 100 umole scale is performed using Lys-(Ne-MPA)-NH.2TFA manual solid-phase synthesis, a Symphony Peptide Synthe sizer and Fmoc protected Rink Amide MBHA. The follow 35 Solid phase peptide synthesis of a modified Kringle-5 ing protected amino acids are sequentially added to resin: peptide on a 100 umole scale was performed using manual Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc-Gly-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc Fmoc protected Rink Amide MBHA. The following pro Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc tected amino acids are sequentially added to resin: Fmoc Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Gly 40 Lys(Aloc)-OH, Fmoc-Tyr(tEu)-OH, Fmoc-Asp(teu)-OH, OH, Fmoc-GluctBu)-OH, Fmoc-Arg(PbD-OH, Fmoc-Leu Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ser(thBu)-OH, Fmoc-Leu Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH. They were dissolved in OH, Fmoc-GluctBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr N,N-dimethylformamide (DMF) and, according to the (thBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly sequence, activated using O-benzotriazol-1-yl-N,N,N',N'- OH, Fmoc-Asp(tPu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-His 45 tetramethyl-uronium hexafluorophosphate (HBTU) and (Boc)-OH. They are dissolved in N,N-dimethylformamide Diisopropylethylamine (DIEA). Removal of the Fmoc pro (DMF) and, according to the sequence, activated using tecting group is achieved using a solution of 20% (VfV) O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium piperidine in N,N-dimethylformamide (DMF) for 20 min hexafluorophosphate (HBTU) and Diisopropylethylamine utes (step 1). At the end of the synthesis Acetic Anhydride (DIEA). Removal of the Fmoc protecting group is achieved 50 was added to acetylate the N-terminal. The selective depro using a solution of 20% (V/V) piperidine in N,N-dimethyl tection of the Lys (Aloc) group is performed manually and formamide (DMF) for 20 minutes (Step 1).The selective accomplished by treating the resin with a solution of 3 eq of deprotection of the Lys (Abc) group is performed manually Pd(PPh.) dissolved in 5 mL of CHCl:NMM:HOAc (18: and accomplished by treating the resin with a solution of 3 1:0.5) for 2 h (Step 2). The resin is then washed with CHC1. eq of Pd(PPh.) dissolved in 5 mL of CHCl:NMM:HOAc 55 (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), (18:1:0.5) for 2 h (Step 2). The resin is then washed with and DMF (6x5 mL). The synthesis is then re-automated for CHCl (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM the addition of the 3-maleimidopropionic acid (Step 3). (6x5 mL), and DMF (6x5 mL). The synthesis is then Between every coupling, the resin is washed 3 times with re-automated for the addition of the 3-maleimidopropionic N,N-dimethylformamide (DMF) and 3 times with isopro acid (Step 3). Between every coupling, the resin is washed 60 panol. The peptide is cleaved from the resin using 85% 3 times with N,N-dimethylformamide (DMF) and 3 times TFA/5% TIS/5% thioanisole and 5% phenol, followed by with isopropanol. The peptide is cleaved from the resin precipitation by dry-ice cold EtO (Step 4). The product is using 85% TFA/5% TIS/5% thioanisole and 5% phenol, purified by preparative reverse phase HPLC using a Varian followed by precipitation by dry-ice cold EtO (Step 4). The (Rainin) preparative binary HPLC system: gradient elution product is purified by preparative reverse phase HPLC using 65 of 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in a Varian (Rainin) preparative binary HPLC system: gradient CHCN (B)) over 180 min at 9.5 mL/min using a Phenom elution of 30-55% B (0.045% TFA in HO (A) and 0.045% enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV US 7,256.253 B2 69 70 detector (Varian Dynamax UVD II) at 214 and 254 nm to was performed using cleavage mixture as described above. afford the desired DAC in >95% purity, as determined by The product was isolated by precipitation and purified by RP-HPLC. preparative HPLC to afford the desired product as a white Solid upon lyophilization. Example 17 The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin Modification of Kringle-5 at the e-Amino Group of with a solution of 3 eq of Pd(PPh) dissolved in 5 mL of the Added C-terminus Lysine Residue CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin was then washed with CHC1 (6x5 mL), 20% HOAc in Preparation of NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys 10 DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The Leu-Tyr-Asp-Tyr-Lys-(Ne-MPA)-NH2TFA synthesis was then re-automated for the addition of the 3-maleimidopropionic acid (Step 3). Resin cleavage and Using automated peptide synthesis, the following pro product isolation was performed using 85% TFA/5% TIS/ tected amino acids were sequentially added to Rink Amide 5% thioanisole and 5% phenol, followed by precipitation by MBHA resin: Fmoc-Lys.(Aloc)-OH, Fmoc-Tyr(tEu)OH, 15 dry-ice cold EtO (Step 4). The product was purified by Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, preparative reversed phased HPLC using a Varian (Rainin) Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, preparative binary HPLC system: gradient elution of Fmoc-ASn(Trt)-OH, Fmoc-Thr(tEu)-OH, Fmoc-Thr(tEu)- 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in OH, Fmoc-Tyr?tBu)OH (step 1). Deblocking of the Fmoc CHCN (B)) over 180 min at 9.5 mL/min using a Phenom group the the N-terminal of the resin-bound amino acid was enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV performed with 20% piperidine in DMF for about 15-20 detector (Varian Dynamax UVD II) at 214 and 254 nm. minutes. Final cleavage from the resin was performed using cleavage mixture as described above. The product was Example 19 isolated by precipitation and purified by preparative HPLC to afford the desired product as a white solid upon lyo 25 Modification of Kringle-5 at the e-Amino Group of philization The selective deprotection of the Lys(Aloc) the Added C-terminus Lysine Residue group was performed manually and accomplished by treat ing the resin with a solution of 3 eq of Pd(PPh.) dissolved Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Gly-Gly-Pro-Trp-Lys-(Ne-MPA)-NH.TFA The resin was then washed with CHCl (6x5 mL), 20% 30 HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 Using automated peptide synthesis, the following pro mL). The synthesis was then re-automated for the addition tected amino acids were sequentially added to Rink Amide of the 3-maleimidopropionic acid (Step 3). Resin cleavage MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Trp-OH, Fmoc and product isolation was performed using 85% TFA/5% Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, TIS/5% thioanisole and 5% phenol, followed by precipita 35 Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)- tion by dry-ice cold EtO (Step 4). The product was purified OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Pbf)- by preparative reversed phased HPLC using a Varian (Rai OH (Step 1). The selective deprotection of the Lys(Aloc) nin) preparative binary HPLC system: gradient elution of group was performed manually and accomplished by treat 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in ing the resin with a solution of 3 eq of Pd(PPh.) dissolved CHCN (B)) over 180 min at 9.5 mL/min using a Phenom 40 in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV The resin was then washed with CHCl (6x5 mL), 20% detector (Varian Dynamax UVD II) at 214 and 254 nm. HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis was then re-automated for the addition Example 18 of the 3-maleimidopropionic acid (Step 3). Resin cleavage 45 and product isolation was performed using 85% TFA/5% Modification of Kringle-5 at the e-Amino Group of TIS/5% thioanisole and 5% phenol, followed by precipita the Added C-terminus Lysine Residue tion by dry-ice cold EtO (Step 4). The product was purified by preparative reversed phased HPLC using a Varian (Rai Preparation of NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val nin) preparative binary HPLC system: gradient elution of Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg 50 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Lys-Leu-Tyr-Asp-Tyr-Lys-(Ne-MPA)-NH.3TFA CHCN (B)) over 180 min at 9.5 mL/min using a Phenom enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV Using automated peptide synthesis, the following pro detector (Varian Dynamax UVD II) at 214 and 254 nm. tected amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-Lys.(Aloc)-OH, Fmoc-Tyr(tEu)OH, 55 Example 20 Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Modification of Kringle-5 at the e-Amino Group of Fmoc-ASn(Trt)-OH, Fmoc-Thr(tEu)-OH, Fmoc-Thr(tEu)- the Added C-terminus Lysine Residue OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val 60 Preparation of NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp (Ne-MPA)-NH2TFA (OtBu)-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg (Pbf)-OH (step 1). Deblocking of the Fmoc group the the Using automated peptide synthesis, the following pro N-terminal of the resin-bound amino acid was performed tected amino acids were sequentially added to Rink Amide with 20% piperidine in DMF for about 15-20 minutes. 65 MBHA resin: Fmoc-Lys.(Aloc)-OH, Fmoc-Tyr(tEu)OH, Coupling of the acetic acid was performed under conditions Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, similar to amino acid coupling. Final cleavage from the resin Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). US 7,256.253 B2 71 72 Deblocking of the Fmoc group the N-terminal of the resin Example 22 bound amino acid was performed with 20% piperidine in DMF for about 15-20 minutes. Coupling of the acetic acid Modification of Kringle-5 at the e-Amino Group of was performed under conditions similar to amino acid the Added C-terminus Lysine Residue coupling. Final cleavage from the resin was performed using 5 cleavage mixture as described above. The product was Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr isolated by precipitation and purified by preparative HPLC Lys-(Ne-AEEA-MPA)-NH2TFA to afford the desired product as a white solid upon lyo philization The selective deprotection of the Lys(Aloc) Using automated peptide synthesis, the following pro 10 tected amino acids were sequentially added to Rink Amide group was performed manually and accomplished by treat MBHA resin: Fmoc-Lys.(Aloc)-OH, Fmoc-Tyr(tEu)OH, ing the resin with a solution of 3 eq of Pd(PPh.) dissolved Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH The resin was then washed with CHCl (6x5 mL), 20% (Step 1). Deblocking of the Fmoc group at the N-terminal of HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 15 the resin-bound amino acid was performed with 20% pip mL). The synthesis was then re-automated for the addition eridine in DMF for about 15-20 minutes. Coupling of the of the 3-maleimidopropionic acid (Step 3). Resin cleavage acetic acid was performed under conditions similar to amino and product isolation was performed using 85% TFA/5% acid coupling. The selective deprotection of the Lys(Aloc) TIS/5% thioanisole and 5% phenol, followed by precipita group was performed manually and accomplished by treat tion by dry-ice cold EtO (Step 4). The product was purified ing the resin with a solution of 3 eq of Pd(PPh.) dissolved by preparative reversed phased HPLC using a Varian (Rai in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). nin) preparative binary HPLC system: gradient elution of The resin was then washed with CHCl (6x5 mL), 20% 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 CHCN (B)) over 180 min at 9.5 mL/min using a Phenom mL). The synthesis was then re-automated for the addition. enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV 25 The synthesis was then re-automated for the addition of the detector (Varian Dynamax UVD II) at 214 and 254 nm. AEEA (aminoethoxyethoxyacetic acid) groupand of the 3-maleimidopropionic acid (MPA) (Step 3). Resin cleavage Example 21 and product isolation was performed using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipita 30 tion by dry-ice cold EtO (Step 4). The product was purified Modification of Kringle-5 at the e-Amino Group of by preparative reversed phased HPLC using a Varian (Rai the Added C-terminus Lysine Residue nin) preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys CHCN (B)) over 180 min at 9.5 mL/min using a Phenom (Ne-MPA)-NH2TFA 35 enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm. Using automated peptide synthesis, the following pro tected amino acids were sequentially added to Rink Amide Example 23 MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tEu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, 40 Modification of Kringle-5 at the e-Amino Group of Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). Deblocking of the Added C-terminus Lysine Residue the Fmoc group the N-terminal of the resin-bound amino acid was performed with 20% piperidine in DMF for about Preparation of NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr 15-20 minutes. Coupling of the acetic acid was performed Lys-(Ne-AEEA-MPA)-NH2TFA under conditions similar to amino acid coupling. Final 45 cleavage from the resin was performed using cleavage Using automated peptide synthesis, the following pro tected amino acids were sequentially added to Rink Amide mixture as described above. The product was isolated by MBHA resin: Fmoc-Lys.(Aloc)-OH, Fmoc-Tyr(tEu)OH, precipitation and purified by preparative HPLC to afford the Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, desired product as a white Solid upon lyophilization 50 Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf-OH, Fmoc-Pro-OH The selective deprotection of the Lys(Aloc) group was (Step 1). Deblocking of the Fmoc group at the N-terminal of performed manually and accomplished by treating the resin the resin-bound amino acid was performed with 20% pip with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of eridine in DMF for about 15-20 minutes. Coupling of the CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin acetic acid was performed under conditions similar to amino was then washed with CHCl (6x5 mL), 20% HOAc in 55 acid coupling. DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The The selective deprotection of the Lys(Aloc) group was synthesis was then re-automated for the addition of the performed manually and accomplished by treating the resin 3-maleimidopropionic acid (Step 3). Resin cleavage and with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of product isolation was performed using 85% TFA/5% TIS/ CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin 5% thioanisole and 5% phenol, followed by precipitation by 60 was then washed with CHCl (6x5 mL), 20% HOAc in dry-ice cold EtO (Step 4). The product was purified by DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The preparative reversed phased HPLC using a Varian (Rainin) synthesis was then re-automated for the addition. The Syn preparative binary HPLC system: gradient elution of thesis was then re-automated for the addition of n AEEA 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in (aminoethoxyethoxyacetic acid) groups and of the 3-male CHCN (B)) over 180 min at 9.5 mL/min using a Phenom 65 imidopropionic acid (MPA) (Step 3). Resin cleavage and enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV product isolation was performed using 85% TFA/5% TIS/ detector (Varian Dynamax UVD II) at 214 and 254 nm. 5% thioanisole and 5% phenol, followed by precipitation by US 7,256, 253 B2 73 74 dry-ice cold EtO (Step 4). The product was purified by OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- preparative reversed phased HPLC using a Varian (Rainin) OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- preparative binary HPLC system: gradient elution of OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu CHCN (B)) over 180 min at 9.5 mL/min using a Phenom- 5 enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV (OtBu)-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc detector (Varian Dynamax UVD II) at 214 and 254 nm. Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1) The selective deprotection of the Lys(Aloc) group was Example 24 performed manually and accomplished by treating the resin 10 with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of Modification of GLP-1 at the e-Amino Group of CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin the Added C-terminus Lysine Residue was then washed with CHC1 (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The Preparation of GLP-1 (1-36)-Lys7(Ne-MPA)- synthesis was then re-automated for the addition of the NH5TFA 15 3-maleimidopropionic acid (Step 3). Resin cleavage and product isolation was performed using 85% TFA/5% TIS/ His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr 5% thioanisole and 5% phenol, followed by precipitation by Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln dry-ice cold EtO (Step 4). The product was purified by Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly preparative reverse phase HPLC using a Varian (Rainin) Arg-Lys(Ne-MPA)-NH.5TFA 2O preparative binary HPLC system: gradient elution of Using automated peptide synthesis, the following pro 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in tected amino acids were sequentially added to Rink Amide CHCN (B)) over 180 min at 9.5 mL/min using a Phenom MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH, Fmoc- 25 detector (Varian Dynamax UVD II) at 214 and 254 nm. Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, The product had >95% purity as determined by RP-HPLC Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(tBoc)- mass spectrometry using a Hewlett Packard LCMS-1100 OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, series spectrometer equipped with a diode array detector and Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, using electro-spray ionization. These steps are illustrated in Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tPu)- the schematic diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 r

Boc-HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Lys.(Aloc)-PS Step 2 piersMM/HOAc/CHCI Boc-HDEFERHAEGTFTSDVSSYLEGQAAKEFLAWLVKGR-Lys-PS

Step 3 | 3-maleimidopropionic acid

HN -N- N O /

PS Boc-HDEFERHAEGTFTSDVSSYLEGQAAKEFLAWLVKGR- N

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol US 7,256.253 B2 75 76

-continued

HN --> N O /

TFA TFA TFA HN-HDEFERHAEGTFTSDVSSYLEGQAAKEFLAWLVKGR- N TFA O GLP-1 (1-36)-Lys7 (E-MPA)-NH2

Example 25 The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin Modification of GLP-1 at the e-Amino Group of with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of the Added C-terminus Lysine Residue CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin Preparation of GLP-1 (1-36)-Lys7(Ne-AEEA was then washed with CHC1 (6x5 mL), 20% HOAc in AEEA-MPA)-NH.5TFA 25 DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis was then re-automated for the addition of the two His-Asp-Glu-Phe-Glu-Arg-His-Ala-Glu-Gly-Thr AEEA (aminoethoxyethoxyacetic acid) groups and the Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln 3-maleimidopropionic acid (Step 3). Resin cleavage and Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly product isolation was performed using 85% TFA/5% TIS/ Arg-Lys(Ne-AEEA-AEEA-MPA)-NH.5TFA 30 5% thioanisole and 5% phenol, followed by precipitation by Using automated peptide synthesis, the following pro dry-ice cold Et2O (Step 4). The product was purified by tected amino acids were sequentially added to Rink Amide preparative reverse phase HPLC using a Varian (Rainin) MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf-OH, preparative binary HPLC system: gradient elution of Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH, Fmoc 35 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, CHCN (B)) over 180 min at 9.5 mL/min using a Phenom Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(tBoc)- enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, detector (Varian Dynamax UVD II) at 214 and 254 nm. Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, The product had >95% purity as determined by RP-HPLC Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tPu)- 40 OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- mass spectrometry using a Hewlett Packard LCMS-1100 OH, Fmoc-Thr(tEu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- series spectrometer equipped with a diode array detector and OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, using electro-spray ionization, ESI-MS m/z for Boc-His(N-Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Glu C.H.N.O. (MH), calcd3868, found M+H," 1934, (OtBu)-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-OH, Fmoc M+H" 1290, M+H"967. These steps are illustrated in Asp(OtBu)-OH, Boc-His(N-Trt)-OH (step 1). the schematic diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 sers

Boc-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Lys(Aloc)- PS Step 2 parlinMM/HOAc/CHCI

Boc-HAEGTFTSDVSSYLEGQAAKEFLAWLVKGR-Lys-PS

Step 3 Fmoc-AEEA-OH (2 times) 2.3-maleimidopropionic acid US 7,256.253 B2 77 78

-continued

O O H N HN N-1-1-N 2 N O O

Boc-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR- N

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

O O H N HN N-1-1--N 2 N O O

TFA TFA TFA

0 0 0 NH2 HN-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR- N TFA O

CC-1051 GLP-1 (7-36-K (AEEA-MPA))-NH

Example 26 OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- 45 OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Modification of GLP-1 at the e-Amino Group of Boc-His(N-Trt)-OH (Step 1). the Added C-terminus Lysine Residue The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin Preparation of GLP-1 (7-36)-Lys7(Ne-MPA)- with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of NH4TFA 50 CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl (6x5 mL), 20% HOAc in His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis was then re-automated for the addition of the Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala 3-maleimidopropionic acid (Step 3). Resin cleavage and Trp-Leu-Val-Lys-Gly-Arg-Lys(Ne-MPA)-NH.4TFA 55 product isolation was performed using 85% TFA/5% TIS/ 5% thioanisole and 5% phenol, followed by precipitation by Using automated peptide synthesis, the following pro dry-ice cold EtO (Step 4). The product was purified by tected amino acids were sequentially added to Rink Amide preparative reverse phase HPLC using a Varian (Rainin) MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, preparative binary HPLC system: gradient elution of Fmoc-Gly-OH, Fmoc-Lys(tEoc)-OH, Fmoc-Val-OH, Fmoc 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in CHCN (B)) over 180 min at 9.5 mL/min using a Phenom Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(tBoc)- detector (Varian Dynamax UVD II) at 214 and 254 nm. OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, The product had >95% purity as determined by RP-HPLC Fmoc-Gly-OH, Fmoc-GlucOtBu)-OH, Fmoc-Leu-OH, is mass spectrometry using a Hewlett Packard LCMS-1100 Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tPu)- series spectrometer equipped with a diode array detector and OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- using electro-spray ionization. US 7,256.253 B2 79 80 Example 27 Example 28 Modification of GLP-1 at the e-Amino Group of Modification of D-Ala GLP-1 at the e-Amino the Added C-terminus Lysine Residue Group of the Added C-terminus Lysine Residue Preparation of GLP-1 (7-36)-Lys7(Ne-AEEA Preparation of D-Ala GLP-1 (7-36)-Lys'(Ne AEEA-MPA)-NH4TFA MPA)-NH4TFA His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser His-d-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala 10 Ser-Tyr-Leu-Glu-Gly-Gin-Ala-Ala-Lys-Glu-Phe-Ile Trp-Leu-Val-Lys-Gly-Arg-Lys(Ne-AEEA-AEEA Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys(Ne-MPA)- MPA)-NH4TFA NHh.4TFA Using automated peptide synthesis, the following pro Using automated peptide synthesis, the following pro tected amino acids were sequentially added to Rink Amide 15 tected amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf-OH, MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH, Fmoc Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH, Fmoc Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(tBoc)- Fmoc-Phe-OH, Fmoc-GlucOtBu)-OH, Fmoc-Lys(tBoc)- OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(Pbf-OH, Fmoc-Ser(tPu)-OH, Fmoc-Ser(tPu)- Fmoc-Tyr(Pbf-OH, Fmoc-Ser(tPu)-OH, Fmoc-Ser(tPu)- OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- OH, Fmoc-Thr(tEu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, 25 OH, Fmoc-Gly-OH, Fmoc-Glu? OtBu)-OH, Fmoc-d-Ala Boc-His(N-Trt)-OH (Step 1). OH, Boc-His(N-Trt)-OH (Step 1). The selective deprotection of the Lys(Aloc) group was The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin 30 CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin was then washed with CHCl (6x5 mL), 20% HOAc in was then washed with CHCl (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis was then re-automated for the addition of the two synthesis was then re-automated for the addition of the AEEA (aminoethoxyethoxyacetic acid) groups and the 3-maleimidopropionic acid (Step 3). Resin cleavage and 3-maleimidopropionic acid (Step 3). Resin cleavage and 35 product isolation was performed using 85% TFA/5% TIS/ product isolation was performed using 85% TFA/5% TIS/ 5% thioanisole and 5% phenol, followed by precipitation by 5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold EtO (Step 4). The product was purified by dry-ice cold EtO (Step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system: gradient elution of preparative binary HPLC system: gradient elution of 40 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in CHCN (B)) over 180 min at 9.5 mL/min using a Phenom CHCN (B)) over 180 min at 9.5 mL/min using a Phenom enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm. detector (Varian Dynamax UVD II) at 214 and 254 nm. The product had >95% purity as determined by RP-HPLC The product had >95% purity as determined by RP-HPLC 45 mass spectrometry using a Hewlett Packard LCMS-1100 mass spectrometry using a Hewlett Packard LCMS-1100 series spectrometer equipped with a diode array detector and series spectrometer equipped with a diode array detector and using electro-spray ionization. These steps are illustrated in using electro-spray ionization. the schematic diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 |rs

Boc-HaEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Lys.(Aloc)- PS Step 2 pensioecil, Boc-HaEGTFTSDVSSYLEGQAAKEFLAWLVKGR-Lys-PS

Step 3 3-maleimidopropionic acid US 7,256.253 B2 81 82

-continued

Boc-HaEGTFTSDVSSYLEGQAAKEFIAWLVKGR- N

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

HN --> N O /

TFA TFA TFA HN-HaEGTFTSDVSSYLEGQAAKEFIAWLVKGR- N TFA O

D-Ala-GLP-1 (7-36)-Lys (E-MPA)- NH

40 Example 29 The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin Modification of D-Ala GLP-1 at the e-Amino with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of Group of the Added C-terminus Lysine Residue 45 CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin Preparation of D-Ala GLP-1 (7-36)-Lys'(Ne was then washed with CHCl (6x5 mL), 20% HOAc in AEEA-AEEA-MPA)-NH4TFA DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis was then re-automated for the addition of the two His-D-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser 50 AEEA (aminoethoxyethoxyacetic acid) groups and the Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile 3-maleimidopropionic acid (Step 3). Resin cleavage and Ala-Trp-Leu-Val-Lys-Gly-Arg-Lys (Ne-AEEA product isolation was performed using 85% TFA/5% TIS/ AEEA-MPA)-NH.4TFA 5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold EtO (Step 4). The product was purified by Using automated peptide synthesis, the following pro 55 tected amino acids were sequentially added to Rink Amide preparative reverse phase HPLC using a Varian (Rainin) MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf-OH, preparative binary HPLC system: gradient elution of Fmoc-Gly-OH, Fmoc-Lys(tBoc)-OH, Fmoc-Val-OH, Fmoc 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Leu-OH, Fmoc-Trp-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, CHCN (B)) over 180 min at 9.5 mL/min using a Phenom Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(tBoc)- 60 enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, detector (Varian Dynamax UVD II) at 214 and 254 nm. Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(Pbf)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tPu)- The product had >95% purity as determined by RP-HPLC OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tPu)- mass spectrometry using a Hewlett Packard LCMS-1100 OH, Fmoc-Thr(tEu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- 65 series spectrometer equipped with a diode array detector and OH, Fmoc-Gly-OH, Fmoc-Glu? OtBu)-OH, Fmoc-d-Ala using electro-spray ionization. These steps are illustrated in OH, Boc-His(N-Trt)-OH (Step 1). the schematic diagram below.

US 7,256.253 B2 85 86 3-maleimidopropionic acid (Step 3). Resin cleavage and product isolation was performed using 85% TFA/5% TIS/ 5% thioanisole and 5% phenol, followed by precipitation by dry-ice cold EtO (Step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) 5 preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in CHCN (B)) over 180 min at 9.5 mL/min using a Phenom enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm. 10 The product had >95% purity as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series spectrometer equipped with a diode array detector and using electro-spray ionization. These steps are illustrated in the schematic diagram below.

Fmoc-Rink Amide MBHA Resin

Step 1 || SPPS y

Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Lys.(Aloe)-PS Step 2Pd(PPh3)4/NMM/HOAc/CHCl y Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Lys-PS

Step 3 3-maleimidopropionic acid y O

HN --> N O /

PS Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS- N

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol --> /

TFA TFA TFA PS HN-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS- N TFA Exendin-4 (1-39)-Lys' (E-MPA)-NH US 7,256.253 B2 87 88 Example 31 (thBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gly-OH, Boc-His(Trt)- Modification of Exendin-4(1-39) at the e-Amino OH (Step 1). Group of the Added C-terminus Lysine Residue The selective deprotection of the Lys(Aloc) group was performed manually and accomplished by treating the resin Preparation of Exendin-4 (1–39)-Lys'(Ne-AEEA with a solution of 3 eq of Pd(PPh) dissolved in 5 mL of AEEA-MPA)-NH.5TFA CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser was then washed with CHCl (6x5 mL), 20% HOAC in Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe 1 O DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly synthesis was then re-automated for the addition of the two Ala-Pro-Pro-Pro-Ser-Lys(Ne-AEEA-AEEA-MPA)- AEEA (aminoethoxyethoxyacetic acid) groups and the NH5TFA 3-maleimidopropionic acid (Step 3). Resin cleavage and product isolation was performed using 85% TFA/5% TIS/ Using automated peptide synthesis, the following pro- 1 5 5% thioanisole and 5% phenol, followed by precipitation by tected amino acids were sequentially added to Rink Amide dry-ice cold EtO (Step 4). The product was purified by MBHA resin: Fmoc-Lys(Aloc)-OH, Fmoc-Ser(tBu)-OH, preparative reverse phase HPLC using a Varian (Rainin) Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Ala preparative binary HPLC system: gradient elution of OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Ser(tBu)-OH, 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Asn 2 O CHCN (B)) over 180 min at 9.5 mL/min using a Phenom (Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Trp enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, detector (Varian Dynamax UVD II) at 214 and 254 nm. Fmoc-Leu-OH, Fmoc-Arg(Bpf)-OH, Fmoc-Val-OH, Fmoc The product had >95% purity as determined by RP-HPLC Ala-OH, Fmoc-GlucCtBu)-OH, Fmoc-GlucCtBu)-OH, mass spectrometry using a Hewlett Packard LCMS-1100 Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, 2 5 series spectrometer equipped with a diode array detector and Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, using electro-spray ionization. These steps are illustrated in Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr the schematic diagram below.

Fmoc-Rink Amide MBHA Resin

Step 1 SPPS Y

Boc-HGEGTFTSDLSKQMEEEAVRLF EWLKNGGPSSGAPPPS-Lys.(Aloc)-PS Step 2 Y

Boc-HGEGTFTSDLSKQMEEEAVR LFIEWLKNGGPSSGAPPPS-Lys-PS

1. Fmoc-AEEA-OH (2 times) Step 3 2.3-maleimidopropionic acid Y O O H N HN N-1-1-N 2 N O O

Boc-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSN H

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

US 7,256.253 B2 93 94 and Diisopropylethylamine (DIEA). Removal of the Fmoc enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV protecting group is achieved using a solution of 20% (V/V) detector (Varian Dynamax UVD II) at w 214 and 254 nm to piperidine in N,N-dimethylformamide (DMF) for 20 min afford the desired DAC in >95% purity, as determined by utes (Step 1).The selective deprotection of the Lys (Aloc) RP-HPLC. group is performed manually and accomplished by treating 5 the resin with a solution of 3 eq of Pd(PPh3)4 dissolved in Example 38 5 mL of CHC13:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHC13 (6x5 mL), 20% HOAc Modification of Neuropeptide Y at the e-Amino in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). Group of the Added N-terminus Lysine Residue The synthesis is then re-automated for the addition of the 10 3-maleimidopropionic acid (Step 3). Between every cou Preparation of (N-eMPA)-Lys-Tyr-Pro-Ser-Lys-Pro pling, the resin is washed 3 times with N,N-dimethylforma Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp mide (DMF) and 3 times with isopropanol. The peptide is Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu cleaved from the resin using 85% TFA/5% TIS/5% thioani sole and 5% phenol, followed by precipitation by dry-ice 15 Solid phase peptide synthesis of a modified neuropeptide cold Et2O (Step 4). The product is purified by preparative Y on a 100 umole scale was performed using manual reversed phased HPLC using a Varian (Rainin) preparative solid-phase synthesis, a Symphony Peptide Synthesizer and binary HPLC system: gradient elution of 30-55% B (0.045% Fmoc protected Rink Amide MBHA. The following pro TFA in H2O (A) and 0.045% TFA in CH3CN (B)) over 180 tected amino acids are sequentially added to resin: Fmoc min at 9.5 mL/min using a Phenomenex Luna 10L phenyl Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr hexyl, 21 mmx25 cm column and UV detector (Varian (thBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc Dynamax UVD II) at 214 and 254 nm to afford the desired Ala-OH, Fmoc-Met-OH, Fmoc-Asp(tEu)-OH, Fmoc-Glu (tbu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, DAC in >95% purity, as determined by RP-HPLC. Fmoc-Asp(tBu)-OH, Fmoc-GluctBu)-OH, Fmoc-Gly-OH, Example 37 Fmoc-Pro-OH, Fmoc-Asn (Trt)-OH, Fmoc-Asp(tEu)-OH, 25 Fmoc-Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr?tBu)-OH, Fmoc-Lys(Aloc)-OH. Modification of Neuropeptide Y at the e-Amino They are dissolved in N,N-dimethylformamide (DMF) and, Group of the Added N-terminus Lysine Residue according to the sequence, activated using O-benzotriazol 1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate Preparation of (N-eMPA)-Lys-Tyr-Pro-Ser-Lys-Pro 30 (HBTU) and Diisopropylethylamine (DIEA). Removal of Glu-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp the Fmoc protecting group is achieved using a solution of Met-Ala-Arg-Tyr-Tyr-Ser-Ala-Leu 20% (V/V) piperidine in N,N-dimethylformamide (DMF) for 20 minutes (Step 1).The selective deprotection of the Lys Solid phase peptide synthesis of a modified neuropeeptide (Aloc) group is performed manually and accomplished by Y on a 100LL mole scale was performed using manual 35 treating the resin with a solution of 3 eq of Pd(PPh)4 solid-phase synthesis, a Symphony Peptide Synthesizer and dissolved in 5 mL of CHCl:NMM:HOAc (18:1:0.5) for 2 h Fmoc protected Rink Amide MBHA. The following pro (Step 2). The resin is then washed with CHCl (6x5 mL), tected amino acids are sequentially added to resin: Fmoc 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr (6x5 mL). The synthesis is then re-automated for the addi (thBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc tion of the 3-maleimidopropionic acid (Step 3). Between Ala-OH, Fmoc-Met-OH, Fmoc-Asp(tBu)-OH, Fmoc-Glu 40 every coupling, the resin is washed 3 times with N.N- (thBu)-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Ala-OH, dimethylformamide (DMF) and 3 times with isopropanol. Fmoc-Asp(tEu)-OH, Fmoc-GluctBu)-OH, Fmoc-Gly-OH, The peptide is cleaved from the resin using 85% TFA/5% Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Glu(tBu)-OH, TIS/5% thioanisole and 5% phenol, followed by precipita Fmoc Pro-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(tBu)-OH, tion by dry-ice cold Et2O (Step 4). The product is purified Fmoc-Pro-OH, Fmoc-Tyr?tBu)-OH, Fmoc-Lys(Aloc)-OH. 45 by preparative reverse phase HPLC using a Varian (Rainin) They are dissolved in N,N-dimethylformamide (DMF) and, preparative binary HPLC system: gradient elution of according to the sequence, activated using O-benzotriazol 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in 1-yl-N,N,N',N'-tetramethyl-uroniurn hexafluorophosphate CHCN (B)) over 180 min at 9.5 mL/min using a Phenom (HBTU) and Diisopropylethylamine (DIEA). Removal of enex Luna 10 L phenyl-hexyl, 21 mmx25 cm column and the Fmoc protecting group is achieved using a solution of 50 UV detector (Varian Dynamax UVD II) at 214 and 254 nm 20% (VN) piperidine in N,N-dimethylformamide (DMF) for to afford the desired DAC in >95% purity, as determined by 20 minutes (Step 1).The selective deprotection of the Lys RP-HPLC. (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh.) Example 39 dissolved in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washed with CHCl (6x5 mL), 55 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF Modification of Dyn A 1-13 at the e-Amino Group (6x5 mL). The synthesis is then re-automated for the addi of the Added N-terminus Lysine tion of the 3-maleimidopropionic acid (Step 3). Between Residue Synthesis of (Ne-MPA)-Dyn A 1-13-NH every coupling, the resin is washed 3 times with N.N- dimethylformamide (DMF) and 3 times with isopropanol. 60 (Ne-MPA)-Lys-Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile The peptide is cleaved from the resin using 85% TFA/5% Arg-Pro-Lys-Leu TIS/5% thioanisole and 5% phenol, followed by precipita tion by dry-ice cold EtO (Step 4). The product is purified by Solid phase peptide synthesis of a modified Dyn A 1-13 preparative reverse phase HPLC using a Varian (Rainin) analog on a 100 umole Scale is performed using manual preparative binary HPLC system: gradient elution of 65 solid-phase synthesis, a Symphony Peptide Synthesizer and 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Fmoc protected Rink Amide MBHA. The following pro CHCN (B)) over 180 min at 9.5 mL/min using a Phenom tected amino acids are sequentially added to resin: Fmoc US 7,256.253 B2 95 96 Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc Example 42 Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Arg(Pbf-OH, Fmoc-Leu-OH, Fmoc-Phe Modification of Kringle-5 at the e-Amino Group of OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH, the Added N-Terminus Lysine Residue Fmoc-Lys(Aloc)-OH. They were dissolved in N,N-dimeth ylformamide (DMF) and, according to the sequence, acti Preparation of (Ne-MPA)-Lys-Pro-Arg-Lys-Leu vated using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uro Tyr-Asp-Tyr-NH2TFA nium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc pro tecting group is achieved using a solution of 20% (V/V) Solid phase peptide synthesis of a modified Kringle-5 piperidine in N,N-dimethylformamide (DMF) for 20 min 10 analog on a 100 umole scale was performed using manual utes (Step 1).The selective deprotection of the Lys (Aloc) solid-phase synthesis, a Symphony Peptide Synthesizer and group is performed manually and accomplished by treating Fmoc protected Rink Amide MBHA. The following pro the resin with a solution of 3 eq of Pd(PPh.) dissolved in 5 tected amino acids are sequentially added to resin: Fmoc mL of CHCl:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The Tyr(tEu)-OH, Fmoc-Asp(tEu)-OH, Fmoc-Tyr(tBu)-OH, resin is then washed with CHCl (6x5 mL), 20% HOAC in 15 Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The Fmoc-Pro-OH, Fmoc-Lys(Aloc)-OH. They were dissolved synthesis is then re-automated for the addition of the 3-ma in N,N-dimethylformamide (DMF) and, according to the leimidopropionic acid (Step 3). Between every coupling, the sequence, activated using O-benzotriazol-1-yl-N, N,N',N'- resin is washed 3 times with N,N-dimethylformamide tetramethyl-uronium hexafluorophosphate (HBTU) and (DMF) and 3 times with isopropanol. The peptide is cleaved Diisopropylethylamine (DIEA). Removal of the Fmoc pro from the resin using 85% TFA/5% TIS/5% thioanisole and tecting group is achieved using a solution of 20% (V/V) 5% phenol, followed by precipitation by dry-ice cold EtO piperidine in N,N-dimethylformamide (DMF) for 20 min (Step 4). The product is purified by preparative reverse utes (step 1).The selective deprotection of the Lys (Aloc) phase HPLC using a Varian (Rainin) preparative binary group is performed manually and accomplished by treating HPLC system: gradient elution of 30-55% B (0.045% TFA 25 in HO (A) and 0.045% TFA in CHCN (B)) over 180 min the resin with a solution of 3 eq of Pd(PPh) dissolved in 5 at 9.5 mL/min using a Phenomenex Luna 10L phenyl-hexyl, mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The 21 mmx25 cm column and UV detector (Varian Dynamax resin is then washed with CHCl (6x5 mL), 20% HOAc in UVD II) at 214 and 254 nm to afford the desired DAC in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The >95% purity, as determined by RP-HPLC. synthesis is then re-automated for the addition of the 3-ma 30 leimidopropionic acid (Step 3). Between every coupling, the Example 40 resin is washed 3 times with N,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide is cleaved Modification of Dyn A 2-17-NH at the N-terminus from the resin using 85% TFA/5% TIS/5% thioanisole and Glycine Synthesis of MPA-AEA-Dyn A 5% phenol, followed by precipitation by dry-ice cold EtO 2-17-NH, 35 (Step 4). The product is purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary (MPA-AEA-AEA-AEA)-Gly-Gly-Phe-Leu-Arg HPLC system: gradient elution of 30-55% B (0.045% TFA Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Glu in HO (A) and 0.045% TFA in CHCN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10L phenyl-hexyl, Using automated peptide synthesis, the following pro 40 21 mmx25 cm column and UV detector (Varian Dynamax tected amino acids and maleimide were sequentially added UVD II) at 214 and 254 nm to afford the desired DAC in to Rink Amide MBHA resin: Fmoc-Gln(Trt)-OH, Fmoc-Asn >95% purity, as determined by RP-HPLC. (Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Example 43 Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc 45 Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc Modification of Kringle-5 at the N-Terminus Phe-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-AEA-OH, Proline Fmoc-AEA-OH, Fmoc-AEA-OH, and MPA. The target dynorphin analog was then removed from the resin; the Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr product was isolated by precipitation and purified by pre 50 Asp-Tyr-Lys-NH2TFA parative HPLC to afford the desired product as a pale yellow solid upon lyophilization in a 32% yield. Anal. HPLC Using automated peptide synthesis, the following pro indicated product to be >95% pure with R. 33.44 min. tected amino acids were sequentially added to Rink Amide ESI-MS m/z for CH7NO. (MH), calcd 2436.8, MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, found MH 813.6. Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH,

TFATFA TFA TFA TFA N -n- N Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-ASn-Gln NH2 N 3 US 7,256.253 B2 97 98 Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH 20% in DMF allow for the subsequent addition of the (step 1). The deprotection of the terminal Fmoc group is 3-MPA (Step 3). Resin cleavage and product isolation was accomplished using 20% piperidine (Step 2) followed by the performed using 86% TFA/5% TIS/5% HO/2% thioanisole coupling of Fmoc-AEEA. Deprotection of the resulting and 2% phenol, followed by precipitation by dry-ice cold Fmoc-AEEA-peptide with piperidine 20% in DMF allow for EtO (Step 4). The product was purified by preparative the subsequent addition of the 3-MPA (Step 3). Resin reversed phased HPLC using a Varian (Rainin) preparative cleavage and product isolation was performed using 86% binary HPLC system using a Dynamax Cls, 60 A, 8 um, 21 TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, fol mmx25 cm column equipped with a Dynamax Cls, 60A, 8 lowed by precipitation by dry-ice cold EtO (Step 4). The um guard module, 21 mmx25 cm column and UV detector product was purified by preparative reversed phased HPLC 10 (Varian Dynamax UVD II) at 214 and 254 nm. The using a Varian (Rainin) preparative binary HPLC system product had >95% purity as determined by RP-HPLC mass using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column spectrometry using a Hewlett Packard LCMS-1100 series equipped with a Dynamax Cls, 60 A, 8 um guard module, spectrometer equipped with a diode array detector and using 21 mmx25 cm column and UV detector (Varian Dynamax electro-spray ionization. UVD II) at 214 and 254 nm. The product had >95% purity 15 as determined by RP-HPLC mass spectrometry using a Example 46 Hewlett Packard LCMS-1100 series spectrometer equipped with a diode array detector and using electro-spray ioniza Modification of Kringle-5 at the N-Terminus tion. Tyrosine Example 44 Preparation of (MPA)-Tyr-Thr-Thr-Asn-Pro-Arg Lys-Leu-Tyr-Asp-Tyr-NH.2TFA Modification of Kringle-5 at the N-Terminus Proline Using automated peptide synthesis, the following pro 25 tected amino acids were sequentially added to Rink Amide Preparation of (MPA)-Pro-Arg-Lys-Leu-Tyr-Asp MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, Tyr-Lys-NH.2TFA Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Using automated peptide synthesis, the following pro Fmoc-ASn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tEu)- tected amino acids were sequentially added to Rink Amide 30 OH, Fmoc-Tyr(tBu)OH (Step 1). The deprotection of the MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, terminal Fmoc group is accomplished using 20% piperidine Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tEu)OH, Fmoc-Leu-OH, (Step 2) followed by the coupling of 3-MPA (Step 3). Resin Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH cleavage and product isolation was performed using 86% (step 1). The deprotection of the terminal Fmoc group is TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, fol accomplished using 20% piperidine (Step 2) followed by the 35 lowed by precipitation by dry-ice cold EtO (Step 4). The coupling of 3-MPA (Step 3). Resin cleavage and product product was purified by preparative reversed phased HPLC isolation was performed using 86% TFA/5% TIS/5% HO/ using a Varian (Rainin) preparative binary HPLC system 2% thioanisole and 2% phenol, followed by precipitation by using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column dry-ice cold EtO (Step 4). The product was purified by equipped with a Dynamax Cls, 60 A, 8 um guard module, preparative reversed phased HPLC using a Varian (Rainin) 40 21 mmx25 cm column and UV detector (Varian Dynamax preparative binary HPLC system using a Dynamax Cs, 60 UVD II) at 214 and 254 nm. The product had >95% purity A, 8 um, 21 mmx25 cm column equipped with a Dynamax as determined by RP-HPLC mass spectrometry using a Cls, 60 A, 8 um guard module, 21 mmx25 cm column and Hewlett Packard LCMS-1100 series spectrometer equipped UV detector (Varian Dynamax UVD II) at 214 and 254 with a diode array detector and using electro-spray ioniza nm. The product had >95% purity as determined by RP 45 tion. HPLC mass spectrometry using a Hewlett Packard LCMS 1100 series spectrometer equipped with a diode array detec Example 47 tor and using electro-spray ionization Modification of Kringle-5 at the N-Terminus Example 45 50 Arginine Modification of Kringle-5 at the N-Terminus Preparation of (MPA-AEEA)-Arg-Asn-Pro-Asp Tyrosine Gly-Asp-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro Arg-Lys-Leu-Tyr-Asp-Tyr-NH.3TFA Preparation of (MPA-AEEA)-Tyr-Thr-Thr-Asn 55 Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2TFA Using automated peptide synthesis, the following pro tected amino acids were sequentially added to Rink Amide Using automated peptide synthesis, the following pro MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, tected amino acids were sequentially added to Rink Amide Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, 60 Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Fmoc-ASn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tEu)- Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-ASn(Trt)-OH, Fmoc-Thr(tEu)-OH, Fmoc-Thr(tEu)- Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val OH, Fmoc-Tyr(tBu)OH (Step 1). The deprotection of the OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp terminal Fmoc group is accomplished using 20% piperidine 65 (OtBu)-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg (Step 2) followed by the coupling of Fmoc-AEEA. Depro (Pbf)-OH (step 1). The deprotection of the terminal Fmoc tection of the resulting Fmoc-AEEA-peptide with piperidine group is accomplished using 20% piperidine (Step 2) fol US 7,256.253 B2 99 100 lowed by the coupling of Fmoc-AEEA. Deprotection of the accomplished using 20% piperidine (Step 2) followed by the resulting Fmoc-AEEA-peptide with piperidine 20% in DMF coupling of Fmoc-AEEA. Deprotection of the resulting allow for the subsequent addition of the 3-MPA (Step 3). Fmoc-AEEA-peptide with piperidine 20% in DMF allow for Resin cleavage and product isolation was performed using the subsequent addition of the 3-MPA (Step 3). Resin 86%TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, cleavage and product isolation was performed using 86% followed by precipitation by dry-ice cold EtO (Step 4). The TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, fol product was purified by preparative reversed phased HPLC lowed by precipitation by dry-ice cold EtO (Step 4). The using a Varian (Rainin) preparative binary HPLC system product was purified by preparative reversed phased HPLC using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column using a Varian (Rainin) preparative binary HPLC system equipped with a Dynamax Cls, 60 A, 8 um guard module, 10 using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column 21 mmx25 cm column and UV detector (Varian Dynamax equipped with a Dynamax Cls, 60 A, 8 um guard module, UVD II) at 214 and 254 nm. The product had >95% purity 21 mmx25 cm column and UV detector (Varian Dynamax as determined by RP-HPLC mass spectrometry using a UVD II) at 214 and 254 nm. The product had >95% purity Hewlett Packard LCMS-1100 series spectrometer equipped as determined by RP-HPLC mass spectrometry using a with a diode array detector and using electro-spray ioniza 15 Hewlett Packard LCMS-1100 series spectrometer equipped tion. with a diode array detector and using electro-spray ioniza tion. Example 48 Example 50 Modification of Kringle-5 at the N-Terminus Arginine Modification of Kringle-5 at the N-Terminus Arginine Preparation of (MPA)-Arg-Asn-Pro-Asp-Gly-Asp Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Arg Preparation of (MPA)-Arg-Asn-Pro-Asp-Gly-Asp Lys-Leu-Tyr-Asp-Tyr-N H.3TFA 25 Val-Gly-Gly-Pro-Trp-NH.TFA Using automated peptide synthesis, the following pro Using automated peptide synthesis, the following pro tected amino acids were sequentially added to Rink Amide tected amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, 30 Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)- Fmoc-ASn(Trt)-OH, Fmoc-Thr(tPu)-OH, Fmoc-Thr(tEu)- OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Pbf)- OH, Fmoc-Tyr(tBu)OH, Fmoc-Ala-OH, Fmoc-Trp-OH, OH (Step 1). The deprotection of the terminal Fmoc group Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val is accomplished using 20% piperidine (Step 2) followed by OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp 35 the coupling of 3-MPA (Step 3). Resin cleavage and product (OtBu)-OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg isolation was performed using 86% TFA/5% TIS/5% HO/ (Pbf)-OH (Step 1). The deprotection of the terminal Fmoc 2% thioanisole and 2% phenol, followed by precipitation by group is accomplished using 20% piperidine (Step 2) fol dry-ice cold EtO (Step 4). The product was purified by lowed by the coupling of 3-MPA (Step 3). Resin cleavage preparative reversed phased HPLC using a Varian (Rainin) and product isolation was performed using 86% TFA/5% 40 preparative binary HPLC system using a Dynamax Cs, 60 TIS/5% HO/2% thioanisole and 2% phenol, followed by A, 8 um, 21 mmx25 cm column equipped with a Dynamax precipitation by dry-ice cold EtO (Step 4). The product was Cls, 60 A, 8 um guard module, 21 mmx25 cm column and purified by preparative reversed phased HPLC using a UV detector (Varian Dynamax UVD II) at 214 and 254 Varian (Rainin) preparative binary HPLC system using a nm. The product had >95% purity as determined by RP Dynamax Cls, 60A, 8 um, 21 mmx25 cm column equipped 45 HPLC mass spectrometry using a Hewlett Packard LCMS with a Dynamax Cls, 60A, 8 Lim guard module, 21 mmx25 1100 series spectrometer equipped with a diode array detec cm column and UV detector (Varian Dynamax UVD II) at tor and using electro-spray ionization. 214 and 254 nm. The product had >95% purity as determined by RP-HPLC mass spectrometry using a Hewlett Example 51 Packard LCMS-1100 series spectrometer equipped with a 50 diode array detector and using electro-spray ionization. Modification of Kringle-5 at the N-Terminus Arginine Example 49 Preparation of (MPA-AEEA)-Arg-Lys-Leu-Tyr Modification of Kringle-5 at the N-Terminus 55 Asp-Tyr-NH2TFA Arginine Using automated peptide synthesis, the following pro Preparation of (MPA-AEEA)-Arg-Asn-Pro-Asp tected amino acids were sequentially added to Rink Amide Gly-Asp-Val-Gly-Gly-Pro-Trp-NH.TFA MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, 60 Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, Using automated peptide synthesis, the following pro Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The tected amino acids were sequentially added to Rink Amide deprotection of the terminal Fmoc group is accomplished MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Trp-OH, Fmoc using 20% piperidine (Step 2) followed by the coupling of Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-AEEA. Deprotection of the resulting Fmoc-AEEA Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(OtBu)- 65 peptide with piperidine 20% in DMF allow for the subse OH, Fmoc-Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Arg(Pbf)- quent addition of the 3-MPA (Step 3). Resin cleavage and OH (step 1). The deprotection of the terminal Fmoc group is product isolation was performed using 86% TFA/5% TIS/ US 7,256.253 B2 101 102 5% HO/2% thioanisole and 2% phenol, followed by pre The product had >95% purity as determined by RP-HPLC cipitation by dry-ice cold EtO (Step 4). The product was mass spectrometry using a Hewlett Packard LCMS-1100 purified by preparative reversed phased HPLC using a series spectrometer equipped with a diode array detector and Varian (Rainin) preparative binary HPLC system using a using electro-spray ionization. Dynamax Cls, 60A, 8 um, 21 mmx25 cm column equipped 5 with a Dynamax Cls, 60A, 8 Lim guard module, 21 mmx25 Example 54 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm. The product had >95% purity as Modification of Kringle-5 at the N-Terminus determined by RP-HPLC mass spectrometry using a Hewlett Proline Packard LCMS-1100 series spectrometer equipped with a 10 diode array detector and using electro-spray ionization Preparation of Example 52 (MPA)-Pro-Arg-Lys-Leu-Tyr-Asp-NH2TFA Modification of Kringle-5 at the N-Terminus Using automated peptide synthesis, the following pro Arginine 15 tected amino acids were sequentially added to Rink Amide MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Preparation of Fmoc-Tyr(tEu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, (MPA)-Arg-Lys-Leu-Tyr-Asp-Tyr-NH2TFA Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (Step 1). The deprotec tion of the terminal Fmoc group is accomplished using 20% Using automated peptide synthesis, the following pro piperidine (Step 2) followed by the coupling of 3-MPA (Step tected amino acids were sequentially added to Rink Amide 3). Resin cleavage and product isolation was performed MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tEu)OH, using 86% TFA/5% TIS/5% HO/2% thioanisole and 2% Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tBu)OH., Fmoc-Leu-OH, phenol, followed by precipitation by dry-ice cold EtO (Step Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH (Step 1). The 4). The product was purified by preparative reversed phased deprotection of the terminal Fmoc group is accomplished 25 HPLC using a Varian (Rainin) preparative binary HPLC using 20% piperidine (Step 2) followed by the coupling of system using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm 3-MPA (Step 3). Resin cleavage and product isolation was column equipped with a Dynamax Cls, 60 A, 8 um guard performed using 86% TFA/5% TIS/5% HO/2% thioanisole module, 21 mmx25 cm column and UV detector (Varian and 2% phenol, followed by precipitation by dry-ice cold Dynamax UVD II) at 214 and 254 nm. The product had EtO (Step 4). The product was purified by preparative 30 >95% purity as determined by RP-HPLC mass spectrometry reversed phased HPLC using a Varian (Rainin) preparative using a Hewlett Packard LCMS-1100 series spectrometer binary HPLC system using a Dynamax Cls, 60 A, 8 um, 21 equipped with a diode array detector and using electro-spray mmx25 cm column equipped with a Dynamax Cls, 60 A. ionization. 8 um guard module, 21 mmx25 cm column and UV detector 3. Modification at an Internal Amino Acid (Varian Dynamax UVD II) at 214 and 254 nm. The 35 product had >95% purity as determined by RP-HPLC mass Example 55 spectrometry using a Hewlett Packard LCMS-1100 series spectrometer equipped with a diode array detector and using Synthesis of Lys’(e-MPA)GLP-1 (7-36)-NH, electro-spray ionization. 40 Example 53 Solid phase peptide synthesis of a modified GLP-1 analog on a 100 Limole scale was performed manually and on a Modification of Kringle-5 at the N-Terminus Symphony Peptide Synthesizer using Fmoc protected Rink Proline amide MBHA resin. The following protected amino acids 45 are sequentially added to the resin: Fmoc-Arg(Pbf)-OH, Preparation of (MPA-AEEA)-Pro-Arg-Lys-Leu-Tyr Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc Asp-NH2TFA Leu-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile OH, Fmoc-Phe-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys Using automated peptide synthesis, the following pro (Aloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)- tected amino acids were sequentially added to Rink Amride 50 OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Leu-OH, MBHA resin: Fmoc-Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Tyr(tEu)-QH., Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)- Fmoc-Tyr(tEu)OH., Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, OH, Fmoc-Val-OH, Fmoc-Asp(tEu)-OH, Fmoc-Ser(tBu)- Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH (step 1). The deprotec OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)- tion of the terminal Fmoc group is accomplished using 20% OH, Fmoc-Gly-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, piperidine (Step 2) followed by the coupling of Fmoc 55 Boc-His(Trt)-OH. They are dissolved in N,N-dimethylfor AEEA. Deprotection of the resulting Fmoc-AEEA-peptide mamide (DMF) and, according to the sequence, activated with piperidine 20% in DMF allow for the subsequent using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium addition of the 3-MPA (Step 3). Resin cleavage and product hexafluorophosphate (HBTU) and Diisopropylethylamine isolation was performed using 86% TFA/5% TIS/5% HO/ (DIEA). Removal of the Fmoc protecting group is achieved 2% thioanisole and 2% phenol, followed by precipitation by 60 using a solution of 20% (V/V) piperidine in N,N-dimethyl dry-ice cold EtO (Step 4). The product was purified by formamide (DMF) for 20 minutes (Step 1). Selective depro preparative reversed phased HPLC using a Varian (Rainin) tection of the LyS(Aloc) group is performed manually and preparative binary HPLC system using a Dynamax Cs, 60 accomplished by treating the resin with a solution of 3 eq of A, 8 um, 21 mmx25 cm column equipped with a Dynamax Pd(PPh.) dissolved in 5 mL of CHCl:NMM:HOAc (18: Cls, 60 A, 8 um guard module, 21 mmx25 cm column and 65 1:0.5) for 2h (Step 2). The resin is then washed with CHCl, UV detector (Varian Dynamax UVD II) at 214 and 254 (6x5mL), 20% HOAc in DCM (6x5mL), DCM (6x5mL), . and DMF (6x5mL). The synthesis is then re-automated for US 7,256.253 B2 103 104 the addition of the 3-maleimidopropionic acid (Step 3). Linking from an internal amino acid in the natural sequence Resin cleavage and product isolation is performed using as in Example 5. The free Cysteine residue must be capped 86%TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, or replaced with another amino acid (e.g. Alanine, Methion followed by precipitation by dry-ice cold EtO (Step 4). The ine, etc.). product is purified by preparative reversed phase HPLC Where the peptide contains one cysteine, the cysteine using a Varian (Rainin) preparative binary HPLC system must stay capped after addition of the maleimide. If the using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column cysteine is involved in binding site, assessment has to be equipped with a Dynamax Cls, 60 A, 8 um guard module, made of how much potency is lost is cysteine is capped by 21 mmx25 cm column and UV detector (Varian Dynamax a protecting group. If the cysteine can stay capped, then the UVD II) at 214 and 254 nm to afford the desired DAC in 10 synthetic path is similar to example (i) above. Examples of >95% purity, as determined by RP-HPLC. These steps are therapeutic peptides that contain one cystein include G, (the illustrated in the schematic diagram below. alpha subunit of Githerapeutic peptide binding protein), the 724-739 fragment of rat brain nitric oxide synthase blocking peptide, the alpha subunit 1-32 fragment of human Tyro Fmoc-Rink Amide MBHA Resin 15 inhibin, the 254-274 fragment of HIV envelope protein, and P34.cdc2 kinase fragment. Step 1 es 1. Modification at the N-Terminus Boc-HAEGTFTSDVSSYLEGQAA-Lys(ALOC)-EFLAWLVKGR-PS Example 56 Step 2 pursuitocic, Modification of Inhibin Peptide at the Added N-Terminus Lysine Boc-HAEGTFTSDVSSYLEGQAA-Lys-EFIAWLVKGR-PS Preparation of (Ne-MPA)-Lys-Tyr-Ser-Thr-Pro Step 3 3-maleimidopropionic acid 25 Leu-Met-Ser-Trp-Pro-Trp-Ser-Pro-Ser-Ala-Leu-Arg Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu-Pro-Ala-Ala Ala-His-Ala-Asn-Cys-His-Arg

HN -N- N O Solid phase peptide synthesis of a modified inhibin pep A 30 tide analog on a 100 umole Scale is performed using manual O solid-phase synthesis, a Symphony Peptide Synthesizer and Fmoc protected Rink Amide MBHA. The following pro tected amino acids are sequentially added to resin: Fmoc Arg(Pbf)-OH, Fmoc-His(Boc)-OH, Fmoc-Cys(Trt)-OH, Boc-HAEGTFTSDVSSYLEGQAA EFIAWLWKGRPS N 35 Fmoc-ASn(Trt)-OH, Fmoc-Ala-OH, Fmoc-His(Boc)-OH, H Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Pro-OH, Fmoc-Glu (thBu)-OH, Fmoc-GluctBu)-OH, Fmoc-Pro-OH, Fmoc-Pro OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu OH, Fmoc-Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol 40 Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc Ser(tBu)-OH, Fmoc-Trp.(Boc)-OH, Fmoc-Pro-OH, Fmoc O O Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Met-OH, Fmoc Leu-OH, Fmoc-Pro-OH, Fmoc-Thr(tEu)-OH, Fmoc-Ser HN ---> N (tPu)-OH, Fmoc-Tyr?tBu)-OH, Fmoc-Lys(Aloc)-OH.They 45 are dissolved in N,N-dimethylformamide (DMF) and, O according to the sequence, activated using O-benzotriazol 1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal of HN-HAEGTFTSDVSSYLEGQAA EFLAWLVKGR-NH the Fmoc protecting group is achieved using a solution of N H 50 20% (V/V) piperidine in N,N-dimethylformamide (DMF) O for 20 minutes (Step 1).The selective deprotection of the Lys Lys26 (E-MPA)GLP-1 (7-36)-NH (Aloc) group is performed manually and accomplished by treating the resin with a solution of 3 eq of Pd(PPh.) dissolved in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h 55 (Step 2). The resin is then washed with CHCl (6x5 mL), D. Preparation of Modified Peptides from Peptides Contain 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF ing One Free Cysteine (6x5 mL). The synthesis is then re-automated for the addi Preparation of maleimido peptides from therapeutic pep tion of the 3-maleimidopropionic acid (Step 3). Between tides containing one free Cysteine is exemplified by the every coupling, the resin is washed 3 times with N.N- synthesis of peptides as described below. The peptide may 60 dimethylformamide (DMF) and 3 times with isopropanol. be modified at the N-terminus, the C-terminus, or at an The peptide is cleaved from the resin using 85% TFA/5% amino acid located between the N-terminus and the C-ter TIS/5% thioanisole and 5% phenol, followed by precipita minus. tion by dry-ice cold EtO (Step 4). The product is purified by Preparation of maleimido peptides from peptides contain preparative reverse phase HPLC using a Varian (Rainin) ing multiple protected functional groups and multiple Cys 65 preparative binary HPLC system: gradient elution of teine residues all with one free Cysteine residues (i.e. all 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Cysteine residues, except one, are tied up as disulfides). CHCN (B)) over 180 min at 9.5 mL/min using a Phenom US 7,256.253 B2 105 106 enex Luna 10L phenyl-hexyl, 21 mmx25 cm column and UV amino acids, O-benzotriazol-1-yl-N,N,N',N'-tetramethyl detector (Varian Dynamax UVD II) at 214 and 254 nm to uronium hexafluorophosphate (HBTU) in N,N-dimethylfor afford the desired DAC in >95% purity, as determined by mamide (DMF) solution and activation with N-methyl mor RP-HPLC. pholine (NMM), and piperidine deprotection of Fmoc groups (Step 1). The deprotection of the terminal Fmoc Example 57 group is accomplished using 20% piperidine (Step 2) fol lowed by the coupling of 3-MPA (Step 3). Resin cleavage Modification of RSV Antifusogenic Peptide at the and product isolation was performed using 85% TFA/5% N-Terminus TIS/5% thioanisole and 5% phenol, followed by precipita 10 Preparation of 3-(MPA)-Val-Ile-Thr-Ile-Glu-Leu tion by dry-ice cold EtO (Step 4). The product is purified by Ser-Asn-Ile-Lys-Glu-Asn-Lys-Cys-Asn-Glu-Ala preparative reverse phase HPLC using a Varian (Rainin) Lys-Val-Lys-Leu-Ile-Lys-Glu-Glu-Leu-Asp-Lys-Tyr preparative binary HPLC system: gradient elution of Lys-Asn-Ala-Val 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in 15 CHCN (B)) over 180 min at 9.5 mL/min using a Phenom Initially, (Cysteine (Cys) was replaced with enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV (Met) within the native sequence. Solid phase peptide syn detector (Varian Dynamax UVD II) at w 214 and 254 nm to thesis of a modified anti RSV analog on a 100 mole scale afford the desired DAC in >95% purity, as determined by was performed on a Symphony Peptide Synthesizer using RP-HPLC. These steps are illustrated by the schematic Fmoc protected Rink Amide MBHA resin, Fmoc protected diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 es

Fmoc-VITIELSNIKENKMNGAKVKLIKQELDKYKNAV-PS Step 2 piperidine

HN-VITIELSNIKENKMNGAKVKLIKQELDKYKNAVK-PS

Step 3 3-maleimidopropionic acid

Na 1a - n VITIELSNIKENKMNGAKVKLIKQELDKYKNAV-PS O

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

TFA TFA TFA TFA Na 1 - n VITIELSNIKENKMNGAKVKLIKQELDKYKNAV-NH O O TFA TFA TFA US 7,256.253 B2 107 108 2. Modification at the C-Terminus Example 59 Example 58 Modification of RSV Fusogenic Peptide at the C-Terminus Modification of Inhibin Peptide at the Added Val-Ile-Thr-Ile-Glu-Leu-Ser-Asn-Ile-Lys-Glu-Asn C-Terminus Lysine Lys-Cys-Asn-Gly-Ala-Lys-Val-Lys-Leu-Ile-Lys-Gln Glu-Leu-Asp-Lys-Tyr-Asn-Ala-Val-(AEEA.MPA) Preparation of (Ne-MPA)-Lys-Cys-Asn-Leu-Lys Glu-Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val-Lys 10 Preparation of maleimido peptides from peptides contain ing multiple protected functional groups and one cysteine is exemplified by the synthesis of a modified RSV fusogenic Solid phase peptide synthesis of a modified inhibin pep peptide. The modified RSV fusogenic peptide was synthe tide analog on a 100 umole Scale is performed using manual sized by linking off the C-terminus by the addition of a solid-phase synthesis, a Symphony Peptide Synthesizer and 15 lysine residue to the natural peptide sequence as illustrated Fmoc protected Rink Amide MBHA. The following pro by the schematic diagram below. In cases where a cysteine tected amino acids are sequentially added to resin: Fmoc residue is contained within the peptide sequence and is not Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Asp(tEu)-OH, Fmoc essentially to the biological activity of the peptide, this Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Ser residue must be replaced with another amino acid (e.g. (tBu)-OH, Fmoc-Ile-OH, Fmoc-Gly-OH, Fmoc-Asp(tEu)- alanine, methionine, etc.). In the following synthesis, the OH, Fmoc-GluctBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu cysteine (Cys) was replaced with a methionine (Met) within OH, Fmoc-Asn(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys the native RSV sequence. (Aloc)-OH, They are dissolved in N,N-dimethylformamide Solid phase peptide synthesis of the maleimido RSV (DMF) and, according to the sequence, activated using fusogenic peptide on a 100 umole scale is performed using O-benzotriazol-1-yl-N,N,N',N'-tetramethyl-uronium 25 manual solid-phase synthesis and a Symphony Peptide Syn thesizer using Fmoc protected Rink Amide MBHA resin, hexafluorophosphate (HBTU) and Diisopropylethylamine Fmoc protected amino acids, O-benzotriazol-1-yl-N,N,N', (DIEA). Removal of the Fmoc protecting group is achieved N-tetramethyl-uronium hexafluorophosphate (HBTU) in using a solution of 20% (VN) piperidine in N,N-dimethyl N,N-dimethylformamide (DMF) solution and activation formamide (DMF) for 20 minutes (Step 1).The selective 30 with N-methyl morpholine (NMM), and piperidine depro deprotection of the Lys (Aloc) group is performed manually tection of Fmoc groups (Step 1). The selective deprotection and accomplished by treating the resin with a solution of 3 of the Lys(Aloc) group is performed manually and accom eq of Pd(PPh.) dissolved in 5 mL of CHCl:NMM:HOAc plished by treating the resin with a solution of 3 eq of (18:1:0.5) for2 h (Step 2). The resin is then washed with Pd(PPh), dissolved in 5 mL of CHCl:NMM:HOAc (18:1: CHCl, (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 35 0.5) for 2 h (Step 2). The resin is then washed with CHC1, mL), and DMF (6x5 mL). The synthesis is then re-auto (6x5 mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), mated for the addition of the 3-maleimidopropionic acid and DMF (6x5 mL). The synthesis is then re-automated for (Step 3). Between every coupling, the resin is washed 3 the addition of 3-maleimidopropionic acid (Step 3). Resin times with N.N-dimethylformamide (DMF) and 3 times cleavage and product isolation is performed using 85% with isopropanol. The peptide is cleaved from the resin 40 TFA/5% TIS/5% thioanisole and 5% phenol, followed by using 85% TFA/5% TIS/5% thioanisole and 5% phenol, precipitation by dry-ice cold EtO (Step 4). The product is followed by precipitation by dry-ice cold EtO (Step 4). The purified by preparative reverse phase HPLC using a Varian product is purified by preparative reverse phase HPLC using (Rainin) preparative binary HPLC system: gradient elution a Varian (Rainin) preparative binary HPLC system: gradient of 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in elution of 30-55% B (0.045% TFA in HO (A) and 0.045% 45 CHCN (B)) over 180 min at 9.5 mL/min using a Phenom TFA in CHCN (B)) over 180 min at 9.5 mL/min using a enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV Phenomenex Luna 10L phenyl-hexyl, 21 mmx25 cm column detector (Varian Dynamax UVD II) at w 214 and 254 nm to and UV detector (Varian Dynamax UVD II) at 214 and 254 afford the desired DAC in >95% purity, as determined by nm to afford the desired DAC in >95% purity, as determined RP-HPLC. These steps are illustrated in the schematic by RP-HPLC. diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 es Boc-VITIELSNIKENKMNGAKVKLIKQELDKYKNAVK(Aloc)-PS Step 2 protocic, Boc-VITIELSNIKENKMNGAKVKLIKQELDKYKNAVK-PS

Step 3 | 3-maleimidopropionic acid US 7,256.253 B2 109 110

-continued

Boc-VITIELSNIKENKMNGAKVKLIKQELDKYKNAVS N H

so 85% TFA/5% TIS/5% thioanisole/5% phenol

HN --> N O /

TFA TFA TFA TFA TFA HN- VITIELSNIKENKMNGAKVKLIKQELDKYKNAVS N TFA TFA TFA H

3. Modification at an Internal Amino Acid tion by dry-ice cold EtO (Step 4). The product is purified by preparative reversed phased HPLC using a Varian (Rainin) Example 60 35 preparative binary HPLC system: gradient elution of 30-55% B (0.045% TFA in HO (A) and 0.045% TFA in Modification of Go. Peptide at Cys-Asn-Leu-Lys CHCN (B)) over 180 min at 9.5 mL/min using a Phenom Glu-Asp-Gly-Ile-Ser-Ala-Ala-Lys-Asp-Val enex Luna 10u phenyl-hexyl, 21 mmx25 cm column and UV 40 detector (Varian Dynamax UVD II) at w 214 and 254 nm to Preparation of maleimido peptides from peptides contain afford the desired DAC in >95% purity, as determined by ing multiple protected functional groups and one cysteine is RP-HPLC. exemplified by the synthesis of a modified GO. peptide. The modified GO. peptide is synthesized by linking at an internal E. Preparation of Modified Peptides from Peptides Contain amino acid as described below. 45 ing Two Cysteines in Disulfide Bridge In cases where a cysteine residue is contained within the Where the peptide contains two cysteines as a disulfide peptide sequence and is not essentially to the biological bridge, the peptide is cleaved from the support resin before activity of the peptide, this residue must be capped or addition of the maleimide. We need to add a Lys protected replaced with another amino acid (e.g. alanine, methionine, with a Mitt group in order to selectively deprotect the lysine etc.). Solid phase peptide synthesis of the modified GO. 50 in presence of other t-Boc protected lysine. All protecting peptide on a 100 umole scale is performed using manual groups are present except at the carboxy terminus (which solid-phase synthesis and a Symphony Peptide Synthesizer stays unprotected due to cleavage from the Support resin) using Fmoc protected Rink Amide MBHA resin, Fmoc and at the two cysteines, which need to be deprotected when protected amino acids, O-benzotriazol-1-yl-N,N,N',N'-tet peptide is cleaved from resin. Mild air oxidation yield the ramethyl-uronium hexafluorophosphate (HBTU) in N.N- 55 disulfide bridge, and the peptide can be purified at that stage. dimethylformamide (DMF) solution and activation with Solution phase chemistry is then required to activate the N-methyl morpholine (NMM), and piperidine deprotection C-terminus in presence of the disulfide bridge and add the of Fmoc groups (Step 1). The selective deprotection of the maleimide (through an amino-alkyl-maleimide) to the C-ter Lys(Aloc) group is performed manually and accomplished minus. The peptide is then fully deprotected. Examples of by treating the resin with a solution of 3 eq of Pd(PPh.) 60 therapeutic peptides that contain two cysteins as a disulfide dissolved in 5 mL of CHC1:NMM:HOAc (18:1:0.5) for 2 h bridge include human osteocalcin 1-49, human diabetes (Step 2). The resin is then washed with CHCl (6x5 mL), associated peptide, the 5-28 fragment of human/canine atrial 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF natriuretic peptide, bovine bactenecin, and human Tyro (6x5 mL). The synthesis is then re-automated for the addi cortistatin 29. tion of 3-maleimidopropionic acid (Step 3). Resin cleavage 65 Preparation of maleimido peptides from therapeutic pep and product isolation is performed using 85% TFA/5% tides containing two Cysteines in a disulfide bridge is TIS/5% thioanisole and 5% phenol, followed by precipita exemplified by the synthesis of peptides as described below. US 7,256.253 B2 111 112 The peptide may be modified at the N-terminus, the C-ter using a Varian (Rainin) preparative binary HPLC system minus, or at an amino acid located between the N-terminus using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column and the C-terminus. equipped with a Dynamax Cls, 60 A, 8 um guard module, 1. Modification at the N-Terminus 21 mmx25 cm column and UV detector (Varian Dynamax UVD II) at 214 and 254 nm. The product had >95% purity Example 61 as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series spectrometer equipped Modification of TH-1 Peptide at N-Terminus with a diode array detector and using electro-spray ioniza tion, ESI-MS m/z for CHNOS (MH"), 1646.8. Preparation of (Ne-MPA)NH-Lys-Arg-Gly-Asp Found: 1646.7. These steps are illustrated in the schematic Ala-Cys-Glu-Gly-Asp-Ser-Gly-Gly-Pro-Phe-Cys diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 |rs

Step 2

SS

Step 3 |1.20% 3-maleimidopropionic piperidine acid

S-S

so 86% TFA/5% TIS/5% H2O/2% thioanisole/2% phenol

H TFA S-S N N- Nin KRGDACEGDSGGPFC-NH O 0 O TFA

45 Preparation of thiol cyclized maleimido peptides from Example 62 peptides containing multiple protected functional groups and no free cysteine residues (i.e. all cysteine residues are Synthesis of N-MPA-Ser'-Somatostatin-28 tied up as disulfide bridges). is illustrated by the synthesis of a modified TH-1 peptide. 50 Solid phase peptide synthesis of the DAC:Somatostatin Solid phase peptide synthesis of the modified TH-1 pep 28 analog on a 100 umole scale is performed manually and tide on a 100 umole scale was performed manually and on on a Symphony Peptide Synthesizer using Fmoc protected a Symphony Peptide Synthesizer using Fmoc protected Rink Rink amide MBHA resin. The following protected amino Amide MBHA resin, Fmoc protected amino acids, O-ben acids are sequentially added to the resin: Fmoc-Cys(Acm)- Zotriazol-1-yl-N,N,N',N'-tetramethyl-uronium hexafluoro 55 phosphate (HBTU) in N,N-dimethylformamide (DMF) OH, Fmoc-Ser(tPu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe solution and activation with N-methyl morpholine (NMM), OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp and piperidine deprotection of Fmoc groups (Step 1). The (Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- removal of the Acm group and resulting oxidation of the two OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly Cys residues to form the cyclized on resin DAC was 60 OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)- accomplished using TI(CFCO), (Step 2). The deprotection OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro of the terminal Fmoc group is accomplished using 20% OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc piperidine followed by the coupling of 3-MPA (Step 3). Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc Resin cleavage and product isolation was performed using ASn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH. They are 86%TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, 65 dissolved in N,N-dimethylformamide (DMF) and, according followed by precipitation by dry-ice cold EtO (Step 4). The to the sequence, activated using O-benzotriazol-1-yl-N,N. product was purified by preparative reverse phase HPLC N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) US 7,256.253 B2 113 114 and Diisopropylethylamine (DIEA). Removal of the Fmoc Fmoc-Thr(tEu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp(Boc)- protecting group is achieved using a solution of 20% (V/V) OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-ASn(Trt)-OH, piperidine in N,N-dimethylformamide (DMF) for 20 min Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly-OH, utes (Step 1). Removal of the Acm groups and resulting Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, oxidation of the two Cys residues to form the disulfide Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, bridge is accomplished using iodine (Step 2). Deprotection Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc-Pro of the terminal Fmoc group is accomplished using 20% OH, Fmoc-ASn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn piperidine followed by the coupling of 3-MPA (Step 3). (Trt)-OH, Fmoc-Ala-OH, Boc-Ser(tBu)-OH. They are dis Resin cleavage and product isolation is performed using solved in N,N-dimethylformamide (DMF) and, according to 86%TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, 10 the sequence, activated using O-benzotriazol-1-yl-N,N,N', followed by precipitation by dry-ice cold EtO (Step 4). The N-tetramethyl-uronium hexafluorophosphate (HBTU) and product is purified by preparative reversed phase HPLC Diisopropylethylamine (DIEA). Removal of the Fmoc pro using a Varian (Rainin) preparative binary HPLC system tecting group is achieved using a solution of 20% (V/V) using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm column piperidine in N,N-dimethylformamide (DMF) for 20 min equipped with a Dynamax Cls, 60 A, 8 um guard module, 15 utes (Step 1). The fully protected peptide is cleaved from the 21 mmx25 cm column and UV detector (Varian Dynamax resin by treatment with 1%TFA/DCM (Step 2). Removal of UVD II) at 214 and 254 nm to afford the desired DAC in the Acm groups and resulting oxidation of the two Cys >95% purity, as determined by RP-HPLC. These steps are residues to form the disulfide bridge is accomplished using illustrated in the schematic diagram below. iodine (Step 3). Ethylenediamine and 3-maleimidopropionic

Fmoc-Rink Amide MBHA Resin Step 1 |rs Fmoc-SANSNPAMAPRERKAGC(Acm)KNFFWKTFTSC(Acm)- Resin

Step 2

S-S

Fmoc-SANSNPAMAPRERKAGCKNFFWKTFTSC-Resin

Step 3 |1.20% 3-maleimidopropionic piperidine acid

O

H S S N N- Nn SANSNPAMAPRERKAGCKNFFWKTFTSC-Resin O O

Step 4 85% TFA/5% TIS/5% thioanisole/5% phenol

O

H S S N N- NS SANSNPAMAPRERKAGCKNFFWKTFTSC-OH O O N-MPA-Ser-Somatostatin-28

2. Modification at the C-Terminus 55 acid are then sequentially added to the free C-terminus (Step 4). The protecting groups are then cleaved and the product Example 63 isolated using 86% TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold Synthesis of Somatostatin-28-EDA-MPA EtO (Step 5). The product is purified by preparative 60 reversed phase HPLC using a Varian (Rainin) preparative Solid phase peptide synthesis of the DAC:Somatostatin binary HPLC system using a Dynamax Cls, 60 A, 8 um, 21 28 analog on a 100 umole scale is performed manually and mmx25 cm column equipped with a Dynamax Cls, 60A, 8 on a Symphony Peptide Synthesizer using SASRIN (super um guard module, 21 mmx25 cm column and UV detector acid sensitive resin). The following protected amino acids 65 (Varian Dynamax UVD II) at 214 and 254 nm to afford the are sequentially added to the resin: Fmoc-Cys(Acm)-OH, desired DAC in >95% purity, as determined by RP-HPLC. Fmoc-Ser(thBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, These steps are illustrated in the schematic diagram below. US 7,256.253 B2 115 116

SASRIN Resin Step 1 |rs

Boc-SANSNPAMAPRERKAGC(Acm)KNFFWKTFTSC(Acm)- Resin Step 2 ". TFADCM

Fmoc-SANSNPAMAPRERKAGC(Acm)KNFFWKTFTSC(Acm)-OH Step 3 |

S-S

Boc-SANSNPAMAPRERKAGCKNFFWKTFTSC-OH

Step 4 |2.3-maleimidopropionic ethylendiamine acid

O S — N Boc-SANSNPAMAPRERKAGCKNFFWKTFTSC-O n 1N1 N r N O O

Step 5 85% TFA/5% TIS/5% thioanisole/5% phenol

O S-S HN-SANSNPAMAPRERKAGCKNFFWKTFTSC-O NN 1N1 N CN O O Somatostatin-28-EDA-MPA

3. Modification at an Internal Amino Acid protecting group is achieved using a solution of 20% (VN) 45 piperidine in N,N-dimethylformamide (DMF) for 20 min Example 64 utes (Step 1). Removal of the Acm groups and resulting oxidation of the two Cys residues to form the disulfide Synthesis of Lys'(e-MPA)-Somatostatin-28 bridge is accomplished using iodine (Step 2). Selective deprotection of the Lys(Aloc) group is performed manually Solid phase peptide synthesis of the DAC:Somatostatin 50 and accomplished by treating the resin with a solution of 3 28 analog on a 100 umole scale is performed manually and eq of Pd(PPh.) dissolved in 5 mL of CHC1:NMM.HOAc on a Symphony Peptide Synthesizer using Fmoc protected (18:1:0.5) for 2 h (Step 3). The resin is then washed with Rink amide MBHA resin. The following protected amino CHCl, (6x5mL), 20% HOAc in DCM (6x5mL), DCM acids are sequentially added to the resin: Fmoc-Cys(Acm)- (6x5mL), and DMF (6x5mL). The synthesis is then re OH, Fmoc-Ser(tPu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe 55 OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Trp automated for the addition of the 3-maleimidopropionic acid (Boc)-OH, Fmoc-Phe-OH, Fmoc-Phe-OH, Fmoc-Asn(Trt)- (Step 4). Resin cleavage and product isolation is performed OH, Fmoc-Lys(Boc)-OH, Fmoc-Cys(Acm)-OH, Fmoc-Gly using 86% TFA/5% TIS/5% HO/2% thioanisole and 2% OH, Fmoc-Ala-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)- phenol, followed by precipitation by dry-ice cold EtO (Step OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro 60 5). The product is purified by preparative reversed phase OH, Fmoc-Ala-OH, Fmoc-Met-OH, Fmoc-Ala-OH, Fmoc HPLC using a Varian (Rainin) preparative binary HPLC Pro-OH, Fmoc-ASn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc system using a Dynamax Cls, 60 A, 8 um, 21 mmx25 cm ASn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH. They are column equipped with a Dynamax Cls, 60 A, 8 um guard dissolved in N,N-dimethylformamide (DMF) and, according module, 21 mmx25 cm column and UV detector (Varian to the sequence, activated using O-benzotriazol-1-yl-N,N. 65 Dynamax UVD II) at 214 and 254 nm to afford the desired N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) DAC in >95% purity, as determined by RP-HPLC. These and Diisopropylethylamine (DIEA). Removal of the Fmoc steps are illustrated in the following schematic diagram. US 7,256.253 B2 117 118

Fmoc-Rink Amide MBHA Resin Step 1 |rs Boc-SANSNPAMAPRER-Lys(Aloc)AGC(Acm)KNFFWKTFTSC(Acm)-Resin Step 2 .

S-S Boc-SANSNPAMAPRER-Lys(Aloc)AGCKNFFWKTFTSC-Resin

Step 3 parlinMM/HOAc/CHCI S-S Boc-SANSNPAMAPRERK-Lys-AGCKNFFWKTFTSC-OH

Step 4 3-maleimidopropionic acid

Boc-SANSNPAMAPRER AGCKNFFWKTFTSC-Resin a N O Step 5 85% TFA/5% TIS/5% thioanisole/5% phenol

AGCKNFFWKTFTSC-OH HN-SANSNPAMAPRER a N H O Lys'(E-MPA)-Somatostatin-28

F. Preparation of Modified Peptides from Peptides Contain Leu-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Acm)-OH, Fmoc ing Multiple Cysteines 55 Phe-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH, Fmoc-Cys 1. Modification at the N-Terminus (thBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Lys(Boc)-OH, Example 65 Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(tEu)- Synthesis of N-MPA-Cys-Endothelin-1 (1-21)-OH 60 OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Acm)-OH. They are dissolved in N,N-dimethylformamide (DMF) and, according Solid phase peptide synthesis of a modified Endothelin-1 to the sequence, activated using O-benzotriazol-1-yl-N,N. analog on a 100 umole Scale is performed manually and on N',N'-tetramethyl-uronium hexafluorophosphate (HBTU) a Symphony Peptide Synthesizer using Fmoc protected Rink and Diisopropylethylamine (DIEA). Removal of the Fmoc Amide MBHA resin. The following protected amino acids 65 protecting groups is achieved using a solution of 20% (VN) are sequentially added to the resin: Fmoc-Trp(Boc)-OH, piperidine in N,N-dimethylformamide (DMF) for 20 min Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc-Asp(OtBu)-OH, Fmoc utes (Step 1). The removal of the Acm groups and resulting US 7,256.253 B2 119 120 oxidation of the first two Cys residues to form the first preparative reverse phase HPLC using a Varian (Rainin) disulfide bridge on resin is accomplished using iodine (Step preparative binary HPLC system using a Dynamax Cs, 60 2). The removal of the tBu groups and resulting oxidation of A, 8 um, 21 mmx25 cm column equipped with a Dynamax the other two Cys residues to form the second disulfide Cls, 60 A, 8 um guard module, 21 mmx25 cm column and bridge on resin is accomplished using thallium (III) trifluo UV detector (Varian Dynamax UVD II) at 214 and 254 nm roacetate (Step 3). The deprotection of the terminal Fmoc to afford the desired DAC in >95% purity, as determined by group is accomplished using 20% piperidine followed by the RP-HPLC. These steps are illustrated in the schematic coupling of 3-MPA (Step 4). Resin cleavage and product diagram above. isolation is performed using 86% TFA/5% TIS/5% HO/2% 10 2. Modification at the C-Terminus thioanisole and 2% phenol, followed by precipitation by dry-ice cold EtO (Step 5). The product is purified by Example 66 Synthesis of Endothelin-1 Fmoc-Rink Amide MBHA Resin 15 (1-21)Lys’-(Ne-MPA)-OH

Step 1 |rs Solid phase peptide synthesis of a modified Endothelin-1 analog on a 100 umole Scale is performed manually and on a Symphony Peptide Synthesizer using Fmoc protected Rink Amide MBHA resin. The following protected amino acids are sequentially added to the resin: Fmoc-Lys(Aloc)-OH, Step 2 |: Fmoc-Trp(Boc)-OH, Fmoc-Ile-OH, Fmoc-Ile-OH, Fmoc 25 Asp(OtBu)-OH, Fmoc-Leu-OH, Fmoc-His(Trt)-OH, Fmoc Cys(Acm)-OH, Fmoc-Phe-OH, Fmoc-Tyr?tBu)-OH, Fmoc S-S Val-OH, Fmoc-Cys(tEu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc Lys(Boc)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Met-OH, Fmoc-CSC(tBu)SSLMDKEC(tBu)VYFCHLDIIW-Resin 30 Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(tEu)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Acm)- Step 3 |alin (III) trifluoracetate OH. They are dissolved in N,N-dimethylformamide (DMF) and, according to the sequence, activated using O-benzot 35 riazol-1-yl-N, N,N',N'-tetramethyl-uronium hexafluoro phosphate (HBTU) and Diisopropylethylamine (DIEA). S-S Removal of the Fmoc protecting group is achieved using a Fmoc-CSCSSLMDKECWYFCHLDIW-Resin solution of 20% (VN) piperidine in N,N-dimethylformamide S-S (DMF) for 20 minutes (Step 1). The removal of the Acm 40 groups and resulting oxidation of the first two Cys residues 1.20% piperidine to form the first disulfide bridge on resin is accomplished Step 4 | 3-maleimidopropionic acid using iodine (Step 2). The removal of the tRu groups and resulting oxidation of the other two Cys residues to form the 45 second disulfide bridge on resin is accomplished using thallium (III) trifluoroacetate (Step 3). Selective deprotec O tion of the Lys(Aloc) group is performed manually and 7 s-s accomplished by treating the resin with a solution of 3 eq of N NN 50 Pd(PPh3)4 dissolved in 5 mL of CHC1:NMM:HOAc (18: O N- csstuntwich.Diw -Resin 1:0.5) for 2 h (Step 4). The resin is then washed with CHC13 O S-S (6x5mL), 20% HOAc in DCM (6x5 mL), DCM (6x5 mL), and DMF (6x5 mL). The synthesis is then re-automated for 859%TFA59%, TIS.5% the addition of the 3-maleimidopropionic acid (Step 5). Step 5 Rs. phenol 55 Resin cleavage and product isolation is performed using 86% TFA/5% TIS/5% HO/2% thioanisole and 2% phenol, followed by precipitation by dry-ice cold EtO (Step 5). The O product is purified by preparative reverse phase HPLC using / S-S 60 a Varian (Rainin) preparative binary HPLC system using a N NN Dynamax Cls, 60A, 8 um, 21 mmx25 cm column equipped with a Dynamax Cls, 60A, 8 Lim guard module, 21 mmx25 O N- csstuntwich.Diw-on O S-S cm column and UV detector (Varian Dynamax UVD II) at N-MPA-Cys-Endothelin-1 (1-21)-OH 65 214 and 254 nm to afford the desired DAC in >95% purity, as determined by RP-HPLC. These steps are illustrated in the schematic diagram below.

US 7,256.253 B2 123 124 3. Modification at an Internal Amino Acid resulting oxidation of the first two Cys residues to form the first disulfide bridge on resin is accomplished using iodine Example 67 (Step 2). The removal of the tRu groups and resulting oxidation of the other two Cys residues to form the second Synthesis of Lys'(Ne-MPA)Sarafotoxin disulfide bridge on resin is accomplished using thallium (III) B(1-21)-OH trifluoroacetate (Step 3). Selective deprotection of the Lys Solid phase peptide synthesis of a modified Sarafotoxin-B (Aloc) group is performed manually and accomplished by analog on a 100 umole Scale is performed manually and on treating the resin with a solution of 3 eq of Pd(PPh3)4 a Symphony Peptide Synthesizer using Fmoc protected Rink 10 dissolved in 5 mL of CHCl:NMM:HOAc (18:1:0.5) for 2 h Amide MBHA resin. The following protected amino acids (Step 4). The resin is then washed with CHC13 (6x5mL). are sequentially added to the resin: Fmoc-Trp(Boc)-OH, 20% HOAc in DCM (6x5mL), DCM (6x5mL), and DMF Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Asp(OtBu)-OH, Fmoc (6x5mL). The synthesis is then re-automated for the addition Gln(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Acm)-OH, of the 3-maleimidopropionic acid (Step 5). Resin cleavage Cys(tEu)-OH, Fmoc-GlucCtBu)-OH, Fmoc-Lys(Boc)-OH, and product isolation is performed using 86% TFA/5% Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Met-OH, TIS/5% HO/2% thioanisole and 2% phenol, followed by Fmoc-Asp(OtBu)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Cys precipitation by dry-ice cold EtO (Step 5). The product is (thBu)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Acm)-OH. They purified by preparative reverse phase HPLC using a Varian are dissolved in N,N-dimethylformamide (DMF) and, 20 (Rainin) preparative binary HPLC system using a Dynamax according to the sequence, activated using O-benzotriazol Cls, 60 A, 8 um, 21 mmx25 cm column equipped with a 1-yl-N,N,N',N'-tetramethyl-uronium hexafluorophosphate Dynamax Cls, 60 A, 8 Lim guard module, 21 mmx25 cm (HBTU) and Diisopropylethylamine (DIEA). Removal of column and UV detector (Varian Dynamax UVD II) at 214 the Fmoc protecting group is achieved using a solution of and 254 nm to afford the desired DAC in >95% purity, as 20% (V/V) piperidine in N,N-dimethylformamide (DMF) determined by RP-HPLC. These steps are illustrated in the for 20 minutes (Step 1). The removal of the Acm groups and schematic diagram below.

Fmoc-Rink Amide MBHA Resin Step 1 jers

Step 2

S S Boc-CSC(tBu)-Lys(Aloc)-DMTDKEC(tBu)LYFCHQDVIW-Resin

Step 3 |alin (III) trifluoracetate

S S Boc-CSC-Lys.(Aloc)-DMTDKECLYFCHQDVIW-Resin

S S Step 4 | Pd(PPh3)4/NMM/HOAc/CHCls

S S Boc-CSC-Lys-DMTDKECLYFCHQDVIW-Resin

S S

Step 5 minions acid US 7,256.253 B2 126

-continued

HN --> N O /

Boc-CSC YN DMTDKECLYFCHQDVIW-Resin H O S-S S-S Step 6 s TFA/5% TIS/5% thioanisole/5% phenol

O

HN --> N O

O

HN-SGS DMTDKECLYFCHQDVIW-OH N H O S-S S-S Lys (E-MPA)Sarafotoxin B (1-21)-OH

35 F. Peptide Stability Assays HPLC/MS. The results are presented below. The HPLC parameters for detection of K5 peptide in serum were as Example 68 follows. 40 The HPLC method was as follows: A Vydac C18250x4.6 Peptide Stability Assay of K5 mm, 5LL particle size column was utilized. The column temperature was 30° C. with a flow rate of 0.5 ml/min. A peptide stability assay was performed. (MPA)-Pro-Arg Mobile Phase A was 0.1% TFA/water. Mobile Phase B was Lys-Leu-Tyr-Asp-Lys-NH. 2TFA was synthesized as 0.1% TFA/acetonitrite. The injection volume was 10 ul. described above and was identified MPA-K5. The non 45 The gradient was as follows: modified counterpart peptide Pro-Arg-Lys-Leu-Tyr-Asp-Lys was also synthesized as described in Example 20 without the addition of 3-MPA and identified as K5. K5 (MW1260.18,918.12 freebase) was prepared as a 100 Time (Minutes) % A % B 50 O 95 5 mM stock solution in water. MPA-K5 (MW=1411.17, 2O 70 30 1069.11 freebase) was prepared as a 100 mM stock solution 25 10 90 in water. Human Serum Albumin (HSA) was obtained as a 30 10 90 25% solution (ca 250 mg/ml, 3.75 mM) as Albutein(R) 35 95 5 available from Alpha Therapeutic. Human plasma was 45 95 5 obtained from Golden West Biologicals. 55 (1) Stability of K6 in Human Plasma The proteins were detected at 214, 254 and 334 nm. For K5 was prepared as a 1 uM solution and dissolved in 25% mass spectral analysis, the ionization mode was API-elec human serum albumin. The mixture was then incubated at trospray (positive mode) at an M/Z range of 300 to 2000. 37°C. in the presence of human plasma to final concentra The gain was 3.0, fragmentor 120 V, threshold 20, stepsize tion of 160 mMK5. Aliquots of 100 ul were withdrawn from 60 0.1. The gas temp was 350° C. and the drying gas volume the plasma at 0, 4 hours and 24 hours. The 100 ul aliquots was 10.01/min. The Neb pressure was 24 psi and the Vcap were mixed with 100 ul of blocking solution (5 vol. was 3500 V. The HPLC method was as follows: A Vydac 5%ZnSO/3 vol. Acetonitrile/2 vol. Methanol) in order to C18250x4.6 mm, 5u particle size column was utilized. The precipitate all proteins. The sample was centrifuged for 5 column temperature was 30° C. with a flow rate of 0.5 min at 10,000 g and the Supernatant containing the peptide 65 ml/min. Mobile Phase A was 0.1% TFA/water. Mobile Phase was recovered and filtered through a 0.22 um filter. The B was 0.1% TFA/acetonitrite. The injection volume was 10 presence of free intact K5 peptide was assayed by the ul. US 7,256.253 B2

The gradient was as follows: stability of the three forms of albumin in plasma is presented below.

Time (Minutes) % A % B O 95 5 5 Time % capped HSA % SH-Albumin % KS-MPA-HSA 2O 70 30 0 hrs. 61.3 16.6 22.1 25 10 90 4 hrs. 64.6 16.OS 19.35 30 10 90 24 hrs. 63 16.8 2O2 35 95 5 45 95 5 ' The percentage of K5-MPA-HSA remained relatively constant throughout the 24 hour plasma assay in contrast to The proteins were detected at 214, 254 and 334 nm. For unmodified K5 which decreased to 9% of the original mass spectral analysis, the ionization mode was API-elec amount of K5 in only 4 hours in plasma. The results trospray (positive mode) at an M/Z range of 300 to 2000. demonstrate that in contrast to K5 which is quite unstable in The gain was 3.0, fragmentor 120 V, threshold 20, stepsize 15 plasma, K5-MPA-HSA is quite stable from peptidase activ 0.1. The gas temp was 350° C. and the drying gas volume ity in plasma. was 10.01/min. The Neb pressure was 24 psi and the Vcap was 3500 V. Example 70

Time % K5 peptide in Plasma 2O Peptide Stability- Assay of Dynorphin 0 hrs. 100% In order to determine the stability of peptide conjugates in 4 hrs 9% the presence of serum peptidases the serum stability of Dyn 24 hrs O% A-(1-13)-OH, Dyn A-(1-13)-NH and Dyn A 1-13(MPA)- 25 NH were compared. Dyn A-(1-13)-OH, Dyn A-(1-13)-NH and Dyn A 1-13(MPA)-NH were synthesized as described The results demonstrate that unmodified K5 peptide is above. The Dynorphin peptides were mixed with human unstable in - plasma likely as a result of protease activity. heparinizedeparin1Zed plplasma tto a finalIlnal concentrauontrati OIf 4 mg/mLmg/mL. (2) Stability of MPA-K5-HSA Conjugate in Plasma After the required incubation time at 37° C., 0, 20, 20, 60, MPA-K5 (modified K5 peptide) was incubated with 25% to 120, 180,360 and 480 minutes) a 100 uL-aliquot was added HSA for 2 hours at room temperature. The MPA-K5-HSA to 100 uL of blocking solution (5 vol. of a 5% aqueous conjugate was then incubated at 37 in the presence of ZnSO solution, 3 vol. of acetonitrile, 2 vol. of methanol) human plasma at a final concentration of 160 lm. After the that precipitates all proteins. After centrifugation (10,000 g specific incubation period (0, 4 and 24 hours) an aliquot of for 2.5 min), clear supernatant was recovered, filtered 100 ul was withdrawn and filtered through a 0.22 um filter. through a 0.45 um filter and stored on ice until LC/MS The presence of intact conjugate was assayed by HPLC-MS. 35 analysis. The column was an Aquapore RP-300, 250x4.6 mm, 7L The samples were analyzed using an LC at 214 nm to particle size. The column temperature was 50° C. The detect the presence of the different compounds and MS to mobile phase A was 0.1% TFA/water. The mobile phase B determine the identity of the detected compound. The inte was 0.1% TFA/acetonitrile. The injection volume was 1 ul. grated area 96 for each peak from the LC chromatogram was The gradient was as follows: 40 then plotted against time and the relative stabilities deter mined in human plasma. The stability data for Dyn A-(1-13)-OH and Dyn A-(1- Time (minutes) % A % B Flow (ml/min) 13)-NH were consistent with that reported in literature: the O 66 34 O.700 proteolytic breakdown of the dynorphin peptides is quite 1 66 34 O.700 45 rapid. Dyn A-(1-13)-OH had a half life of about 10 minutes. 25 58.8 41.2 O.700 Dyn A-(1-13)-NH had a half life of about 30 minutes. In 30 50 50 O.70 contrast Dyn A 1-13 (MPA)-NH exhibited striking stabili 35 5 95 1.OO Zation in the presence of serum peptidase activity. Unmodi 3. s fied dynorphin peptides are degraded within 60 minutes. In 46 66 34 0.70 so contrast, modified dynorphin peptides (Dyn A 1-13(MPA)- NH) are stable from serum peptidase activity for up to 480 minutes. The peptide was detected at 214 mm for quantification. The stability determination of the dynorphin conjugate is For mass spectral analysis of the peptide, the ionization determined by ELISA. In order to determine if the observed mode was API-electrospray at 1280 to 1500 m/z range, gain 55 signal is due to a dynorphin conjugate and what the conju 1.0, fragmentor 125 V, threshold 100, stepsize 0.40. The gas gate is, LC mass spectrometrytral analysis of the reaction temperature was 350° C. the drying gas was 13.01/min. The mixture after 8 h was performed. The use of mass spec pressure was 60 psi and the Vcap was 6000V. The results are trometry permits a determination of the molecular weight of presented below. the conjugate and allows the determination whether there are Approximately 33% of circulating albumin in the blood- any truncated forms of the dynorphin conjugate. Mass stream is mercaptalbumin (SH-albumin) which is not 9 spectrometry of human plasma shows the two forms of blocked by endogenous Sulfhydryl compounds such as cys- albumin, the free thiol at 66436 Da and the oxidized form at teine or glutathione and is therefore available for reaction 66557 Da. Also, mass spectrometry can distinguish between with maleimido groups. The remaining 66% of the circu- a Dyn 2-13 truncated conjugate (68046 Da) and the intact lating albumin is capped or blocked by Sulfhydryl com- Dyn 1-13 conjugate, (68207 Da) in an equal mixture. pounds. The HPLC MS assay permits the identification of 65 Mass spectrometry analysis of dynorphin samples taken capped-HSA, SH-albumin and K5-MPA-albumin. The MPA from the serum after 480 minutes of exposure to the serum covalently bonds to the free thiol on the albumin. The peptidases identifies only the presence of the intact conju US 7,256.253 B2 129 130 gate (68192 Da) and not the breakdown products thereby demonstrating the stability of the dynorphin conjugate from TABLE 1-continued serum peptidase activity. NATURAL AMINO ACIDS AND THEIRABBREVIATIONS TABLE 1. 3-Letter 1-Letter Protected Amino NATURAL AMINO ACIDS AND THEIRABBREVIATIONS Name Abbreviation Abbreviation Acids

3-Letter 1-Letter Protected Amino Isoleucine Ile I Name Abbreviation Abbreviation Acids 10 Leucine Leu L Lysine Lys K Alanine Ala Methionine Met M Arginine Arg Phenylalanine Phe F Asparagine ASn Proline Pro P Aspartic acid Asp Serine Ser S Cysteine Cys Threonine Thr T Glutamic acid Glu Tryptophan Trp W Glutamine Gln Tyrosine Tyr Y Glycine Gly Valine Wall V Histidine His

SEQUENCE LISTING

The patent 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=US0725.6253B2). 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: 1. A conjugate having the formula:

45 His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu Ser-Arg-Leu-Arg-Glu-Gly-Ala-Arg-Leu-Gln Arg-Leu-Leu-Gln-Gly-Leu-Val

50

55 N NH2 N

60 (SEQ ID NO:437) wherein X is S, O, or NH of an amino acid of said albumin. 65 2. The conjugate of claim 1, wherein X is S.