111111
`
`1111111111111111111111111111111111111111111111111111111111111
`US007414107B2
`
`c12) United States Patent
`Larsen
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,414,107 B2
`Aug. 19, 2008
`
`(54) PHARMACOLOGICALLY ACTIVE PEPTIDE
`CONJUGATES HAVING A REDUCED
`TENDENCY TOWARDS ENZYMATIC
`HYDROLYSIS
`
`(75)
`
`Inventor: Bjarne Due Larsen, Vanlose (DK)
`
`(73) Assignee: Zealand PharmaA/S (DK)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`2007/0004905 A1
`
`112007 Holm eta!.
`
`FOREIGN PATENT DOCUMENTS
`wo 91119735
`wo 95/13085
`wo 97/24445
`wo 98/03192
`wo 98/11126
`wo 98/22577
`wo 98/28427
`wo 98/52614
`
`12/1991
`5/1995
`7/1997
`111998
`3/1998
`5/1998
`7/1998
`1111998
`
`wo
`wo
`wo
`wo
`wo
`wo
`wo
`wo
`
`(21) Appl. No.: 11/007,772
`
`(22)
`
`Filed:
`
`Dec. 7, 2004
`
`(65)
`
`Prior Publication Data
`
`US 2006/0063699 Al
`
`Mar. 23, 2006
`
`Related U.S. Application Data
`
`(63)
`
`Continuation of application No. 09/341,590, filed as
`application No. PCT/DK99/00118 on Mar. 9, 1999.
`
`(30)
`
`Foreign Application Priority Data
`
`Mar. 9, 1998
`
`(DK)
`
`..................................... 0317/98
`
`(51)
`
`Int. Cl.
`C07K 1104
`(2006.01)
`(52) U.S. Cl. ....................... 530/334; 530/333; 530/324;
`530/325; 530/326; 530/327
`(58) Field of Classification Search ................. 530/333,
`530/334, 326, 327, 324, 325
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`4,018,754 A
`4,081,434 A
`4,542,124 A
`4,707,468 A
`4,724,229 A
`4,833,125 A
`4,847,240 A
`5,194,586 A
`5,330,971 A
`5,376,530 A
`5,545,719 A
`5,646,120 A
`5,652,122 A
`5,688,760 A
`5,723,129 A
`5,831,001 A
`5,968,513 A
`6,126,939 A
`7,176,282 B1
`
`4/1977 Inouye eta!.
`3/1978 Li
`9/1985 Huffman
`1111987 Yoshino et a!.
`2/1988 Ali
`5/1989 Neer eta!.
`7/1989 Ryser
`3/1993 Maeda eta!.
`7/1994 Wells et al.
`12/1994 De The eta!.
`8/1996 Shashoua
`7/1997 Smith eta!.
`7/1997 Frankel
`1111997 Kemp eta!.
`3/1998 Potter eta!.
`1111998 Twist eta!.
`10/1999 Gallow eta!.
`10/2000 Eisenbach-Schwartz eta!.
`2/2007 Holmet al.
`
`OTHER PUBLICATIONS
`
`Barany (Peptide & Protein Research 30, 705-739, 1987).*
`Sai eta!., 1998, Amer. J. Physiol. 275:G514-G520.
`Wakamiya eta!., 1998, Bull. Chern. Soc. Jpn. 71:699-709.
`Thomas eta!., 1997, J. Pharm. Exp. Ther. 281:817-825.
`Prokai, 1997, Exp. Opin. Ther. Patents 7(3):233-245.
`Tarnai eta!., 1997, J. Pharm. Exp. Ther. 280:410-415.
`Tarnai eta!., 1996, Adv. Drug Delivery Rev. 19:401-424.
`Greene et al., 1996, J. Pharm .. Exp. Ther. 277:1366-1375.
`Zhou eta!., 1991, Inti. J. Pharm. 75:97-115.
`Fawell eta!., 1994, Proc. Nat!. Acad. Sci. USA 91:664-668.
`Duguay, 1995, J. Bioi. Chern. 270:17566.
`Steiner, 1992, J. Bioi. Chern. 267-23435.
`Docherty, 1987, Antimicrob Agents and Chemotherapy 31: 1562.
`Burger, 1951, J. Bioi. Chern. 193:13.
`I. V. Syskov, et al "Structural-Functional Organization Of ACTH:
`Synthesis And Properties Of Analogues Of The ACTH-(11-24)(cid:173)
`Tetradeca- And ACTH-(1-24)- Tetracosapeptides Containing
`Hexa(Amino Acids) In Place Of The Natural ACTH 19-24 Sequence
`Of Amino Acids", Translated fromBioorganicheskaya Khimiya, vol.
`10, No.5, pp. 618-625, May 1984.
`Howells, "Proenkephalin biosynthesis in the rat," NIDA Res. Monogr.
`70:43-65 1986.
`Mascotti eta!., "Thermodynamics of single-stranded RNA binding to
`oligolysines containing tryptophan," Biochemistry. 31:8932-8946
`1992.
`Noguchi et a!., "Dipeptides as inhibitors of the gelation of sickle
`hemoglobin," Mol Pharmacal. 28:40-44 1985.
`Rao et a!. "Molecular cloning, sequence analysis and translation of
`proenkephalin mRNA from rat heart," Regul. Pept. 40:397-408 1992.
`
`(Continued)
`
`Primary Examiner-David Lukton
`(7 4) Attorney, Agent, or Firm-Clark & Elbing LLP; Kristina
`Bieker-Brady
`
`(57)
`
`ABSTRACT
`
`The invention is directed to a pharmacologically active pep(cid:173)
`tide conjugate having a reduced tendency towards enzymatic
`cleavage comprising a pharmacologically active peptide
`sequence (X) and a stabilizing peptide sequence (Z) of 4-20
`amino acid residues covalently bound to X.
`
`8 Claims, No Drawings
`
`SANOFI-AVENTIS Exhibit 1010 - Page 1
`
`IPR for Patent No. 8,951,962
`
`

`
`US 7,414,107 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Rosen eta!., "Isolation and characterization of the rat proenkephalin
`gene," J Bioi Chern. 259:14309-14313 1984.
`Shimohigashi et al., "Sensitivity of opioid receptor-like receptor
`ORL 1 for chemical modification on nociceptin, a naturally occurring
`
`nociceptive peptide," J Bioi Chern. 271:23642-23645 1996.
`Tamiya et al., "Effect of synthetic Lys-Trp on adenosine
`triphosphatase activity of carp andrabbitmyosin Bs," Camp Biochem
`Physiol B. 75:23-25 1983.
`* cited by examiner
`
`SANOFI-AVENTIS Exhibit 1010 - Page 2
`
`IPR for Patent No. 8,951,962
`
`

`
`US 7,414,107 B2
`
`1
`PHARMACOLOGICALLY ACTIVE PEPTIDE
`CONJUGATES HAVING A REDUCED
`TENDENCY TOWARDS ENZYMATIC
`HYDROLYSIS
`
`CROSS-REFERENCES TO RELATED
`APPLICATIONS
`
`The present application is a continuation of co-pending
`application U.S. Ser. No. 09/341,590 as filed on Jul. 12, 1999,
`which application is a national phase entry under 35 USC
`§371 ofPCT/DK99/00118 as filed on Mar. 9, 1999, which
`application claims the benefit of Danish application no. 0317 I
`98 as filed on Mar. 9, 1998. The disclosures of the U.S. Ser.
`No. 09/341,590, PCT/DK99/00118, and Danish application
`no. 0317/98 are each incorporated herein by reference.
`
`FIELD OF THE INVENTION
`
`2
`a!., 1996, Adv. Drug Delivery Rev. 19:401-404, discloses that
`E2078, a dynorphin analog was chemically modified to make
`it more stable to enzyme degradation by adding anN-methyl
`group at the amino-terminus of Arg and replacing D-Leu with
`5 L-Leu and adding ethylamine at the carboxy-terminal.
`A different approach involves the formation of chimeric
`peptides. This approach involves coupling the peptide that is
`not normally transported through the blood-brain barrier to
`peptide or protein 'vectors' that undergo receptor-mediated or
`10 adsorptive-mediated transcytosis.
`WO 98/22577 discloses a method for increasing the resis(cid:173)
`tance of a "core protein" to proteolytic degradation by linking
`or inserting a "stabilizing polypeptide" having the formula
`[(GlyJX(Gly6)Y[(Glyc)Z]n. X, Y, and Z may be alanine,
`15 serine, valine, isoleucine, leucine, methionine, phenylala(cid:173)
`nine, proline, and threonine.
`U.S. Pat. No. 5,545,719 discloses molecules comprising
`protein fragments homologous to an active region of protein
`fragments capable of stimulating nerve growth (neu(cid:173)
`ronotrophic proteins such as epidermal growth factor, tubu(cid:173)
`lin, nerve growth factor, laminin, fibronectin, ncam and
`ependymin) no greater than 80 amino acids long connected to
`a secondary molecule which can be a second protein fragment
`derived from the original protein, from another protein or
`25 from a non-proteinaceous moiety. This secondary molecule
`facilitates the transport of the peptide across the blood-brain
`barrier. It is stated in colunm 3, lines 3-7, "Upon entering the
`central nervous system, prodrug can remain intact or the
`chemical linkage between the carrier and the protein frag-
`30 ment may be hydrolyzed thereby separating the carrier from
`the fragment to release the nerve growth-stimulating frag(cid:173)
`ment". A preferred method for facilitating the coupling of the
`secondary molecule to the protein fragment is via one or more
`basic amino acids, preferably a pair of Lys residues, an Arg
`35 residue, or Arg-Lys.
`Fawell eta!., 1994, Proc. Natl.Acad. Sci. USA 91: 664-668
`discloses chemically crosslinking various Tat peptide frag(cid:173)
`ments to ~-galactosidase, RNAse A and domain III of
`pseudomonas exotoxin A. These included Tat-(37-72), Tat-
`40 (37-58) and Tat-(47-58). All of these peptides appeared to
`promote uptake of galactosidase, RNAse and domain III into
`cells. It was stated that this is the basic region of Tat. Conju(cid:173)
`gates containing poly (L-lysine) or poly (L-arginine) were not
`taken up by the cells.
`WO 97/24445 discloses fusion proteins of albumin and
`growth hormone or variants thereof. It is stated in the speci(cid:173)
`fication that variants of albumin should have the oncotic,
`ligand-binding and non-immunogenic properties of full
`length albumin and that variants of growth hormone should
`50 have its non-immunogenicity and ability to bind and activate
`the growth hormone receptor.
`W098/28427 discloses an Fc-OB fusion protein. Fe is an
`immunoglobulin fragment and OBis leptin. It has been found
`that such conjugates are more stable than OB alone. The Fe
`55 fragment is 3 78 amino acids in length. The Fe fragment can be
`conjugated directly or via a linker to OB or an OB fragment.
`A further approach involves preparing peptide analogs
`with increased stability and/or activity by adding a peptide
`tail. Greene eta!., J. Pharm. Exp. Therap. 277:1366-1375,
`60 discloses results of studies with various enkephalin analog
`prodrugs of [D-Pen2, D-Pen5
`] enkephalin (DPDPE) and
`[D-Pen2, L-Cys5
`] enkephalin (DPLCE) (SEQ ID NO: 1),
`specifically DPLCE-Arg-Pro-Ala (SEQ ID NO: 2), DPDPE(cid:173)
`Phe (SEQ ID NO: 3), DPLCE-Phe (SEQ ID NO: 4), DPDPE-
`65 Arg-Gly (SEQ ID NO: 5), DPLCE-Arg-Gly (SEQ ID NO: 6),
`DPDPE-Phe-Ala-NH---C6H 13 (SEQ lD NO 7), DPDPE-Phe(cid:173)
`Ala-CONH2 (SEQ ID NO: 7). The half lives of most of the
`
`The present invention relates to pharmacologically active 20
`peptide conjugates having a reduced tendency towards enzy(cid:173)
`matic cleavage.
`
`BACKGROUND OF THE INVENTION
`
`There exist a large number of pharmacologically active
`peptides, e.g., naturally occurring in man or in animals, or
`synthetic analogues of such peptides. An illustrative example
`of such a peptide is the analgetically active peptide enkepha(cid:173)
`lin that has given rise to a vast number of synthetic analogues.
`However, due to precisely their peptic nature, the routes of
`administration thereof have been rather limited. Thus, pep(cid:173)
`tides are rapidly and very effectively degraded by enzymes,
`generally with half-lives in the range of minutes. Proteases
`and other proteolytic enzymes are ubiquitous, particularly in
`the gastro-intestinal tract, and therefore peptides are usually
`susceptible to degradation in multiple sites upon oral admin(cid:173)
`istration, and to some extent in the blood, the liver, the kidney,
`and the vascular endothelia. Furthermore, a given peptide is
`usually susceptible to degradation at more than one linkage
`within the backbone; each locus ofhydrolysis is mediated by
`a certain protease. Even if such obstacles are overcome, for
`neuropeptides in particular, difficulties have been encoun(cid:173)
`tered in their transport across the blood-brain barrier.
`There has been a number of attempts to protect peptides 45
`against premature degradation (reviewed in Prokai, 1997,
`Exp. Opin. Ther. Patent 7:233-245, Tarnai eta!., 1996, Adv.
`Drug Delivery Rev. 19:401-424 and Zhou eta!., 1991, Int. J.
`Pharm. 7 5:97 -115). One approach includes osmotically alter(cid:173)
`ing the blood-brain barrier by infusion of hypertonic solu(cid:173)
`tions of mannitol, arabinose, lactamide, saline, urea, glycerol
`and radiographic contrast agents. However, there could be
`toxic side effects.
`Another approach involves the use of protease inhibitors
`(reviewedinZhou eta!., 1991, Int. J. Pharm. 75:97-115). This
`approach has yielded mixed results.
`A third approach has involved the use of absorption
`enhancers in peptide formulations (reviewed in Zhou et a!.,
`1991, Int. J. Pharm. 75:97-115). Examples include fatty acids
`and bile salts. However, varying results have been obtained
`regarding efficacies and the value of a particular enhancer is
`dependent on the route of administration used.
`Another approach for enhancing the absorption of peptides
`involves chemically modifYing the peptide by, for example,
`attaching a liphophilic moiety. It has also been found that
`attaching a pyroglutamyl residue at theN-terminal end can
`render a compound relatively resistant to hydrolysis. Tarnai et
`
`SANOFI-AVENTIS Exhibit 1010 - Page 3
`
`IPR for Patent No. 8,951,962
`
`

`
`US 7,414,107 B2
`
`3
`analogs, except for DPDPE-Arg-Gly are less than the parent
`compounds. It is stated on page 13 72, column 2 that "the ideal
`CNS-targeted prodrug would have a long half-life in the
`serum and a short half-life in the brain." U.S. Pat. No. 4,724,
`229 discloses vasopressin antagonists which have a tripeptide 5
`side chain having three basic amino acids, such as arginine,
`lysine or ornithine which have potent antagonistic activity.
`U.S. Pat. No. 4,542,124, discloses vasopressin antagonists
`which have a dipeptide side chain having two amino acids,
`one of which is basis which has potent vasopressin antago- 10
`nistic activity.
`In the international patent application PCT/DK97/00376
`(Bjame Due Larsen and Arne Holm) prodrugs of pharmaco(cid:173)
`logically active peptides are described, wherein the pharma(cid:173)
`cologically active peptide is coupled at its C-terminal to a 15
`peptide pre-sequence via a linker, the linker typically being an
`a-hydroxy carboxylic acid. These special peptide derivatives
`were found to have a prolonged half-life in the presence of
`proteolytic enzymes such as carboxypeptidase A, leucine
`aminopeptidase, pepsin A and a-chymotrypsin. In addition, 20
`PCT/DK97/00376 discloses (as reference compounds) four
`different peptides equipped with a peptide pre-sequence but
`without linker, namely DSIP-(Lys-Glu)3 (SEQ ID NO: 8),
`DSIP-(Glu), (SEQ ID NO: 9), Leu-enkephalin-(Glu)6 (SEQ
`ID NO: 1 0) and Leu-enkephalin-(Lys )6 (SEQ ID NO: 11 ).
`It is evident that there is a need for a peptide conjugate
`which contains a pharmacologically active peptide and a sta(cid:173)
`bilising protein that is relatively simple to synthesize, retains
`its activity even without removing the stabilising peptide, is
`stable in plasma or serum and is relatively resistant to enzyme 30
`degradation. Therefore, it is an object of the invention to
`provide a peptide conjugate comprising a pharmacologically
`active peptide and stabilising peptide that is relatively resis(cid:173)
`tant to enzyme degradation.
`
`25
`
`4
`wherein R 1 and R 2 are selected from the group consisting of
`hydrogen, cl-6-alkyl, phenyl, and phenyl-methyl, wherein
`C 1_6-alkyl is optionally substituted with from one to three
`substituents selected from halogen, hydroxy, amino, cyano,
`nitro, sulfono, and carboxy, and phenyl and phenyl-methyl is
`optionally substituted with from one to three substituents
`selected from cl-6 alkyl, c2-6-alkenyl, halogen, hydroxy,
`amino, cyano, nitro, sulfono, and carboxy, or R 1 and R2
`together with the carbon atom to which they are bound form
`a cyclopentyl, cyclohexyl, or cycloheptyl ring, e.g., 2,4-di(cid:173)
`aminobutanoic acid (Dbu) and 2,3-diaminopropanoic acid
`(Dpr); and
`wherein the ratio between the half-life of said peptide
`conjugate and the half-life of the corresponding pharmaco(cid:173)
`logically active peptide sequence, X, when treated with car(cid:173)
`boxypeptidase A or leucine aminopeptidase in about 50 mM
`phosphate buffer solution at about pH 7.4 at about 3 7° C. or in
`plasma or serum is at least about 2, preferably at least about 3,
`such as at least about 5, more preferably at least about 7, such
`as at least about 9, e.g., at least about 10 and the ratio between
`the half-life of said peptide conjugate or when the pharmaco-
`logically active peptide is not orally absorbed, said conjugate
`is orally absorbed or a salt thereof.
`In one embodiment, the pharmacologically active peptide
`is not selected from the group consisting of H-Trp-Ala-Gly(cid:173)
`Gly-Asp-Ala-Ser-Gly-Glu-(Lys-GlukOH (SEQ ID NO: 8),
`H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-(GlukOH (SEQ
`ID NO: 9), H-Tyr-Gly-Gly-Phe .. Leu-(Glu)6-0H (SEQ ID
`NO: 10) and H-Tyr-Gly-Gly-Phe .. Leu-(LyskOH (SEQ ID
`NO: 11).
`The present invention also relates to a composition, e.g., a
`pharmaceutical composition, comprising said pharmacologi(cid:173)
`cally active peptide conjugate and a pharmaceutically accept(cid:173)
`able carrier, to a pharmacologically active peptide conjugate
`35 for use in therapy, a method of treating a disorder and to the
`use of a pharmacologically active peptide conjugate for the
`manufacture of a pharmaceutical composition for use in
`therapy. Specifically, the invention is directed to a method for
`inhibiting neurons from transmitting pain impulses to the
`40 spinal cord, comprising administering to a subject in need
`thereof a conjugate comprising enkephalin and Z in an
`amount effective to inhibit neurons from transmitting pain
`impulses, as well as the use of said conjugate for the manu(cid:173)
`facture of a pharmaceutical composition for use in treatment
`45 of pain; a method for stimulating the release of growth hor(cid:173)
`mone from the pituitary comprising administering to a sub(cid:173)
`ject in need thereof a conjugate comprising growth hormone
`releasing hormone or growth hormone releasing peptide and
`Z in an amount effective to stimulate the release of growth
`50 hormone as well as the use of said conjugate for the manu(cid:173)
`facture of a pharmaceutical composition for use in stimulat(cid:173)
`ing the release of growth hormone; a method for increasing
`hemoglobin levels comprising administering to a subject in
`need thereof a conjugate comprising EMP-1 (SEQ ID NO:
`55 117) (erythropoietin mimetic protein-!) and Z in an amount
`effect to increase hemoglobin levels as well as the use of said
`conjugate for the manufacture of a pharmaceutical composi(cid:173)
`tion for use in increasing treating anemia by increasing hemo(cid:173)
`globin levels; a method for treating or preventing bone loss by
`60 altering the balance between osteoclastic (bone resorption)
`and osteoblast activity comprising administering to a patient
`in need thereof a conjugate comprising parathyroid hormone
`and Z in an amount effective to treat or prevent bone loss as
`well as use of a said conjugate for the manufacture of a
`65 pharmaceutical composition for use in treating or preventing
`osteoporosis; a method for reducing blood glucose levels
`comprising administering to a subject in need thereof a con-
`
`SUMMARY OF THE INVENTION
`
`It has now surprisingly been found that by conjugating a
`pharmacologically active peptide, for example, at its C-ter(cid:173)
`minal, at its N-terminal or at its C- and N-terminal, with a
`suitable stabilising peptide sequence, it is possible to render
`the resulting peptide conjugate significantly less susceptible
`to degradation by proteases compared to the corresponding
`free pharmacologically active peptide. Without being bound
`to any specific model for this effect, it is believed that the
`presence of the stabilising peptide sequence induces a degree
`of structuring, based on hydrogen bonds, of the pharmaco(cid:173)
`logically active peptide, whereby the peptide conjugate is less
`susceptible to proteases in contrast to peptides in the random(cid:173)
`coil conformation. As a result of the structuring, the peptide
`conjugate is much more difficult for a protease to degrade.
`Moreover, the resulting peptide conjugate is still pharmaco(cid:173)
`logically active, i.e. the peptide conjugate possesses the abil(cid:173)
`ity to exert the pharmacological function of the free pharma(cid:173)
`cologically active peptide.
`Thus, in a first aspect the invention relates to a pharmaco(cid:173)
`logically active peptide conjugate having a reduced tendency
`towards enzymatic cleavage, said peptide conjugate com(cid:173)
`prises: a pharmacologically active peptide sequence (X) and
`a stabilising peptide sequence (Z) of 4-20 amino acid residues
`covalently bound to X, each amino acid residue in said stabi(cid:173)
`!ising peptide sequence (Z) being independently selected
`from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn, Gin,
`Asp, Glu, Lys, Arg, His, Met, Om, and amino acid residues of
`the general formula I
`
`-NH---{:(R1)(R2)-C(=0)-
`
`(I)
`
`SANOFI-AVENTIS Exhibit 1010 - Page 4
`
`IPR for Patent No. 8,951,962
`
`

`
`US 7,414,107 B2
`
`5
`jugate compnsmg glucagon-like peptide-! and Z in an
`amount effective to reduce blood sugar levels, as well as use
`of said conjugate in the treatment of diabetes; a conjugate
`comprising delta sleep inducing peptide and Z in an amount
`effective to prevent convulsions, act as a neuroprotectant
`during ischemia and act as a detoxification agent of an opiod
`as well as a use of said conjugate for the manufacture of a
`pharmaceutical composition for use in treating sleep disor(cid:173)
`ders; a method for regulating production of sex hormones
`comprising administering to a subject in need thereof a con(cid:173)
`jugate comprising gonadotropin releasing hormone and Z in
`an amount effective to regulate production of sex hormones as
`well as use of said conjugate for the manufacture of a phar(cid:173)
`maceutical composition for use in regulating the level of sex
`hormones.
`In another aspect the present invention relates to the use of
`a peptide conjugate, as defined herein, for the manufacture of
`a pharmaceutical composition for the treatment or prophy(cid:173)
`laxis of a condition or disorder, where the peptide sequence
`X, when not bound to Z, is able to interact with a receptor (or
`a receptor system) involved with the condition or disorder in
`question, and where the interaction between X, when not
`bound to Z, and the receptor (or receptor system) has a thera(cid:173)
`peutic or prophylactic effect on the condition or disorder.
`The present invention also relates to methods for the prepa- 25
`ration of said pharmacologically active peptide conjugate, by
`means of recombinant DNA-technology comprising the steps
`of (a) introducing a nucleic acid sequence encoding said
`conjugate into a host cell and (b) culturing said host cell and
`(c) isolating said conjugate from the culture or (a) culturing a 30
`recombinant host cell comprising a nucleic acid sequence
`encoding said conjugate under conditions permitting the pro(cid:173)
`duction of said conjugate and (b) isolating said conjugate
`from the culture.
`The method also relates to methods for the preparation of
`said pharmacologically active peptide conjugate in which the
`pharmacologically active peptide X is obtained via recombi(cid:173)
`nant DNA methods by isolating said peptide or from com(cid:173)
`mercial sources. X is then conjugated to Z which is attached
`to a solid support or has been prepared by solid phase syn(cid:173)
`thetic methods.
`Furthermore, the invention relates to the use of a stabilising
`peptide sequence (Z) for the preparation of a pharmacologi(cid:173)
`cally active peptide conjugate.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`Peptide Conjugates
`In the present context, the term "amino acid residue" as
`used in connection with X means any naturally occurring or
`synthetic a, ~'or y-amino acid (whether in the L-form or the
`D-form) as well as side-chain modified amino acids such as
`modified tyrosines wherein the aromatic ring is further sub(cid:173)
`stituted with e.g., one or more halogens, sulfono groups, nitro
`groups etc., and/or the phenol group is converted into an ester
`group, etc, including side-chain protected amino acids,
`wherein the amino acid side-chains are protected in accor(cid:173)
`dance with methods known to the person skilled in peptide
`chemistry, such as described in, e.g., M. Bodanszky and A.
`Bodanszky, "The Practice of Peptide Synthesis", 2. Ed,
`Springer-Verlag, 1994, and J. Jones, "The Chemical Synthe(cid:173)
`sis ofPeptides", Clarendon Press, 1991.
`In the present context, the term "pharmacologically active
`peptide sequence" or "free peptide" as applied to X is
`intended to mean any peptide or peptide-containing structure,
`either naturally occurring or synthetic which is therapeuti-
`
`6
`cally or prophylactically active without the stabilising
`sequence Z covalently bound thereto. As defined herein, a
`peptide sequence is "therapeutically active" if it can be used
`for the treatment, remission, or attenuation of a disease state,
`physiological
`condition,
`symptoms
`or
`etiological
`indication(s) or evaluation or diagnosis thereof. A peptide
`sequence is "prophylactically active" if it can be used to
`prevent a disease state, physiological condition, symptoms or
`etiological indications. A pharmacologically active agent is
`10 also physiologically or biologically active.
`Pharmacological activity measures the effect of a sub(cid:173)
`stance (peptide) on physiological or biological systems in
`vitro, in vivo or ex vivo and may be assayed using standard in
`vitro, in vivo or ex vivo assays known in the art for a particular
`15 peptide or a peptide with a similar physiological function
`Peptides are utilised in a number of processes, e.g., cell(cid:173)
`to-cell communication, some being present in the autonomic
`and central nervous system. Some of the latterpeptides, and a
`number of other peptides, exert important effects on vascular
`20 and other smooth muscles. In a preferred embodiment, X has
`at the most 75 amino acid residues (or a structure correspond(cid:173)
`ing to at the most 75 amino acid residues). Alternatively, X
`consists of at most 65, 60, 55, 53, 50, 45, 40, 35, 30, 25, 20, 15,
`or at the most 1 0 amino acid residues and consists of at least
`2, preferably 5 and more preferably 10 amino acid residues.
`In the present context, the pharmacologically active pep(cid:173)
`tide sequence X can be any peptide which in its native form is
`present as the C-terminal free carboxylic acid, such as Leu(cid:173)
`enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) (SEQ ID NO: 12),
`or is present in its native form as a C-terminal amide, such as
`oxytocin
`( Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu -Gly-NH2 )
`(SEQ ID NO: 13), or is present in its native form as a C-ter(cid:173)
`minal ester. Furthermore, the pharmacologically active pep(cid:173)
`tide may also contain other special structural features such as
`35 disulfide bridges as in the case insulin.
`The pharmacologically active peptide may be selected
`from the group consisting of enkephalin, Leu-enkephalin
`(SEQ ID NO: 12), Met-enkephalin, angiotensin I, angio(cid:173)
`tensin II, vasopressin, endothelin, vasoactive intestinal pep-
`40 tide, neurotensin, endorphins, insulin, gramicidin, paracelsin,
`delta-sleep inducing peptide, gonadotropin-releasing hor(cid:173)
`mone (SEQ ID NO: 115), human parathyroid hormone
`(1-34), truncated erythropoietin analogues described in
`Wrighton et a!., 1996, Science 273:458-463), specifically
`45 EMP-1 (SEQ ID NO: 117), Atrial natriuretic peptide (ANP,
`ANF), human brain natriuretic peptide (hBNP), cecropin,
`kinetensin, neurophysins, elafin, guamerin, atriopeptin I,
`atriopeptin II, atriopeptin III, deltorphin I, deltorphin II, vaso(cid:173)
`tocin, bradykinin, dynorphin, dynorphin A, dynorphin B,
`50 growth hormone release factor, growth hormone, growth hor(cid:173)
`mone releasing peptide, oxytocin, calcitonin, calcitonin
`gene-related peptide, calcitonin gene-related peptide II,
`growth hormone releasing peptide, tachykinin, adrenocorti(cid:173)
`cotropic hormone (ACTH), brain natriuretic polypeptide,
`55 cholecystokinin, corticotropin releasing factor, diazepam
`binding inhibitor fragment, FMRF -amide, galanin, gastric
`releasing polypeptide, gastric inhibitory polypeptide, gastrin,
`gastrin releasing peptide, glucagon, glucagon-like peptide-!,
`glucagon-like peptide-2, LHRH, melanin concentrating hor-
`60 mane, melanocyte stimulating hormone (MSH), alpha-MSH,
`morphine modulating peptides, motilin, neurokinin A, neu(cid:173)
`rokinin B, neuromedin B, neuromedin C, neuromedin K,
`neuromedin N, neuromedin U, neuropeptide K, neuropeptide
`Y, pituitary adenylate cyclase activating polypeptide
`65 (PACAP), pancreatic polypeptide, peptide YY, peptide histi(cid:173)
`dine-methionine amide (PHM), secretin, somatostatin, sub(cid:173)
`stance K, thyrotropin-releasing hormone (TRH), kyotorphin,
`
`SANOFI-AVENTIS Exhibit 1010 - Page 5
`
`IPR for Patent No. 8,951,962
`
`

`
`US 7,414,107 B2
`
`7
`melanostatin (MIF -1 ), thrombopoeitin analogs, in particular
`AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala(cid:173)
`Ala-Arg-Ala) (SEQ ID NO: 14), insulin-like growth factor I
`(57 -70)
`(Ala-Leu-Leu-Glu-Thr-Tyr-Cys-Ala-Thr-Pro-Ala(cid:173)
`Lys-Ser-Glu) (SEQ ID NO: 15), insulin-like growth factor I 5
`(30-41) (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro -Gln(cid:173)
`Thr) (SEQ ID NO: 16), insulin-like growth factor I(24-41)
`(Tyr-Phe-Asn-Lys-Pro
`-Thr-Gly-Tyr-Gly-Ser-Ser-Ser-Arg(cid:173)
`Arg-Ala-Pro-Gln-Thr) (SEQ ID NO: 17), insulin-like growth
`factor II(33-40) (Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) (SEQ 10
`ID NO: 18), insulin-like growth [tyro] factor II (33-40) (Tyr(cid:173)
`Ser-Arg-Val-Ser-Arg-Arg-Ser-Arg) (SEQ ID NO: 19), insu(cid:173)
`lin-like growth factor II (69-84) (Asp-Val-Ser-Thr-Pro-Pro(cid:173)
`Thr-Val-Leu -Pro-Asp-Asn-Phe-Pro-Arg-Tyr) (SEQ ID NO:
`20), growth hormone (GH)-releasing peptide-6 (GHRP-6) 15
`(His-DTrp-Ala-Trp-DPhe-Lys-NH2) (SEQ ID NO: 21 ), beta(cid:173)
`Interleukin I (163-171) (Val-Gln-Gly-Glu-Glu-Ser-Asn(cid:173)
`Asp-Lys) (SEQ ID NO: 22), beta-Interleukin II (44-56) (Ile(cid:173)
`Leu-Asn-Gly-Ile-Asn-Asn-Tyr-Lys-Asn-Pro-Lys-Leu)
`(SEQ ID NO: 23), Interleukin II (60-70) (Leu-Thr-Phe-Lys- 20
`Phe-Tyr-Met-Pro-Lys-Lys-Ala) (SEQ ID NO: 24), exendin-4
`(GLP-1 analog)
`(His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp
`-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe(cid:173)
`Ile-Glu-Trp-Leu-Lys-Asn
`-Gly-Gly-Pro-Ser-Ser-Gly-Ala(cid:173)
`Pro-Pro-Pro-Ser-NH2) (SEQ ID NO: 25), exendin-3 (GLP-1 25
`analog)
`(His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser(cid:173)
`Lys-Gln-Met-Glu-Glu-Glu -Ala-Val-Arg-Leu-Phe-Ile-Glu(cid:173)
`Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-4Ala-Pro-Pro
`-Pro-Ser) (SEQ ID NO: 26), [Cys(Acm)20,31] epidermal
`growth factor (20-31) Cys(Acm)-Met-His-Ile-Glu-Ser-Leu- 30
`Asp-Ser-Tyr-Thr-Cys(Acm) (SEQ ID NO: 27), bivalirudin
`(D-Phe-Pro-Arg-Pro-(Gly)4 -Asn-Gly-Asp-Phe(cid:173)
`(Hirulog)
`Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu) (SEQ ID NO: 28), hiru(cid:173)
`log-1 (D-Phe-Pro-Arg-Pro-(Gly)4 -Asn-Gly -Asp-Phe-Glu(cid:173)
`ID NO: 29), C-type 35
`Glu-Ile-Pro-Glu-Tyr-Leu
`(SEQ
`natriuretic peptide (1-53) (CNP) (Asp-Leu-Arg-Val-Asp(cid:173)
`Thr-Lys-Ser-Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln -Glu(cid:173)
`His-Pro-Asn-Ala-Arg-Lys-Tyr-Lys-Gly-Ala-Asn-Lys-Lys-
`Gly-Leu-Ser-Lys-Gly
`-Cys-Phe-Gly-Leu-Lys-Leu-Asp-
`Arg-Ile-Gly-Ser-Met-Ser-Gly-Leu-Gly-Cys;
`Disulfide 40
`bridge: Cys37-Cys53) (SEQ ID NO: 30), "MiniANP" (Met(cid:173)
`Cys-His-cyclohexy!Ala-Gly
`-Gly-Arg-Met-Asp-Arg-Ile(cid:173)
`Ser-Cys-Tyr-Arg, disulfide bridge cys2-cys 13) (SEQ ID NO:
`31), Melanotan-II (also known as MT-II, alpha-MSH4-10-
`NH2, or Ac-Nle4-Asp5-His6-D-Phe7-Arg8-Trp9-Lys10) 45
`(SEQ ID NO: 32), thymosin alpha! (TAl) (Ac-Ser-Asp-Ala
`-Ala-Val-Asp-Thr-Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu(cid:173)
`Lys-Glu-Lys-Lys-Glu-Val-Val
`-Glu-Glu-Ala-Glu-Asn)
`(SEQ ID NO: 33), ornipressin (also known as 8-ornithine(cid:173)
`vasopressin, (POR-8), [Phe2,Ile3,0rn8]vasopressin), Cys- 50
`Phe-Ile-Gln-Asn-Cys-Pro-Orn -Gly-NH2, Disulfide bridge:
`Cysl-Cys6) (SEQ ID NO: 34), octreotide (201-995) (DPhe
`-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol; disulfide bridge: Cys2-
`Cys7) (SEQ ID NO: 35), eptifibatide (INTEGRILIN), calci(cid:173)
`tonin gene-related peptide (CGRP) (Ala-Cys-Asp-Thr -Ala- 55
`Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg(cid:173)
`Ser-Gly-Gly-Val-Val-Lys -Asn-Asn-Phe-Val-Pro-Thr-Asn(cid:173)
`Val-Gly-Ser-Lys-Ala-Phe-NH2 (SEQ ID NO: 36); Disulfide
`bridge: Cys2-Cys7), endomorphin-1 (Tyr-Pro-Trp-Phe-NH2
`(SEQ ID NO: 37); endomorphin-2 Tyr-Pro-Phe-Phe-NH2 60
`(SEQ ID NO: 38), nociceptin (SEQ ID NO: 39) (also known
`as Orphanin FQ), Phe-Gly-Gly-Phe-Thr-Gly-Ala-Arg-Lys(cid:173)
`Ser-Ala-Arg-Lys
`-Leu-Ala-Asn-Gln),
`angiotensinogen
`(1-13) (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu -Val-lie(cid:173)
`His) (SEQ ID NO 40), adrenomodullin (1-12) (Tyr-Arg-Gln- 65
`Ser-Met-Asn-Asn -Phe-Gln-Gly-Leu-Arg) (SEQ ID NO:
`41), antiarrhytmic peptide (AAP) (Gly-Pro-Hyp -Gly-Ala-
`
`8
`Gly) (SEQ ID NO: 42), Antagonist G (Arg-DTrp-(nMe)Phe(cid:173)
`DTrp-Leu-Met -NH2) (SEQ ID NO 123), indolicidin (lie(cid:173)
`Leu-Pro-Trp-Lys-Trp-Pro-Trp -Trp-Pro-Trp-Arg-Arg-NH2)
`(SEQ ID NO: 43), osteocalcin (37-49) (Gly -Phe-Gln-Glu(cid:173)
`Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val) (SEQ ID NO: 44),
`cortistatin 29 (1-13) (Glp)-Glu-Arg-Pro-Pro-Leu-Gln-Gln(cid:173)
`Pro-Pro-His-Arg-Asp) (SEQ ID NO: 45), cortistatin 14 Pro(cid:173)
`Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Ser-Ser-Cys-Lys
`(SEQ ID NO: 46); Disulfide bridge: Cys2-Cys 13, PD-145065
`(Ac-D-B