CORRECTED
`VERSION‘
`
`WORLD INTELLI-:CrUAL, PROPERTY ORGANIZATION
`lntemattonal Bureau
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(51) International Patent Classification 6 :
`
`(11) International Publication Number:
`
`WO 99/46283
`
`C07K 1/107, A61K 47/48
`
`(43) International Publication Date:
`
`16 September 1999 (l6.09.99)
`
`(21) International Application Number:
`
`PCT/DK99/00118
`
`(22) International Filing Date:
`
`9 March 1999 (0903.99)
`
`(30) Priority Data:
`0317/98
`
`9 March 1998 (09.03.98)
`
`DK
`
`(71) Applicant (for all designated States except US): ZEALAND
`PHARMACEUTICALS A/S [DK/DK];
`Innovationshuset,
`Agem A116 3, DK—2970 Harsholm (DK).
`
`.
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): LARSEN, Bjame, Due
`[DK/DK]; Arildsgérd 5, l.th., DK—2700 Brmshej (DK).
`
`(74) Agent: PDOUGHMANN, VINGTOFI‘ & PARTNERS A/S:
`Sankt Anne Plads 11, PO. Box 3007, DK—102l Copen-
`hagen K (DK).
`
`(81) Designated States: AL, AM, AT, AT (Utility model), AU, AZ,
`BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, CZ (Utility
`model), DE, DE (Utility model), DK, DK (Utility model),
`EB, EE (Utility model), ES, F1, F1 (Utility model), GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR. KZ, LC, LK, LR. LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI,
`SK, SK (Utility model), SL, TJ, TM, TR, Tl", UA, UG, US,
`UZ, VN, YU. ZW, ARIPO patent (GH, GM, KE. LS, MW.
`SD, SL, SZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG,
`KZ, MD, RU, TJ, TM), European patent (AT, BE, CH, CY,
`DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT,
`SE), OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW,
`ML, MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(54) Title: PHARMACOLOGICALLY ACTIVE PEPTIDE CONJUGATES HAVING A REDUCED TENDENCY TOWARDS ENZY-
`MATIC HYDROLYSIS
`
`(57) Abstract
`
`The invention is directed to a pharmacologically active peptide conjugate having a reduced tendency towards enzymatic cleavage
`comprising a phannacologically active peptide sequence (X) and a stabilising peptide sequence (Z) of 4-20 amino acid residues covalently
`bound to X.
`
`‘(Referred to in PCT Gazette No. 46/ I999, Section II)
`
`SANOFI-AVENTIS Exhibit 1009 - Page i
`IPR for Patent No. 8,951 ,962
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`

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`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`cz
`DE
`DK
`EE
`
`Albania
`Armenia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Ci\te d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`UG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`VietNam
`Yugoslavia
`Zimbabwe
`
`SANOFI-AVENTIS Exhibit 1009 - Page ii
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`1
`PHARMACOLOGICALLY ACTIVE PEPTIDE CONJUGATES HAVING A
`
`REDUCED TENDENCY TOWARDS ENZYMATIC HYDROLYSIS
`
`FIELD OF THE INVENTION
`
`The present invention relates to pharmacologically active peptide conjugates having a
`
`reduced tendency towards enzymatic cleavage.
`
`BACKGROUND OF THE INVENTION
`
`5
`
`10
`
`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 enkephalin that has given rise to a vast number
`
`of synthetic analogues. However, due to precisely their peptic nature, the routes of
`
`1 5 administration thereof have been rather limited. Thus, peptides 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 administration, and to some extent in the blood, the liver, the kidney, and the vascular
`
`20 endothelia. Furthermore, a given peptide is usually susceptible to degradation at more than
`
`one linkage within the backbone; each locus of hydrolysis is mediated by a certain protease.
`
`Even if such obstacles are overcome, for neuropeptides in particular, difficulties have been
`
`encountered in their transport across the blood-brain barrier.
`
`25 There has been a number of attempts to protect peptides against premature degradation
`
`(reviewed in Prokai, 1997, Exp. Opin. Ther. Patent 7:233-245, Tarnai et al., 1996, Adv.
`
`Drug Delivery Rev. 19:401-424 and Zhou et al., 1991, Int. J. Pharm. 75:97-115). One
`
`approach includes osmotically altering the blood-brain barrier by infusion of hypertonic
`
`solutions of mannitol, arabinose, lactamide, saline, urea, glycerol and radiographic contrast
`
`30 agents. However, there could be toxic side effects.
`
`SUBSTITUTE SHEET (RULE 26)
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`2
`Another approach involves the use of protease inhibitors (reviewed in Zhou et al., 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
`
`5 (reviewed in Zhou et al., 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
`
`10 the peptide by, for example, attaching a liphophilic moiety.
`
`It has also been found that
`
`attaching a pyroglutamyl residue at the N-terminal end can render a compound relatively
`
`resistant to hydrolysis. Tarnai et al., 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-
`
`15 Leu with 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 adsorptive-mediated
`
`20 transcytosis.
`
`WO 98/22577 discloses a method for increasing the resistance of a "core protein" to
`
`proteolytic degradation by linking or inserting a "stabilizing polypeptide" having the
`
`formula [(Glya)X(Glyb)Y[(Glyc)Z]n. X, Y, and Z may be alanine, serine, valine, isoleucine,
`
`25 leucine, methionine, phenylalanine, proline, and threonine.
`
`U.S. Patent No. 5,545,719 discloses molecules comprising protein fragments homologous to
`
`an active region of protein fragments capable of stimulating nerve growth (neuronotrophic
`
`proteins such as epidermal growth factor, tubulin, nerve growth factor, laminin, fibronectin,
`
`30 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
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 2
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`3
`another protein or from a non-proteinaceous moiety. This secondary molecule facilitates the
`
`transport of the peptide across the blood-brain barrier. It is stated in column 3, lines 3-7,
`
`"Upon entering the central nervous system, prodrug can remain intact or the chemical
`
`linkage between the carrier and the protein fragment may be hydrolyzed thereby separating
`
`5 the carrier from the fragment to release the nerve growth-stimulating fragment". 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 residue, or Arg(cid:173)
`
`Lys.
`
`1 0 Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91: 664-668 discloses chemically
`
`crosslinking various Tat peptide fragments to p-galactosidase, RNAse A and domain III of
`
`pseudomonas exotoxin A. These included Tat-(37-72), Tat -(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. Conjugates containing poly (L-lysine)
`
`1 5 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 specification that variants of albumin should have the oncotic, ligand(cid:173)
`
`binding and non-immunogenic properties of full length albumin and that variants of growth
`
`20 hormone should have its non-imrnunogenicity and ability to bind and activate the growth
`
`hormone receptor.
`
`W098/28427 discloses an Fc-OB fusion protein. Fe is an immunoglobulin fragment and
`
`OB is leptin. It has been found that such conjugates are more stable than OB alone. The
`
`Fe fragment is 378 amino acids in length. The Fe fragment can be conjugated directly or via
`
`25 a linker to OB or an OB fragment.
`
`A further approach involves prepanng peptide analogs with increased stability and/or
`
`activity by adding a peptide tail. Greene et al., J. Pharm. Exp. Therap. 277:1366-1375,
`discloses results of studies with various enkephalin analog prodrugs of [o-Pen2
`, o-Pen5
`30 enkephalin (DPDPE) and [o-Pen2
`, L-Cys5
`Pro-Ala, DPDPE-Phe, DPLCE-Phe, DPDPE-Arg-Gly, DPLCE-Arg-Gly, DPDPE-Phe-Ala-
`
`] enkephalin (DPLCE), specifically DPLCE-Arg(cid:173)
`
`]
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 3
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`4
`NH-C6H 13, DPDPE-Phe-Ala-CONH2. The half lives of most of the analogs, except for
`DPDPE-Arg-Gly are less than the parent compounds. It is stated on page 1372, 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. Patent No. 4,724,229 discloses vasopressin antagonists which
`
`5 have a tripeptide side chain having three basic amino acids, such as arginine, lysine or
`
`ornithine which have potent antagonistic activity. U.S. Patent 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 antagonistic activity.
`
`10 In the international patent application PCT/DK97/00376 (Bjarne Due Larsen and Arne
`
`Holm) prodrugs of pharmacologically active peptides are described, wherein
`
`the
`
`pharmacologically active peptide is coupled at its C-terminal to a 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
`
`1 5 such as carboxypeptidase A, leucine aminopeptidase, pepsin A and a-chymotrypsin. In
`
`addition, PCT/DK97/00376 discloses (as reference compounds) four different peptides
`
`equipped with a peptide pre-sequence but without linker, namely DSIP-(Lys-Glu)3, DSIP(cid:173)
`(Glu)6, Leu-enkephalin-(Glu)6 and Leu-enkephalin-(Lys)6.
`
`20 It is evident that there is a need for a peptide conjugate which contains a pharmacologically
`
`active peptide and a stabilising 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 degradation. Therefore, it is an object of the invention to
`
`provide a peptide conjugate comprising a pharmacologically active peptide and stabilising
`
`25 peptide that is relatively resistant to enzyme degradation.
`
`SUMMARY OF THE INVENTION
`
`It has now surprisingly been found that by conjugating a pharmacologically active peptide,
`
`30 for example, at its C-terminal, 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
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 4
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`5
`
`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 pharmacologically active peptide, whereby the
`
`peptide conjugate is less susceptible to proteases in contrast to peptides in the random-coil
`
`5 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 pharmacologically
`
`active, i.e. the peptide conjugate possesses the ability to exert the pharmacological function
`
`of the free pharmacologically active peptide.
`
`1 0 Thus, in a first aspect the invention relates to a pharmacologically active peptide conjugate
`
`having a reduced tendency towards enzymatic cleavage, said peptide conjugate comprises: 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 stabilising
`
`peptide sequence (Z) being independently selected from the group consisting of Ala, Leu,
`
`15 Ser, Thr, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, His, Met, Om, and amino acid residues ofthe
`
`general formula I
`
`(I)
`
`20 wherein R1 and R2 are selected from the group consisting of hydrogen, C 1_6-alkyl, phenyl,
`and phenyl-methyl, wherein C1_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
`25 carboxy, or R1 and R2 together with the carbon atom to which they are bound form a
`cyclopentyl, cyclohexyl, or cycloheptyl ring, e.g., 2,4-diaminobutanoic 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
`
`30 corresponding pharmacologically active peptide sequence, X, when
`
`treated with
`
`carboxypeptidase A or leucine aminopeptidase in about 50 mM phosphate buffer solution at
`
`about pH 7.4 at about 3TC or in plasma or serum is at least about 2, preferably at least
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 5
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`6
`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 pharmacologically active peptide is not orally absorbed, said conjugate is orally
`
`absorbed or a salt thereof.
`
`5
`
`In one embodiment, the pharmacologically active peptide is not selected from the group
`
`consisting ofH-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-(Lys-Glu)3-0H,
`H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu-(Glu)6-0H,
`H-Tyr-Gly-Gly-Phe-Leu-(Glu)6-0H and
`10 H-Tyr-Gly-Gly-Phe-Leu-(Lys)6-0H.
`
`The present invention also relates to a composition, e.g., a pharmaceutical composition,
`
`comprising said pharmacologically active peptide conjugate and a pharmaceutically
`
`acceptable carrier, to a pharmacologically active peptide conjugate for use in therapy, a
`
`1 5 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 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
`
`20 pain impulses, as well as the use of said conjugate for the manufacture of a pharmaceutical
`
`composition for use in treatment of pain; a method for stimulating the release of growth
`
`hormone from the pituitary comprising administering to a subject 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 hormone as well as
`
`25 the use of said conjugate for the manufacture of a pharmaceutical composition for use in
`
`stimulating the release of growth hormone; a method for increasing hemoglobin levels
`
`comprising administering to a subject in need thereof a conjugate comprising EMP-1
`
`(erythropoietin mimetic protein-1) and Z in an amount effect to increase hemoglobin levels
`
`as well as the use of said conjugate for the manufacture of a pharmaceutical composition for
`
`30 use in increasing treating anemia by increasing hemoglobin levels; a method for treating or
`
`preventing bone loss by altering the balance between osteoclastic (bone resorption) and
`
`osteoblast activity comprising administering to a patient in need thereof a conjugate
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 6
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`7
`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 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 conjugate comprising glucagon-like
`
`5 peptide- I 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 disorders; a
`
`10 method for regulating production of sex hormones comprising administering to a subject in
`
`need thereof a conjugate 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 pharmaceutical composition for use in regulating the level of sex
`
`hormones.
`
`15
`
`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
`
`prophylaxis 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
`
`20 disorder in question, and where the interaction between X, when not bound to Z, and the
`
`receptor (or receptor system) has a therapeutic or prophylactic effect on the condition or
`
`disorder.
`
`The present invention also relates to methods for the preparation of said pharmacologically
`
`25 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
`
`recombinant host cell comprising a nucleic acid sequence encoding said conjugate under
`
`conditions permitting the production of said conjugate and (b) isolating said conjugate from
`
`30 the culture.
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`SUBSTITUTE SHEET (RULE 26)
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`8
`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
`
`recombinant DNA methods by isolating said peptide or from commercial sources. X is then
`
`conjugated to Z which is attached to a solid support or has been prepared by solid phase
`
`5 synthetic methods.
`
`Furthermore, the invention relates to the use of a stabilising peptide sequence (Z) for the
`
`preparation of a pharmacologically active peptide conjugate.
`
`1 0 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, p, or y-amino acid (whether in the L-form or the D-
`15 form) as well as side-chain modified amino acids such as modified tyrosines wherein the
`
`aromatic ring is further substituted 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(cid:173)
`
`chain protected amino acids, wherein the amino acid side-chains are protected in accordance
`
`with methods known to the person skilled in peptide chemistry, such as described in, e.g.,
`
`20 M. Bodanszky and A. Bodanszky, "The Practice of Peptide Synthesis", 2. Ed, Springer(cid:173)
`
`Verlag, 1994, and J. Jones, "The Chemical Synthesis 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,
`
`25 either naturally occurring or synthetic which is therapeutically 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
`
`30 be used to prevent a disease state, physiological condition, symptoms or etiological
`
`indications. A pharmacologically active agent is also physiologically or biologically active.
`
`Pharmacological activity measures the effect of a substance (peptide) on physiological or
`
`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 8
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`9
`biological systems in vitro, in vivo or ex vivo and may be assayed using standard in vitro. in
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`vivo or ex vivo assays known in the art for a particular peptide or a peptide with a similar
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`physiological function
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`5 Peptides are utilised in a number of processes, e.g., cell-to-cell communication, some being
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`present in the autonomic and central nervous system. Some of the latter peptides, and a
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`number of other peptides, exert important effects on vascular and other smooth muscles. In
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`a preferred embodiment, X has at the most 75 amino acid residues (or a structure
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`corresponding to at the most 75 amino acid residues). Alternatively, X consists of at most
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`1 0 65, 60, 55, 53, 50, 45, 40, 35, 30, 25, 20, 15, or at the most 10 amino acid residues and
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`consists of at least 2, preferably 5 and more preferably 10 amino acid residues.
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`In the present context, the pharmacologically active peptide sequence X can be any peptide
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`which in its native form is present as the C-terminal free carboxylic acid, such as Leu-
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`15 enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH), or is present in its native form as a C-terminal
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`amide, such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2), or is present in its
`native form as a C-terminal ester. Furthermore, the pharmacologically active peptide may
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`also contain other special structural features such as disulfide bridges as in the case insulin.
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`20 The pharmacologically active peptide may be selected from the group consisting of
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`enkephalin, Leu-enkephalin, Met-enkephalin, angiotensin I, angiotensin II, vasopressin,
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`endothelin, vasoactive intestinal peptide, neurotensin, endorphins, insulin, gramicidin, para(cid:173)
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`celsin, delta-sleep inducing peptide, gonadotropin-releasing hormone, human parathyroid
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`hormone (1-34), truncated erythropoietin analogues described in Wrighton et al., 1996,
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`25 Science 273:458-463), specifically EMP-1, Atrial natriuretic peptide (ANP, ANF), human
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`brain natriuretic peptide (hBNP), cecropin, kinetensin, neurophysins, elafin, guamerin,
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`atriopeptin I, atriopeptin II, atriopeptin III, deltorphin I, deltorphin II, vasotocin, bradykinin,
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`dynorphin, dynorphin A, dynorphin B, growth hormone release factor, growth hormone,
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`growth hormone releasing peptide, oxytocin, calcitonin, calcitonin gene-related peptide,
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`30 calcitonin gene-related peptide II, growth hormone releasing peptide,
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`tachykinin,
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`adrenocorticotropic hormone (ACTH), brain natriuretic polypeptide, cholecystokinin,
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`corticotropin releasing factor, diazepam binding inhibitor fragment, FMRF-amide, galanin,
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`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 9
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`IPR for Patent No. 8,951,962
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`wo 99/46283
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`PCT/DK99/00118
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`10
`gastric releasing polypeptide, gastric inhibitory polypeptide, gastrin, gastrin releasing
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`peptide, glucagon, glucagon-like peptide- I, glucagon-like peptide-2, LHRH, melanin
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`concentrating hormone, melanocyte stimulating hormone (MSH), alpha-MSH, morphine
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`modulating peptides, motilin, neurokinin A, neurokinin B, neuromedin B, neuromedin C,
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`5 neuromedin K, neuromedin N, neuromedin U, neuropeptide K, neuropeptide Y, pituitary
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`adenylate cyclase activating polypeptide (PACAP), pancreatic polypeptide, peptide YY,
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`peptide histidine-methionine amide
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`(PHM),
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`secretin,
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`somatostatin,
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`substance K,
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`thyrotropin-releasing hormone (TRH), kyotorphin, melanostatin (MIF -1 ), thrombopoeitin
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`analogs, in particular AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-
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`1 0 Ala),
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`insulin-like growth factor I (57 -70) (Ala-Leu-Leu-Glu-Thr-Tyr-Cys-Ala-Thr-Pro(cid:173)
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`Ala-Lys-Ser-Glu), insulin-like growth factor 1(30-41) (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg(cid:173)
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`Ala-Pro-Gln-Thr), insulin-like growth factor 1(24-41 )(Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr(cid:173)
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`Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr), insulin-like growth factor II (33-40) (Ser-Arg(cid:173)
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`Val-Ser-Arg-Arg-Ser-Arg), insulin-like growth [tyro] factor II (33-40) (Tyr-Ser-Arg-Val-
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`15 Ser-Arg-Arg-Ser-Arg), insulin-like growth factor II (69-84) (Asp-Val-Ser-Thr-Pro-Pro-Thr(cid:173)
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`Val-Leu-Pro-Asp-Asn-Phe-Pro- Arg-Tyr), growth hormone (GH)-releasing peptide-6
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`(GHRP-6) (His-DTrp-Ala-Trp-DPhe-Lys-NH2), beta-Interleukin I (163-171) (Val-Gln-Gly(cid:173)
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`Glu-Glu-Ser-Asn-Asp-Lys ), beta-Interleukin II ( 44-56) (Ile-Leu-Asn-Gly-Ile-Asn-Asn-Tyr(cid:173)
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`Lys-Asn-Pro-L ys-Leu), Interleukin II ( 60-70) (Leu-Thr-Phe-L ys-Phe-Tyr-Met-Pro-Lys-
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`20 Lys-Ala), exendin-4 (GLP-1 analog) (His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys(cid:173)
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`Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser(cid:173)
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`Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2),
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`exendin-3 (GLP-1 analog) (His-Ser-Asp-Gly-Thr(cid:173)
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`Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp(cid:173)
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`Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser), [Cys(Acm)20,31] epidermal
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`25 growth factor (20-31) Cys(Acm)-Met-His-Ile-Glu-Ser-Leu-Asp-Ser-Tyr-Thr-Cys(Acm),
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`bivalirudin (Hirulog) (D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu(cid:173)
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`Glu-Tyr-Leu), hirulog-1 D-Phe-Pro-Arg-Pro-(Gly)4-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro(cid:173)
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`Glu-Tyr-Leu, C-type natriuretic peptide (1-53) (CNP) (Asp-Leu-Arg-Val-Asp-Thr-Lys-Ser(cid:173)
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`Arg-Ala-Ala-Trp-Ala-Arg-Leu-Leu-Gln-Glu-His-Pro-Asn-Ala-Arg-L ys-Tyr-L ys-G ly-Ala-
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`30 Asn-Lys-Lys-Gly-Leu-Ser-Lys-Gly-Cys-Phe-Gly-Leu-Lys-Leu-Asp-Arg-Ile-Gly-Ser-Met(cid:173)
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`Ser-Gly-Leu-Gly-Cys; Disulfide bridge: Cys37-Cys53), "Mini ANP" (Met-Cys-His(cid:173)
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`cyclohexylAla-Gly-Gly-Arg-Met-Asp-Arg-Ile-Ser-Cys-Tyr-Arg. disulfide bridge cys2-
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`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 10
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`IPR for Patent No. 8,951,962
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`11
`cys13), Melanotan-II (also known as MT-II, alpha-MSH4-10-NH2, or Ac-Nle4-Asp5-His6-
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`D-Phe7-Arg8-Trp9-Lys10), thymosin alphal (TAl) (Ac-Ser-Asp-Ala-Ala-Val-Asp-Thr(cid:173)
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`Ser-Ser-Glu-Ile-Thr-Thr-Lys-Asp-Leu-Lys-Glu-Lys-Lys-Glu-Val-Val-Glu-Glu-Ala-Glu-
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`Asn),
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`om1pressm
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`(also
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`known
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`as
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`8-omithine-vasopressin,
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`(POR-8),
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`5 [Phe2,Ile3, Om8 ]vasopressin),
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`Cys-Phe-Ile-G In-Asn-Cys-Pro-Om-G 1 y-NH2, Disulfide
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`bridge: Cys 1-Cys6), octreotide
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`(20 1-995) (DPhe-Cys-Phe-DTrp-Lys-Thr-Cys-Thr-ol;
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`disulfide bridge: Cys2-Cys7), eptifibatide (INTEGRILIN), calcitonin gene-related peptide
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`(CORP) (Ala-Cys-Asp-Thr-Ala-Thr-Cys-Val-Thr-His-Arg-Leu-Ala-Gly-Leu-Leu-Ser-Arg(cid:173)
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`Ser-Gly-Gly-Val-Val-Lys-Asn-Asn-Phe-Val-Pro-Thr-Asn-Val-Gly-Ser-Lys-Ala-Phe-NH2;
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`1 0 Disulfide bridge: Cys2-Cys7), endomorphin-1 Tyr-Pro-Trp-Phe-NH2; endomorphin-2 Tyr(cid:173)
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`Pro-Phe-Phe-NH2, nociceptin (also known as Orphanin FQ, Phe-Gly-Gly-Phe-Thr-Gly(cid:173)
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`Ala-Arg-Lys-Ser-Ala-Arg-Lys-Leu-Ala-Asn-Gln), angiotensinogen (1-13) (Asp-Arg-Val(cid:173)
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`Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His), adrenomodullin (1-12) (Tyr-Arg-Gln-Ser-Met(cid:173)
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`Asn-Asn-Phe-Gln-Gly-Leu-Arg), antiarrhytmic peptide (AAP) (Gly-Pro-Hyp-Gly-Ala-
`15 Gly), Antagonist G (Arg-DTrp-(nMe)Phe-DTrp-Leu-Met-NH2), indolicidin (Ile-Leu-Pro(cid:173)
`Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2), osteocalcin (37-49) (Gly-Phe-Gln-Glu(cid:173)
`Ala-Tyr-Arg-Arg-Phe-Tyr-Gly-Pro-Val), cortistatin 29 (1-13) (Glp)-Glu-Arg-Pro-Pro-Leu(cid:173)
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`Gln-Gln-Pro-Pro-His-Arg-Asp), cortistatin 14
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`Pro-Cys-Lys-Asn-Phe-Phe-T rp-L ys-Thr(cid:173)
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`Phe-Ser-Ser-Cys-Lys; Disulfide bridge: Cys2-Cysl3, PD-145065 (Ac-D-Bhg-Leu-Asp-Ile-
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`20 Ile-Trp), PD-142893 (Ac-D-Dip-Leu-Asp-Ile-Ile-Trp), fibrinogen binding inhibitor peptide
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`(His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val), leptin (93-1 05) (Asn-Val-Ile-Gln(cid:173)
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`Ile-Ser-Asn-Asp-Leu-Glu-Asn-Leu-Arg), GR 83074
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`(Boc-Arg-Ala-DTrp-Phe-DPro-Pro(cid:173)
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`Nle-NH2), Tyr-W -MIF -1
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`(Tyr-Pro-Trp-Gly-NH2), parathyroid hormone related peptide
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`(107-111) (Thr-Arg-Ser-Ala-Trp), angiotensinogen (1-14) Asp-Arg-Val-Tyr-Ile-His-Pro-
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`25 Phe-His-Leu-Val-Ile-His-Asn, Leupeptin (Ac-Leu-Leu-Arg-CHO), and any modified or
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`truncated analogue thereof.
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`It is well known that many pharmacologically active peptides also exert their desired
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`pharmaceutical effect when present in a modified or truncated form. In the case of, for
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`30 example, insulin, porcine insulin differ from human insulin by only one amino acid residue,
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`the B30 amino acid in porcine insulin being Ala and the B30 amino acid in human insulin
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`being Thr. · Despite this difference, porcine insulin has been used as an effective diabetes
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`SUBSTITUTE SHEET (RULE 26)
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`SANOFI-AVENTIS Exhibit 1009 - Page 11
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`IPR for Patent No. 8,951,962
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`drug for many years. In a similar way it has been found that the essential features for
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`activity in the heptadecapeptide Porcine gastrin I are all contained in the C-terminal
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`tetrapeptide and that essentially all pharmaceutical effects of neurotensin are associated with
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`the C-terminal hexapeptide. Furthermore, pharmacologically active peptides, wherein one
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`5 or more amide bonds have been modified, e.g., reduced, often exhibit a similar or even
`enhanced pharmaceutical activity; for example the C