E7:
`'3: "'
`3;:
`¢\-3%? Attorney Docket‘No.:PPT—20479-US
`
`C
`
`516 nec'dPtQIO 13 JUL 1999
`o9/34159
`PATENT
`
`‘
`:
`
`"5’; V
`33-3
`
`[N THE UNITED STATES DESIGNATED/ELECTED OFFICE (DO/E0/US)
`"
`INTERNATIONAL APPLICATION NO. PCT/DK99/00118
`
`INTERNATIONAL FILING DATE: March 9, 1999
`
`PRIORITY DATE: March 9, 1998
`
`TITLE:
`
`PI-IARMACOLOGICALLY ACTIVE PEPTIDE CONJUGATES HAVING A
`
`.
`
`REDUCED TENDENCY TOWARDS ENZYMATIC HYDROLYSIS
`
`APPLICANT(S) FOR R0/US: Bjame Due Larsen
`
`EXPRESS MAIL CERTIFICATE
`
`Assistant Commissioner for Patents
`Box PCT
`
`Washington, D.C. 20231
`
`_"
`K
`
`Sir:
`Express Mail Label No. EJ445393l27US
`Date of Deposit July 12, 1222
`
`I hereby certify that the following attached paper(s) or fee
`
`OO\IO\£II-#935-)'—'
`
`Transmittal Letter to the DO/E0/US
`
`Preliminary Amendment
`Check for $807.00
`Copy of international application
`Declaration and Power of Attorney
`Assignment Recordation Cover Sheet
`Assignment
`Verified Statement Claiming Small Entity Status
`
`are being deposited with the United States Postal Service “Express Mail Post Office to Addressee
`under 37 C.F.R. 1.10 on the date indicated above and is addresed to the Assistant Commissioner
`
`for Patents, Box PCT, Washington, DC 20231.
`
`Chegl H. Agris
`(Name of person mailing paper(s) or fee)
`
`(signafiofperso%mailing paper(s) or fee)
`
`Mailing address:
`
`P.O. Box 806
`
`Pelham, N.Y. 10803
`(9l4) 712-0093
`
`SANOFI-AVENTIS Exhibit 1013 - Page i
`IPR for Patent No. 8,951,962
`
`

`
`FVJRH HO-U90
`(RIV. L90)
`
`(L8. XPARHER 0? (E13
`FATE?!‘ AND ‘l'lADD¢A.|'ll OFFICE
`
`TRANSMITTAL LETTER TO THE UNITED STATES
`DESIGNATED/ELECTED OFFICE (DO/E0/US)
`
`':93\fl
`
`09/341590
`PCTIPTO
`I 3 JUL 1999
`
`516 '
`
`ATTORNEY'S DOCKET NUMBER
`PPT-20479-US
`
`
`
`INTERNATIONAL APPLICATION NO.
`PCT/DK99/00118
`
`INTERNATIONAL FILING DATE
`March 9. 1999
`
`PRIORITY DATE CLAIMED
`March 9, 1998
`
`TITLE OF INVENTION
`PHARMACOLOGICALLY ACTIVE PEPTIDE CONJUGATES HAVING A REDUCED TENDENCY TOWARDS ENZYMATIC HYDROLYSIS
`
`APPLICANT(S) FOR DO/E0/US
`Bjarne Due Larsen
`
`Applicant herewith submits to the United States Designated/Elected Office (D0/E0/US)
`and other information:
`’///
`This is a PIRST submission of
`l.
`[x]
`_
`
`items concerning a filing under 35 U.S.C. 371.
`
`the following items
`
`[
`
`1 This is a SICOND or SUBSBQUINT submission of
`
`items concerning a filing under 35 U.S.C. 371.
`
`to immediately begin national examination procedures (35 U.S.C. 371(f)) at
`[x) This express request
`any time rather than delay examination until
`the expiration of
`the applicable time limit set
`in 35
`U.S.C. 37l(b) and PCT Articles 22 and 39(1).
`
`[]
`
`(x)
`a.
`b.
`c.
`
`A proper Demand for International Preliminary Examination was made by the 19th month from the
`earliest claimed priority date.
`
`the International Application as filed (35 U.S.C. 371(c)(2))
`A copy of
`[x]
`is transmitted herewith (required only if not
`transmitted by the International Bureau).
`[
`] has been transmitted by the International Bureau.
`[
`]
`is not required. as the application was filed in the United States Receiving Office (R0/US).
`
`A translation of the International Application into English (35 U.S.C. 371(c)(2)).
`]
`[
`[x] Amendments
`to the claims of the International Application under PCT Article 19
`(35 U.S.C.
`371(c)(3))
`transmitted by the International Bureau).
`[
`] are transmitted herewith (required only if not
`[
`] have been transmitted by the International Bureau.
`[
`] have not been made; however,
`the time limit for making such amendments has NOT expired.
`[x] have not been made and will not be made.
`
`a.
`b.
`c.
`d
`
`(35 U.S.C. 371(c)(3)).
`
`the amendments to the claims under PCT Article 19
`A translation of
`1
`(
`[x] An oath or declaration of
`the inventor(s)
`(35 U.S.C. 371(c)(4)).
`[
`1
`A translation of
`the annexes to the International Preliminary Examination Report under PCT Article
`36
`(35 U.S.C. 371(c)(5)).
`the present application
`J Documents
`forming the basis for the examination of
`a.
`(
`]
`the documents of
`the international application published by the International Bureau.
`b.
`[
`]
`the documents of
`the international application published by the International Bureau as
`amended under Article 19 PCT before the International Bureau.
`[
`I Amendments
`to the claims of the International Application under PCT Article 19 are
`transmitted herewith.
`(35 U.S.C. 371(c)(3)).
`I A translation of
`the amendments to the claims under PCT Article 19
`I
`the documents of
`the international application published by the International Bureau as
`amended under Article 34 PCT in the procedure before the International Preliminary Examining
`Authority.
`to the International Preliminary
`I
`] Amendments under Article 34 PCT which are the Annexes
`Examination Report
`(35 U.S.C. 371(c)(5)) are transmitted herewith.
`] A translation of
`the Annexes to the International Preliminary Examination Report under PCT
`Article 36 (35 U.S.C. 371(c)(5)).
`
`[
`
`c
`
`(
`
`I
`
`[
`
`2.
`
`3.
`
`gq.
`fifi
`ti‘:
`5
`%-
`g:
`f_
`ya
`
`.
`
`;§.
`of.
`Fe
`E3
`g
`Fa
`as
`at
`ig.
`ifi.
`§O.
`_5§
`fie
`11.
`
`E §
`
`Items 12.
`
`to 17. below concern other documont(a) or information included:
`
`12.
`
`(
`
`I An Information Disclosure Statement under 37 CPR 1.97 and 1.98.
`
`13.
`
`14.
`
`15.
`
`16.
`17.
`
`[xi An assignment document for recording.
`3.31 is included.
`
`A separate cover sheet
`
`in compliance with 37 CPR 3.28 and
`
`[x]
`[
`]
`I
`]
`
`A FIRST Preliminary Amendment.
`A SECOND or SUBSEQUENT preliminary amendment.
`A substitute specification.
`
`‘
`
`__
`
`.y,[‘
`
`-
`
`[
`[
`
`A change of power of attorney and/or address letter.
`]
`] other items or information:
`
`‘v“7
`
`SANOFI-AVENTIS Exhibit 1013 - Page ii
`IPR for Patent No. 8,951,962
`
`

`
`CLAIMS
`
`(1) FOR
`
`(2) NUMBER FILED
`
`NUMBER EXTRA
`
`(4)
`
`(5)
`CALCULATION
`
`s
`L 1.
`L— s
`
`ATTORNEY'S DOCKET NUMBER
`
`PPT-20479-US
`
`BASIC NATIONAL FEB (37 CPR 1.492(a)(1)-(4)):
`. ..$ 840
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`.
`[
`] Search Report has been prepared by the EPO or JPO .
`I
`]
`International preliminary examination fee paid to USPTO (37 CPR l.48..$ 670
`[
`1 No international preliminary examination fee paid to USPTO (37 CPR 1.482)
`but
`international Search fee paid to USPTO (37 CPR 1.445(a)(2)) .
`.
`.
`.
`. ..$
`[x] Neither
`international preliminary examination fee (37 CPR 1.482) nor
`international search fee (37 CPR 1.445(a)(2)) paid to USPTO .
`.
`.
`.
`.
`.
`.
`.
`. ..$
`International preliminary examination fee paid to USPTO (37 CFR 1.482)
`and all claims satisfied provisions of PCT Article 33(2)
`to (4) .
`.
`.
`.
`. ..$‘
`
`760
`
`970
`
`[
`
`]
`
`96
`
`Surcharge of $130.00 for furnishing the National fee or oath or declaration later
`than [
`] 20 [
`]
`30 mos.
`from the earliest claimed priority date (37 CFR l.492(e)).
`
`TOTAL OF ABOVE CALCULATIONS
`
`=
`
`1534.00
`
`Reduction by 1/2 for filing by small entity,
`filed also.
`(Note 37 CFR 1.9. 1.27. 1.28)
`
`if applicable. Affidavit must be
`
`767.00
`
` 767.00
`Processing fee of $130.
`for furnishing the English Translation later than
`[
`] 20 (
`] 30 mos.
`from the earliest claimed priorit
`date (37 CPR 1.492(f)).
`— mm. mom. m
`Fee for recording the enclosed assignment
`(37 CFR l.21(h)).
`
`s moo
`40.00
`
`+
`
`[x]
`
`A check in the amount of
`
`S 392.99
`
`to cover the above fees is enclosed.
`
`[
`[
`
`c.
`d.
`
`19. The above checked items are being transmitted
`a.
`[x] before the 18th month publication.
`b.
`[
`] after publication and the Article 20 communication but before 20 months from the
`priority date.
`(surcharge and/or processing fee included).
`] after 20 months but before 22 months
`] after 22 months
`(surcharge and/or processing fee included).
`note: Petition to revive (37 CPR 1.137(a) or
`(b))
`is necessary if 35 U.s.C. 371
`requirements submitted after 22 months and no proper demand for International
`Preliminary Examination was made by 19 months from the earliest claimed priority
`date.
`by 30 months and a proper demand for International Preliminary Examination was made by
`the 19th month from the earliest claimed priority date.
`after 30 months but before 32 months and a proper demand for International Preliminary
`Examination was made by the 19th month from the earliest claimed priority date
`(surcharge and/or processing fee included).
`after 32 months
`(surcharge and/or processing fee included).
`Note:Petition to revive (37 CPR l.137(a) or
`(b))
`is necessary if 35 U.S.C. 371
`requirements submitted after 32 months and a proper demand for International
`Preliminary Examination was made by 19 months from the earliest claimed priority
`date.
`
`SEND ALL CORRESPONDENCE T0:
`
`Cheryl H. Agris, Ph.D.
`Attorney at Law
`P.O. Box 806
`
`Pelham, N.Y. 10803
`(914) 712-0093
`
`REGISTRATION NUMBER
`
`SANOFI-AVENTIS Exhibit 1013 - Page iii
`IPR for Patent No. 8,951,962
`
`

`
`1
`_
`,4
`.
`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
`
`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
`
`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
`
`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.
`
`There has been a number of attempts to protect peptides against premature degradation
`
`(reviewed in Prokai, 1997, Exp. Opin. Ther. Patent 7:233-245, Tamai et al., l996, Adv.
`
`Drug Delivery Rev.
`
`l9:401-424 and Zhou et al., 1991, Int. J. Pharm. 75:97-ll5). One
`
`approach includes osmotically altering the blood-brain barrier by infusion of hypertonic
`
`solutions of mannitol, arabinose, lactamide, saline, urea, glycerol and radiographic contrast
`
`agents. However, there could be toxic side effects.
`
`'in‘‘_‘
`
`'::'3
`
`F“:
`
`I 3T=E
`
`3 5
`
`'~"§am}
`
`as
`
`5.?
`32%}
`
`SANOFI-AVENTIS Exhibit 1013 - Page 1
`IPR for Patent No. 8,951,962
`
`

`
`20479P~1!""""--
`
`.~
`
`2
`Another approach involves the use of protease inhibitors (reviewed in Zhou et al., 1991,
`
`Int. J. Pharrn. 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. Phann. 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 [(Giy8)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
`
`SANOFI-AVENTIS Exhibit 1013 - Page 2
`
`IPR for Patent No. 8,951,962
`
`

`
`••
`
`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.
`
`10 Fawell et al., 1994, Proc. Natl. Acad. Sci. USA 91: 664-668 discloses chemically
`
`crosslinking various Tat peptide fragments to fl-galactosidase, RNAse A and domain Ill 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-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
`
`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 preparing 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 prodru_gs of [o-Pe!(, o-Pen5
`]
`(.S~q .l'> NO· lJ
`
`30 enkephalin IDPDPE) and [o-Pen2; L-Cys51 enkee..halin (DPLCE), specifically DPLCE-Arg-
`\.S£.Q l.O NO. "t\ G&!O ~o ~&)
`(.St.Q ...I.O 1-\0 • .).)
`Pro-Ala,I\DPDPE-Phe.J>PLCE-Pne~\ DPDPE-Arg-Gly J\ DPLCE-Arg-Gly \JDPDPE-Phe-Ala-
`(lt~ 10 Nl). l)
`(S\:Q .lO No. b)
`
`SANOFI-AVENTIS Exhibit 1013 - Page 3
`
`IPR for Patent No. 8,951,962
`
`

`
`,-..
`20479PC.
`
`(Stq .lll NO. i)
`(&~Q ~ No.l)
`NH-C6H13~ DrFU·~~-~1)0NHk The half lives of most of the analogs, except for
`DPDPE-Arg-Gl~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 differef~{:Jf8e~" t)
`equigped with a peptide !'re-sequence but without linker, namely DSIP-(Lys-Giu)3.. DSIP-
`ttO. '9
`"
`(&Co U
`lSC' .lO NO,\). ~CQ 'lO N.O. \0)
`(Glu)% Leu-enkephhlin-(Giu)~and Leu-eilkephalin-(LysJ%
`
`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
`
`SANOFI-AVENTIS Exhibit 1013 - Page 4
`
`IPR for Patent No. 8,951,962
`
`

`
`r--
`20479PC.
`
`.5
`
`free phannacologically 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 phanna~ologically 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 phannacologically
`
`active, i.e. the peptide conjugate possesses the ability to exert the phannacological function
`
`of the free phannacologically active peptide.
`
`1 0 Thus, in a first aspect the invention relates to a phannacologically active peptide conjugate
`
`having a reduced tendency towards enzymatic cleavage, said peptide conjugate comprises: a
`
`phannacologically 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
`
`1 5
`
`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
`
`(I)
`
`20
`
`wherein R 1 and R2 are selected from the group consisting of hydrogen, C1-6-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
`
`~
`
`~ .. ~-;-J
`
`~!;
`~,Y
`
`selected from cl-6-alkyl, c2-6-alkenyl, halogen, hydroxy, amino, cyano, nitro, sulfono, and
`25 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-diaminobutanoic acid (Dbu) and 2,3-
`_\f/ ~
`/1\,
`
`diaminopropanoic acid (Dpr); and
`
`wherein the ratio between the half-life of said peptide conjugate and the half-life of the
`
`30 corresponding phannacologically 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 37·c or in plasma or serum is at least about 2, preferably at least
`
`SANOFI-AVENTIS Exhibit 1013 - Page 5
`
`IPR for Patent No. 8,951,962
`
`

`
`.~
`
`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 of H-Trp-A1a-Gly-Gly-Asp-Ala-Ser-Gly-Glu-(Lys-Glu)3-0H,
`H-Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gjy-Glu-(Glu)6-0H,
`C~~Q :tO N(). lO)
`H-Tyr-Gly-Gly-Phe-Leu-(Glu)6·0li\and
`(S.tQ J..P loU. H)
`1 0 H-Tyr-Gly-Gly-Phe-Leu-(Lys)6·0J:\.
`
`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
`15 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
`
`SANOFI-AVENTIS Exhibit 1013 - Page 6
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`IPR for Patent No. 8,951,962
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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 phannaceutical 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 phannaceutical composition for use in treating sleep disorders; a
`1 0 method for regulating production of sex honnones 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 phannaceutical 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 phannaceutical 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 phannacologically
`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 nennittino tht'! nrndnrtinn nf ~jtf 1'1\nitiO!ltP !lntf fh\ ie!nlotinn .,..;..1 ,...,. .. :.,r.n~"' f .. ,. .....
`
`SANOFI-AVENTIS Exhibit 1013 - Page 7
`
`IPR for Patent No. 8,951,962
`
`

`
`z—.
`
`a
`
`204'/sec.
`
`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
`
`synthetic methods.
`
`Furthermore, the invention relates to the use of a stabilising peptide sequence (2) for the
`
`preparation of a pharmacologically 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 at, B, 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 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-
`
`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.,,
`
`M. Bodanszky and A. Bodanszky, “The Practice of Peptide Synthesis”, 2. Ed, Springer-
`
`Verlag, 1994, and J. Jones, “The Chemical Synthesis of Peptides”, 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 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
`
`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
`
`SANOFI-AVENTIS Exhibit 1013 - Page 8
`IPR for Patent No. 8,951,962
`
`
`
`
`
`'li“tliii.’tlzlu
`
`"'3"1
`'3
`i’‘=7
`
`5 5
`
`"“!ass
`
`:33
`
`tlifuliifiuEEK"i
`
`

`
`C!
`
`'i;i.
`
`d"! = a. ~ w
`~
`a 1
`F
`H'i
`~
`
`9
`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 peptide or a peptide with a similar
`
`physiological function
`
`5 Peptides are utilised in a number of processes, e.g., cell-to--cell communication, some being
`
`present in the autonomic and central nervous system. Some of the latter peptides, and a
`
`number of other peptides, exert important effects on vascular and other smooth muscles. In
`a preferred embodiment, X has at the most 75 amino acid residues (or a structure
`corresponding to at the most 75 amino acid residues). Alternatively, X consists of at most
`10 65, 60, 55, 53, 50, 45, 40, 35, 30, 25, 20, 15, or at the most 10 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 peptide sequence X can be any peptide
`
`which in its native form is present~ the C-terminal free carboxylic acid, such as Leu(cid:173)
`W\Q JO NO. 1~)
`15 enkephalin (H· Tyr-Gly-Gly-Phe-Leu-OHk or is present in its native form as a C-terminal
`(.StQ l9 ~~ 1.{)
`amide, such as oxytocin (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-N"H2~, or is present in its
`native form as a C-terminal ester. Furthermore, the pharmacologically active peptide may
`
`also contain other special structural features such as disulfide ~ridges as in the case insulin.
`
`20 The pharmacologically active peptide may be selected from the group consisting of
`enkephalin, Leu-enkephalin, Met-enkephalin, angiotensin I, angiotensin II, vasopressin,
`
`endothelin, vasoactive intestinal peptide, neurotensin, endorphins, insulin, gramicidin, para(cid:173)
`celsin, delta-sleep inducing peptide, gonadotropin-releasing hormone, human parathyroid
`
`hormone (1-34), truncated erythropoietin analogues described in Wrighton et al., 1996,
`
`25 Science 273:458-463), specifically EMP-1, 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, vasotocin, bradykinin,
`
`dynorphin, dynorphin A, dynorphin B, growth hormone release factor, growth hormone,
`
`·~
`
`~
`
`:: =
`=
`C:
`; ~
`w
`
`~~
`
`:.f~
`~
`~D
`
`SANOFI-AVENTIS Exhibit 1013 - Page 9
`
`IPR for Patent No. 8,951,962
`
`

`
`-·
`
`10
`gastric releasing polypeptide, gastric inhibitory polypeptide, gastrin, gastrin releasing
`
`peptide, glucagon, glucagon-like peptide-I, glucagon-like peptide-2, LHRH, melanin
`
`concentrating honnone, melanocyte stimulating honnone (MSH), alpha-MSH, morphine
`modulating peptides, motilin, neurokinin A, neurokinin B, neuromedin B, neuromedin C,
`5 neuromedin K, neuromedin N, neuromedin U, neuropeptide·K, neuropeptide Y, pituitary
`
`adenylate cyclase activating polypeptide (P ACAP), pancreatic polypeptide, peptide YY,
`peptide histidine-methionine amide
`(PHM), secretin, somatostatin, substance K,
`thyrotropin-releasing honnone (TRH), kyotorphin, melanostatin (MIF-1 ), thrombopoeitin
`anQ.Iogs, in particqlar AF 12505 (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg·
`ts~Q lD ~. 1'1)
`insulin-like growth f~ctor I (57-70) (Ala-Leu-Leu-Glu-Thr· Tyr-Cys-Ala-Thr-Pro-
`10 Ala),,
`CllQ r-~ No. lS;
`11
`Ala-Lys-Ser-Gtu), ·insulin-liKe growth factor 1(3041} (Gly-Tyr-Gly-Ser-Ser-Ser-Arg-Arg-
`
`,
`
`#'<
`
`.. I
`
`Ala-Pro-Gln~Thr), insulin-like growth factor 1(24'"41 )(Tyr-Phe-Asn-Lys-Pro-Thr-Gly-Tyr(cid:173)
`
`Gly-Ser-Ser-Ser-Arg-Arg-Ala-Pro-Gln-Thr), insulin-like growth factor II (33-40) (Ser-Arg·
`c&' Q ll> ~o '')
`Val-Ser-Arg-Arg-Ser-Arg), insulin-like growth [tyro] factor II (3340) (Tyr-Ser-Arg-Val-
`Ui Q .fb ~o. '~
`15 Ser-Arg-Arg-Ser-Argk insulin-like growth factor II (69-84)~sp Vel Ser 'Fhr Pre Pr.G llv-
`Val-Leu-Pro-Asp-Asn-Phe-Pro· Arg-Tyr}, growth honnone (GH)-releasing peptide-6
`(GHRP-6) {Hts::DTrp-Ala-Trp-DPhe-tys ~U 12}, beta-Interleukin I (163-171) (V al-Gin-Gly(cid:173)
`CSlQ J.» NO.~
`Glu-Glu-Ser-Asn-Asp-Lys), beta-lnterleuk.in II .(44-56) (lle-Leu-Asn-Gly-Ile-Asn-Asn-Tyr(cid:173)
`CS~Q ~~o. d-3)
`Lys-Asn-Pro-Lys-Leu1, lnterleuk.in II (60-70) (Leu· Thr-Phe-Lys-Phe· Tyr-Met-Pro-Lys(cid:173)
`(S'o ;z,o N~. ~ ~
`20 Lys-Ala~ exendin-4 (GLP-1 analog){Hisc6ly.OIU-6ly.fht-Phe-Tin•Ser Asp·beu Ser•Lys·
`. .Qla hfet-etlo-Gio-61u-Ma•.Yai-AtgaJ5eu=Phe-lto.6hi- rrp-Lea-Lys-7\ss Gly Gly Pre Ser
`~- Ser-Gly-Ala-Pro-Pro-Pro·Ser-NH2)~ exendin-3 (GLP-1 analog) (His-Ser-Asp-Gly-Thr-
`
`Phe· Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Gjy-Ala-V al-Ar~·Leu-Phe-Ile-Glu-Trp·
`()~Q :r,o .-.o. ~-)
`Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser)~ [Cys(Acm)20,31] epidennal "" \
`(StQ :J.I) NO·""')
`25 growth factor (20-31) Cys(Acm)-Met-His-lle-Glu-Ser-Leu·Asp-Ser-Tyr-Thf-C