Claims for Patent: 6,849,710

Summary for Patent: 6,849,710

Title:	Method for the synthesis of analogs of parathyroid hormone and parathyroid hormone related peptide
Abstract:	A fragment condensation process for the synthesis of analogs of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP), in which amino acid residues (22-31) form a synthetic amphipathic .alpha.-helix, is provided.
Inventor(s):	Arzeno; Humberto (Cupertino, CA)
Assignee:	F. Hoffmann-La Roche AG (Basle, CH)
Application Number:	08/903,124
Patent Claims:	1. A process for the synthesis of a synthetic polypeptide analog of parathyroid hormone (PTH) or parathyroid hormone related peptide (PTHrP), or salt thereof, in which amino acid residues (22-31) form an amphipathic .alpha.-helix, said residues (22-31) selected from the group consisting of (SEQ ID NOS: 85, 86, 26, 27, 28, 29, and 30), which process comprises: a) independently synthesizing precursor peptide fragments of the desired polypeptide on a solid resin support; b) cleaving all but the intended ultimate C-terminal precursor peptide fragment of the desired polypeptide from their respective resin supports; c) sequentially condensing said cleaved precursor peptide fragments with the resin bound C-terminal peptide fragment to form the desired polypeptide product; d) removing side chain protecting groups; and e) cleaving the polypeptide product from the resin support, wherein at least two of the fragments are coupled via leucine-leucine bonding. 2. A process of claim 1 in which the polypeptide product is prepared from three precursor peptide fragments: an N-terminus, a middle, and a C-terminus fragment. 3. A process of claim 2 in which the N-terminus fragment has a C-terminal glycine, the middle fragment has a C-terminal leucine, and the C-terminus fragment has an N-terminal leucine. 4. A process of claim 3 in which the final polypeptide product comprises a PTH or PTHrP analog of the formula: Term, wherein: Xaa.sup.1 is absent or is Ala; Xaa.sup.2 is absent or is Val; Xaa.sup.3 is absent or is Ser; Xaa.sup.4 is absent or is Glu or Glu(OCH.sub.3); Xaa.sup.5 is absent or is His or Ala; Xaa.sup.6 is absent or is Gln; Xaa.sup.7 is absent or is Leu; Xaa.sup.11 is Lys, Arg, or Leu; Xaa.sup.13 is Lys, Arg, Tyr, Cys, Leu, Cys(CH.sub.2 CONH(CH.sub.2).sub.2 NH(biotinyl)), Lys(7-dimethylamino-2-oxo-2H-1-benxopyran-4-acetyl), or Lys(dihydrocinnamoyl); Xaa.sup.20 is Arg or Leu; Xaa.sup.19 and Xaa.sup.21 are independently Lys, Ala, or Arg; Xaa.sup.22-31 is selected from (SEQ ID NOS:85, 86, 26, 27, 28, 29, or 30); Xaa.sup.32 is His, Pro, or Lys; Xaa.sup.33 is absent, or is Pro, Thr, Glu, or Ala; Xaa.sup.34 is absent, or is Pro, Arg, Met, Ala, hSer, hSer lactone, Tyr, or Leu; Xaa.sup.35 is absent or is Pro, Glu, Ser, Ala, or Gly; Xaa.sup.36 is absent or is Ala, Arg, or Ile; Xaa.sup.37 is absent or is Arg, Trp, or 3-(-2-naphthyl)-L-alanine; Xaa.sup.38 is absent or is Ala or hSer or Xaa.sup.38-42 is Thr Arg Ser Ala Trp; and Term is OR or NR.sub.2 where each R is independently H, (C.sub.1 -C.sub.4)alkyl or phenyl(C.sub.1 -C.sub.4)alkyl; and the pharmaceutically acceptable salts thereof. 5. A process of claim 4 in which the polypeptide product is prepared from three precursor peptide fragments: an N-terminus, a middle, and a C-terminus fragment. 6. A process of claim 1 in which the polypeptide analog of PTH or PTHrP comprises the formula: Term, wherein: Xaa.sup.1 is Ser or Ala; Xaa.sup.7 is Leu or Phe; Xaa.sup.8 is Leu, Met, or Nle; Xaa.sup.11 is Leu or Lys; Xaa.sup.16 is Asn or Ser; Xaa.sup.18 is Leu, Met, or Nle; Xaa.sup.19 is Glu, Thr, or Arg; Xaa.sup.21 is Val, Ser, or Arg; Xaa.sup.22-31 is selected from (SEQ ID NOS: 26, 27, 28, 29, or 30); Xaa.sup.34 is Phe, hSer, or Tyr; Term is OR or NR.sub.2, where R is H or a (C.sub.1 -C.sub.4)alkyl; and the pharmaceutically acceptable salts thereof. 7. A process of claim 6 in which the polypeptide product is prepared from three precursor peptide fragments: an N-terminus, a middle, and a C-terminus fragment. 8. A process of claim 1 in which the PTH or PTHrP analog is selected from the group consisting of: AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH.sub.2 (SEQ ID NO:7) AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-OH (SEQ ID NO:6) AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA-OH (SEQ ID NO:15) AVSEHQLLHDRGRSIQDLRRRELLERLLERLHTA-OH (SEQ ID NO:16) AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH (SEQ ID NO:17) AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA-OH (SEQ ID NO:5) AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAGRR-OH (SEQ ID NO:10) AVSEAQLLHDLGKSIQDLRRRELLEKLLEKLHAL-OH (SEQ ID NO:14) AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL-NH.sub.2 (SEQ ID NO:11) AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY-NH.sub.2 (X=Nle, SEQ ID NO:23) AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY-NH.sub.2 (X=Nle, SEQ ID NO:24) AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA-NH.sub.2 (SEQ ID NO:20) AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA-NH.sub.2 (SEQ ID NO:12) AVSEHQLLHDKGKSIQDLRRAELLEKLLEKLHTA-NH.sub.2 (SEQ ID NO:13) AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA-NH.sub.2 (SEQ ID NO:21) AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA-NH.sub.2 (SEQ ID NO:22) AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH.sub.2 (SEQ ID NO:35) AVSEHQLLHD KGKSIQDLKL KELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:38) AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTA-NH.sub.2 (SEQ ID NO:39) AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAP-OH (SEQ ID NO:40) AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRR-OH (SEQ ID NO:41) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTY-NH.sub.2 (SEQ ID NO:43) AVSEHQLLHD KGYSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:44) AVSEHQLLHD KGCSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:45) AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:46) (X=Cys(CH.sub.2 CONH(CH.sub.2).sub.2 NH(biotinyl)) AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:47) (X=Lys(7-dimethylamino-2-oxo-2H-1-benxopyran-4-acetyl)) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAG-OH (SEQ ID NO:48) AVSX.sub.1 HQLLHX.sub.2 KGKSIQX.sub.2 LRR RX.sub.1 LLX.sub.1 KLLX.sub.1 K LHA-OH (SEQ ID NO:49) (X.sub.1 =Glu(OCH.sub.3); X.sub.2 =Asp(OCH.sub.3)) AVSX.sub.1 HQLLHX.sub.2 KGKSIQX.sub.2 LRR RX.sub.1 LLX.sub.1 KLLX.sub.1 K LHA-OCH.sub.3 (SEQ ID NO:50) (X.sub.1 =Glu(OCH.sub.3); X.sub.2 =Asp(OCH.sub.3)) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAP-OH (SEQ ID NO:52) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-OH (SEQ ID NO:53) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-NH.sub.2 (SEQ ID NO:54) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH.sub.2 (SEQ ID NO:55) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH.sub.2 (SEQ ID NO:56) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-OH (SEQ ID NO:57) AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-OH (SEQ ID NO:58) AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-NH.sub.2 (SEQ ID NO:59) AVSEHQLLHD RGXSIQDLRR RELLERLLER LHTAGRRTRSAW-OH (SEQ ID NO:60) (X=Lys(dihydrocinnamoyl)) AVSEIQFXHN LGKHLSSXTR SAWLRKKLQD VHNY-NH.sub.2 (SEQ ID NO:61) (X=norleucine) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTMA-NH.sub.2 (SEQ ID NO:62) AVSEHQLLHD KGKSIQDLRR RFFLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:64) AVSEHQLLHD KGKSIQDLRR RELLHKLLEK LHTA-NH.sub.2 (SEQ ID NO:65) AVSEHQLLHD KGKSIQDLRR RELLEHLLEK LHTA-NH.sub.2 (SEQ ID NO:66) AVSEHQLLHD KGKSIQDLRR RELLEKLIAK LHTA-NH.sub.2 (SEQ ID NO:67) AVSEHQLLHD KGKSIQDLRR RELLEKLLEE IHTA-NH.sub.2 (SEQ ID NO:68) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH.sub.2 (SEQ ID NO:72) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAX-OH (SEQ ID NO:73) (X=Nal(2)=3-(2-naphthyl)-L-alanine) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTASAW-OH (SEQ ID NO:74) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIRA-OH (SEQ ID NO:75) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIR-OH (SEQ ID NO:76) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEI-OH (SEQ ID NO:77) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAE-OH (SEQ ID NO:78) SEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:80) LLHD KGKSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:81) LHD KGKSIQDLRR RELLEKLLEK LHTA-NH.sub.2 (SEQ ID NO:82) SEHQLLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH (SEQ ID NO:83) LLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH (SEQ ID NO:84) AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX (X=hSerlac, SEQ ID NO:9) AVSEIQFX.sub.1 HN KGKHLSSX.sub.1 ER VEWLRKKLQD VHNX.sub.2 (SEQ ID NO:79) (X.sub.1 =L-norleucine; X.sub.2 =homoserine lactone) AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX-NH.sub.2 (X=hSer, SEQ ID NO:8) AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNX-NH.sub.2 (SEQ ID NO:36) (X=homoserine) AVSEIQFLHN KGKHLSSLRR RELLEKLLEK LHNX-NH.sub.2 (SEQ ID NO:37) (X=homoserine) AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRRX-NH.sub.2 (SEQ ID NO:42) (X=homoserine) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH.sub.2 CH.sub.3 (SEQ ID NO:69) (X=homoserine) AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH.sub.2 CH.sub.2 C.sub.6 CH.sub.5 (SEQ ID NO:70) (X=homoserine), and AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-OH (SEQ ID NO:51) (X=homoserine). 9. A process of claim 8 in which the polypeptide product is prepared from three precursor peptide fragments: an N-terminus, a middle, and a C-terminus fragment. 10. A process for the synthesis of the polypeptide of SEQ ID NO:7, AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH.sub.2, which process comprises: a) independently synthesizing precursor peptide fragments of SEQ ID NO:7 on a solid resin support; b) cleaving all but the intended ultimate C-terminal precursor peptide fragment of SEQ ID NO:7 from their respective resin supports; c) sequentially condensing said cleaved precursor peptide fragments with the resin bound C-terminal peptide fragment to form the polypeptide of SEQ ID NO:7; d) removing side chain protecting groups; and e) cleaving the polypeptide of SEQ ID NO:7 from the resin support, wherein at least two of the fragments are coupled via leucine-leucine bonding. 11. A process of claim 10 in which the polypeptide product is prepared from three precursor peptide fragments: an N-terminus, a middle, and a C-terminus fragment. 12. A process of claim 11 wherein said first fragment comprises AVSEHQLLHDKG (SEQ ID No. 87) said second fragment comprises KSIQDLRRREL (SEQ ID No. 88), and said third fragment comprises LEKLLEKLHTA (SEQ ID No. 89). 13. A process of claim 12 wherein said third fragment is formed by the condensation of LEKL, LEKL, and HTA. 14. The synthetic polypeptide of the sequence AVSEHQLLHDKG (SEQ ID No. 87). 15. The synthetic polypeptide of the sequence KSIQDLRRREL (SEQ ID No. 88). 16. The synthetic polypeptide of the sequence LEKLLEKLHTA (SEQ ID No. 89). 17. The synthetic polypeptide of the formula KSIQDLRRRE (SEQ ID No. 90). 18. The synthetic polypeptide of the formula LLEKLLEKLHTA (SEQ ID No. 91). 19. The process of claim 1 wherein the resin bound C-terminal peptide fragment comprises between 6 and 12 amino acids. 20. The process of claim 10 wherein the resin bound C-terminal peptide fragment comprises between 6 and 12 amino acids.

Details for Patent 6,849,710

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Nps Pharmaceuticals, Inc.	NATPARA	parathyroid hormone	For Injection	125511	01/23/2015		2016-07-30