US005434246A United States Patent (19) 11 Patent Number: 5,434,246 Fukuda et al. 45 Date of Patent: Jul.18, 1995 Biochemistry, 17(16):3188-3191 (1978). 54 PARATHYROID HORMONEDERIVATIVES F. Cohen et al., The Journal of Biological Chemistry, 75) Inventors: Tsunehiko Fukuda, Kyoto; Shizue Nakagawa; Shigehisa Taketomi, both 266(3):1997-2004 (1991). of Osaka, all of Japan Primary Examiner-Howard E. Schain Assistant Examiner-Phynn Touzeau 73) Assignee: Takeda Chemical Industries, Ltd., Attorney, Agent, or Firm-David G. Conlin; Peter F. Osaka, Japan Corless (21) Appl. No.: 33,099 57 ABSTRACT 22 Filed: Mar. 16, 1993 Parathyroid hormone (PTH) derivatives represented by (30) Foreign Application Priority Data the general formula: Mar. 19, 1992 JP Japan .................................. 4-063517 Ser-Val-R1-Glu-Ile-Gln-Leu-Met-His-Asn-Leu Feb. 18, 1993 JP Japan .................................. 5-029283 Gly-Lys-R2-Met-Glu-Arg-Val-Glu-Trp-Leu-R3 51) Int. Cl......................... C07K 14/00; C07K 4/00; Leu-Gln-Asp-Val-His-Asn-R4 A61K 38/23 52 U.S. Cl. .................................................... 530/324 or a salt thereof, wherein R1 represents Ser or a D-a- 58) Field of Search ............... 530/324;930/DIG. 10; amino acid residue of 4 or less carbon atoms; 514/12 R2 represents a tetrapeptide chain which contains at 56) References Cited least one water-soluble a-amino acid residue; U.S. PATENT DOCUMENTS R3 represents a tripeptide chain which contains at least one water-soluble d-amino acid residue; and 4,086, 196 4/1978 Tregear ............................... 530/324 4,086,198 4/1978 Tregear ............................... 530/324 R4 represents an aromatic amino acid residue or an 4,656,250 4/1987 Morita et al. ....................... 530/324 amide thereof, 4,968,669 11/1990 Rosenblatt ............................ S14/12 except that R1 is Ser when R2 is His-Leu-Asn-Ser, R3 is FOREIGN PATENT DOCUMENTS E-F-G 0477885A2 4/1992 European Pat. Off. wherein E is Arg or His, F is Lys or His, G is Lys, Leu WO92/00753 1/1992 WIPO . or Gln, are disclosed. The parathyroid hormone derivatives of the present OTHER PUBLICATIONS invention are stable and have high biological activity, Reeve et al., British Medical J., 7 Jun. pp. 1340-1344 therefore they are useful as drugs for bone diseases and (1980). the like. Lehninger, “Principles of Biochemistry”, Worth Pub. pp. 100-101 (1982). 23 Claims, No Drawings 5,434,246 1. 2 PTH(1-34) fragment, however, no derivative similarly PARATHYROD HORMONE DERVATIVES substituted has been known. BACKGROUND OF THE INVENTION SUMMARY OF THE INVENTION The present, invention relates to novel parathyroid 5 In order to solve the above described problems, the hormone derivatives useful in hormonetherapy. inventors previously substituted one or more amino Parathyroid hormone (PTH) is synthesized in the acid residues of human PTH(1-34) by chemical synthe parathyroid, and plays an important role in controlling sis and proposed several human PTH(1-34) dervatives blood calcium concentrations or phosphoric acid ion by (1) amino acid residue site, considering the concentrations by acting on the bone and the kidney 10 resistance to various proteases, (2) e ancement in ac which are its target organs. PTH is a peptide hormone tivity of the hormone according to the amino acid eS1 consisting of 84 amino acids, and the biological activity due substitution considering the expected two-dimen thereof can be reproduced by a peptide fragment of an sional structure as well as hydrophilic/hydrophobic or N-terminal (1 through 34 amino acid) portion G. W. ionic media, and (3) substitution of the amino acid resi Tregear et al, Endocrinology 93, 1349-1353 (1973). 15 due unstable to acidic, basic or oxidation conditions Th fth t of with an amino acid residue stable to these conditions the te is E. e Epis it. (European Patent Publication No. 477885). As a result PTH (thi ti inaf of further intensive investigation, the inventors have E. Epipes. af now discovered that substitution of the 3-position, 14 1 2 3 4 5 6 7 8 9 10 1 2 3 H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys 14 15 16 17 18. 19 20 2 22 23 24 25 26 His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys 27 28 29 30 3 32 33 34 Lys-Leu-Gln-Asp-Val-His-Asn-Phe-OH (SEQED NO: 1) From the biological action of PTH, it is expected that the use of PTH as a drug will provide a drug useful for 30 position, 15-position, 16-position, 17-position, 25-posi various bone diseases and the like. However, the fol- tion, 26-position, 27-position or 34-position amino acid, lowing properties of the peptide are obstacles to its or combinations thereof provide peptide derivatives efficacious use as a therapeutic agent: having excellent activity. (1) The peptide is easily decomposed by various en- In particular, the present invention provides a peptide zymes within the body; 35 represented by the amino acid sequence: (2) The absorption efficiency of the peptide into the body through various routes is very low; and Ser-Val-R-Glu-Ile-Gln-Leu-Met-His-Asn-Leu (3) The peptide is unstable to various physico-chemi- Gly-Lys-R-2-Met-Glu-Arg-Val-Glu-Trp-Leu-R3 cal conditions such as oxidation. Leu-Gln-Asp-Val-His-Asn-R4 In order to solve such problems and understand the 40 relationship between structure and activity of the above or a salt thereof wherein R1 represents Ser or a D-a- hormone, various derivatives have been synthesized for amino acid residue of 4 or less carbon atoms; the PTH(1-34) fragment. While a number of syntheses R2 represents a tetrapeptide chain which contains at have been conducted for bovine PTH(1-34), few exam- least one water-soluble a-amino acid residue; ples are known for human PTH(1-34). For example in 45 R3 represents a tripeptide chain which contains at one such derivatives, when the C-terminus Phe of least one water-soluble a-amino acid residue; and human PTH(1-34) is converted to Phe-NH2, an in- R4 represents an aromatic amino acid residue or an crease in activity is observed (Japanese Patent Unexam- amide thereof, except that R1 is Ser when R2 is His ined Publication No. 58-96052). This increase in activity Leu-Asn-Ser, is believed to be due to inhibition of carboxypeptidase 50 R3 is E-F-G wherein E is Arg or His, F is Lys or His, which decomposes the hormone. Further, human G is Lys, Leu or Gln. PTH(1-34) contains two Met residues. A molecule in which these residues are substituted with Nile residues DESCRIPTION OF THE INVENTION prevents the hormone from losing its activity due to In the present specification, a water-soluble a-amino oxidation (Japanese Patent Unexamined Publication 55 acid means a naturally occurring or non-natural type No. 61–24598). hydrophilic a-amino acid which has a polar group at Furthermore, F. E. Cohen et al. substituted the 3- the side chain. A naturally occurring water-soluble position Ser of bovine PTH(1-34) with various L-amino a-amino acid is especially preferable. Among them, the acids, but the activity was markedly reduced by the naturally occurring amino acid means a water-soluble amino acid substitution, except that the Ala substituted 60 amino acid constituting a natural protein originating peptide exhibited an activity approximately similar to from animals, plants or microorganisms and an amino that of the natural type peptide The Journal of Biologi- acid which is a metabolite thereof. They may be acidic-, cal Chemistry 226, 1997–2004 (1991)). S. Reppe et al. neutral- and basic- amino acid depending on the polar showed that for the human PTH(1-84) protein in which groups such as a carboxyl, amino, guanidino, carboxam the 26-position Lys was substituted with Gln, the pro- 65 ide, imidazole and hydroxyl group at the side chain. tein had an activity similar to that of the natural type The basic amino acid residue is preferably an L- or protein The Journal of Biological Chemistry 226, D-a amino acid residue represent by the following for 14198-1420 (1991). As to the active human mula: 5,434,246 4 tives, whereby elimination of the peptide from the resin and removal of all protective groups can be achieved in H. , one step. Z-(CH2)-CHCOOH The resulting crude peptide can be purified by known means for purifying peptides or proteins. Examples of wherein Z represents NH2, NHC(NH)NH2 or an imid azole ring, n represents the integer of 1 to 5. such means include column chromatography under Examples of the D-a-amino acids of 4 or less carbon various principles such as gel filtration, ion exchange atoms represented by R1 include neutral amino acids chromatography using a cation exchange resin or an such as D-Ala, D-Asn, D-Cys, D-Ser and D-Thr, and anion exchange resin, hydrophobic chromatography preferably D-a-amino acids of 3 or less carbon atoms O and partition adsorption chromatography, and high such as D-Ser and D-Ala. performance liquid chromatography(HPLC). When R2 of a tetrapeptide chain having at least one The peptides of the present invention can be obtained water-soluble amino acid is represented by A-B-C-D, A in various salt forms. Examples of the salts include salts represents His or a water-soluble amino acid other than of inorganic acids, salts of organic acids such as formic His; B represents Leu or a water-soluble amino acid; C 15 acid, acetic acid, tartaric acid and citric acid, salts of represents Asn or a water-soluble amino acid other than inorganic bases such as sodium and ammonium, and Asn; and D represents Ser or a water-soluble amino acid salts of organic bases such as triethylamine, ethylamine other than Ser.