EP0539491A1 - An positionen 3,6 oder 9 veränderte parathyroidhormonanaloga - Google Patents

An positionen 3,6 oder 9 veränderte parathyroidhormonanaloga

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Publication number
EP0539491A1
EP0539491A1 EP91913697A EP91913697A EP0539491A1 EP 0539491 A1 EP0539491 A1 EP 0539491A1 EP 91913697 A EP91913697 A EP 91913697A EP 91913697 A EP91913697 A EP 91913697A EP 0539491 A1 EP0539491 A1 EP 0539491A1
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Prior art keywords
ser
val
glu
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ala
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English (en)
French (fr)
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EP0539491A4 (de
Inventor
Fred A. Cohen
Robert A. Nissenson
Gordon J. Strewler
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University of California
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University of California
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to polypeptide analogs which have agonist or antagonist properties relative to parathyroid hormone, (PTH), parathyroid hormone-like protein (PLP) or parathyroid-related protein (PTHrP).
  • PTH parathyroid hormone
  • PBP parathyroid hormone-like protein
  • PTHrP parathyroid-related protein
  • the serine amino acid at position 3, the glutamine amino acid at position 6, or the histidine amino acid at position 9, or combinations thereof, are substituted by other natural or synthetic amino acids.
  • a human PTH fragment of about 34 amino acids is sufficient for useful pharmacological activity.
  • potent PTH agonists and/or antagonists have been intensive.
  • the availability of potent and specific antagonists would provide a powerful research tool for the study of the mechanism of action and physiological and/or pathological role for PTH.
  • Some research efforts have resulted in in vitro PTH antagonists. However, during in vivo evaluation of these polypeptides, they often did not have any clear antagonist properties.
  • PTH Parathyroid hormone
  • PTH(1-34) is a full agonist of the native 84 amino-acid hormone with respect to adenylate cyclase activation in canine renal membranes (See Ref. 1 below.
  • the letters used are the conventional ones to describe an amino acid sequence).
  • PTH-related protein is found in a variety of human and animal tumors, and evidence suggests that the protein plays a pathogenetic role in the hypercalcemia that frequently accompanies malignant disease (Ref. 19,20).
  • hPTH or bPTH, etc. analogs have been reported where the amino acids are substituted in the 1-9 amino acid positions.
  • the present invention provides a method to select and to produce novel hPTH or bPTH, etc. analogs where the amino acids at positions 3 and/or 6 and/or 9 have been replaced using the natural or synthetically made unnatural (unusual) amino acids.
  • These analogues replaced at the 3, 6, or 9 positions have surface side chains which are useful to modulate receptor binding and activity.
  • analogues are useful as agonists or antagonists in the treatment of a number of disease conditions, particularly osteoporosis.
  • the present invention concerns a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the formula:
  • amino acid B at position 3 is independently selected from L-serine or those other natural or synthetic
  • D or L amino acids having a spatial volume comparable to or greater than serine, with the proviso that B is not glycine,
  • amino acid R at position 6 is independently selected from L-glutamine or from other natural or synthetic D or L amino acids or mixtures of the D and L amino acids,
  • the amino acid X at position 9 is independently selected from L-histidine or other natural or synthetic D or L-amino acids, with the proviso that when group B is L-serine and group J is L-glutamine, Group X is not histidine, when group B is L-serine and group X is histidine, J is not glutamine, and when group J is glutamine and group X is histidine, group B is not L-serine, and
  • amino acid at position 1 is serine
  • amino acid at position 7 is leucine
  • amino acid at position 16 is Asn
  • amino acid at position 18 is methionine, i.e. a modified analog of human PTH.
  • the amino acid at position 1 is alanine
  • the amino acid at position 7 is phenylalanine
  • the amino acid at position 16 is serine
  • the amino acid at position 18 is methionine, i.e. a modified analog of bovine PTH.
  • Z, B or J are independently selected as follows:
  • B is a synthetic amino acid
  • B is a naturally occurring amino acid
  • J is a synthetic amino acid
  • J is a naturally occurring amino acid
  • B is L-serine and J is selected from Leu, Phe, Ala, Glu, Ser or Phe; or
  • J is L-glutamine and B is independently selected from Ala, Phe, Gln, Glu, Lys, His, Leu, or Tyr.
  • the invention also relates to a compound of wherein J is independently selected from L+ Serine, Ala, Phe, Gin, Glu, Lys, His or Tyr.
  • J is independently selected from L-glutamine, Leu, Phe, Ala, Glu, Ser or Phe.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of structure I or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable excipient.
  • the present invention relates to pharmaceutical compositions comprising a compound of the structure for hPTH(1-34) wherein Z is selected -COOH or
  • the invention also relates to a method of treatment of a mammal in need of therapeutic treatment, which method comprises administration of a therapeutically effective amount of the peptide analog of a compound of Structure I or a pharmaceutically acceptable salt thereof in admixture with a pharmaceutically acceptable excipient.
  • the invention also relates to administration of a therapeutically effective amount of a compound of structure I by oral, parenteral, subcutaneous, intramuscular, intravenous, vaginal, rectal, buccal, sublingual or intranasal means.
  • the invention also relates to a method of therapeutic treatment, wherein the compound of structure I is used to treat cancer, osteoporosis, hypercalcemia or hyperparathyroid conditions in a human being.
  • the present invention relates to a method for selecting polypeptide sequences modified at the 3, 6, 9 position or combinations thereof of a PTH or PTH(1- 34) useful in pharmaceutical compositions, which method comprises:
  • Figure 1 is a representation of aligned sequences of PTH(1-34) from five species of mammals and human and chicken PTHrP (1-34). Positions of sequence identity are highlighted with solid lines.
  • Figure 1A is representation of the 1-84 amino acids of human PTH, bovine PTH and porcine PTH.
  • Figure 2 is a representation of secondary structural features of the 1-34 peptides of bPTH(l-34) and human PTHrP(1-34) as predicted by statistical and pattern-based theoretical approaches.
  • the arrows indicate predicted alpha-helical structure, and the wavy line represents a possible beta-turn.
  • Figure 3 is the circular dichroism (CD) spectra of peptides in the presence and absence of 45% trifluoroethanol. Nadirs at 208 nm and 222 nm are characteristic of a-helical structure.
  • the peptides analyzed are: Figure 3A, bPTH(l-34); Figure 3B [Tyr 34 ]bPTH(7-34)amide; and Figure 3C, hPTHrP(l-34)amide.
  • Figure 4 is a representation of the effect of trifluoroethanol on the alpha-helical content of bPTH(l-34) (0), [Tyr 34 ]bPTH(7-34) amide ( ⁇ ), and hPLP (1-34) amide ( ⁇ ).
  • the alpha-helical content was determined by deconvolution of circular dichroism spectra as described by Taylor and Kaiser (Ref. 29).
  • Figure 5 is a schematic representation of the paired helix model for bPTH(1-34) and hPTHrP(1-34). Positions of relative sequence conservataon are shown. The hydrophobic core, binding region, and trigger region are designated.
  • Figure 6 shows graphs of the biologic activity in canine renal plasma membranes of analogues of PTH(1-34) containing substitutions at positions 3,6, and 9.
  • Figure 6A shows adenylate cyclase (AC) activation produced by bPTH(1-34) (o), and [Glu 3 ]bPTH(l-34) ( ⁇ ), [His 3 ]bPTH(1-34) (v), [Lys 3 ]bPTH(1-34) (Y), and [Gln 3 ]bPTH (1-34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 6B shows adenylate cyclase (AC) activation produced by bPTH(1-34) (o), [Ala 3 ]bPTH( 1-34) (•), [Phe 3 ] bPTH( 1-34) ( ⁇ ), [Leu 3 ] bPTH( 1-34) ( ⁇ ), and [Tyr 3 ]bPTH(l34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 6C shows adenylate cyclase activation produced by bPTH(1-34) (o), [Ala 6 ] bPTH( 1-34) (•), [Phe 6 ] bPTH( 1-34) ( ⁇ ) [Leu 6 ]bPTH(1-34) ( ⁇ ), [Ser 6 ]bPTH(1-34) ( ⁇ ), [Glu 6 ]bPTH(1 ⁇ 34) ( ⁇ ), and [Phe 6 ] bPTH( 1-34) ( ⁇ ).
  • Figure 6D shows competitive binding to PTH receptors by bPTH(1-34) (o), [Ala 3 ] bPTH( 1-34) (•), [Phe 3 ]bPTH(1-34) ( ⁇ ), [Leu 3 ]bPTH(1-34) ( ⁇ ), and [Tyr 3 ]bPTH(1-34) ( ⁇ ).
  • Figure 6E shows competitive binding to PTH receptors by bPTH(1-34) (o), [GlU 3 ]bPTH(1-34) ( ⁇ ), [His 3 ] bPTH( 1-34) ( ⁇ ), [Lys 3 ]bPTH(1-34) ( ⁇ ), and [Gln 3 ]bPTH(1-34) ( ⁇ ).
  • Figure 6F shows competitive binding to PTH receptors by bPTH(1-34) (o), [Ala 6 ] bPTH( 1-34) (•), [Phe 6 ] bPTH( 1-34) ( ⁇ ), [Leu 6 ] bPTH( 1-34) ( ⁇ ), [Ser 6 ]bPTH(1-34) ( ⁇ ), and [GlU 6 ]bPTH(1-34) ( ⁇ ).
  • Figure 7 shows graphs of the biologic activity in UMR 106-H5 cells of analogues of PTH (1-34) containing substitutions at positions 3, 6, and 9.
  • Figure 7A shows adenylate cyclase (AC) activations produced by bPTH(1-34) (o), [Ala 3 ] bPTH( 1-34) (•), [Phe 3 ]bPTH(1-34) ( ⁇ ) , [Leu 3 ] bPTH( 1-34) ( ⁇ ), and [Tyr 3 ]bPTH(1-34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 7B shows adenylate cyclase (AC) activation produced by bPTH(1-34) (o), [His 3 ]bPTH(1-34) ( ⁇ ), [Lys 3 ] bPTH(1-34) ( ⁇ ), and [Gln 3 ]bPTH(1-34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 7C shows adenylate cyclase (AC) activation produced by bPTH(1-34) (o), [Ala 6 ] bPTH( 1-34) (•), [Phe 6 ]bPTH(1-34) ( ⁇ ), [Leu 6 ] bPTH( 1-34) ( ⁇ ), [ Ser 6 ] bPTH( 1-34) ( ⁇ ), and [Glu 6 ]bPTH(1-34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 7D shows competitive binding to PTH receptors by bPTH(1-34) (o), [Ala 3 ]bPTH(1-34) (•) [Phe 3 ] bPTH( 1-34) ( ⁇ ), [Leu 3 ]bPTH(1-34) ( ⁇ ), and [Tyr 3 ]bPTH( 1-34) ( ⁇ ).
  • Figure 7E shows competitive binding to PTH receptors by bPTH(1-34) (o), [Glu 3 ]bPTH(1-34) ( ⁇ ), [His 3 ] bPTH( 1-34) ( ⁇ ), [Lys 3 ] bPTH( 1-34) ( ⁇ ), and [Gln 3 ]bPTH(1-34) ( ⁇ ).
  • Figure 7F shows competitive binding to PTH receptors by bPTH(l-34) (o), [Ala 6 ]bPTH(1-34) (•), [Phe 6 ]bPTH(1-34) ( ⁇ ), [Leu 6 ]bPTH(1-34) ( ⁇ ), [Ser 6 ]bPTH(1-34) ( ⁇ ), [Glu 6 ]bPTH(1-34) ( ⁇ ), and [Phe 9 ]humanPTH(1-34) ( ⁇ ).
  • Figure 8 shows inhibition of bPTH(1-34)-stimulated adenylate cyclase activity by the partial agonist [Phe 6 ]bPTH(1-34).
  • Figure 8A shows adenylate cyclase (AC) activation in canine renal plasma membranes produced by bPTH(l-34) (o), [Phe 6 ]bPTH(l-34) (•), and bPTH(1-34) (5nM) in the presence of varying concentrations of [Phe 6 ]bPTH(1-34) ( ⁇ ).
  • AC adenylate cyclase
  • Figure 8B shows adenylate cyclase (AC) activation in
  • UMR 106-H5 cells produced by [Phe 6 ]bPTH(1-34) (o) and by 0.2 nM bPTH(1-34) in the presence of varying concentrations of [Phe 6 ]bPTH(1-34) (•).
  • Tyr 34 refers to the amino acid at position 34 of PTH be tyrosine.
  • B 3 refers to the amino acid B at position 3 of PHT, etc.
  • J 6 refers to the amino acid J at position 6 of PHT, etc.
  • X r9 y refers to the amino acid X at position 9 of PHT, etc.
  • bPHT refers to the bovine sequence of PTH having a specific sequence of 1-84 amino acids.
  • bPHT(1-34) refers to a shortened bovine PHT sequence, the sequence of active 1-34 amino acids.
  • CPTH refers to the 1-84 chicken sequence for PTH.
  • hPTH refers to the 1-84 human sequence for PTH.
  • hPTH(1-34) refers to the shortened amino acid sequence for human PTH.
  • hPTHrP refers to the natural human parathyroid hormone related protein of 139-141 amino acids.
  • bPTHrP is derived from bovine cells.
  • hPTHrP is derived from human cells.
  • pPTHrP is derived from porcine cells.
  • hPTHrP(1-34) refers to the active 1-34 amino acids of natural human parathyroid hormone related protein.
  • PTH refers to the 1-84 porcine anmino acid sequence for PTH.
  • pPTH(1-34) refers to the active 1-34 normal porcine amino acid sequence for pPTH.
  • polypeptides are identified as the
  • Natural amino acids refer to those well known in the art. They are listed and standard abbreviations are provided in the U.S.P.T.O. publication. Trademark Official Gazette, published May 15, 1990, p. 33 at 46. These amino acids and abbreviations are specifically incorporated herein by reference.
  • amino acids include those substituted amino acids which are further substituted on the molecule with another group, such as alkyl or hydroxyl.
  • Typical substituted amino acids include, for example, 4-hydroxy-L-proline, sarcosine ("Sar” also known as N-methylglycine), D-3-(2-naphthylalanine) "D-Nal”, N 5 - (aminocarboxyl)-or-nithine "Cit”, pyro-glutamic acid, ornithine ; pmp (1-B-mercapto-beta,beta-pentamethylene propionic acid; Tyr (Et), tyrosine ethylated at the 4-hydroxyl position.
  • phenyl may or may not be substituted and that the description includes both unsubstituted phenyl and phenyl wherein there is substitution;
  • “optionally followed by converting the free base to the acid addition salt” means that said conversion may or may not be carried out in order for the process described to fall within the invention, and the invention includes those processes wherein the free base is converted to the acid addition salt and those processes in which it is not.
  • salts refers to salts that retain the desired biological activity of the parent compound and do not impart any undesired toxicological effects.
  • examples of such salts are (a) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; and salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid; (b) salts with metal (M) cations such as sodium, potassium, zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or with an organic cations
  • the present invention combines available or derived structure-activity data with predictive and/or experimental determinations regarding the molecular structure of PTH, or PTHrP to identify key amino acid residues in the 1-34 (or larger) amino acid sequences expected to play critical roles in high affinity receptor binding and activation.
  • the analogs containing amino acid substitutions at the 3, 6, or 9 positions or combinations thereof are synthesized and evaluated for the influence of side-chain polarity, charge and size or biological activity.
  • the in vitro bioassay is described for PTH receptor binding and adenylate cyclase activity in canine renal membrane and in rat bone and human bone cells. The results of these in vitro assays are predictive of in vivo biological activity.
  • the putative COOH-region helix contains residues thought to play a direct role in hormone binding to its receptor, whereas the NH 2 -region helix would include either part of or all of the 1-6 peptide segment required for activation for the receptor (induction of coupling to the stimulatory GTPbinding component of adenylate cyclase, G s .
  • two helical segments are predicted for PTHrP(1-34). These would comprise 74% (23/34) to 94% (32/34) of the peptide.
  • Chou-Fasman (Ref. 22) calculations suggest a beta-turn in positions 10-13 of PTH(1-34), and strongly predict a betaturn in the corresponding region of PTHrP.
  • Circular dichroism (CD) spectra for bPTH(1-34), [Tyr 34 ]bPTH(7-34)amide, and hPTHrP(1-34) amide were obtained in an aqueous buffer in the presence and absence of trifluoroethanol (TFE).
  • TFE trifluoroethanol
  • bPTH(l-34) yielded a spectrum with a nadir at 208 nm and a broad shoulder in the region of 222 nm ( Figure 3).
  • Figure 3 In the presence of 45% TFE, the nadir at 208 nm deepened, and a second minimum appeared at 222 nm.
  • PTH(7-34) contained 7 to 8 fewer alpha-helical residues than either bPTH(l-34) or hPTHrP(1-34)amide, providing direct evidence to suggest that the amino-terminal 1-6 domain of PTH assumes or induces alpha-helical structure in the presence of TFE. This result was confirmed in a second experiment in which [Tyr 34 ]bPTH(1-34) amide and [Tyr 34 ]bPTH(7-34)amide were calculated to display, respectively 24 and 18 alpha-helical residues in the presence of 30% TFE.
  • bPTH(l-34), hPTHrP(1-34) amide, and [Tyr 34 ]bPTH(7-34)amide are calculated to have 7-10 alpha-helical residues, consistent with a second alpha-helical domain that persists under strictly polar solvent conditions. This latter domain is predicted to lie between residues 17-34 of PTH and PTHrP. These results are similar to previous estimates of less than 12 alpha-helical residues for bPTH(1-34) and hPTH(1-34) (37-39), and 9 alpha-helical resides for bPTH(7-34) (Ref. 37) under aqueous conditions in the absence of amphiphiles.
  • the effect of TFE on the secondary structure of the PTH(7-34) analogue may reflect extension of the putative COOH-region helical domain under conditions of increasing solvent amphiphilicity.
  • conserved substitutions at positions 4, 7, and 8 as well as 20, 23, 24, 28, and 31 involve residues buried in the hydrophobic core. Presumably these residues contribute to the stabilization of the folded structure.
  • conserved solventexposed residues at positions 3, 6, 9, and 12 are likely to participate in critical interactions at the ligand-receptor interface.
  • the potency and activity of bPTH(134) is designated as 100%, and the peptide analogues are described in percent relative to the bPTH(1-34).
  • the renal membrane assay values listed reflect the effect of the polypeptide analogue with kidney tissue receptors.
  • the UMR-106-H5 assay values reflect the effect of the peptide analogue with rat bone tissue receptors.
  • the Sa05-2 assay values reflect the effect of the peptides for human bone tissue receptors.
  • Activity is defined as the intrinsic ability to stimulate adenylate cyclase so that a full agonist would have 100% activity.
  • a full antagonist would have 0% activity.
  • a partial agonist would have >0 and ⁇ 100% activity.
  • An ideal antagonist in either assay would have 100% binding (B) and 0% activity (AC).
  • a superagonist would have greater than 100% (e.g. 200%) binding (B), and 100% activity (AC) in either assay.
  • a preferred embodiment is a bone specific peptide analogue which would have binding (B) which approaches 0% and 0% activity in soft renal membranes, and approaches or is greater than 100% binding and activity in the UMR 106-H5 assay.
  • analog [Tyr 3 bPHT (1-34)] in Table 1 shows a preferred pattern having low binding and activity in renal tissue and high bind and activity in the UMR-106-H5 assay.
  • Table 1A describes the potency and activity of hPTH (1-34) designated as 100% and the peptide analogs are described in percent relative to hPTH (1-34) (human).
  • hPTH(1-34) 100 100 100 100 100 100 100 100 100 100 100 35 91
  • AC is the concentration of modified peptide half maximal enzyme activation (i.e. the mid-point of the curve transition - can be determined from the enclose d
  • AC* defines the enzyme activity of large doses of modified peptide relative to PTH reference at 100%.
  • the proposed model yields a domain structure that includes a hydrophobic core, hydrophilic faces of each helix, and loop between helices.
  • a relatively parallel arrangement is expected (see Figure 5). This packing angle is commonly found in helical proteins (Ref. 44,45).
  • An analogous paired helical structure has been demonstrated for 28-residue avian pancreatic polypeptide by X-ray crystallography (Ref. 46).
  • Helical structure was assigned to the amino-terminal region instead of the carboxyl-terminal region based on an analysis of the 3 J ⁇ CHNH coupling constants and the CD spectra of PTHrP analogues.
  • the region identified by Barden and Kemp as helical (residues 3-9) has an average 3JaCHNH of 5.4 Hz. Residues 24-30 in the C-terminal region have a similar average 3J ⁇ CHNH of 6.1 Hz.
  • the CD spectra of PTHrP(1-34), PTHrP(1-25), and PTHrP(7-34) in water were interpreted as showing evidence of an amino-terminal helix only.
  • the CD spectra for bPTH(13-34) and bPTH(19-34) also indicated little if any alpha-helical structure in these peptides under aqueous condition (Ref. 37).
  • a C-terminal amide stabilizes the helix, perhaps increasing biological potency.
  • Lys 13 (within the proposed loop region) stabilizes the dipole of the putative N-terminal helix in PTH an dPTHrP. Indeed, deletion of Lys 13 from PTH(1-34) results in a peptide with markedly reduced bioactivity (Ref. 55).
  • oxidation of Met18 is not expected to destabilize interhelical interactions, and [SOMet 18 ]bPTH(1-84) retains nearly 50% of the activity of the native hormone (Ref. 49).
  • a combination of spectro-scopic and predictive methods are used to develop a structural model for PTH (1-34) (F.E. Cohen et al., J. Biol. Chem. 266:1997, 1991).
  • the peptide is envisaged to consist of two amphipathic ⁇ -helices separated by a loop.
  • the amino-region ⁇ -helix initiates at or near the amino-terminus and extends to approximately residue 10.
  • the second ⁇ -helix initiates at about residue 18 and extends to the carboxyl-terminus.
  • the helices are suggested to fold back on one another in an antiparallel fashion, resulting in formation of a hydrophobic core with externally-facing polar residues that are expected to be centrally involved in receptor binding.
  • Circular dichroism is used in the art and in this invention to assess the effects of amino acid substitutions on the secondary structure of PTH(1-34).
  • Under aqueous solvent conditions virtually all of single-substituted analogs display 30-40% ⁇ -helical content.
  • the only exception is [ 3 Glu]PTH(1-34) which displays only 19% ⁇ -helical content, and shows a marked reduction in biological activity.
  • the aforementioned loss of intrinsic activity are not attributed to a gross change in the secondary structure of the analogs.
  • combined substitutions at residues 3 and 6 have a profound effect on receptor-active structure and on biological activity.
  • Substitutions are in positions 3 and 6 which individually do not impact markedly upon activity are introduced in pairs to create a series of analogs substituted in both positions.
  • Lys 3 ala 6 b-PHT and his 3 ala 6 b-PHT show low receptor affinity and low cyclase-stimulating activity in bone cells and in membranes from bone cells and kidney. Since single substitutions of alanine or glutamic acid for glutamine at position 6 or of lysine or histamine for serine at position 3 have minimal effects on intrinsic activity, the result suggests that effective activation of the PTH receptor involves an interaction between positions 3 and 6 in the ligand.
  • the amino acid in position 9 is the natural one histidine or is preferably one which will easily accept a positive charge, and thus modify the helical structure, e.g. tyrosine, tryptophan, phenylalanine, diaminobutyric acid, D-Nal, ornithine, citrulline, 3,4 or 5-fluoro-histidine and the like.
  • step (i) in step (d) (i) the high binding and high activity in the specific tissues, membranes or cells and a high specific bone cell binding and high activity are each about 50% or greater than that of the reference peptide.
  • step (d) (ii) the high binding and high activity as agonists for medical treatment in the specific tissues, membranes, or cells is about 50% or greater than that of the reference peptide and the low specific bone cell binding is 0% and low activity of bone cell is about 10% or less.
  • step (d) (iii) the low binding abo ut 5%
  • the low activity is less than 10% and the low activity is 0% in specific tissue, and high specific bone cell binding and the high activity is about 50% or greater, for disease conditions of the bone.
  • step (d) (iv) the high binding
  • 5 to is greater than 50% and the low activity is essentia ⁇ ly 0% in soft tissues, membranes or cells or in bone, for use as pharmaceutical antagonists in the medical treatment of hormonal disorders and cancers.
  • the soft tissue etc. are derived from human or bovine kidey.
  • amide is useful as an agonist for soft tissue or for bone, (d), (i).
  • phe 6 bPTH(1-34) amide is selected as being useful as a specific agonist for disease conditions in seoft tissue, etc., (d), (ii).
  • phe 3 bPTH(1-34) amide is selected as being useful as a specific agonist for disease conditions in bone (d) (iii).
  • ser 6 bPTH(1-34) amide is selected as an antagonist for disease conditions in soft tissue and bone (d), (iv).
  • the present invention describes a model for the structures of PTH(1-34) and PTHrP(1-34) in which their biologically active conformations at the receptor consist of N- and C-terminal amphiphilic helices connected by a loop of approximately 12 residues. Interhelical interactions result in a hydrophobic core with externallyfacing hydrophilic residues that presumably include determinants of receptor binding and activation.
  • the synthesis of analogues substituted in two such externallyfacing positions permits the demonstration that amino acid positions 3, 6 or 9 contribute important determinants of receptor binding and activation. Further delineation of the structural constraints at these positions will facilitate the rational design of potent PTH antagonists.
  • amino acid at position 1 is alanine
  • amino acid at position 3 is varied:
  • amino acid at position 1 is alanine and position 3 is serine, and position 6 is a variety of amino acids.
  • amino acid at position 1 is serine
  • amino acid at position 3 is varied and the amino acid at position 6 is varied and position 9 is His.
  • amino acid at position 1 is alanine
  • amino acid at position 3 is serine
  • amino acid J at position 6 is glutamine
  • amino acid X at position 9 is different.
  • D' is defined above:
  • amino acid at position 1 is alanine
  • X is phe and only the amino acids (B,J) at positions 3 and 6 are different. is defined above:
  • polypeptides of the present invention are prepared by the synthesis techniques known in this art.
  • teachings of the U.S. patents 4,318,905 and 3,531,258 are specifically incorporated herein by reference.
  • a key feature of the present invention is the preparation of biologically active synthetic polypeptides wherein at least the 3 amino acid B, or the 6 amino acid, or the 9 amino acid X, or combinations thereof are replaced a with natural, unusual or synthetic amino acid analogue.
  • polypeptides of the present invention may be synthesized by any techniques that are known to those skilled in the peptide art. An excellent summary of the many techniques so available may be found in J.M. Stewart and J.D. Young, “Solid Phase Peptide Synthesis", 2nd ed. , Pierre Chem. Co., Rockford, IL., (1969) and J. Gonhofer, "Hormonal Proteins and Peptides,” Vol. 2, p. 46, Academic Press (New York), 1973 for solid phase peptide synthesis and E. Schroder and K. Lubke, "The Peptides", Vol. 1, Academic Press (New York), 1965 for classical solution synthesis.
  • these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain.
  • a suitable protecting group either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group.
  • the protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having complimentary ( amino or carboxyl) group suitably protected, under conditions suitable for forming the amide linkage.
  • the protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support) are removed sequentially or concurrently, to afford the final polypeptide.
  • a particularly preferred method of preparing compound of the present invention involves solid phase peptide synthesis.
  • the alpha-amino function of the amino acids is protected by an acid or base sensitive group.
  • Such protecting groups should have the properties of being stable to the conditions of peptide linkage formation, while being readily removable without destruction of the growing peptide chain or racemization of any of the chiral centers contained therein.
  • Suitable protecting groups are t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, alpha,alpha-dimethy 1-3.5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl, 2-cyano-t-butyloxycarbonyl, 9-fluoroenylmethyloxycarbonyl and the like, especially t-butyloxcarbonyl (Boc).
  • Particularly preferred side chain protecting groups are, for arginine:nitro, p-toluenesulfonyl, 4-methoxybenzenesufonyl, Cbz, Boc and adamantyloxycarbonyl; for tyrosine:benzyl, o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, cyclohexyl, cyclopentyl and acetyl; for serine:benzyl and tetrahydropyranyl; for histidine; benzyl, p-toluenesulfonyl and 2,4-dinitrophenyl.
  • the C-terminal amino acid is attached to a suitable solid support.
  • suitable solid supports useful for the above synthesis are those materials which are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reactions, as well as being insoluble in the media used.
  • Suitable solid supports are chloromethylpolystyrene-divinylbenzene polymer, hydroxymethyl-polystyrene-divinylbenzene polymer, and the like, especially chloromethyl-polystyrene-1% divinylbenzene polymer.
  • a particularly useful support is the benzhydrylaminopolystyrene-divinylbenzene polymer described by P. Rivaille, et al. Helv. Chim. Acta., 542772 (1971).
  • the attachment to the chloromethyl polystyrene-divinylbenzene type of resin is made by means of the reaction of the N alpha -protected amino acid, especially the Boc-amino acid, as its cesium, tetramethylammonium, triethylammonium, 4,5-diazabicyclo[5.4.0]undec-5-ene, or similar salt in ethanol, acetonitrile, N,N-dimethylformamide (DMF), and the like, especially the cesium salt in DMF, with the chloromethyl resin at an elevated temperature, for example between 40° and 60°C., preferably about 50°C, for from about 12 to 48 hours, preferably about 24 hours.
  • the N alpha -protected amino acid especially the Boc-amino acid
  • the chloromethyl resin at an elevated temperature, for example between 40° and 60°C., preferably about 50°C, for from about 12 to 48 hours, preferably about 24 hours.
  • the N lalpha -Boc-amino acid is attached to the benzhydrylamine resin by means of an N,N'-dicyclohexylcarbodiimide (DCC) /1-hydroxybenzatriazole (HBT) mediated coupling for from about 2 to about 24 hours, preferably about 12 hours at a temperature of between about 10° and 50°C, preferably 25°C, in a solvent such as dichloromethane or DMF, preferably dichlormethane.
  • DCC N,N'-dicyclohexylcarbodiimide
  • HBT /1-hydroxybenzatriazole
  • the coupling of successive protected amino acids can be carried out in an automatic polypeptide synthesizer as is well known in the art.
  • the removal of the N a -protecting groups may be performed in the presence of, for example, a solution of trifluoroacetic acid in methylene chloride, hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or other strong acid solution, preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature.
  • a solution of trifluoroacetic acid in methylene chloride, hydrogen chloride in dioxane, hydrogen chloride in acetic acid, or other strong acid solution preferably 50% trifluoroacetic acid in dichloromethane at about ambient temperature.
  • Each protected amino acid is preferably introduced in approximately 2.5 molar excess and the coupling may be carried out in dichloromethane dichloromethane/DMF mixtures. DMF and the like, especially in methylene chloride at about ambient temperature.
  • the coupling agent is normally DCC in dichloromethane but may be N,N -di-iso-propylcarbodimide or other carbodiimide either alone or in the presence of HBT, N-hydroxysuccinimide, other N-hydroximides or oximes. Alternately protected amino acid active esters (e.g., pnitrophenyl, pentafluorophenyl and the like) or symmetrical anhydrides may be used.
  • the fully protected polypeptide is removed from the resin support is of benzyl ester type, cleavage is by means of aminolysis with alkylamine or fluoroalkylamine for peptides with a proline C-terminus, or by aminolysis with, for example, ammonia/methanol or ammonia/ethanol for peptides with a glycine C-terminus at a temperature between 10° and 50°C., preferably about 25°C, for between about 12 and 24 hours preferably about 18 hours.
  • the peptide may be removed from the resin by transesterification, e.g., with methanol, followed by aminolysis.
  • the protected peptide may be purified at this point by HPLC or by silica gel chromatography.
  • modified peptide analogs of the present invention i.e. those PTH, etc. analogs having modified amino acids at the 3,6 or 9 positions, or combinations of substitution at the 3,6 and 9 positions, are useful as agonists or as antagonists for treatment of medical conditions or diseases involving parathyroid hormone, particularly in a human being.
  • the diseases etc. are described herein.
  • compositions containing as an active ingredient, a compound of the present invention the compositions comprise a compound in admixture with a pharmaceutically acceptable non-toxic carrier (excipient).
  • excipient a pharmaceutically acceptable non-toxic carrier
  • such compositions may be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for oral or buccal administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols.
  • compositions may conveniently be administered in unit dosage form and may be prepared by any of the methods well-known in the pharmaceutical art, for example as described in Remington's Pharmaceutical Sciences. Mack Publishing Company, Easton, Pa., 1970.
  • Formulations for parenteral administration may contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
  • Formulations for vaginal or rectal administration, e.g. suppositories may contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.
  • Formulations for inhalation administration may be solid and contain as excipients, for example, lactose or may be aqueous or oily solutions for administration in the form of nasal drops.
  • excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like.
  • a dosage form a may contain a pharmaceutically acceptable non-toxic salt of the compounds which has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'-dibenzyl- ethylened
  • the compounds of the present invention or, preferably, a relatively insoluble salt such as those just described may be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
  • Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
  • Another type of slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,9-19.
  • the compounds or, preferably, relatively insoluble salts such as those described above may also be formulated in cholesterol matrix silastic pellets, particularly for use in animals. Additional slow release, depot or implant formulations, e.g. liposomes, are well known in the literature. See, for example, "Sustained and Controlled Release Drug Delivery Systems", J.R. Robinson ed., Marcel Dekker, Inc., New York, 1978.
  • MATERIALS- - Synthetic bovine PTH (1-34) was obtained from Bachem, Inc., Torrance, California.
  • Synthetic human PTHrP (1-34) amide was obtained from Merck Sharp and Dohme, West Point, Pennsylvania.
  • Synthetic bovine [Tyr 34 ]PTH(7-34) amide and [Tyr 34 ]PTH(7-34) amide were obtained from Peninsula Laboratories, Inc., Belmont, California. The purity of these peptides exceeded 95% by HPLC. Appropriate peptide composition was verified by quantitative amino-acid analysis.
  • CD SPECTROSCOPY Far ultraviolet circular dichroism (CD) spectra were obtained in phosphate buffer (ph 7.0) at 25 ⁇ 1°C in a 1.0 mm cell on an Instruments SA Jobin Yvon circular dichrograph calibrated with (+) 10-camphorsulfonic acid and epiandosterone. Peptides were analyzed at concentrations ranging from 0.1-0.3 mg/ml. The mean molar ellipticity per residue at 222 nm ([ ⁇ ]222 nm-, deg cm 2 /dmol) was used to derive estimates of alpha-helicity according to the method of Taylor and Kaiser (ref. 29).
  • PEPTIDE SYNTHESIS AND PURIFICATION Peptide synthesis was carried out using an Applied Biosystems Model 43OA Peptide Synthesizer, Foster City, California.
  • a t-Boc-PheOCH2- Pam resin was used as the solid support, and the following t-Boc (tert-butyloxycarbonyl) amino acid derivatives were employed: Arg(Tos), Asp(OBzl), Glu(OBzl), His(DNP), His(Z), Lys(Cl-Z), Ser(Bzl), Thr(Bzl), Trp(CHO), and Tyr(Br-Z).
  • a standard program pre-formed symmetric anhydrides and pre-formed HOBt esters
  • the general synthesis of the peptides is found, for example, in U.S.Patent No. 4,318,905 which is specifically incorporated herein by reference in its entirety.
  • Standard t-Boc cycles exist for the activation, solvent exchange, and coupling processes; called ACT, CONC, and RV cycles, respectively.
  • the set of vessel cycl assignments in a standard t-Boc static run file is designated for each amino acidon the basis of testing and synthesis optimization performed at Applied biosystems.
  • CONC Concentrator Cycles
  • RV Reaction Vessel Cycles
  • the amino acid solution is transferred from the ACT to the CONC.
  • a complete wash of the transfer line proceeds and follows each transfer between vessels. This procedure ensures that the line is clean both before and after the transfer, and eliminates the possibility of amino acid carry-over between cycles.
  • Transfer times of the activated amino acid solution to the CONC are longer for aboc 4 cycle than for the aboc 1, aboc 2 or aboc 3; again because of the larger solution volume handled in the aboc 4 ACT cycle.
  • Two DCM rinses of the ACT are transferred into the CONC in all single couple cycles.
  • the time listed in Table 2 includes the initial activation time and purging time.
  • the amino acid solution is ready for transfer to the CONC.
  • a complete wash of the transfer line precedes and follows each transfer between vessels. This procedure ensures that the line is clean both before and after the transfer to eliminate the possibility of carry-over between cycles.
  • the activated amino acid solution is transferred to the CONC along with one subsequent DMF rinse of the ACT.
  • the HOBt-ester activation cycles produce twice as much DCU as the symmetric anhydride cycles; therefore, a more extensive washing process is required after the transfer.
  • This washing consists of two 50:50 DCM:MeOH washes followed by three DCM washes.
  • the first DCM:MeOH wash almost fills the vessel; any DCU carried to the top of the vessel during the purging will be dissolved in this wash.
  • the second DCM:MeOH solution ensures the removal of any DCU adhering to the frit.
  • the residual MeOH is rinsed away with three subsequent DCM washes.
  • ** aboc 4 is used to His(Tos)DCHA after the completion of the ion exchange procedure
  • ** aboc 4d is used to double couple His(Tos)DCHA after the completion of the ion exchange procedure
  • the c recouple cycle is present on the standard, but is not assigned in the standard t-Boc run. This cycle is simply a transfer cycle designed to recouple any amino acid using DCM as the coupling solvent. This cycle does not include purging or heater use because the initial solvent DCM, is not exchanged for a different coupling solvent, DMF. The amino acid solution is transferred to the RV along with two additional DCM rinses of CONC.
  • the c recpl cycle should be used in conjunction with the normal single couple ACT cycle, and either recp!2 or recp!2r. the DCM RV recoupled cycles.
  • Resin-sampling cycles rinse the sampling line several times from the lower RV valve block up through the bulkhead fitting and into the RV. This rinse prevents accumulation of resin, TFA, or coupling solution in the line.
  • Resin-sampling cycles have an "r" at the end of the cycle name (i.e., rboc 1r . If the cycle has "r” at the end (i.e., rboc 1rr), then two resin samples are taken after the coupling. The "rr” cycles are included on this disk for installation purposes.
  • the RV cycles with the same number designation, such as rboc 1 and rboc 1r, are the same cycles, except that the resin sampler is used in the latter cycles.
  • the first half of the doubling couple cycles are similar to the single couple cycles.
  • the first six processes in the list above are exactly the same as the equivalent processes in a single couple RV cycle. However, there is a longer total coupling time for the double couple cycles.
  • the rboc 1d and rboc 1dr cycles each have a 42-minute (first) coupling period; the rboc 2d and rboc 2dr cycles each have a 26-minute (first) coupling period.
  • Peptide resins were treated with thiophenol in dimethylformamide to deblock histidine residues (ref. 30).
  • the peptides are contacted with HF:anisole:DMS(10:1:1) at -5 to 0°C for 6 minutes.
  • the HF is easily evaporated with the aid of nitrogen flow within ten to fifteen minutes. To prevent side reactions during this process, it is important to keep the reaction vessel at -5°C to 0oC.
  • ether is added to the reaction vessel and the peptide-resin-scavenger mixture is mixed for about 30 seconds. The ether solution is then filtered through a sintered glass funnel. Repeated two more time, this ether wash removes most of the scavengers.
  • the peptide is extracted from the peptide-resin mixture by stirring the mixture in 30% acetic acid. For those peptides not soluble in 30% acetic acid, a higher concentration of acetic acid is recommended. Typically, for one gram of peptide resin approximately 30 mL of 30% acetic acid is used. The acetic acid extract is filtered through the same sintered glass funnel used to the ether extraction, but into a different filter flask. To ensure complete extraction of the peptide, repeat the extraction procedure using approximately 30 mL of 10% acetic acid (2 times). Dilute the acetic acid solution with water before freeze drying. A more dilute solution of acetic acid in water will remain frozen while a concentrated solution of acetic acid in water may melt during lyophilization.
  • the solution is then lyophilized to obtain crude peptide.
  • N-O shift may occur during HF cleavage.
  • Remaining formyl groups were eliminated by treatment with 1 M ethanolamine in 6 M guanidine-HC1 at 0°C for 5 min.
  • the formyl (CHO) protecting group of tryptophan is an HF-trifluoromethane sulfonic acid stable species
  • Deformylation of a peptide synthesized by using TRP (CHO requires a separate step after strong acid cleavage. Since the deformylation of Trp (CHO) can pose problems to even with the most skilled the following procedure should be strictly followed.
  • the peptide can be desalted on a gel filtration column or dialyzed to remove the guanidine and ethanolamine. Alternatively, the solution is loaded onto a preparative high performance liquid chromatography (HPLC) system for purification.
  • HPLC high performance liquid chromatography
  • the deformylated crude peptide preparations were desalted on a reverse-phase column (Delta Pak C 18 , 300 Ao) using a gradient of acetonitrile in 0.1% trifluoroacetic acid (0-60%, 24 ml/min) followed by cation-exchange chromatography on a TSK 535 CM column (7.5 x 150 mm) eluted with a NaCl gradient (0 - 0.5 M) in 20 mM sodium phosphate buffer, pH 6.4.
  • the instrument for the ion-exchange HPLC consisted of an LKB 2150 HPLC pump, an LKB 2152 HPLC controller, LKB 2040 gradient-mixing valve, an LKB 2154 HPLC injector and an LKB 2151 HPLC variable-wavelength detector connected to an LKB 2210 recorder.
  • the separation was carried out on an LKB UltroPac TSK 535 CM cation-exchanger column (150 ⁇ 7.5 mm I.D.
  • Receptor binding assays were carried out in canine renal plasma membranes and in UMR 106-H5 rat osteosarcoma cells as previously described in detail (Ref. 14,32), using 1 25 I-labeled PTH(1-34) amide as the radioligand.
  • Binding potency (IC 50 ) was quantified as the concentration of unlabeled peptide required for half-maximal displacement of the radioligand.
  • Non-specific binding defined as residual radioligand binding in the present of a greater than 1, microM unlabeled bPTH(l-34), was subtracted from all binding values.
  • the ratio of specific:non-specific binding of 125 I-PTHrP(1-34)amide average >10:1 and 3:1 in the renal membrane, and UMR 106-H5 assays, respectively.
  • Adenylate cyclase activity was assessed in canine renal membranes by the conversion of [ ⁇ -32 p ]ATP to [ 32 P]cyclic AMP, essentially as described, except for the addition of 100 micromolar GTP (Ref. 33,34).
  • Adenylate cyclase activity in UMR 106-H5 cells was assessed essentially as described (Ref. 35). In brief, cells were incubated with [3H]adenine 1 micro Ci/ml of serum-free MEM for two hours at 37oC to label the endogenous ATP pool.

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US5434246A (en) * 1992-03-19 1995-07-18 Takeda Chemical Industries, Ltd. Parathyroid hormone derivatives
US5814603A (en) * 1992-06-12 1998-09-29 Affymax Technologies N.V. Compounds with PTH activity
US5589452A (en) * 1992-07-14 1996-12-31 Syntex (U.S.A.) Inc. Analogs of parathyroid hormone and parathyroid hormone related peptide: synthesis and use for the treatment of osteoporosis
US5977070A (en) * 1992-07-14 1999-11-02 Piazza; Christin Teresa Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis
US6235288B1 (en) 1992-08-26 2001-05-22 The Scripps Research Institute Peptides for inducing cytotoxic T lymphocyte responses to hepatitis B virus
US5840688A (en) * 1994-03-22 1998-11-24 Research Corporation Technologies, Inc. Eating suppressant peptides
CO4410206A1 (es) * 1994-07-28 1997-01-09 Sandoz Ag DERIVADOS DE PTH o PTHrP, SU PREPARACION Y COMPOSICIONES FARMACEUTICAS QUE LAS COMPRENDEN
CA2178894A1 (en) * 1995-06-15 1996-12-16 Tsunehiko Fukuda Parathyroid hormone derivatives and their use
EP1166796A3 (de) 1996-02-01 2002-03-06 Chugai Seiyaku Kabushiki Kaisha Medikamente zur Behandlung oder Vorbeugung von Thrombocytopenie
US5908400A (en) * 1996-06-20 1999-06-01 Hisamitsu Pharmaceutical Co., Inc. Device structure for iontophoresis
TW505654B (en) * 1996-07-30 2002-10-11 Hoffmann La Roche Synthesis of analogs of PTH and PTHrP
HUP9904177A3 (en) 1996-09-26 2001-10-29 Chugai Pharmaceutical Co Ltd Antibody against human parathormone related peptides
IL132896A0 (en) 1997-05-15 2001-03-19 Chugai Pharmaceutical Co Ltd Cachexia remedy
PL345117A1 (en) * 1998-06-26 2001-12-03 Chugai Pharmaceutical Co Ltd Remedies for hypercalcemic crisis
TWI255718B (en) 1999-07-02 2006-06-01 Chugai Pharmaceutical Co Ltd Ameliorative agent for low vasopressin concentration
EP1197225A4 (de) * 1999-07-02 2006-02-22 Chugai Pharmaceutical Co Ltd MITTEL FÜR ERKRANKUNGEN DIE VON PTH ODER PTHrH VERURSACHT WERDEN
PL353407A1 (en) * 1999-07-06 2003-11-17 Chugai Seiyaku Kabushiki Kaisha Remedies for drug-resistant hypercalcemia
WO2001054725A1 (fr) * 2000-01-25 2001-08-02 Chugai Seiyaku Kabushiki Kaisha Remèdes et agents prophylactiques pour maladies dentaires
WO2001064249A1 (en) * 2000-02-28 2001-09-07 Chugai Seiyaku Kabushiki Kaisha Tissue decomposition inhibitor
ATE420661T1 (de) 2000-04-28 2009-01-15 Chugai Pharmaceutical Co Ltd Zellproliferation-inhibitoren
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