Parathyroid hormone analogues for the treatment of osteoporosis

A technology of parathyroid hormone and analogues, applied in the field of analogues of human parathyroid hormone

Inactive Publication Date: 2004-11-24
NAT RES COUNCIL OF CANADA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These analogues are thought to be effective in the treatment of osteoporosis with minimal effects on

Method used

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  • Parathyroid hormone analogues for the treatment of osteoporosis
  • Parathyroid hormone analogues for the treatment of osteoporosis
  • Parathyroid hormone analogues for the treatment of osteoporosis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Example 1 Synthesis and purification of linear hPTH-(1-31) amide analogs

[0087] During the coupling process, the α-amino group of the amino acid is protected by 9-fluorenylmethoxycarbonyl (Fmoc). With 1-hydroxybenzotriazole (HOBt), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) and diiso A mixture of propylethylamine (DIPEA) was used for coupling. Asn, Gln, His, Val and Ile residues were added by double coupling using a 4-fold excess of the activated amino acid. The coupling time was increased from 30 minutes to 60 minutes with the addition of Arg and Gly. The first residue (Val 31 ) coupled to the carrier (Tentagel * R, Rapp Polymere, Tubingen, Germany). All other steps in PerSeptive Biosystems * Model 9050 Plus automatic peptide synthesizer. The side chains are protected as follows: Arg (2,2,5,7,8-pentamethylchroman-6-sulfonyl); Glu, Asp and Ser (t-butyl); His, Gln and Asn (trityl); Trp (t-butoxycarbonyl).

[0088] After remova...

Embodiment 2

[0090] Example 2 Synthesis and purification of cyclic analogs

[0091] [Leu 27 ] Ring (Glu 22 -Lys 26 )-hPTH-(1-31)-NH 2 Peptides were synthesized as described in Example 1. Lys-Alloc and Glu-OAII were substituted at positions 26 and 22, respectively. Join Fmoc-Ser 17 Afterwards, the peptide-resin was transferred from the column to a reaction tube (Minivial * , AppliedScience), and suspended under argon in 1.7 ml dichloromethane (DCM) containing tetrakis (triphenylphosphine) palladium (0.24 mmol), 5% acetic acid and 2.5% N-methylmorpholine (NMM) solution, followed by shaking at 20°C for 6 hours to remove the allyl and alloc protecting groups [Sole, N.A. et al. (1993), in Peptides: Chemistry, Structure and Biology, Smith, J. and Hodges, R.( eds), ESCOM, pp. 93-94, cited here by reference]. The peptide-resin was then washed with DMF (50ml) containing 0.5% diethyldithiocarbamate (DEDT), 0.5% NMM, followed by DMF (50ml) and DCM (50ml). The peptide (0.06 mmol) was cyclized...

Embodiment 3

[0093] Example 3 Determination of adenylyl cyclase

[0094] The ability of hPTH to mimic a signaling mechanism coupled to receptor binding and its activation of adenylyl cyclase was assayed on the differentiation-competent osteoblast-like ROS17 / 2 rat osteosarcoma (RUS) cell line. This activity is known to be tightly coupled with the analog's ability to remodel bone mass in ovariectomized rats. Stimulate the activity of adenylyl cyclase by using [ 3 [H]-adenine pre-labels cellular ATP pools and then measures the 10 min period beginning after exposure to the specific analog by [ 3 H]-ATP produced [ 3 H]-cyclic AMP was evaluated. This is based on the method described in Whitfied et al., J. Cell Physiol., 150, 299-303, 1992, incorporated herein by reference.

[0095] The adenylyl cyclase results are presented in Table 2 below as the concentration required to achieve a half-maximal increase in AC activity. This data is also presented in Figure 4. In Figure 4, the solid circle...

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Abstract

This invention describes analogues of human parathyroid hormone which have increased activities in bone restoration, and increased bioavailabilities. The peptides described are derivatives of hPTH-(1-31) which are cyclized for example, by formation of Lactams between either Glu<22> and Lys<26> or Lys<26> and Asp<30>. In addition, the natural Lys<27> may be substituted by either a Leu or other hydrophobic residues, such as Ile, norleucine, Met, Val, Ala, Trp, or Phe. Typically, these analogues have enhanced abilities to stimulate adenylyl cyclase in rat osteosarcoma cells, and show increased activities in bone restoration, using the ovariectomized rat model. The analogues also show enhanced activities and bioavailabilities, as demonstrated by their hypotensive effects in the rat. An assay which correlates hypotensive activity with osteogenic activity is also described.

Description

field of invention [0001] The present invention relates to analogs of human parathyroid hormone and has been found to be effective in the treatment of osteoporosis. Background of the invention [0002] Osteoporosis is the leading cause of disability in older adults, especially older women. Human parathyroid hormone (hPTH) and some of its analogs have recently been recognized as stimulators of bone growth and are useful in the treatment of osteoporosis. Osteoporosis is a progressive disease leading to total bone loss and increased bone fragility. This often results in spontaneous fractures of the weight-bearing bones and the physical and mental deterioration typical of activity injuries. Postmenopausal osteoporosis is caused by estrogen depletion, which triggers a long-term acceleration of bone metabolism, increasing the imbalance between old bone resorption and new bone formation. This can lead to thinning, increased porosity, and decreased trabeculae in weight-bearing bo...

Claims

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Application Information

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IPC IPC(8): A61K38/22A61K38/00A61K38/29A61P19/10C07K14/635
CPCC07K14/635A61K38/29A61P19/00A61P19/10A61P5/14A61P5/20
Inventor J-R·巴比尔P·默雷W·纽杰鲍尔V·罗斯J·辉特菲尔德G·E·威利克
Owner NAT RES COUNCIL OF CANADA
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