Yonsei Med J.  2006 Apr;47(2):214-222. 10.3349/ymj.2006.47.2.214.

In Vitro and In Vivo Effect of Parathyroid Hormone Analogue (1-14) Containing alpha-amino-iso-butyric Acid Residue (Aib)1,3

• Rhee Y
• Lee W
• Lee EJ
• Ma S
• Park SY
• Lim SK

• KMID: 1110745
• DOI: http://doi.org/10.3349/ymj.2006.47.2.214

Abstract

Firstly, parathyroid hormone (1-14) [PTH (1-14)] analogue containing various alpha-amino-iso-butyric acid residue (Aib) was synthesized by exchanging the 1st and 3rd Ala residues of alpha carbon of PTH (1-14). This analogue revealed to have the quite tight and stable alpha-helical structure using the nuclear magnetic resonance (NMR) analysis. The biological activities of these analogues were examined using a cAMP-generating assay in LLC-PK1 cell lines stably transfected with the wild-type human PTH1 receptor. Only the PTH analogue substituted with methyl moiety without acetylation showed significant cAMP generating action with 15.0 +/- 3.414 of EC50. Then, we used an ovariectomized rat model system to compare the in vivo effects of parathyroid hormone analogue with that of PTH (1-84). Daily subcutaneous administration of the unacetylated Aib1,3PTH (1-14) for 5 weeks in 30 nM/kg subcutaneously with positive control group receiving PTH (1-84) with 8 nM/ kg were performed. However, there was no significant change in spinal or femoral bone mineral density assessed by dual x-ray absorptiometry (DXA) in the Aib1,3PTH (1-14) group where definite increase of these parameters shown in the PTH (1-84) group (p < 0.001). Assessment of bone strength was evaluated with no significant differences among all groups. It was quite disappointing to see the actual discrepancies between the result of significant pharmacokinetic potency and the in vivo clinical effect of the Aib1,3PTH (1-14). However, there are several limitations to mention, such as the short duration of treatment, matter of dosage, and insufficient effect of tight alpha-helical structures with absence of C-terminus. In conclusion, our findings suggest that unacetylated Aib1,3PTH (1-14) did not exhibit any anabolic effects at the bones of ovariectomized rats.

Keyword

Parathyroid hormone analogue; alpha-helical structure; ovariectomized rat; bone mineral density; bone strength

MeSH Terms

Transfection
Time Factors
Structure-Activity Relationship
Stress, Mechanical
Spectrometry, X-Ray Emission
Rats
Protein Structure, Tertiary
Protein Structure, Secondary
Protein Conformation
Protein Binding
Peptides/chemistry
Parathyroid Hormone/*analogs & derivatives/chemistry/*metabolism
Molecular Sequence Data
Molecular Conformation
Models, Statistical
Models, Molecular
Magnetic Resonance Spectroscopy
LLC-PK1 Cells
Humans
Female
Dose-Response Relationship, Drug
Densitometry
Cyclic AMP/metabolism
Cell Line
Bone and Bones/metabolism
Bone Density
Biomechanics
Animals
Aminoisobutyric Acids/*metabolism
Amino Acid Sequence
Alanine/chemistry

Figure

• 

  Fig. 1 Schematic structures of PTH analogs.

• 

  Fig. 2 cAMP formation of various PTH analogues in LLC-PK1 cells stably transfected with hPTH1R. (A) The effect of 1st and 3rd residue substitution in PTH (1-14) with methyl, ethyl and propyl moiety on cAMP activity. (B) The effects of acetylation in the AiBPTH (1-14) analogue. Each experiment was performed in duplicate and repeated three times. The symbols are defined in the figure key, and the curves were fitted to the data points by non-linear regression analysis, as described in Materials AND Methods.

• 

  Fig. 3 Two-dimensional NOESY spectrum of modifications of PTH (1-14) showing NH regions with a mixing time 400 ms. Modification PTH (1-14) dissolved in 30% TFE solution at pH 7.2, 288 K. A continuous stretch of sequential dNN (i, i+1) NOEs is observed in the helical region.

• 

  Fig. 4 Summary of NMR data for modifications of PTH (1-14) collected in 30% TFE solution at 288 K, showing the sequential and short-range NOE contacts. Slowly exchanging amide protons (□), backbone vicinal coupling constants (●; 3JHN(<6 Hz), and chemical shift indices (CSI) for the CαH chemical shift are indicated.

• 

  Fig. 5 Solution structures of Aib1,3 PTH (1-14). (A) Super-imposition of the final 15 k structures of PTH (1-14) analogue upon the energy-minimized average structure (k) (Cα traces) (B) Ribbon diagram of the PTH (1-14) analogue. Modified Aib1, Aib3, Nle8 residues are displayed using ball and stick model.

• 

  Fig. 6 Bone mineral density (BMD, g/cm2) of the right femur (A). *p<0.05 vs. PTH14, **p<0.001 vs. OVX, PTH 14; lumbar spinal BMD (B); *p<0.005 vs. OVX, PTH14, **p<0.001 vs. OVX, PTH14.

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