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.
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