Hypoparathyroidism causes severe hypocalcaemia and defective skeletal metabolism. Treatment with calcium and vitamin D supplementation can cause kidney failure whilst native parathyroid hormone (PTH) requires repeated injections and causes renal impairment paralleling high peak and low trough PTH levels. A long-acting PTH, providing constant physiological levels, is needed. LA-PTH, a hybrid of PTH and PTH related peptide, prolongs cAMP responses via altered receptor mechanisms.1 Protein fusion to growth hormone binding protein (GHBP) delays clearance.2 Fusing LA-PTH to GHBP could generate an improved long-acting PTH.
Methods: LA-PTH fusions were gene-synthesised and stable CHO clones generated using Invitrogens Flp-In system. Large-scale roller bottle protein expression was followed by purification using ion-exchange and affinity chromatography. Sample analysis by Coomassie staining & western blotting followed SDS-PAGE. A Dual Luciferase Reporter Assay in rat Osteosarcoma (UMR-106) cells assessed potency. The ligands ability to generate cAMP at times after ligand washout was assessed via competitive ELISA following cAMP generation assays.
Results: Two fusions were constructed and their sequences confirmed: LA-PTH-1 is LA-PTH linked to GHBP and PTH receptor extracellular domain; LA-PTH-2 is LA-PTH linked to GHBP. Western blotting on stably transfected CHO cell media, using an anti-GHBP antibody, confirmed expression: LA-PTH-1 and LA-PTH-2 separated as diffuse bands between 100-75 kDa and 50-37 kDa respectively. 1.49 mg/mL LA-PTH-1 and 0.42 mg/mL LA-PTH-2 was expressed. LA-PTH-1 (EC50 mean±SD: 222±133 nM) was ˜30-fold less potent than PTH-1-34 (8±3), however, LA-PTH-2 (14±2) was approximately equipotent. Fusion longevity of action profiles resembled PTH-1-34, however LA-PTH-2 accumulated ˜3-fold PTH-1-34 cAMP levels (equimolar challenge, 100 nM); area under the curve ˜15 000 vs. ˜5000 pmol/well.
Conclusion: It is possible to express and purify two PTH hybrid analogues in a mammalian cell line whilst retaining in-vitro bioactivity. Longevity of action testing is ongoing; future work will examine in-vivo bioactivity and pharmacokinetics.