Development of a Hypoparathyroid Male Rodent Model for Testing Delayed-Clearance PTH Molecules.

CONTEXT: Parathyroid hormone (PTH) replacement is a promising approach in the management of hypoparathyroidism but long-acting analogues need to be developed. To date, animal models for testing PTH required parathyroidectomy by surgery. We have developed a nonsurgical rodent hypoparathyroid model and tested a delayed-clearance PTH molecule (DC-PTH). OBJECTIVE: The aim of this study was to use cinacalcet to suppress calcium levels in normal rats and to reverse these effects with the administration of PTH or PTH analogues. METHODS: Male Wistar rats were gavaged with either 30 mg/kg cinacalcet-HCl (cinacalcet) or vehicle only. Animals were then dosed with either single or repeated subcutaneous doses of PTH 1-34 or a DC-PTH at 20 nmol/kg. Control animals received vehicle only. Serum samples were analyzed for ionized calcium (iCa), phosphate, PTH, and DC-PTH. A pharmacokinetic-pharmacodynamic (PK-PD) model was built for cinacalcet, PTH 1-34, and DC-PTH using Phoenix64. RESULTS: Cinacalcet reduced iCa levels between 2 and 24 hours, returning to baseline by 72 hours post dose with nadir at 8 hours (analysis of variance P < .001), associated with a fall in rat PTH. For phosphate there was a variable biphasic response. Single-dose PTH abrogated the cinacalcet-induced fall in iCa for up to 2 hours. DC-PTH prevented the fall in iCa from 4 hours post dose and gave a prolonged response, with iCa levels quicker to return to baseline than controls. DC-PTH has a half-life of 11.5 hours, approximately 44 times longer than human PTH 1-34. The PK-PD models defined the reproducible effect of cinacalcet on iCa and that DC-PTH had prolonged biological activity. CONCLUSION: The administration of cinacalcet provides a robust and reproducible nonsurgical animal model of hypoparathyroidism. DC-PTH holds promise for the treatment of hypoparathyroidism in the future.

A long-acting GH receptor antagonist through fusion to GH binding protein.

Acromegaly is a human disease of growth hormone (GH) excess with considerable morbidity and increased mortality. Somatostatin analogues are first line medical treatment but the disease remains uncontrolled in up to 40% of patients. GH receptor (GHR) antagonist therapy is more effective but requires frequent high-dose injections. We have developed an alternative technology for generating a long acting potent GHR antagonist through translational fusion of a mutated GH linked to GH binding protein and tested three candidate molecules. All molecules had the amino acid change (G120R), creating a competitive GHR antagonist and we tested the hypothesis that an amino acid change in the GH binding domain (W104A) would increase biological activity. All were antagonists in bioassays. In rats all antagonists had terminal half-lives >20 hours. After subcutaneous administration in rabbits one variant displayed a terminal half-life of 40.5 hours. A single subcutaneous injection of the same variant in rabbits resulted in a 14% fall in IGF-I over 7 days. IN CONCLUSION: we provide proof of concept that a fusion of GHR antagonist to its binding protein generates a long acting GHR antagonist and we confirmed that introducing the W104A amino acid change in the GH binding domain enhances antagonist activity.

Expression of a glycosylphosphatidylinositol-anchored ligand, growth hormone, blocks receptor signalling.

We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain to the cell surface using a native lipid GPI (glycosylphosphatidylinositol) anchor. We also investigated the effect of tethering GH, the ligand itself, to the cell surface and demonstrated that tethering either the ecGHR (extracellular domain of GHR) or the ligand itself to the cell membrane via a GPI anchor greatly attenuates signalling. To elucidate the mechanism for this antagonist activity, we used confocal microscopy to examine the fluorescently modified ligand and receptor. GH-GPI was expressed on the cell surface and formed inactive receptor complexes that failed to internalize and blocked receptor activation. In conclusion, contrary to expectation, tethering an agonist to the cell surface can generate an inactive hormone receptor complex that fails to internalize.

Immunogenicity, toxicology, pharmacokinetics and pharmacodynamics of growth hormone ligand-receptor fusions.

A fundamental concern for all new biological therapeutics is the possibility of inducing an immune response. We have recently demonstrated that an LR-fusion (ligand-receptor fusion) of growth hormone generates a potent long-acting agonist; however, the immunogenicity and toxicity of these molecules have not been tested. To address these issues, we have designed molecules with low potential as immunogens and undertaken immunogenicity and toxicology studies in Macaca fascicularis and pharmacokinetic and pharmacodynamic studies in rats. Two variants of the LR-fusion, one with a flexible linker (GH-LRv2) and the other without (GH-LRv3), were tested. Comparison was made with native human GH (growth hormone). GH-LRv2 and GH-LRv3 demonstrated similar pharmacokinetics in rats, showing reduced clearance compared with native GH and potent agonist activity with respect to body weight gain in a hypophysectomized rat model. In M. fascicularis, a low level of antibodies to GH-LRv2 was found in one sample, but there was no other evidence of any immunogenic response to the other fusion protein. There were no toxic effects and specifically no changes in histology at injection sites after two repeated administrations. The pharmacokinetic profiles in monkeys confirmed long half-lives for both GH-LRv2 and GH-LRv3 representing exceptionally delayed clearance over rhGH (recombinant human GH). The results suggest that repeated administration of a GH LR-fusion is safe, non-toxic, and the pharmacokinetic profile suggests that two to three weekly administrations is a potential therapeutic regimen for humans.

A ligand-receptor fusion of growth hormone forms a dimer and is a potent long-acting agonist.

Cytokine hormones have a short plasma half-life and require frequent administration. For example, growth hormone replacement involves daily injections. In common with other cytokines, the extracellular domain of the growth hormone receptor circulates as a binding protein, which naturally prolongs the biological half-life of growth hormone. Here we have studied the biological actions of a ligand-receptor fusion of growth hormone and the extracellular domain of its receptor. The genetically engineered ligand-receptor fusion protein was purified from mammalian cell culture. In rats, the ligand-receptor fusion had a 300-times reduced clearance as compared to native growth hormone, and a single injection promoted growth for 10 d, far exceeding the growth seen after administration of native growth hormone. The ligand-receptor fusion forms a reciprocal, head-to-tail dimer that provides a reservoir of inactive hormone similar to the natural reservoir of growth hormone and its binding protein. In conclusion, a ligand-receptor fusion of cytokine to its extracellular receptor generates a potent, long-acting agonist with exceptionally slow absorption and elimination. This approach could be easily applied to other cytokines.