Compounds in the diazo/hydrazino acid class require expression of Tpo receptor (TpoR) for activity. These compounds have demonstrated a remarkable species specificity in their Tpo receptor agonist activity, i.e., the activation of signalling pathways by this class of compounds has only been demonstrated in human and chimpanzee platelets, but not in platelets of other species such as cynomolgus macaques. In order to demonstrate in vivo activity of SB-497115, a single dose and 5 daily dose pharmacology and safety study in chimpanzees was conducted. All procedures involving the care and use of animals were reviewed and approved by the appropriate Institutional Animal Care and Use Committee. Female chimpanzees were administered vehicle or SB-497115 at doses of 0.1 to 10 mg/kg/day by oral gavage. SB-497115 was well tolerated in chimpanzees at all doses tested. Following 5 daily doses of 10 mg/kg/day SB-497115, there was a 1.3- to 2.4-fold increase in circulating platelet counts in 3/3 chimpanzees. A similar change in reticulated platelet counts was observed preceding this increase. To elucidate the mechanism by which this species specificity occurs, HepG2 cells were transiently transfected with a STAT-activated reporter gene along with chimeric and mutated receptors and treated with SKF-57626, a representative compound from the diazo class. A series of cyno and human TpoR chimeras were constructed in which the complement receptor homology region 1 (CRH1), CRH2 and the transmembrane (TM) and cytoplasmic domains were interchanged. Functionality of all chimeric receptors was confirmed by response to rhTpo. The minimal human composition of the chimeric TpoR activated by SKF-57626 was composed of human sequence within the membrane proximal region of the CRH2 domain and the TM domain. Two amino acids in this region are different between cyno and human, a Thr to Ala change in the extracellular CRH2 domain and a Leu at residue 499 in cynos, rather than His in humans, in the TM domain. Sequencing of the TpoR transmembrane domain confirmed that chimpanzee TpoR is similar to human and contains His499. To verify the requirement for His499 in the TM domain, a point mutation replacing only Leu499 with His in the cyno TpoR conferred activity when treated with compound. Further experiments utilized mutations in the murine G-CSF receptor (mGCSFR). Human TpoR and mGCSFR have little homology in their TM domains and there is no detectable signalling in GCSFR expressing cells by either compound or rhTpo. However, a mGSFR mutated to contain a His residue nine amino acids into the hydrophobic TM domain of GCSFR (corresponding to His499 in the human TpoR) responded to SB-497115. These results suggest a model in which these TpoR agonist compounds interact with His499 to change the conformation of TpoR or to induce dimerization, resulting in activation of the signal transduction pathways of TpoR and imparting biologically relevant function.
[Proc Amer Assoc Cancer Res, Volume 46, 2005]