Lactam formation increases receptor binding, adenylyl cyclase stimulation and bone growth stimulation by human parathyroid hormone (hPTH)(1-28)NH2

Human parathyroid hormone (1-28)NH2 [hPTH(1-28)NH2] is the smallest of the PTH fragments that can fully stimulate adenylyl cyclase in ROS 17/2 rat ost eoblast-like osteosarcoma cells. This fragment has an IC50 of 110 nM for di splacing I-125-[Nle(8,18)Tyr(34)]bovine PTH(1-34)NH2 from HKRK B7 porcine k idney cells, which stably express 950,000 human type 1 PTH/PTH-related prot ein (PTHrP) receptors (PTH1Rs) per cell. It also has an EC50 of 23.9 nM for stimulating adenylyl cyclase in ROS 17/2 cells. Increasing the amphiphilic ity of the alpha-helix in the residue 17-28 region by replacing Lys(27) wit h Leu and stabilizing the helix by forming a lactam between Glu(22) and Lys (26) to produce the [Leu(27)]cyclo(Glu(22)-Lys(26))hPTH(1-28)NH2 analog dra matically reduced the IC50 for displacing I-125-[Nle(8,18),Tyr(34)]bPTH(1-3 4)NH2 from hPTH1Rs from 110 to 6 nM and dropped the EC50 for adenylyl cycla se stimulation in ROS 17/2 cells from 23.9 to 9.6 nM, These modifications a lso increased the osteogenic potency of hPTH(1-28)NH2, Thus, hPTH(1-28)NH2 did not significantly stimulate either femoral or vertebral trabecular bone growth in rats when injected daily at a dose of 5 nmol/100 g body weight f or 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stim ulated the growth of trabeculae in the cancellous bone of the distal femurs and L5 vertebrae when injected at 25 nmol/100 g body weight. By contrast [ Leu(27)]cyclo(Glu(22)-Lys(26))hPTH(1-28)NH2 significantly stimulated trabec ular bone growth when injected at 5 nmol/100 g of body weight. Thus, these modifications have brought the bone anabolic potency of hPTH(1-28)NH2 consi derably closer to the potencies of the larger PTH peptides and analogs.