V. Ross

STIMULATION OF THE GROWTH OF FEMORAL TRABECULAR BONE IN OVARIECTOMIZED RATS BY THE NOVEL PARATHYROID HORMONE FRAGMENT, HPTH-(1-31)NH2 (OSTABOLIN)

The human parathyroid hormone, hPTH-(1–84), and its hPTH-(1–34) fragment are promising anabolic agents for treating osteoporosis because they can strongly stimulate the production of biomechanically effective cortical and trabecular bone in osteopenic ovariectomized (OVX) rats and trabecular bone in osteoporotic postmenopausal humans. The ideal PTH fragment for treating osteoporosis would be the smallest and functionally simplest fragment that activates only one signal mechanism and still strongly stimulates trabecular bone growth. A new PTH fragment, hPTH-(1–31)NH2, which only stimulates adenylyl cyclase instead of stimulating both adenylyl cyclase and phospholipase-C as do hPTH-(1–84) and hPTH-(1–34), is this minimum, high-potency anabolic fragment. hPTH-(1–31)NH2 (which we have named Ostabolin) can greatly thicken trabeculae and increase the dry weight and calcium content of trabecular bone in the distal femurs of osteopenic, young, sexually mature OVX Sprague-Dawley rats when injected subcutaneously each day for 6 weeks at doses between 0.4 and 1.6 nmole/100 g of body weight.

Comparison of the ability of recombinant human parathyroid hormone, rhPTH-(1-84), and hPTH-(1-31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats

A recombinant human parathyroid hormone, rhPTH-(1–84), which is currently in Phase II clinical trial, and hPTH-(1–31)NH2 (Ostabolin) are promising anabolic agents for treating osteoporosis because they can stimulate cortical and trabecular bone growth in osteopenic, ovariec-tomized (OVX) rats and in osteoporotic, postmenopausal women when injected subcutaneously and intermittently at low doses. We have now found that, despite their different sizes and signaling properties (rhPTH-(1–84) stimulates adenylyl cyclase and phospholipase C; hPTH-(1–31)NH2 only stimulates adenylyl cyclase), they are equally osteogenic in OVX rats. Thus daily subcutaneous injections of 0.6 nmol/ 100 g of body weight of rhPTH-(1–84) or hPTH-(1–31)NH2 into 3-month-old OVX rats for 6 weeks starting 2 weeks after OVX equally reduced the otherwise large OVX-triggered loss of femoral trabecular bone. Daily subcutaneous injections of 0.4 or 0.8 nmol/100 g of body weight of the two agents for 6 weeks also equally increased the mean thickness of the remaining femoral trabeculae in 3-month-old and 1-year-old OVX rats to 20 to 80% above the value in normal animals when started 9 weeks after ovariectomy.

Cyclization by a specific lactam increases the ability of human parathyroid hormone (hPTH)-(1-31)NH2 to stimulate bone growth in ovariectomized rats

Human parathyroid hormone (hPTH)‐(1–31)NH2 (Ostabolin), which only stimulates adenylyl cyclase (AC) instead of AC and phospholipase‐C as do hPTH(1–84) and hPTH(1–34), strongly stimulates femoral cortical and trabecular bone growth in ovariectomized (OVX) rats. Two side‐chain lactams have been introduced in the hydrophilic face of the receptor‐binding region of the fragments Ser17‐Val31 amphiphilic α‐helix in an attempt to develop improved analogs for the treatment of osteoporosis. Replacing the polar Lys27 with an apolar Leu on the hydrophobic face of this α‐helix and stabilizing the helix with a lactam between Glu22 and Lys26 produced a fragment, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2, which had six times the AC‐stimulating ability of hPTH(1–31)NH2 in ROS 17/2 rat osteosarcoma cells, but the other helix‐stabilizing lactam derivative [Leu27]‐cyclo(Lys26‐Arg30)‐hPTH(1–31)NH2 did not have a greater AC‐stimulating ability than hPTH(1–31)NH2, to stimulate AC in ROS 17/2 rat osteosarcoma cells. As expected from AC stimulation being responsible for PTHs anabolic action, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2 was, depending on the experimental design, a 1.4 to 2 times better stimulator of trabecular bone growth in the OVX rat model than either hPTH(1–31)NH2 or [Leu27]‐cyclo(Lys26‐Arg30)‐hPTH(1–31)NH2. Thus, there is now a more potently anabolic derivative of hPTH(1–31)NH2, [Leu27]‐cyclo(Glu22‐Lys26)‐hPTH(1–31)NH2, which might ultimately prove to be one of the more effective therapeutics for osteoporosis.

Restoration of severely depleted femoral trabecular bone in ovariectomized rats by parathyroid hormone-(1–34)

It is commonly believed that the parathyroid hormones (PTHs) main function in bone is to stimulate osteoclastic resorption. However, intermittent injections of small doses of PTH holoprotein, but more often its bioactive hPTH-(1–34) fragment, have been shown to stimulate bone growth in animals and humans through their ability to stimulate adenylyl cyclase and not their ability to independently activate a protein kinases-C stimulating mechanism. This anabolic action suggests that PTH might be an effective therapeutic for osteoporosis. If so, the hormone must be able to restore severely depleted trabecular bone, and the goal of this study was to find out if it can. To do this, we started a multiweek program of daily subcutaneous injections of 0.8 nmoles of hPTH-(1–34)/100 g body weight into rats at 4, 8, or 16 weeks after ovariectomy (OVX) and the increasingly severe selective loss of trabecular bone. These injections strongly stimulated femoral trabecular bone to grow and mineralize at the same rate regardless of how much of it had been lost before the injections were started. Thus, the progressively depleting trabecular bone in the femurs of OVX rats does not lose its anabolic responsiveness to PTH. This finding is another indication of the likelihood of small, adenylyl cyclase-stimulating PTH fragments being effective therapeutics for osteoporosis.

The effect of monocyclic and bicyclic analogs of human parathyroid hormone (hPTH)-(1-31)NH2 on bone formation and mechanical strength in ovariectomized rats

The [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 lactam is a stronger stimulator of adenylyl cyclase activity and a better stimulator of trabecular bone in the ovariectomized (OVX) rat model of osteopenia than hPTH-(1-31)NH2. This enhanced activity is due in large part to the stabilization of the amphiphilic receptor-binding α-helix in the Ser17-Gln29 region. The goal of the present study was to determine whether further cyclization could produce a more active hPTH analog. To this end, we compared the relative bioactivities of the bicyclic hPTH analog [Glu17,Leu27]cyclo(Lys13-Glu17,Glu22-Lys26)-hPTH-(1-31)NH2, made by replacing Ser17 with Glu17 and introducing a second lactam linkage between Lys13 and Glu17. The relative EC50 for adenylyl cyclase stimulation by the bicyclic hPTH analog was similar to the EC50 of the monocyclic [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, but the bicyclic analog was still more active than hPTH-(1-31)NH2. As expected from adenylyl cyclase stimulation being responsible for PTH’s anabolic action, the bicyclic hPTH analog [Glu17, Leu27]cyclo(Lys13-Glu17, Glu22-Lys26)-hPTH-(1-31)NH2 was able to increase femoral trabecular volume and thickness and mechanical strength in OVX rats, but it was no more effective than [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 when injected once daily in a dose of 0.8 nmol/100 g body weight. Thus, further constraint of the conformation of hPTH-(1-31)NH2 by introducing two lactam linkages between Lys13-Glu17 and Glu22-Lys26 did not raise the osteogenicity above that of the monocyclic analog.

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 osteoblast‐like osteosarcoma cells. This fragment has an IC50 of 110 nM for displacing 125I‐[Nle8,18, Tyr34]bovine PTH(1–34)NH2 from HKRK B7 porcine kidney cells, which stably express 950,000 human type 1 PTH/PTH‐related protein (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 amphiphilicity of the α‐helix in the residue 17–28 region by replacing Lys27 with Leu and stabilizing the helix by forming a lactam between Glu22 and Lys26 to produce the [Leu27]cyclo(Glu22‐Lys26)hPTH(1–28)NH2 analog dramatically reduced the IC50 for displacing 125I‐[Nle8,18, Tyr34]bPTH(1–34)NH2 from hPTHIRs from 110 to 6 nM and dropped the EC50 for adenylyl cyclase stimulation in ROS 17/2 cells from 23.9 to 9.6 nM. These modifications also 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 for 6 weeks, beginning 2 weeks after ovariectomy (OVX), but it strongly stimulated 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 [Leu27]cyclo(Glu22‐Lys26)hPTH(1–28)NH2 significantly stimulated trabecular 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 considerably closer to the potencies of the larger PTH peptides and analogs. (J Bone Miner Res 2000;15:964–970)

The Stimulation of Vertebral and Tibial Bone Growth by the Parathyroid Hormone Fragments, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2

Abstract. The native human parathyroid hormone, hPTH-(1-84), and certain carboxyl truncated analogs such as hPTH-(1-34) and even smaller fragments such as hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-30)NH2 stimulate femoral trabecular and cortical bone growth in ovariectomized (OVX) rats. Here we show that when injected once daily for 6 weeks starting 2 weeks after OVX in doses of 1 or 2 nmol/100 g of body weight, hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 prevented the loss of trabecular volume in the L5 vertebrae induced by OVX. In fact, by the end of the sixth week of injections (i.e., the eighth week after OVX) the fragments had increased the volume and trabecular thickness significantly above the values in vehicle-injected sham-operated rats. hPTH-(1-30)NH2 can stimulate vertebral bone growth as much as the larger fragments, but 10–25 times more of it was needed to do so. The same daily doses of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)hPTH-(1-31)NH2, and hPTH-(1-34)NH2 also raised the trabecular volume and thickness in the L5 vertebrae of rats well above the values in vehicle-treated animals when the injections were started 9 weeks after OVX. This restoration of trabecular bone in the L5 vertebrae in estrogen-deprived animals was accompanied by a significant increase in the bone mineral density (BMD) of the L1–L4 vertebrae and tibias. However, there was no significant drop in the pelvic BMD in the estrogen-deprived animals and the effects of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-(Lys) hPTH-(1-31)NH2, and hPTH-(1-34)NH2 on the pelvic BMD were equivocal.

Stimulation of Femoral Trabecular Bone Growth in Ovariectomized Rats by Human Parathyroid Hormone (hPTH)-(1-30)NH2

Abstract. It has been proposed that intermittent bursts of adenylyl cyclase and the surges of cyclic AMP (cAMP) they produce can trigger PTHs bone anabolic action without the activation of phospholipase-C (PLC). This was based on the osteogenic action in ovariectomized (OVX) rats of hPTH-(1-31)NH2, which can stimulate adenylyl cyclase but not PLC in ROS 17/2 rat osteosarcoma cells, and the osteogenic impotence of fragments such as 1-desamino-hPTH-(1-34) and hPTH-(8-84) which strongly stimulate PLC but not adenylyl cyclase. But this seems to have been disproven by the inability of hPTH-(1-30)NH2 to stimulate bone growth despite its having hPTH-(1-31)NH2s ability to strongly stimulate adenylyl cyclase but not PLC in cells with rat type1 PTH/PTHrP receptors. Because of the importance of hPTH-(1-30)NH2s apparent osteogenic impotence for knowing how PTH triggers bone growth, we have reinvestigated the fragments ability to stimulate trabecular bone growth in the femurs of young OVX rats and have found it to be strongly osteogenic at doses 2–10 times higher than the highest dose used previously. Thus, 6 weeks of once-daily subcutaneous injections of 10–50 nmol of hPTH-(1-30)NH2/100 g of body weight into young rats starting 2 weeks after OVX significantly increased the femoral trabecular volume and mean thickness of individual trabeculae above those in sham-operated control rats. In OVX rats treated with 50 nmol of hPTH-(1-30)NH2/100 g of body weight, the trabecular volume was 2.6 times higher and the mean trabecular thickness nearly 4 times higher than in the sham-operated control rats. This very large increase in the mean trabecular thickness was as much as the increase induced by 2 nmol/100 g of body weight of hPTH-(1-31)NH2, [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, hPTH-(1-34)NH2 and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-34)NH2. These results have removed a major objection to the proposal that PTHs osteogenic action in rats can be triggered solely by intermittent surges of cAMP and the bursts of cAMP-dependent protein kinase activity they cause.

Comparison of the Abilities of Human Parathyroid Hormone (hPTH)-(1-34) and [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 to Stimulate Femoral Trabecular Bone Growth in Ovariectomized Rats

Abstract. hPTH-(1-31)NH2, so far the smallest of the potently anabolic N-terminal fragments of the human parathyroid hormone, stimulates trabecular growth in the distal femurs of ovariectomized (OVX) rats as strongly as hPTH-(1-34) when injected at a high daily dose such as 1 nmol/100 g of body weight, but it is only about 70% as effective as hPTH-(1-34) when injected at the suboptimal 0.6 nmol/100 g of body weight. A lactam derivative of hPTH-(1-31)-NH2, [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2, is a much more effective stimulator of adenylyl cyclase in ROS 17/2 rat osteoblast-like cells and a significantly more effective stimulator of femoral trabecular growth in OVX rats than hPTH-(1-31)NH2. We have now shown that [Leu27]-cyclo(Glu22-Lys26)-hPTH-(1-31)NH2 prevents the OVX-induced loss of femoral trabeculae significantly more effectively than hPTH-(1-34) and stimulates the thickening of the trabeculae remaining in severely depleted femoral trabecular bone of OVX rats as effectively as hPTH-(1-34) when injected at 0.6 nmol/100 g of body weight.

The Hypotensive Actions of Osteogenic and Nonosteogenic Parathyroid Hormone Fragments

Parathyroid hormone (PTH), hPTH-(1-84), and its hPTH-(1-34), hPTH-(1-31)NH2, and hPTH-(1-30)NH2 fragments reduced the tail artery pressure in anesthetized female Sprague-Dawley rats by 42.4–67.1% within about 1 minute after injection into a femoral vein, but reduced the pressure by only 8.5–36.2% 2–19 minutes after subcutaneous injection. hPTH-(1-84) and hPTH-(1-34) stimulate both adenylyl cyclase and phospholipase-C in their target cells, but the hypotensive action must have been stimulated specifically by adenylyl cyclase activation, because hPTH-(1-30)NH2 and hPTH-(1-31)NH2, which can only stimulate adenylyl cyclase, were potently hypotensive when injected intravenously whereas hPTH-(7-84), which can only stimulate phospholipase-C, was not significantly hypotensive when injected intravenously. Since PTHs osteogenic action is also mediated by adenylyl cyclase stimulation, it was expected that the hypotensive response might be used to screen new PTH constructs for possible osteogenicity. Indeed, the osteogenic activities of subcutaneously injected hPTH-(1-31)NH2, hPTH-(1-34), and hPTH-(1-84) correlated closely to their hypotensive activities, with hPTH-(1-34) being much more hypotensive and significantly more osteogenic than the other two molecules. hPTH-(1-31)NH2 and hPTH-(1-84) were equally osteogenic and hypotensive. However, this correlation broke down with hPTH-(1-30)NH2 which does not stimulate bone formation, but in the present study it stimulated adenylyl cyclase and reduced tail artery pressure almost as much as hPTH-(1-31)NH2 and hPTH-(1-34). Nevertheless, the ability to significantly reduce arterial pressure is a common property of osteogenic PTH and PTH fragments and is thus a rapidly determinable preliminary indicator of in vivo bioactivity of PTH fragments.