Contributions of parathyroid hormone (PTH)/PTH-related peptide receptor signaling pathways to the anabolic effect of PTH on bone

PTH regulates osteoblastic function by activating PTH/PTHrP receptors (PTH1Rs), which trigger several signaling pathways in parallel, including cAMP/protein kinase A (PKA) and, via both phospholipase-C (PLC)-dependent and PLC-independent mechanisms, protein kinase C (PKC). These signaling functions have been mapped to distinct domains within PTH(1–34), but their roles in mediating the anabolic effect of intermittent PTH in vivo are unclear. We compared the anabolic effects in mice of hPTH(1–34) with those of two analogs having restricted patterns of PTH1R signaling. [G¹,R¹⁹]hPTH(1–28) lacks the 29–34 domain of hPTH(1–34) needed for PLC-independent PKC activation, incorporates a Gly¹ mutation that prevents PLC activation, and stimulates only cAMP/PKA signaling. [G¹,R¹⁹]hPTH(1–34) retains the 29–34 domain and activates both cAMP/PKA and PLC-independent PKC.

Human PTH(1–34) (40 μg/kg), [G¹,R¹⁹]hPTH(1–34) (120 μg/kg), and [G¹,R¹⁹]hPTH(1–28) (800 μg/kg), at doses equipotent in elevating blood cAMP at 10 min and cAMP-dependent gene expression in bone at 6 h after s.c. injection, were administered to 10-week-old female C57BL/6J mice 5 days/week for 4 weeks. Acute blood cAMP responses, retested after 4 weeks, were not reduced by the preceding PTH treatment. The three PTH peptides induced equivalent increases in distal femoral bone mineral density (BMD), and, by microCT analysis, distal femoral and vertebral bone volume and trabecular thickness and mid-femoral cortical endosteal apposition. [G¹,R¹⁹]hPTH(1–34) and hPTH(1–34) increased distal femoral BMD more rapidly and augmented total-body BMD and bone volume of proximal tibial trabeculi to a greater extent than did [G¹,R¹⁹]hPTH(1–28).

We conclude that cAMP/PKA signaling is the dominant mechanism for the anabolic actions of PTH in trabecular bone and that PLC-independent PKC signaling, attributable to the PTH(29–34) sequence, appears to accelerate the trabecular response and augment BMD at some skeletal sites. PTH1R PLC signaling pathway is not required for an anabolic effect of intermittent PTH(1–34) on bone.     

间歇皮下注射人甲状旁腺激素不同片段(hFTH1-34及 hFTH1-84)对去卵巢大鼠股骨及腰椎骨量的影响

目的 比较间歇皮下注射人甲状旁腺激素不同片段（hPTH1-34)及（hPTH1-84)对完整雌性（Non-OVX)大鼠和去卵巢（OVX）大鼠股骨及腰椎1－4骨矿物含量（BMC）和骨密度（BMD）的影响。方法 Wistar雌性大鼠176只，分为hPTH1-34和hPTH1-84两大组（各80只及96只），每大组及各自分4组（每组各20只或24只），分别为：两组安慰剂组（未切卵巢及切卵巢）用安慰剂（PBS）进行皮下注射，每周3次，共2周；两组治疗组（未切卵巢及切卵巢）用hPTH1-34或hPTH1-84，皮下注射，每周3次，共2周。结果 1．卵巢切除术后3个月大鼠股骨及腰椎1－4BMC和BMD明显下降；2．两种片段的甲状旁腺激素（hPTH1-34及pPTH1-84)间歇注射均能使Non-OVX大鼠和OVX大鼠股骨及腰椎1－4BMC和BMD较相应对照组明显升高；且腰椎1－4较股骨的BMC和BMD升高更明显；3．OVX大鼠治疗后股骨与腰椎1－4BMC和BMD的升高率较Non-OVX大鼠更明显；OVX大鼠在治疗后股骨及腰椎骨量能恢复到去卵巢前水平；4．hPTH1-34较hPTH1-84更明显的使完整大鼠和OVX大鼠股骨BMC和BMD升高。结论 间歇皮下注射人甲状旁腺激素对大鼠股骨及腰椎骨量均有增高作用，尤其对腰椎的骨量以及对去卵巢大鼠骨量升高作用更明显；hPTH1-34片段对大鼠股骨骨量的增高作用强于hPTH1-84片段。     

Daily nasal spray of hPTH(1–34) for 3 months increases bone mass in osteoporotic subjects: a pilot study

Introduction Although intermittent parathyroid hormone (PTH) injection can lead to strong anabolic effects on bone, daily subcutaneous injection is a disadvantage for patient acceptance. We have developed a nasal spray formula of parathyroid peptide [hPTH(1–34)] with peak serum hPTH(1–34) concentrations by nasal spray of 1,000 μg similar to those by subcutaneous injections of 20 μg hPTH(1–34).Methods To determine the clinical efficacy and safety of nasal hPTH(1–34) spray, a randomized, open-labeled clinical trial was conducted in subjects with osteoporosis. Ninety osteoporotic subjects age 52–84 years (mean 66.5 years) were randomly assigned to receive either 250 μg (PTH250, n=31), 500 μg (PTH500, n=30), or 1,000 μg (PTH1000, n=29) of daily nasal hPTH(1–34) spray for 3 months. All received daily supplements of 300 mg calcium and 200 IU vitamin D₃.Results Daily nasal hPTH(1–34) spray for 3 months increased lumbar bone mineral density (L-BMD) in a dose-dependent manner, and the PTH1000 group showed a 2.4% increase in L-BMD from baseline. Only the 1,000-μg dose produced consistent and statistically significant changes in markers of bone turnover; after 3 months, median serum type I procollagen N-propeptide (PINP) and osteocalcin increased 14.8% and 19.4% from baseline, while urinary type I collagen N-telopeptide (NTX) decreased 16.4%. Seven subjects developed transient hypercalcemia at 3 h after nasal hPTH(1–34) spray, but none of the subjects developed sustained hypercalcemia.Conclusion These observations demonstrate that nasal hPTH(1–34) spray is safe and well tolerated and can rapidly increase L-BMD. The results warrant further studies to examine its long-term efficacy on bone mass and fractures.     

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 alpha-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 hPTH1Rs 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.     

Loss of the anabolic effect of parathyroid hormone on bone after discontinuation of hormone in rats

We have previously reported that low doses of hPTH 1–34 given daily to rats exert an anabolic effect on bone. The objective of this study was to determine if the anabolic effect of PTH was dependent upon continued daily administration of the hormone. Young, male rats were given daily subcutaneous injections of either vehicle or 8 μg/100g bw hPTH 1–34 for 12, 16, 20, or 24 days. Additional groups were treated with 8 μg/100g bw hPTH for 12 days followed by vehicle for the next 4, 8, or 12 days, or 8 μg/100g bw hPTH 1–34 for 16 days followed by 4 days of vehicle. We measured calcium (Ca), dry weight (DW), and hydroxyproline (Hyp) of the distal femur, percent of osteoblast (Ob.S/BS) and osteoclast (Oc.S/BS) surface, mineral apposition rate (MAR), double label surface (DLS/BS), and bone formation rate (BFR) in the metaphysis of the proximal tibia, and serum calcium and phosphate. Trabecular and cortical bone Ca and DW and the histologic measures of bone formation increased in all PTH-treated rats. Serum calcium and phosphate were comparable in all rats. The PTH-stimulated bone mass was lost 12 days after discontinuation of PTH. Discontinuation of PTH administration for 4, 8, or 12 days, respectively, resulted in a 72%, 68%, or 50% decrease in Ob.S/BS from the 2- to 3-fold increase associated with PTH treatment (p < .05). Oc.S/BS increased compared to controls after 4 days of PTH withdrawal (NS), but was comparable to controls 8 days after withdrawal of PTH. The loss of new bone mass after discontinuation of hormone was due to an inhibition of PTH-stimulated bone formation and an initial transient phase of increased resorption. Thus, the anabolic effect of PTH was dependent upon daily administration of the hormone in our model.     

Effects of cyclic versus daily hPTH(1-34) regimens on bone strength in association with BMD, biochemical markers, and bone structure in mice.

We developed a cyclic PTH regimen with repeated cycles of 1-week on and off daily PTH injection and explored its effects on bone strength, BMD, bone markers, and bone structure in mice. Cyclic protocols produced 60-85% of the effects achieved by daily protocols with 57% of the total PTH given, indicating more economic use of PTH. The study supports further exploration of cyclic PTH regimens for the treatment of osteoporosis. INTRODUCTION: To minimize the cost and the catabolic action of hPTH(1-34), a cyclic PTH regimen with repeated 3-month cycles of on-and-off daily injection of hPTH(1-34) was developed in humans and shown to be as effective as a daily regimen in increasing vertebral BMD. However, changes in BMD may not adequately predict changes in bone strength. A murine model was developed to explore the efficacy of a cyclic PTH regimen on bone strength in association with other bone variables. MATERIALS AND METHODS: Twenty-week-old, intact, female C57BL/J6 mice (n = 7/group) were treated with (1) daily injection with vehicle for 7 weeks (control); (2) daily injection with hPTH(1-34) (40 microg/kg/day) for 7 weeks (daily PTH); and (3) daily injection with hPTH(1-34) and vehicle alternating weekly for 7 weeks (cyclic PTH). BMD was measured weekly by DXA, and serum bone markers, bone structure, and strength were measured at 7 weeks. RESULTS: Daily and cyclic PTH regimens increased BMD at all sites by 16-17% and 9-12%, respectively (all p < 0.01). The most dramatic effect of cyclic PTH occurred during the second week of treatment when PTH was off, with femoral and tibial BMD continuing to increase to the same extent as that produced by daily PTH. Both daily and cyclic PTH regimens significantly increased osteocalcin (daily, 330%; cyclic, 260%), mTRACP (daily, 145%; cyclic, 70%), femoral cortical width (daily, 23%; cyclic, 13%), periosteal circumference (daily, 5%; cyclic, 3.5%), and bone strength (max load: daily, 48%; cyclic, 28%; energy absorbed: daily, 103%; cyclic, 61%), respectively. Femoral bone strength was positively correlated with BMD, bone markers, and cortical structure. Neither regimen had an effect on vertebral bone strength. Although actual effects of cyclic PTH were 60-85% of those produced by daily PTH, the effects of cyclic PTH per unit amount administered were slightly greater than those of daily PTH for most measures. CONCLUSIONS: PTH-enhanced femoral bone strength is positively correlated with its effects on femoral BMD, bone markers, and bone structure. Cyclic PTH regimens represent a potential economic use of PTH and warrant further study.     

Effect of parathyroid hormone (hPTH[1-84]) treatment on bone mass and strength in ovariectomized rats.

Skeletal fragility in osteoporotic patients is a prominent underlying cause of low-trauma fractures of most bone sites in humans. Clinical research is now focused on developing treatment strategies, including anabolic agents such as parathyroid hormone (PTH), to recover osteoporosis-related bone loss. Female Sprague-Dawley rats (4.5 mo old) were allowed to become osteopenic for 10 wk postovariectomy. Eight rats were killed at the time of ovariectomy (-10 wk) as a baseline control; sham and ovariectomized (OVX) groups were killed at wk 0. Eight rats per group (sham, OVX + vehicle, OVX + hPTH [5 d/wk], and OVX + hPTH [3 d/wk]) were killed after 4, 8, 14, and 20 wk of treatment with 50 microg/kg of human parathyroid hormone (hPTH[1-84]). Bone mineral content and density were measured only in the vertebral body. Bone strength was evaluated in the vertebral body, femoral diaphysis, femoral neck, and distal femur. Significant, lasting osteopenia developed in the vertebral body of OVX rats by 10 wk postovariectomy. Bone mineral density of the vertebral body partially recovered by 8 wk and fully recovered to that seen in sham animals only by 20 wk posttreatment with either a 5 or 3 d/wk dosing schedule of PTH[1-84]. Therefore, hPTH[1-84] (50 microg/kg) given either 3 or 5 d/wk fully restores vertebral and femoral bone strength in osteopenic OVX rats.     

Treatment with Parathyroid Hormone hPTH(1-34), hPTH(1-31), and Monocyclic hPTH(1-31) Enhances Fracture Strength and Callus Amount After Withdrawal Fracture Strength and Callus Mechanical Quality Continue to Increase

The influence of intermittent hPTH(l-34)NH₂, hPTH(1-31)NH₂, and monocyclic [Leu²⁷]cyclo(Glu²²-Lys²⁶)hPTH(1-31)NH₂ treatment on callus formation, mechanical strength, and callus tissue mechanical quality of tibial fractures in rats was investigated after 8 and 16 weeks of healing. In the 8 weeks of healing animals, the PTH-peptides were injected subcutaneously during the entire observation period (15 nmol/kg/day [hPTH(1-34)NH₂: 15 nmol = 60 µg]), and control animals with fractures were given vehicle. In the 16 weeks of healing animals, the PTH-peptides were injected only during the first 8 weeks of healing (15 nmol/kg/day), after which the animals were left untreated during the rest of the healing period. After the first 8 weeks of healing, increased fracture strength and callus volume were seen in the PTH-treated rats (ultimate load 66%, ultimate stiffness 58%, callus volume 28%), and the three peptides were equally effective. No difference in callus tissue mechanical quality was found between PTH and vehicle animals. After 16 weeks of healing, no differences in fracture strength, callus volume, or callus tissue mechanical quality were seen between PTH and vehicle. When comparing PTH-treated animals at 8 and 16 weeks, fracture strength and callus tissue mechanical quality continued to increase after the withdrawal of PTH (ultimate load 23%, ultimate stress 88%, elastic modulus 87%) and external callus volume declined during this period (27%).     

Dexamethasone-treated ROS 17/2.8 rat osteosarcoma cells are responsive to human carboxylterminal parathyroid hormone peptide hPTH (53–84): Stimulation of alkaline phosphatase

Summary We tested the effects of various parathyroid hormone (PTH) peptides on alkaline phosphatase (ALP) activity in the osteoblastic cell line ROS 17/2.8. In dexamethasonetreated ROS 17/2.8 cells there was a dose-related increase in ALP activity due to treatment with hPTH (53–84). ALP activity was stimulated by 10 nM hPTH (53–84) by a mean of 1.51±0.07-fold (P<0.001) in nine experiments, whereas the same dose of bPTH (1–34) and bPTH (1–84) inhibited enzyme activity to 0.36±0.02-fold (P<0.001) and 0.37±0.03-fold (P<0.001), respectively. Significant stimulation of ALP activity occurred with doses of hPTH (53–84) as low as 0.01 nM. There was no stimulation of enzyme activity by hPTH (53–84) in the absence of dexamethasone; the maximum ALP response to hPTH (53–84) occurred between 96 and 144 hours, and no significant effect was seen at time periods less than 96 hours. The optimum dose of dexamethasone required to enable the response to hPTH (53–84) was 10 nM. Carboxylterminal PTH fragments had a specific stimulatory effect on ALP activity in dexamethasone-treated ROS 17/2.8 cells, but the aminoterminal PTH effect appeared to be dominant, as the equimolar combination of bPTH (1–34) and hPTH (53–84) resulted in inhibition of ALP activity. Thus, in order for the effects of carboxylterminal fragments to be manifest, the cells would have to be stimulated under conditions in which the aminoterminal receptor is unoccupied; this could occur under somein vivo conditions. The physiological significance of these observations is unknown, but the enabling effect of dexamethasone on hPTH (53–84) stimulation of ALP suggests that osteoblastic cells are responsive to this hormonal peptide at a certain stage of differentiation.     

Trabecular bone volume and microdamage accumulation in the femoral heads of women with and without femoral neck fractures

Prostaglandin E₂ (PGE₂) possesses significant anabolic properties when administered systemically (i.e., it increases bone formation and, consequently, bone mass). We recently characterized the effects of a 3 week administration of 6 mg/kg PGE₂ into young rats and showed it increases cortical and cancellous bone mass and mechanical strength in long bones and bone density in the calvaria. We also found that a single dose of PGE₂ induces the expression of early-response genes (c-fos, c-jun, and egr-1) in bone marrow cells within these two types of bone. These observations, together with findings by others of new cancellous bone formation in PGE₂-treated animals, suggested that recruitment of osteoblasts from their precursors is a major mechanism of the anabolic effect of PGE₂. To test this hypothesis directly, we injected PGE₂ (6 mg/kg) or vehicle into 4-week-old rats for 2 weeks and then assessed the osteogenic potential of bone marrow in an ex vivo culture system. Primary and first-passage bone marrow cultures were established in the presence of β-glycerophosphate, ascorbate, and dexamethasone, and osteogenic differentiation was measured by bone nodule formation and alkaline phosphatase activity. This regimen increased bone mass expressed as femoral ash weight by 4.7% and tibial cancellous bone area by 38.3%. Nodule formation at 21 days was increased in both primary and first-passage cultures from PGE₂-treated rats despite seeding of the same number of marrow cells. Alkaline phosphatase activity was elevated in both primary and first-passage cultures from PGE₂-treated rats beginning 6–10 days after culture initiation. Cell proliferation was only slightly elevated in cultures from PGE₂-treated rats. These data strongly suggest that in vivo administration of PGE₂ induces the proliferation or differentiation of osteoprogenitor cells in bone marrow, and this effect takes a major part in its anabolic effect in vivo.     

Human parathyroid hormone (1-34) increases mass and structure of the cortical shell,with resultant increase in lumbar bone strength,in ovariectomized rats

Estrogen deficiency causes reduction of bone mass and abnormal bone microarchitecture, consequently reducing bone strength. Human parathyroid hormone (hPTH) (1-34) increases bone mass and strength. To clarify the factors that determine the recovery of bone strength in the lumbar vertebrae of ovariectomized rats by intermittent hPTH administration, we analyzed the relationship between skeletal measurements and bone strength. Human PTH (1-34) administration resulted in recovery of cortical bone mineral content (BMC) and cortical bone area to sham the levels, but in resulted in a less pronounced recovery of trabecular BMC and no increase in the total cross-sectional area of the vertebral body. Of the three-dimensional (3D) trabecular bone parameters, hPTH (1-34) increased trabecular thickness (Tb.Th). The cortical shell area of L4, determined by histomorphometry, was also increased. In hPTH-treated rats, the only determinant of the compressive load of L5 was the cortical shell BMC, in the early recovery period (days 42–84). Our data suggest that increased cortical bone mass contributes more than trabecular bone mass and structure to the recovery of bone strength in response to hPTH therapy in the rat lumbar vertebral body after ovariectomy.     

Maintenance of cancellous bone in ovariectomized, human parathyroid hormone [hPTH(1-84)]-treated rats by estrogen, risedronate, or reduced hPTH

This study compares effects of maintenance doses of human parathyroid hormone [hPTH(1-84)], 17beta-estradiol (E2), and risedronate on distal femur bone mineral density and proximal tibia cancellous bone histomorphometry in ovariectomized (ovx), osteopenic rats previously administered a higher dose of hPTH. Nine groups (n = 8) of 3.5-month-old ovx or intact Sprague-Dawley rats were left untreated for 11 weeks to allow for the development of cancellous osteopenia in the ovx groups. Next, the ovx rats received subcutaneous injections of hPTH (75 microg/kg per day, three times per week) or vehicle for 12 weeks. Treatments were then changed to E2 (10 microg/kg per day, two times per week), risedronate (Ris; 3 microg/kg per day, three times per week), low-dose hPTH(1-84) (LowPTH; 25 microg/kg per day, three times per week), or vehicle, and administered for 36 weeks. The intact control group remained untreated for the duration of study. Femora and tibiae were collected at weeks -11 (baseline); 0 (ovx effect); 12 (hPTH effect), and 24, 36, and 48 (maintenance effects). Endpoints evaluated included distal femur bone mineral density (BMD) and proximal tibia cancellous bone volume (BV/TV), osteoclast surface (Oc.S), mineralizing surface (MS), mineral apposition rate (MAR), and bone formation rate (BFR). Ovariectomy had a negative effect on distal femur BMD and proximal tibia BV/TV. Treatment of ovx rats with hPTH for 12 weeks resulted in higher BMD in comparison to intact controls, and higher cancellous BV/TV in comparison to ovx controls. Discontinuation of hPTH resulted in loss of gained BMD within 24 weeks and loss of gained BV/TV within 12 weeks. Treatment of ovx rats with hPTH for 12 weeks followed by E2 treatment left BMD and BV/TV similar to vehicle-treated ovx rats by week 48 (36 weeks after commencement of the E2 maintenance treatment). Maintenance treatment with risedronate resulted in BMD and BV/TV similar to that of intact controls. Maintenance treatment with low-dose hPTH resulted in greater BMD and similar BV/TV in comparison to intact controls. MS and BFR were highest after low-dose hPTH administration. MS and BFR were lowest after E2 or risedronate, whereas Oc.S was lowest after risedronate administration. Thus, in osteopenic rats, the increment in distal femur BMD and proximal tibia BV/TV gained by 12 weeks of hPTH treatment was lost within 24 and 12 weeks of treatment termination, respectively. Low-dose hPTH maintained BMD and BV/TV after hPTH treatment by stimulating bone formation, whereas risedronate maintained BMD and BV/TV by reducing bone resorption. E2 in a maintenance dose failed to maintain BMD and BV/TV after withdrawal of hPTH treatment.     

Assessment of maintenance therapy with reduced doses of PTH(1-34) in combination with a raloxifene analogue (LY117018) following anabolic therapy in the ovariectomized rat.

This experiment was designed to evaluate the ability of a raloxifene analogue (RA), LY117018, with or without reduced dosing of human parathyroid hormone (hPTH)(1-34) to maintain gains in bone mass after a fully anabolic treatment regimen given to aging osteopenic rats. Six-month-old rats were ovariectomized (ovx) or sham-operated (sham). After 1 month, ovx rats were treated with an anabolic regimen consisting of subcutaneous hPTH(1-34) 80 microg/kg/day and oral raloxifene 3 mg/kg/day, each given 5 days/week for 3 months. Thereafter, the treated ovx rats went on to an 8 week maintenance phase of treatment with either RA alone at the same dose, hPTH(1-34) at a reduced dosing interval (twice a week), or a combination of the two. Bone mineral density (BMD) was measured ex vivo at four skeletal sites, lumbar spine (L2-4), proximal hemipelvis, whole femur, and tibia, by dual-energy X-ray densitometry. All four sites showed a similar pattern of response. After the 3 month anabolic phase, the sham group had significantly higher BMD values than ovx rats at all skeletal sites (p < or = 0.002). The ovx rats treated with PTH + RA during the anabolic phase of the protocol had significantly higher BMD than the sham group in the femur, tibia, and spine (p < or = 0.02) and higher but not significantly different values in the pelvis. Following the 2 month maintenance phase, comparisons were made with the PTH-RA group at the end of the anabolic phase. Decrements in BMD were seen in all three maintenance therapy groups, but they were not statistically significant in the RA plus reduced PTH dose group. However, reduced hPTH(1-34) dosing and RA alone resulted in significant reductions of bone mass measurements at several skeletal sites during the maintenance phase. We conclude that the raloxifene analogue LY117018 may be useful in maintaining bone mass in aging ovx rats following anabolic therapy with hPTH(1-34) and raloxifene analogue, but that this strategy only allows for dose reduction of hPTH(1-34) rather than its discontinuation.     

Effects of intermittent administration of low dose human PTH(1–34) on cancellous and cortical bone of lumbar vertebral bodies in adult beagles

This study assessed the effect of low dose human parathyroid hormone [hPTH(1–34)] administration on cancellous and cortical bone of lumbar vertebrae in intact male beagles. 16 19–20-month-old beagle dogs were randomized into four groups: in group 1, the vehicle control group, saline was injected daily; in group 2, the sequential group, 0.375 μg/kg of PTH was injected daily for 4 weeks, then off 8 weeks, and this sequence was once repeated for another 4 and 8 weeks; in group 3, the same dose of PTH was injected once per week for 24 weeks; and, in group 4, PTH was injected three times per week for 24 weeks. Histomorphometric assessment on cancellous and cortical bone (both ventral and dorsal shell) and two-dimensional node-strut analysis were done on the fifth lumbar vertebral bodies after calcein double bone labeling. In intact adult beagles, on the group treated with 0.375 μg/kg per day three times per week (group 4): (1) had a higher mean value in cancellous bone formation parameters [osteoid surface (+74%), osteoid volume (twofold), mineral apposition rate (+21%), and bone formation rate (two-fold)]; (2) exhibited no effect on cortical thickness and porosity in both the ventral and dorsal shell; and (3) showed a lower mean value of node to termini (0.11 ± 0.02 vs. 0.22 ± 0.09) and a higher mean value of cortex to node (0.18 ± 0.06 vs. 0.08 ± 0.02), but not in trabeculae to trabeculae node, than age-related controls. In conclusion, we found that a low dose of PTH administration: (1) stimulated cancellous bone formation; (2) improved connectivity of trabeculae joined to the cortex; (3) did not decrease cortical thickness; and (4) did not increase cortical porosity in both ventral and dorsal cortexal shell of the lumbar vertebrae during this dosage and period in intact male beagles.     

Both hPTH(1-34) and bFGF increase trabecular bone mass in osteopenic rats but they have different effects on trabecular bone architecture.

Osteoporosis is a syndrome of excessive skeletal fragility that results from both the loss of trabecular bone mass and trabecular bone connectivity. Recently, bFGF has been found to increase trabecular bone mass in osteoporotic rats. The purpose of this study was to compare how trabecular bone architecture, bone cell activity, and strength are altered by two different bone anabolic agents, bFGF and hPTH(1-34), in an osteopenic rat model. MATERIALS AND METHODS: Six-month-old female Sprague-Dawley rats (n = 74) were ovariectomized (OVX) or sham-operated (sham) and maintained untreated for 2 months. Then OVX rats were subcutaneously injected with basic fibroblast factor (bFGF; 1 mg/kg, 5 days/week), human parathyroid hormone [hPTH(1-34); 40 microg/kg, 5 days/week], or vehicle for 60 days (days 60-120). Sham-operated and one group of OVX animals were injected with vehicle. Biochemical markers of bone turnover (urinary deoxypyridinoline cross-links; Quidel Corp., San Diego, CA, USA) and serum osteocalcin (Biomedical Technologies, Stroughton, MA, USA) were obtained at study days 0, 60, 90, and 120 and analyzed by ELISA. At death, the right proximal tibial metaphysis was removed, and microcomputed tomography was performed for trabecular bone structure and processed for histomorphometry to assess bone cell activity. The left proximal tibia was used for nanoindentation/mechanical testing of individual trabeculae. The data were analyzed with Kruskal Wallis and post hoc testing as needed. RESULTS: Ovariectomy at day 60 resulted in about a 50% loss of trabecular bone volume compared with sham-treated animals. By day 120 post-OVX, OVX + vehicle treated animals had decreased trabecular bone volume, connectivity, number, and high bone turnover compared with sham-operated animals [p < 0.05 from sham-, hPTH(1-34)-, and bFGF-treated groups]. Treatment of OVX animals with bFGF and hPTH(1-34) both increased trabecular bone mass, but hPTH(1-34) increased trabecular thickness and bFGF increased trabecular number and connectivity. Histomorphometry revealed increased mineralizing surface and bone formation rate in both bFGF and hPTH(1-34) animals. However, osteoid volume was greater in bFGF-treated animals compared with both the hPTH(1-34) and OVX + vehicle animals (p < 0.05). Nanoindentation by atomic force microscope was performed on approximately 20 individual trabeculae per animal (three animals per group) and demonstrated that elastic modulus and hardness of the trabeculae in bFGF-treated animals were similar to that of the hPTH-treated and sham + vehicle-treated animals. CONCLUSION: Both hPTH(1-34) and bFGF are anabolic agents in the osteopenic female rat. However, hPTH(1-34) increases trabecular bone volume primarily by thickening existing trabeculae, whereas bFGF adds trabecular bone mass through increasing trabecular number and trabecular connectivity. These results suggest the possibility of sequential treatment paradigms for severe osteoporosis.     

Transient Increased Calcium and Calcitriol Requirements After Discontinuation of Human Synthetic Parathyroid Hormone 1‐34 (hPTH 1‐34) Replacement Therapy in Hypoparathyroidism

Synthetic human PTH 1‐34 (hPTH 1‐34) replacement therapy in hypoparathyroidism maintains eucalcemia and converts quiescent bone to high‐turnover bone. However, the skeletal and metabolic effects of drug discontinuation have not been reported. Nine subjects with hypoparathyroidism received subcutaneous injections of hPTH 1‐34 two to three times daily for 19.8 to 61.3 months and then transitioned back to calcium and calcitriol. Biochemistries and bone mineral density (BMD) by dual‐energy X‐ray absorptiometry (DXA) were assessed at baseline, while on treatment, and at follow‐up 3 to 12 months after drug discontinuation. Two subjects developed hypocalcemia when hPTH 1‐34 was abruptly discontinued. Thus, to avoid hypocalcemia, subjects were slowly weaned from hPTH 1‐34 over several weeks. When hPTH 1‐34 was stopped, subjects were requiring two to three times pretreatment doses of calcitriol and calcium to maintain blood calcium levels. Doses were gradually reduced over many weeks until calcium levels were stable on doses similar to baseline. Bone‐specific alkaline phosphatase (BSAP), N‐telopeptide (NTX), and osteocalcin (OC) increased significantly with hPTH 1‐34; at follow‐up, BSAP and NTX had returned to baseline while OC was still slightly elevated. During treatment, BMD was unchanged at the hip and lateral spine but declined at the anterior‐posterior (AP) spine, radius, and total body. During weaning, BMD increased, with the hip and lateral spine exceeding pre‐hPTH 1‐34 values and the whole body returning to baseline. AP spine was increased non‐significantly compared to baseline at follow‐up. hPTH 1‐34 must be gradually weaned in hypoparathyroid patients with high doses of oral medications given to avoid hypocalcemia. The transient increased requirements accompanied by increased BMD after long‐term hPTH 1‐34 therapy suggest a reversal of the expanded remodeling space favoring bone formation as the skeleton returns to a low‐turnover state, reminiscent of the hungry bone syndrome. Further study and close monitoring is required to ensure safe transition to conventional therapy and to elucidate the physiological mechanism of this phenomenon. © 2015 American Society for Bone and Mineral Research.     

Human parathyroid hormone (1-34) accelerates natural fracture healing process in the femoral osteotomy model of cynomolgus monkeys

Several studies in rats have demonstrated that parathyroid hormone accelerates fracture healing by increasing callus formation or stimulating callus remodeling. However the effect of PTH on fracture healing has not been tested using large animals with Haversian remodeling system. Using cynomolgus monkey that has intracortical remodeling similar to humans, we examined whether intermittent treatment with human parathyroid hormone [hPTH(1–34)] accelerates the fracture healing process, especially callus remodeling, and restores geometrical shapes and mechanical properties of osteotomized bone.

Seventeen female cynomolgus monkeys aged 18–19 years were allocated into three groups: control (CNT, n = 6), low-dose PTH (0.75 μg/kg; PTH-L, n = 6), and high-dose PTH (7.5 μg/kg; PTH-H, n = 5) groups. In all animals, twice a week subcutaneous injection was given for 3 weeks. Then fracture was produced surgically by transversely cutting the midshaft of the right femur and fixing with stainless plate. After fracture, intermittent PTH treatment was continued until sacrifice at 26 weeks after surgery. The femora were assessed by soft X-ray, three-point bending mechanical test, histomorphometry, and degree of mineralization in bone (DMB) measurement. Soft X-ray showed that complete bone union occurred in all groups, regardless of treatment. Ultimate stress and elastic modulus in fractured femur were significantly higher in PTH-H than in CNT. Total area and percent bone area of the femur were significantly lower in both PTH-L and PTH-H than in CNT. Callus porosity decreased dose-dependently following PTH treatment. Mean DMB of callus was significantly higher in PTH-H than in CNT or PTH-L. These results suggested that PTH decreased callus size and accelerated callus maturation in the fractured femora.

PTH accelerates the natural fracture healing process by shrinking callus size and increasing degree of mineralization of the fracture callus, thereby restoring intrinsic material properties of osteotomized femur shaft in cynomolgus monkeys although there were no significant differences among the groups for structural parameters.     

Enhancement of graft bone healing by intermittent administration of human parathyroid hormone (1-34) in a rat spinal arthrodesis model

Bone grafting is commonly used to treat skeletal disorders associated with large bone defect or unstable joint. It can take several months, however, to achieve a solid union and bony fusion sometimes delays or fails especially in osteoporosis patients. Therefore, we used a rat spinal arthrodesis model to examine whether intermittent administration of human PTH(1-34) accelerates bone graft healing. Eighty-two male Sprague-Dawley rats underwent posterolateral spinal arthrodesis surgery using autologous bone grafts. Animals were given daily subcutaneous injections of hPTH(1-34) (40 microg/kg/day PTH group) or 0.9% saline vehicle (control group) from immediately after surgery till death. Five rats each were killed 2, 4, 7, and 14 days after the surgery, and mRNA expression analysis was performed on harvested grafted bone. Seven rats each were killed 14, 28, and 42 days after the surgery, and the lumbar spine, which contained the grafted spinal segment, was subjected to fusion assessment, microstructural analysis using three-dimensional micro-computed tomography, and histologic examination. Serum bone metabolism markers were analyzed. The results indicated that PTH administration decreased the time required for graft bone healing and provided a structurally superior fusion mass in the rat spinal arthrodesis model. PTH administration increased the fusion rate on day 14 (14% in the control group and 57% in the PTH group), accelerated grafted bone resorption, and produced a larger and denser fusion mass compared to control. mRNA expression of both osteoblast- and osteoclast-related genes was upregulated by PTH treatment, and serum bone formation and resorption marker levels were higher in the PTH group than in the control group. Histologically calculated mineral apposition rate, mineralized surface and osteoclast surface were also higher in the PTH group than in the control group. These findings suggest that intermittent administration of PTH(1-34) enhanced bone turn over dominantly on bone formation at the graft site, leading to the acceleration of the spinal fusion. Based on the results of this study, intermittent injection of hPTH(1-34) might be an efficient adjuvant intervention in spinal arthrodesis surgery and all other skeletal reconstruction surgeries requiring bone grafts.     

Effects of cyclic vs. daily treatment with human parathyroid hormone (1-34) on murine bone structure and cellular activity

Previously, we demonstrated that the human parathyroid hormone (1-34) fragment (hPTH(1-34)) increased bone strength in proportion to its effects on BMD and cortical bone structure in the murine femur by comparing cyclic vs. daily administration of hPTH(1-34). Both cyclic and daily regimens increased vertebral BMD similarly at 7 weeks. Here, we have examined the effects of daily and cyclic PTH regimens on bone structure and cellular activity by static and dynamic histomorphometry. Twenty-week-old, intact female C57BL/J6 mice were treated with the following regimens (n=7 for each group): daily injection with vehicle for 7 weeks [control]; daily injection with hPTH(1-34) (40 microg/kg/day) for 7 weeks [daily PTH]; and daily injection with hPTH(1-34) (40 microg/kg/day) and vehicle alternating weekly for 7 weeks [cyclic PTH]. At days 9 and 10, and 2 and 3 prior to euthanasia, calcein (10 mg/kg) was injected subcutaneously. At the end of study, the lumbar vertebrae 1-3 and the left femora were excised, cleaned, and processed for histomorphometry. In the lumbar vertebrae, daily and cyclic PTH regimens significantly increased cancellous bone volume (BV/TV), trabecular number, trabecular osteoclast and osteoblast perimeters, trabecular mineral apposition rate (MAR) and bone formation rate (BFR), and periosteal MAR and BFR compared to control, with no significant difference between the two PTH-treated groups. Increased trabecular tunneling was observed in both PTH-treated groups. Both regimens tended to increase vertebral cortical bone formation parameters with the effects at the periosteum site being more marked than those at the endosteum site, resulting in a significant increase in cortical width. In the femur, the effects of cyclic PTH on BV/TV, trabecular width and number, trabecular and endocortical osteoblast and osteoclast perimeters, cortical width, and trabecular and periosteal BFR were less marked than those of daily PTH. A cyclic PTH regimen was as effective as a daily regimen in improving cancellous and cortical bone microarchitecture and cellular activity in the murine vertebra.     

hPTH 1–34 treatment of osteoporosis with added hormone replacement therapy: Biochemical,kinetic and histological responses

Twelve patients with vertebral fracture osteoporosis were recruited into a trial of treatment with hPTH 1–34 by daily injection for 1 year combined (from the 5th month) with an anti-resorptive agent (oestrogen, n=9; nandrolone, n=3). Treatment outcomes were monitored by biochemical and radiotracer measurements together with histomorphometry of transiliac biopsies before and at the end of treatment following double in vivo pre-labelling with demethylchlortetracyc-line. Indices of whole body bone formation, obtained from the analysis of⁸⁵Sr data, showed substantial increases (P<0.005) for all three indices measured) while biochemical (hydroxyproline) and kinetic measurements of bone resorption showed modest and equivocal changes only. As a result calcium balance improved. Gastrointestinal calcium absorption showed a tendency to improve, while urine calcium decreased; but these changes were statistically not significant except for radiocalcium absorption in the oestrogen treated subgroup. Histomorphometry revealed substantial increases in cancellous bone volume as reported previously with hPTH 1–34 given alone. However, iliac (as distinct from whole body) indices related to bone formation and resorption appeared to have returned towards pre-treatment values by the time of the second biopsy under the influence of the anti-resorptive agent given with the hPTH 1–34. It is confirmed that hPTH 1–34 therapy can increase iliac cancellous bone mass (as well as spinal cancellous bone mass as reported earlier) without a long-term increment in whole body bone resorption, providing the hPTH is combined with an anti-resorptive agent.