Six Weeks of Daily Abaloparatide Treatment Increased Vertebral and Femoral Bone Mineral Density, Microarchitecture and Strength in Ovariectomized Osteopenic Rats

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to fractures at multiple skeletal sites, most often at the spine, hip or wrist [1]. Osteoporosis is estimated to affect >20 million Americans, with 1.5 million osteoporotic fractures occurring in the USA every year [2]. As the population ages, the prevalence of osteoporosis and the incidence of osteoporotic fracture is increasing. Parathyroid hormone (PTH) and PTH(1-34) (teriparatide) are the only anabolic agents currently approved for treatment of osteoporosis. PTH is secreted by the parathyroid glands in response to low plasma calcium and acts directly on bone and kidneys to restore blood calcium levels [3]. PTH and PTH(1-34) were shown to be safe and effective in increasing bone mass and reducing fracture risk in postmenopausal women with osteoporosis [4, 5]. The anabolic mechanism of PTH and PTH(1-34) involves the stimulation of new bone formation, but this action is typically coupled by increased bone resorption activity [3, 6‐8], and recent clinical data indicate that this resorptive response may significantly limit bone mineral density (BMD) gains achieved with intermittent PTH(1-34) therapy, notably at cortical sites [9]. Moreover, increased bone resorption with PTH(1-34) is associated with and may contribute to increase serum calcium levels [10, 11].

Signaling mediated via the PTH 1 receptor (PTHR1) plays an important role in skeletal development and homeostasis. Although both PTH and PTH-related peptide (PTHrP) act through the common PTHR1, mounting evidence suggests distinct skeletal roles for PTH and PTHrP. While both ligands can stimulate bone formation pharmacologically, PTHrP is uniquely involved in maintaining post-natal bone mass. For example, mice genetically deficient in PTHrP develop osteoporosis [12‐14], whereas mice deficient in PTH exhibit increased bone mass that may be indirectly mediated by a local compensatory induction of PTHrP [13]. These findings suggest that PTHrP is a naturally occurring skeletal anabolic agent, the lack of which is not physiologically compensated by endogenous PTH [12‐16]. Distinct receptor binding properties may be responsible for the differential effects of PTH and PTHrP [17‐19]. Consistent with the genetic evidence for PTHrP as an important endogenous anabolic factor, a pharmacology study in adult ovariectomized (OVX) rats showed that 6 months of daily administration of native PTHrP(1-36) markedly enhanced bone mass and biomechanical properties [20]. Short‐term studies with limited numbers of human volunteers also indicate that treatment with native PTHrP(1-36) increased bone formation markers [21‐24] and BMD [24], which has driven interest over the past several years in testing PTHrP(1-36) as a potential treatment for osteoporosis. The results of these studies have been mixed, with some suggesting that intermittent administration of high-dose native PTHrP(1-36) increases bone formation without concomitant stimulation of bone resorption and others reporting measurable stimulation of bone resorption and significant hypercalcemia [21‐24].

Abaloparatide (ABL; previously referred to as BA058) is a novel 34 amino acid peptide selected for its potent and selective activation of PTHR1 signaling. Abaloparatide has 41 % homology to PTH(1-34) and 76 % homology to PTHrP(1-34) and was selected to retain potent bone anabolic activity but with a limited effect on bone resorption and low calcium-mobilizing potential [25]. In a complete Phase 2 clinical trial in postmenopausal woman with osteoporosis, daily subcutaneous injection of abaloparatide for 24 weeks increased BMD of the lumbar spine, total hip and femoral neck in a dose-dependent manner, in association with increased biochemical markers of bone formation. Moreover, the abaloparatide-induced BMD increases at the total hip were greater than achieved with teriparatide treatment [25]. These results are consistent with the bone anabolic activity and profile of abaloparatide.

The current work represents the first published nonclinical data on the effects of abaloparatide on bone. This study assessed the effects of 6-week daily administration of 2 doses levels of abaloparatide on bone in adult osteopenic OVX rats. The OVX rat model of postmenopausal osteoporosis allowed for high-resolution imaging of various skeletal sites to quantify microarchitectural and geometric changes in cancellous and cortical bone. Destructive ex vivo biomechanical testing was also performed to assess changes in the strength of the femur shaft, femur neck and lumbar vertebra. Results of the current study indicated a favorable efficacy and bone safety profile with 6 weeks of abaloparatide administration, which serves as a foundation for future bone quality, efficacy and safety studies of this novel agent.

Abaloparatide is a novel peptide activator of the PTH1 receptor signaling pathway that is currently in late-stage clinical development for the treatment of postmenopausal women with osteoporosis. The primary goal of osteoporosis therapies is to reduce fracture risk by improving bone strength, and the gold-standard approach for assessing treatment effects on bone strength is destructive ex vivo biomechanical testing. The primary objective of this study was to combine destructive bone strength testing with high-resolution CT imaging in order to assess the effects of a 6-week regimen of daily abaloparatide injections in adult OVX rats. We hypothesized that abaloparatide treatment would reverse bone loss and the deterioration of bone mechanical properties associated with OVX-induced osteopenia by promoting gains in bone mass and restoration of bone microarchitecture. The current results, the first that describe the effects of abaloparatide on bone strength, indicate that treatment with abaloparatide fully reversed OVX-induced bone loss and increased bone strength at the lumbar spine, femur diaphysis and femur neck of OVX rats. In many cases, bone mass and strength parameters in the OVX-ABL groups exceeded those of ovary-intact sham controls, which further highlights the robust efficacy potential of abaloparatide. These observations of marked gains in bone mass following treatment with ABL in osteopenic OVX rats are consistent with the robust BMD gains seen in a Phase 2 clinical trial with abaloparatide treatment in postmenopausal woman with osteoporosis [25].

Abaloparatide administration to osteopenic OVX rats fully and dose-dependently restored aBMD at the lumbar spine and femur by 6 weeks, indicating a rapid onset of effects in cortical and cancellous compartments. Gains in spine and femur aBMD appear to be mediated primarily by increases in trabecular and cortical bone volume. Improvements in cortical bone were evident from increased aBMD of the mid-femur diaphysis, a purely cortical site, and from µCT-derived evidence for increases in cortical bone volume and cortical thickness. Improvements in trabecular bone with abaloparatide were revealed by µCT assessments of L4 and the distal femur metaphysis, which demonstrated increases in trabecular bone volume and vBMD, and improved trabecular microarchitecture. Microarchitectural improvements with abaloparatide included more numerous and thicker trabeculae, with reduced spacing and a more plate-like arrangement, each of which may have contributed to the observed improvements in vertebral strength. High-dose abaloparatide was also associated with reduced trabecular connectivity density values for L4 and the distal femur, and analyses of human vertebrae suggest that reduced connectivity density may be associated with greater structural strength [28]. This seemingly paradoxical relationship does not indicate that reduced connectivity per se is biomechanically advantageous, but that trabecular structures with more connections can in some cases be less robust.

Abaloparatide-related gains in bone mass and improvements in bone microarchitecture parameters were associated with improved biomechanical parameters. Evidence that BMD gains with abaloparatide leads to improved bone strength in this preclinical osteoporosis model extends and complements recently reported clinical study results where abaloparatide treatment increased BMD in lumbar spine and total hip in a Phase 2 clinical study of postmenopausal women with osteoporosis [25]. BMD gains with abaloparatide were rapid in both studies, reversing the catabolic effects of 8 weeks of OVX-induced estrogen deficiency within 6 weeks in these rats, and significantly increasing lumbar spine and total hip BMD within 12 weeks in women with osteoporosis [25]. In the Phase 2 study, BMD gains with abaloparatide were greater than those observed with teriparatide [rhPTH(1-34)] at both the 12- and 24-week time points [25]. Data on the effect of abaloparatide on vertebral and nonvertebral fracture risk are starting to emerge from a Phase 3 clinical trial [29], which support the notion that rapid and substantial BMD gains with abaloparatide are accompanied by substantial fracture risk reduction.

The effects of abaloparatide treatment were seen in all regions of the femur, suggesting that the effect on BMD potentially includes positive effects on both the trabecular and cortical bone compartments. The pharmacological mechanisms underlying the BMD effects in the cortical bone observed with abaloparatide treatment are not entirely clear. Additional studies will be required to further evaluate the potential effects on both the trabecular and cortical compartments. The current study provides the first animal data indicating increased cortical BMD with abaloparatide, along with increases in trabecular bone microarchitecture parameters, which were associated with increased bone strength.

Destructive biomechanical assessments of bone strength revealed substantial improvements in structural strength in abaloparatide-treated animals. The effects of abaloparatide included greater maximum load for L4, the femur diaphysis and femur neck relative to either OVX-Veh or Sham controls. It may be noteworthy from a bone quality perspective that abaloparatide-induced increments in maximum load of L4 and the femur diaphysis were well aligned with the magnitude of gains in bone mass parameters measured at the same skeletal site, which suggests that bone matrix that accrues during abaloparatide therapy has good material properties. Femur BMD values were positively and similarly correlated with maximum load values in the two Veh control groups and in the two abaloparatide groups, suggesting that abaloparatide had no deleterious effects on bone quality. Bone quality was also assessed by adjusting structural strength parameters for bone geometry (femur diaphysis) or for specimen size and bone volume fraction (L4 vertebral body). By this approach, both abaloparatide groups showed similar material properties compared with OVX-Veh control groups for both skeletal sites, indicating maintenance of material strength properties. Based on these findings, it is reasonable to conclude that increased bone structural strength with abaloparatide is largely a function of increased bone mass, density and volume. These initial bone quality assessments will be followed by additional studies conducted with longer treatment durations to better assess the impacts of abaloparatide on bone strength and bone quality.

The molecular mechanisms by which abaloparatide exerts its effects on bone are not fully understood but may be related to confirmation-selective binding to the PTH1 receptor relative to other ligands including PTH and PTHrP. PTH and PTHrP share some sequence homology and may have arisen by duplication of a common ancestral gene, but each plays a distinct role in bone physiology. PTH, which is secreted by the parathyroid glands, acts in a classical endocrine manner to promote osteoclastic bone resorption and calcium mobilization. In contrast, PTHrP functions as a paracrine regulator of bone formation. Despite these differences, PTH and PTHrP both increase intracellular cAMP concentrations by activating the same PTHR1, a G protein-coupled receptor (GPCR). However, continuous administration of PTH leads to bone resorption over formation, whereas continuous PTHrP administration preferentially stimulates formation [30, 31]. Recent studies have provided a basis for the divergent actions of PTH and PTHrP in bone. Specifically, PTHrP activity at PTHR1 is restricted to the cell surface and yields a brief intracellular cAMP burst, whereas the receptor conformation associated with PTH stabilizes its binding to the receptor and its coupled G protein and moves to internalized compartments of the cell, leading to persistent cAMP generation [17, 19, 32, 33]. The significance of ligands that form more stable complexes and more cAMP responses is a more catabolic response resulting in elevated blood calcium levels [18]. In contrast, ligands such as PTHrP transiently produce cAMP and mobilize calcium, yet results in greater anabolic action than PTH. Consistent with these reports, a recent study evaluated the binding of abaloparatide to two distinct PTHR1 conformations. The findings suggested that the enhanced bone anabolic activity seen with abaloparatide treatment may arise from a more selective binding to the R0 PTHR1 than the RG conformation, compared to PTH (1-34), a long-acting PTH (LA-PTH) analog or PTHrP [34]. Further studies are required to elucidate the specific molecular mechanisms of abaloparatide that cause increased anabolic activity.

This study has several strengths, including a robust number of animals that provided good statistical power for the ex vivo assessments of bone architecture and strength. Many novel bone therapeutics were initially tested in healthy animals with normal bone mass, whereas this index pharmacology report for abaloparatide involved ovariectomized rats with established osteopenia, which represents a reasonably high hurdle for efficacy and proof of concept for osteoporosis therapy. The study also involved bone strength assessments at multiple skeletal sites that correspond to areas at increased risk of fracture in postmenopausal women. The study also has several limitations, some of which relate to the study goal as an initial pharmacology assessment, including the use of young growing animals, the absence of data for histomorphometry and for bone turnover markers. The study also lacked µCT data on the proximal femur including the femur neck, and therefore, the compartment-specific structural changes associated with improved femur neck strength in the abaloparatide groups remain unknown. Future longer-term studies will address these questions.

In summary, 6 weeks of abaloparatide administration to OVX osteopenic rats increased bone mass and microarchitecture parameters and increased bone strength. Abaloparatide was well tolerated, and there was no evidence for impairment of bone quality or reductions in bone material properties. The overall data indicate that abaloparatide administration results in rapid and robust gains in bone mass and bone strength, and support the continued investigation of abaloparatide as potential therapy for the treatment of postmenopausal women with osteoporosis.