In Vivo Bone Effects of a Novel Bisphosphonate‐EP4a Conjugate Drug (C3) for Reversing Osteoporotic Bone Loss in an Ovariectomized Rat Model
Sheikh Z, Chen G, Al-Jaf F, et al. In Vivo Bone Effects of a Novel Bisphosphonate-EP4a Conjugate Drug (C3) for Reversing Osteoporotic Bone Loss in an Ovariectomized Rat Model. JBMR Plus. September 2019
Pathological bone loss is a regular feature of postmenopausal osteoporosis and the microstructural changes along with the bone loss make the individual prone to getting hip, spine and wrist fractures. We have developed a new conjugate drug named C3, that has a synthetic, stable EP4 agonist (EP4a) covalently linked to an inactive alendronate (ALN) that binds to bone and allows physiological remodeling. After losing bone for 12 weeks, seven groups of rats were treated for 8 weeks via tail vein injection. The groups were: C3 conjugate at low and high doses, vehicle‐treated ovariectomy (OVX) and sham, C1 (a similar conjugate but with active alendronate at high dose), inactive ALN alone and mixture of unconjugated ALN and EP4a to evaluate the conjugation effects. Bone turnover was determined by dynamic and static histomorphometry; MicroCT was employed to determine bone microarchitecture; and bone mechanical properties were evaluated via biomechanical testing. Treatment with C3 significantly increased trabecular bone volume and vertebral bone mineral density versus OVX controls. There was also significant improvement in the vertebral load bearing abilities and stimulation of bone formation in femurs after C3 treatment. This preclinical research revealed that C3 resulted in significant anabolic effects on trabecular bone and EP4a and ALN conjugation components is vital to conjugate anabolic efficacy. A combined therapy using an EP4 selective agonist anabolic agent linked to an inactive ALN is presented here that produces significant anabolic effects, allows bone remodeling and has the potential for treating post‐menopausal osteoporosis or other diseases where bone strengthening would be beneficial.
A Novel Anabolic Conjugate (C3) in the Matrix of Dicalcium Phosphate Onlay Block Grafts for Achieving Vertical Bone Augmentation: An Experimental Study on Rabbit Calvaria
Sheikh Z, Chen G, Thévenin M, Young R, Grynpas M, Glogauer M. Int J Oral Maxillofac Implants. 2019;34(4)
PURPOSE: Achieving successful and predictable alveolar ridge augmentation in the vertical dimension is extremely challenging. Several materials have been investigated to achieve vertical ridge augmentation; however, the results are highly unpredictable. The collaborative team presenting this research has developed brushite- and monetite-based grafts that incorporate in their matrix a novel bone anabolic conjugate (C3) of a bisphosphonate and a potent bone-activating EP4 receptor agonist. The study objective was to investigate the potential of these graft formulations to achieve rapid, enhanced, and clinically significant bone regeneration in the vertical dimension.MATERIALS AND METHODS: Brushite and monetite grafts were fabricated and characterized for phase purity, porosity, compressive strength, and microstructural morphology. They were implanted in 12 rabbit calvaria for 12 weeks. Each group (n = 6): brushite control, brushite with C3, monetite control, and monetite with C3. Postmortem samples were retrieved and processed for analysis. The percentage bone volume, vertical bone height gained, and graft resorption were calculated and assessed.RESULTS: The brushite and monetite grafts containing C3 integrated well onto the calvarial bone surface, with new bone extending through the graft area (36% and 80%, respectively), while the C3 lacking grafts showed decreased surface integration and bone infiltration (28% and 38%, respectively). The C3 containing brushite and monetite grafts demonstrated bone growth vertically (1.8 mm and 2.7 mm, respectively) and improved graft resorption.CONCLUSION: The brushite and monetite-based grafts loaded with the C3 conjugate resulted in greater de novo bone formation in the vertical dimension when compared with the grafts without the drug. However, the monetite grafts produced much more and predictable vertical height gain than was achieved with brushite grafts. The advantages of this new graft drug formulation in future would be to provide more predictable vertical bone regeneration, which will ultimately benefit patients undergoing dental implant placement.
Targeting therapeutics to bone by conjugation with bisphosphonates
Young RN, Grynpas MD. Curr Opin Pharmacol. 2018;40:87-94
Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This targeting ability has been exploited to design and prepare bisphosphonate conjugate prodrugs to deliver a wide variety of drug molecules selectively to bones. It is important that conjugates be stable in the blood stream and that conjugate that is not taken up by bone is eliminated rapidly. The prodrugs should release active drug at a rate appropriate so as to provide efficacy. Radiolabelling is the best method to quantify and evaluate pharmacokinetics, tissue distribution, bone uptake and release of the active drug(s). Recent reports have described bisphosphonate conjugates derived from the antiresorptive drug, alendronic acid and anabolic prostanoid drugs that effectively deliver prostaglandins and prostaglandin EP4 receptor agonists to bone and show enhanced anabolic efficacy and tolerability compared to the drugs alone. These conjugate drugs can be dosed infrequently (weekly or bimonthly) whereas the free drugs must be dosed daily.
In vivo effects of two novel ALN-EP4a conjugate drugs on bone in the ovariectomized rat model for reversing postmenopausal bone loss
Hu S, Liu CC, Chen G, Willett T, Young RN, Grynpas MD. Osteoporos Int. 2016;27(2):797-808.
Two alendronate-EP4 agonist (ALN-EP4a) conjugate drugs, C1 and C2, which differ in structure by a short linker molecule, were evaluated in ovariectomized (OVX) rats for their anabolic effects. We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. Introduction EP4as were covalently linked to ALN to create ALN-EP4a conjugate anabolic bone drugs, C1 and C2, which differ in structure by a short linker molecule in C1. When administered systemically, C1 and C2 are delivered to bone through targeted binding of ALN, where local hydrolytic enzymes liberate EP4a from ALN to exert anabolic effects. Here, we compare effects of C1 to C2 in a curative in vivo study. Methods Three-month-old female Sprague Dawley rats were OVX or sham operated and allowed to lose bone for 3 months. Animals were then treated via tail vein injections for 3 months and sacrificed. Treatment groups were as follows: C1L (5 mg/kg biweekly), C1H (5 mg/kg weekly), C2L (15 mg/kg monthly), C2H (15 mg/kg biweekly), OVX and sham control (phosphate-buffered saline (PBS) biweekly), and ALN/EP4a- unconjugated mixture (0.75 mg/kg each biweekly). Results MicroCT analysis showed that C1H treatment significantly increased vertebral bone mineral density (vBMD) and trabecular bone volume versus OVX controls while C2 treatments did not. Biomechanical testing showed that C1H treat- ment but not C2 treatments led to significant improvement in the load bearing abilities of the vertebrae compared to OVX controls. C1 stimulated endocortical bone formation and increased load bearing in femurs, while C2 did not. Conclusions We showed that C1 led to significant anabolic effects on cortical and trabecular bone while anabolic effects associated with C2 were minimal. These results led us to hypothesize a mode of action by which presence of a linker is crucial in facilitating the anabolic effects of EP4a when dosed as a prodrug with ALN.
Determination of the Rat in Vivo Pharmacokinetic Profile of a Bone- Targeting Dual-Action Pro-Drug for Treatment of Osteoporosis
Chen G, Arns S, Young RN. Bioconjug Chem. 2015;26(6):1095-1103
The in vivo hydrolytic pathway of a dual-function bone-targeting EP4 receptor agonist-bisphosphonate pro-drug was deduced from radiolabeling experiments. A 14C labeled pro-drug was used to monitor liberation of the bisphosphonate and results were compared to parallel studies where the EP4 receptor agonist was labeled with 3H. The bone-adsorption of the 14C pro-drug following an IV bolus was about 10% compared to 7.8% for the tritiated pro-drug. The difference in release half-life (5.2 and 19.7 days from 3H and 14C experiments, respectively) indicated that, after binding to bone, the initial hydrolysis occurred at the ester moiety of the linker releasing the EP4 agonist. The conjugate was found to concentrate in more porous, high-surface- area regions of the long bones. Both 3H and 14C experiments indicated a short circulating half-life (1−2 h) in blood. 1. Chen G, Arns S, Young RN. Determination of the rat in vivo pharmacokinetic profile of a bone-targeting dual-action pro-drug for treatment of osteoporosis. Bioconjug Chem. 2015;26(6):1095-1103. doi:10.1021/acs.bioconjchem.5b00160
Novel EP4 Receptor Agonist-Bisphosphonate Conjugate Drug (C1) Promotes Bone Formation and Improves Vertebral Mechanical Properties in the Ovariectomized Rat Model of Postmenopausal Bone Loss
Liu CC, Hu S, Chen G, et al. J Bone Miner Res. 2015;30(4):670-680
Current treatments for postmenopausal osteoporosis aim to either promote bone formation or inhibit bone resorption. The C1 conjugate drug represents a new treatment approach by chemically linking the antiresorptive compound alendronate (ALN) with the anabolic agent prostanoid EP4 receptor agonist (EP4a) through a linker molecule (LK) to form a conjugate compound. This enables the bone-targeting ability of ALN to deliver EP4a to bone sites and mitigate the systemic side effects of EP4a, while also facilitating dual antiresorptive and anabolic effects. In vivo hydrolysis is required to release the EP4a and ALN components for pharmacological activity. Our study investigated the in vivo efficacy of this drug in treating established bone loss using an ovariectomized (OVX) rat model of postmenopausal osteopenia. In a curative experiment, 3-month-old female Sprague-Dawley rats were OVX, allowed to lose bone for 7 weeks, then treated for 6 weeks. Treatment groups consisted of C1 conjugate at low and high doses, vehicle-treated OVX and sham, prostaglandin E2 (PGE2), and mixture of unconjugated ALN-LK and EP4a to assess the effect of conjugation. Results showed that weekly administration of C1 conjugate dose-dependently increased bone volume in trabecular bone, which partially or completely reversed OVX-induced bone loss in the lumbar vertebra and improved vertebral mechanical strength. The conjugate also dose-dependently stimulated endocortical woven bone formation and intracortical resorption in cortical bone, with high-dose treatment increasing the mechanical strength but compromising the material properties. Conjugation between the EP4a and ALN-LK components was crucial to the drug’s anabolic efficacy. To our knowledge, the C1 conjugate represents the first time that a combined therapy using an anabolic agent and the antiresorptive compound ALN has shown significant anabolic effects which reversed established osteopenia. © 2014 American Society for Bone and Mineral Research.
Design and synthesis of novel bone-targeting dual-action pro-drugs for the treatment and reversal of osteoporosis
Arns S, Gibe R, Moreau A, Monzur Morshed M, Young RN. Bioorg Med Chem. 2012;20(6):2131-2140.
There is an important medical need for effective therapies to redress the general bone loss associated with advanced osteoporosis. Prostaglandin E2 and related EP4 receptor agonists have been shown to stimulate bone regrowth but their use has been limited by systemic side effects. Herein is described the design and synthesis of novel dual-action bone-targeting conjugate pro-drugs where two classes of active agents, a bone growth stimulating prostaglandin E2 EP4 receptor subtype agonist (5 or 6) and a bone resorption inhibitor bisphosphonate, alendronic acid (1), are coupled using metabolically labile carbamate or 4-hydroxyphenylacetic acid based linkers. Radiolabelled conjugates 9, 11a/b and 25 were synthesized and evaluated in vivo in rats for uptake of the conjugate into bone and subsequent release of the EP4 agonists over time. While conjugate 11a/b was taken up (9.0% of initial dose) but not released over two weeks, conjugates 9 and 25 were absorbed at 9.4% and 5.9% uptake of the initial dose and slowly released with half-lives of approximately 2 weeks and 5 days respectively. These conjugates were well tolerated and offer potential for sustained release and dual synergistic activity through their selective bone targeting and local release of the complimentary active components.
A nonprostanoid EP4 receptor selective prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats
Ke HZ, Crawford DT, Qi H, et al. J Bone Miner Res. 2006;21(4):565-575
CP432 is a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 agonist. CP432 stimulates trabecular and cortical bone formation and restores bone mass and bone strength in aged ovariectomized rats with established osteopenia. INTRODUCTION: The purpose of this study was to determine whether a newly discovered, nonprostanoid EP4 receptor selective prostaglandin E2 (PGE2) agonist, CP432, could produce bone anabolic effects in aged, ovariectomized (OVX) rats with established osteopenia. MATERIALS AND METHODS: CP432 at 0.3, 1, or 3 mg/kg/day was given for 6 weeks by subcutaneous injection to 12-month-old rats that had been OVX for 8.5 months. The effects on bone mass, bone formation, bone resorption, and bone strength were determined. RESULTS: Total femoral BMD increased significantly in OVX rats treated with CP432 at all doses. CP432 completely restored trabecular bone volume of the third lumbar vertebral body accompanied with a dose-dependent decrease in osteoclast number and osteoclast surface and a dose-dependent increase in mineralizing surface, mineral apposition rate, and bone formation rate-tissue reference in OVX rats. CP432 at 1 and 3 mg/kg/day significantly increased total tissue area, cortical bone area, and periosteal and endocortical bone formation in the tibial shafts compared with both sham and OVX controls. CP432 at all doses significantly and dose-dependently increased ultimate strength in the fifth lumber vertebral body compared with both sham and OVX controls. At 1 and 3 mg/kg/day, CP432 significantly increased maximal load in a three-point bending test of femoral shaft compared with both sham and OVX controls. CONCLUSIONS: CP432 completely restored trabecular and cortical bone mass and strength in established osteopenic, aged OVX rats by stimulating bone formation and inhibiting bone resorption on trabecular and cortical surfaces. 2.
Prostaglandin Receptor EP4 Mediates the Bone Anabolic Effects of PGE
Machwate M, Harada S, Leu CT, et al. Mol Pharmacol. 2001;60(1):36-41.
Prostaglandin (PG) E2 is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE2 is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP4 , one of the PGE2 receptors, on PGE2 -induced bone formation in vivo. We first determined the ability of EP4 A, an EP4 -selective ligand, to act as an antagonist. PGE2 increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP4 A, suggesting that EP4 A acts as an EP4 antagonist in the cells at concentrations consistent with its in vitro binding to Prostaglandins, especially PGE2, have multiple effects on bone, including stimulation of both resorption and formation (Raisz at al., 1993; reviewed in Bergmann and Schoutens, 1995). PGE2 administered to rats in vivo increases cortical as well as trabecular bone mass (Jee et al., 1985, 1987; Mori et al., 1990; Suponitzky and Weinreb, 1998). PGE1, an alternate agonist with the same activity spectrum as PGE2, was shown to stimulate bone formation and cause hyperostosis in infants (Ueda et al., 1980; Ringel et al., 1982). Despite extensive documentation of in vivo bone anabolic effects, the cellular and molecular mechanisms that mediate PGE2 action remain unclear. In organ culture of fetal rat calvaria, PGE2 stimulates DNA synthesis in the periosteum, but suppresses collagen production (Raisz and Koolmans- Beynen, 1974). In the mouse MC3T3-E1 osteoblastic cell line, low concentrations of PGE2 increase cell proliferation, and high concentrations stimulate differentiation (Hakeda et al., 1986). These effects correlate with an increase in intracellular calcium and cAMP, respectively. In cultures of adult rat calvaria cells, PGE2 stimulates nodule formation, via a Ca2 dependent pathway (Kaneki et al., 1999). In rat RP-11 periosteal cells (Machwate et al., 1998), PGE2 increases cell This paper is available online at http://molpharm.aspetjournals.org EP4. We then examined the effect of EP4 on bone formation induced by PGE2 in young rats. Five- to 6-week-old rats were treated with PGE2 (6 mg/kg/day) in the presence or absence of EP4A (10 mg/kg/day) for 12 days. We found that treatment with EP4A suppresses the increase in trabecular bone volume induced by PGE2. This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE2 in rats is mediated by the EP4 receptor.
Prostaglandin E2-Bisphosphonate Conjugates: Potential Agents for Treatment of Osteoporosis
Gil L, Han Y, Opas EE, et al. Bioorganic Med Chem. 1999;7(5):901-919.
Conjugates of bisphosphonates (potential bone resorption inhibitors) and prostaglandin E2 (a bone formation enhancer) were prepared and evaluated for their ability to bind to bone and to liberate, enzymatically, free PGE2. The conjugate 3, an amide at C-1 of PGE2 proved to be too stable in vivo while conjugate 6, a thioester, was too labile. Several PGE2, C-15 ester-linked conjugates (18, 23, 24 and 31) were prepared and conjugate 23 was found to bind effectively to bone in vitro and in vivo and to liberate PGE2 at an acceptable rate. A 4-week study in a rat model of osteoporosis showed that 23 was better tolerated and more e ective as a bone growth stimulant than daily maximum tolerated doses of free PGE2.