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

Abstract

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 E 2 (PGE 2 ), 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.

In vivo effects of two novel ALN-EP4a conjugate drugs on bone in the ovariectomized rat model for reversing postmenopausal bone loss

“…The appropriate dosage was determined based on the results from our earlier study [31] as well as the pharmacokinetic parameters of the C1 conjugate as determined by radiolabeling studies, which allows the calculation that a 5 mg/kg weekly dose of C1H should provide a sustained release of EP4a at approximately 14 μg/kg/day. This is comparable to the rate of PGE 2 release in a previous conjugate study shown to be effective in reversing bone loss in OVX rats [35].…”

“…Following sacrifice, tibiae were processed and stained for dynamic and static histomorphometry analysis according to procedures described in our previous study [31]. All static and dynamic histomorphometric analyses were performed using the Bioquant Osteo 11.2.6 MIR software (Bioquant Image Analysis Corporation).…”

“…The sixth lumbar vertebrae and left femurs were scanned using the SkyScan 1174 Compact Desktop Micro-CT machine (Bruker microCT, Belgium) to determine volumetric bone mineral density (vBMD) and bone microarchitecture based on the setup described previously [31]. All scans were performed using the following settings: X-ray voltage=5 kV, X-ray current=800 μA, and frame averaging=2, with a 0.25-mm aluminum filter to remove image noise and an isotropic voxel size of 11.6 μm.…”

“…Past studies have shown that ovariectomy-induced bone loss in rats shares many similar characteristics with postmenopausal bone loss in humans, making it a suitable animal model for the current study [33]. Previously, our laboratory conducted a 6-week long treatment study examining the effects of the C1 conjugate on OVX rats, which was shown to exert robust anabolic effects on the lumbar vertebrae and femurs [31]. The current study aims to confirm and further characterize the effects of C1 conjugate on bone in a longer, 3-month drug treatment study and to investigate the anabolic effects of the more stable C2 conjugate in comparison to C1 on reversing bone loss due to ovariectomy.…”

“…Since these chemical groups have been shown to be required for the biological functions of ALN [1] and EP4a [30], respectively, the conjugate drugs are not expected to exert drug effects until they are cleaved to yield the ALN and EP4a components. Past in vivo experiments conducted by our laboratory with C1 have shown efficacy for bone growth stimulation in rats, and that cleavage of the LK is required for ALN to fully exert its antiresorptive effects on bone [31].…”