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


BACKGROUND
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 treatment 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.
Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, ON, M5S 1A8, Canada.|Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.
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Hu
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.|Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada.
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Liu
Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
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Chen
Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada.|Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada.
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Willett
Department of Chemistry, Simon Fraser University, Burnaby, BC, V5A 1S6, Canada.
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Young
Department of Laboratory Medicine and Pathology, University of Toronto, Toronto, ON, M5S 1A8, Canada. grynpas@mshri.on.ca.|Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, M5G 1X5, Canada. grynpas@mshri.on.ca.|Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, M5S 3G9, Canada. grynpas@mshri.on.ca.|, 25 Orde St., Suite 417, Toronto, ON, M5T 3H7, Canada. grynpas@mshri.on.ca.
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Grynpas
D019386:Alendronate / Q000627:therapeutic use*; D000818:Animals; D015519:Bone Density / Q000187:drug effects; D050071:Bone Density Conservation Agents / Q000627:therapeutic use*; D016723:Bone Remodeling / Q000187:drug effects; D004195:Disease Models, Animal; D004338:Drug Combinations; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005260:Female; D006801:Humans; D008159:Lumbar Vertebrae / Q000187:drug effects / Q000503:physiopathology; D015663:Osteoporosis, Postmenopausal / Q000188:drug therapy* / Q000503:physiopathology; D010052:Ovariectomy; D017207:Rats, Sprague-Dawley; D058309:Receptors, Prostaglandin E, EP4 Subtype / Q000819:agonists* / Q000737:chemistry / Q000627:therapeutic use; D013329:Structure-Activity Relationship; D016474:Weight-Bearing / Q000502:physiology; D055114:X-Ray Microtomography / Q000379:methods
D050071:Bone Density Conservation Agents; D004338:Drug Combinations; D058309:Receptors, Prostaglandin E, EP4 Subtype; D019386:Alendronate
Anabolic drug; Biomechanical testing; Bisphosphonates; Bone histomorphometry; Conjugate drug; Osteoporosis