An engineered dual function peptide to repair fractured bones
Stewart A Low et al. J Control Release. 2022 Oct.
Abstract
Targeted drug delivery, often referred to as "smart" drug delivery, is a process whereby a therapeutic drug is delivered to specific parts of the body in a manner that increases its concentration at the desired sites relative to others. This approach is poised to revolutionize medicine as exemplified by the recent FDA approval of Cytalux (FDA approves pioneering drug for ovarian cancer surgery - Purdue University News) which is a folate-receptor targeted intraoperative near infrared (NIR) imaging agent that was developed in our laboratories. Fracture-associated morbidities and mortality affect a significant portion of world population. United states, Canada and Europe alone spent $48 billion in treating osteoporosis related fractures although this number doesn't count the economic burden due to loss in productivity. It is estimated that by 2050 ca 21 million hip fractures would occur globally which will be leading cause of premature death and disability. Despite the need for improvement in the treatment for fracture repair, methods for treating fractures have changed little in recent decades. Systemic delivery of fracture-homing bone anabolics holds great promise as a therapeutic strategy in this regard. Here we report the design of a fracture-targeted peptide comprised of a payload that binds and activates the parathyroid hormone receptor (PTHR1) and is linked to a targeting ligand comprised of 20 D-glutamic acids (D-Glu20) that directs accumulation of the payload specifically at fracture sites. This targeted delivery results in reduction of fracture healing times to <1/2 while creating repaired bones that are >2-fold stronger than saline-treated controls in mice. Moreover, this hydroxyapatite-targeted peptide can be administered without detectable toxicity to healthy tissues or modification of healthy bones in dogs. Additionally, since similar results are obtained upon treatment of osteoporotic and diabetic fractures in mice, and pain resolution is simultaneously accelerated by this approach, we conclude that this fracture-targeted anabolic peptide displays significant potential to revolutionize the treatment of bone fractures.
Keywords: Anabolic peptide; Fracture healing; PTHR1 receptor; Targeted delivery.
Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.
PubMed Disclaimer
Conflict of interest statement
Declaration of Competing Interest P.S.L and S.A.L own stock in Quince Tx. S.A.L, C.M.C, M.T. and J.J.N are employees of Quince Tx.
Figures Fig. 1.
Analysis of the biodistribution and pharmacokinetics of D-Glu20 targeted peptides. A. 99mTc-labeled peptides were injected via tail vein into mice with fractured femurs and its biodistribution was imaged 18 h later. B&C. 125I-labeled abaloparatide and 125I-Ab46-D-Glu20 were injected into mice with fractured femurs via tail vein and their clearance from the bloodstream (panel B) and fracture site (panel C) were quantitated. (Each time point represents data on 5 sacrificed mice; error bars = SEM). Fig. 2.
Analysis of the impact of Ab46-D-Glu20 on the healing of fractured femurs containing internal stabilizing rods. A–C. Comparison of microCT scans of representative fractures from mice treated with vehicle (A), abaloparatide (B), and Ab46-D-Glu20 (C) obtained 3 weeks after induction of femur fracture. D and E. Morphometric analysis of the calcified fracture volume (BV; panel D) and BV/TV (panel E). F and G, Mechanical strength analysis of the maximum load (panel F) and work to fracture the repaired femurs (panel G). (n = 10 mice per group; Error bars = SEM). Statistical analyses were made using one way ANOVA (*P < 0.05). Fig. 3.
Evaluation of the impact of Ab46-D-Glu20 on the healing of fractured femurs induced in osteoporotic mice via ovariectomy. A-C. Comparison of microCT scans of representative fractures from mice treated with saline (vehicle, panel A), estrogen (panel B), or Ab46-D-Glu20 (panel C) obtained 4 weeks after induction of femur fracture. D. and E. Morphometric analysis of the calcified fracture volume (BV, panel D) and BV/TV (panel E). F. Comparison of the maximum load before refracture among the different treatment cohorts. (n = 10 mice per group; Error bars = SEM). Statistical analyses were made using one way ANOVA (*P < 0.05; ns, not significant). Fig. 4.
Evaluation of the impact of Ab46-D-Glu20 on the healing of fractured femurs in mice induced to develop diabetes by injection of streptozotocin and maintenance of blood glucose above 250 mg/dL for two months prior to induction of femur fracture. A–C. Comparison of microCT scans of representative fractures from mice treated with saline (vehicle, panel A), insulin (panel B), and Ab46-D-Glu20 (panel C) obtained 4 weeks after the induction of femur fracture. D. and E. Morphometric analysis of the calcified fracture volume (BV, panel D) and BV/TV (panel E). F. and G. Comparison of the maximum load and work to fracture among the treatment cohorts. (n = 10 mice per group; Error bars = SEM). Statistical analyses were made using one way ANOVA (*P ≤ 0.05, **P ≤ 0.01; ***P ≤ 0.001; ns, not significant). Fig. 5.
Evaluation of discomfort experienced by mice with broken femurs treated with saline (●), Ab46-D-Glu20 (■), or ibuprofen (▴). Total distance traveled (panel A), velocity (panel B), and time spent moving (panel C) were videorecorded and analyzed by the program Ethovision XT. (n = 10 mice per group). Fig. 6.
Analysis of the toxicity of Ab46-D-Glu20 in dogs. Beagles were dosed daily for 17 weeks with 0, 0.5, 1.6, or 5 nmol/kg/day of Ab46-D-Glu20 and then sacrificed for toxicology analyses. Based on allometric scaling, the 0.5 nmol/kg/day dose was calculated to be comparable to both the usual human dose (20 nmol/person/day) as well as the dose used in the murine studies above (3.8 nmol/kg/day). Body weights (A), heart rates (B), serum calcium (C) and serum alkaline phosphatase (D) were monitored regularly. Representative histology images of the kidneys (E & F), liver (G & H) and injection site (I & J) from dogs treated with either saline (left panels) or 5 nmol/kg/d Ab46-D-Glu20 (right panels) reveal no obvious toxicities (Fig. S8). (n = 3 dogs in the saline cohort and 2 dogs each in the other three cohorts).
References
Fracture Trends, BMUS: The Burden of Musculoskeletal Diseases in the United States, available at, https://www.boneandjointburden.org/2014-report/via23/fracture-trends.
Karataglis D, Stavridis SI, Petsatodis G, Papadopoulos P, Christodoulou A, New trends in fixation of proximal humeral fractures: a review, Injury 42 (2011) 330–338, 10.1016/j.injury.2010.10.016. - DOI - PubMed
Agarwal A, Nonunions: Diagnosis, Evaluation and Management, Springer, 2017.
Burge R, Dawson-Hughes B, Solomon DH, Wong JB, King A, Tosteson A, Incidence and economic burden of osteoporosis-related fractures in the United States, 2005–2025, J. Bone Miner. Res 22 (2007) 465–475, 10.1359/jbmr.061113. - DOI - PubMed
Nielsen JJ, Low SA, Ramseier NT, Hadap RV, Young NA, Wang M, Low PS, Analysis of the bone fracture targeting properties of osteotropic ligands, J. Control. Release (2020), 10.1016/j.jconrel.2020.09.047. - DOI - PubMed